BIOLOGY LIBRARY
UNIVERSITY OF CALIFORNIA
SCHOOL OF PUBLIC HEALTH
3563 LIFE SCIENCES BUILDING
r. LEGGE, M. D.
6 ROBLE ROAD
BERKELEY, CALIF.
WORKS BY
CHARLES F. BOLDUAN, M.D.
PUBLISHED BY
JOHN WILEY & SONS
Immune Sera.
A concise exposition of the main facts and
theories of infection and immunity, Fourth
edition, rewritten. By Charles F. Bolduan, M.D.
12mo, ix + 226 pages. Cloth, $1.50.
TRANSLATIONS.
The Suppression of Tuberculosis.
Together with Observations concerning Phthisio-
genesis in Man and Animals, and Suggestions con-
cerning the Hygiene of Cow Stables and the Pro-
duction of Milk for Infant Feeding, with Special
Reference to Tuberculosis. By Professor E. von
Behring, University of Marburg. Authorized
Translation by Charles F. Bolduan, M.D. 12mo,
vi + 85 pages. Cloth, $1.00.
Manual of Serum Diagnosis.
By Doctor O. Rostoski, University of Wurzburg.
Authorized Translation by Charles F. Bolduan,
M.D. 12mo, vi + 86 pages. Cloth, $1.00.
Studies in Immunity.
By Professor Paul Ehrlich. Translated by Charles
F. Bolduan, M.D. Second Edition, Revised and
Enlarged. 8vo, xi + 712 pages. Cloth, $6.00.
IMMUNE SERA
A CONCISE EXPOSITION OF THE
MAIN FACTS AND THEORIES OF
INFECTION AND IMMUNITY
BY
DR. CHARLES FREDERICK BOLDUAN
BACTERIOLOGIST, RESEARCH LABORATORY, DEPARTMENT OF HEALTH
CITY OF NEW YORK
FOURTH EDITION, REWRITTEN AND ENLARGED
FIRST THOUSAND
NEW YORK:
JOHN WILEY & SONS
LONDON: CHAPMAN & HALL, LIMITED
1911
9tl
67
COPYRIGHT, 1907, 1908, 1911.
BT CHARLES FREDERICK BOLDUAN
THE SCIENTIFIC PRESS
ROBERT DRUMMOND AND COMPANY
BROOKLYN, N. Y.
TO
HERMANN M. BIGGS, M.D., LL.D.
PIONEER IN SPECIFIC SERUM THERAPY AND SERUM
DIAGNOSIS IN AMERICA, FOUNDER OF THE
FIRST MUNICIPAL SERUM LABORATORY
IN THE WORLD, THIS VOLUME
IS DEDICATED AS A TOKEN
OF GREAT ADMIRATION
AND ESTEEM
PREFACE TO THE FOURTH EDITION
THE favorable reception accorded to the previous
editions of this book, together with the fact that
our knowledge of the subject has increased con-
siderably in the past few years, has led the author
to undertake a thorough revision of the work.
In its first edition, in 1904, this book dealt only
with certain antibodies whose discovery had aroused
a great deal of scientific interest, namely, hsemoly-
sins, cytotoxins, and precipitins. To this was
added, in subsequent editions, a discussion of anti-
toxins, agglutinins, and opsonins. All these topics
were naturally embraced under the title " Immune
Sera." In the present (fourth) edition, while the
old title has been retained, the scope of the sub-
ject matter has been greatly extended, so that now
there is presented an exposition of the main facts
of infection and immunity.
It is but natural that any discussion of the
immunity reactions should center about the ingen-
ious side-chain theory of Ehrlich, which has domi-
nated the work in this field. Its heuristic value
vi PREFACE
has unquestionably been very great. At the same
time it cannot be doubted that some of the deduc-
tions from the theory have led, here and there,
to strained conceptions which apparently violate
established biological facts. While presenting
Ehrlich's views at length, therefore, the author
has endeavored to bring out clearly just why and
wherein certain other investigators differ. The aim
of the book has been to present a broad, clear outline
of the main facts and theories concerning infection
and immunity, and while this may perhaps have
led to the omission of some really excellent studies,
it was felt best not to confuse the beginner with a
mass of apparently contradictory observations.
CHARLES BOLDUAN.
NEW YORK, April, 1911.
CONTENTS
PAGE
Antitoxins i
HISTORICAL ; i
PRESENT METHOD OF PRODUCING DIPHTHERIA ANTI-
TOXIN 2
PRODUCTION OF DIPHTHERIA TOXIN 2
IMMUNIZING THE ANIMALS 3
COLLECTING THE SERUM 4
TESTING THE STRENGTH OF THE SERUM 5
EHRLICH'S THEORY FOR PRODUCTION 6
TOXINS, TOXOIDS 6
RECEPTORS 9
WEIGERT'S OVERPRODUCTION THEORY 10
EXPERIMENTAL EVIDENCE FOR EHRLICH'S THEORY... . 13
ANTIGENS OR HAPTINS 16
NATURE OF ANTITOXINS IN GENERAL 17
TOXINS AND OTHER POISONOUS CELL DERIVATIVES IN
GENERAL 20
RELATIONS BETWEEN TOXIN AND ANTITOXIN 22
"L "and "L t " 23
PARTIAL SATURATION METHOD OF STUDYING TOXINS
TOXONS, TOXOIDS 23
EHRLICH'S " POISON SPECTRA " 24
VIEWS OF ARRHENIUS, BORDET, AND OTHERS 28
Agglutinins 3 2
THE PHENOMENON 32
H^MAGGLUTININS 34
ISOAGGLUTININS 35
PURPOSE OF AGGLUTINATION 36
HISTORICAL 37
PFAUNDLER'S REACTION (THREAD REACTION). . 38
vii
CONTENTS
PAGE
NATURE OF AGGLUTININS AND OF THE AGGLUTINATION
REACTION ...................................... 39
AGGLUTINOIDS ...................................... 39
GROUP AGGLUTININS : ............................... 43
ABSORPTION METHODS FOR DIFFERENTIATING BETWEEN
A MIXED AND A SINGLE INFECTION ................ 46
FORMATION OF AGGLUTININS ACCORDING TO THE ^IDE-
CHAIN THEORY, RECEPTORS OF FIRST, SECOND, AND
THIRD ORDER .................................. 47
Bacteriolysins and Haemolysins .................. 5 o
HISTORICAL ........................................ 50
FFEIFFER'S PHENOMENON ............................ 51
HAEMOLYSIS ........................................ 52
NATURE OF H^MOLYTIC SERA .................... . . 54
ANALOGY BETWEEN THE BACTERIOLYTIC AND HVEMO-
LYTIC PROCESSES ............................... 57
EHRLICH AND MORGENROTH ON THE NATURE OF HAEMO-
LYSIS .......................................... 58
THEIR THREE CLASSIC EXPERIMENTS ................ 59
NOMENCLATURE ................................... 63
ROLE OF THE IMMUNE BODY ............. .- ......... 64
ON WHAT THE SPECIFICITY DEPENDS ................ 65
DIFFERENCE BETWEEN A SPECIFIC SERUM AND A NOR-
MAL ONE ......................... .. ............ 66
DIVERGING VIEWS OF EHRLICH AND BORDET ........... 67
THE SIDE-CHAIN THEORY APPLIED TO THESE BODIES. . . 68
MULTIPLICITY OF COMPLEMENTS ...................... 70
THE BORDET-GENGOU PHENOMENON; NEISSER-
SACHS BLOOD TEST ............................. 71
NORMAL SERUM, ITS H^MOLYTIC AND BACTERIOLYTIC
, ACTION ........................................ 73
ACTIVE AND INACTIVE NORMAL SERUM ....... '. ...... 75
ACTION NOT ENTIRELY SPECIFIC .................... 77
MULTIPLICITY OF THE ACTIVE SUBSTANCES ........... 78
DIFFERENCE BETWEEN A NORMAL AND A SPECIFIC IM-
MUNE SERUM ................................... 79
NATURE OF THE IMMUNE BODY PARTIAL IMMUNE
BODIES OF EHRLICH. . 82
CONTENTS { x
PAGE
METCHNIKOFF'S VIEWS 84
SUPPORT FOR EHRLICH'S VIEW 85
ANTIH^MOLYSINS: THEIR NATURE ANTI-COMPLEMENT
OR ANTI-IMMUNE BODY 86
ANTI-COMPLEMENT 88
FLUCTUATIONS IN THE AMOUNT OF THE ACTIVE SUB-
STANCES IN SERUM 91
SOURCE OF THE COMPLEMENTS LEUCOCYTES AS A
SOURCE OTHER SOURCES 93
STRUCTURE OF THE COMPLEMENTS COMPLEMENTOIDS. . . 94
ISOLYSINS AUTOLYSINS ANTI-ISOLYSINS 97
DEFLECTION OF COMPLEMENT 100
PRACTICAL VALUE OF ANTI-BACTERIAL SERA 104
Precipitins 107
DEFINITIONS 107
BACTERIAL PRECIPITINS 108
LACTOSERUM OTHER SPECIFIC PRECIPITINS 108
SPECIFICITY OF THE PRECIPITINS 109
NATURE OF THE PRECIPITINS in
PRACTICAL APPLICATION 112
THE WASSERMANN-UHLENHUTH BLOOD TEST 113
IMMUNIZING THE ANIMALS 114
COLLECTING THE SERUM 115
THE TEST 116
APPEARANCE OF THE REACTION 117
DELICACY OF THE PRECIPITIN TEST 118
OTHER APPLICATIONS OF THE PRECIPITIN TEST , . . 1 18
ANTIPRECIPITINS ISOPRECIPITINS 119
Cy totoxins 121
DEFINITION, LEUCOTOXIN NATURE OF THE CYTOTOXIN
ANTICYTOTOXIN 121
Nt v UROTOXIN 122
SPERMOTOXIN 123
COMMON RECEPTORS 124
CYTOTOXIN FOR EPITHELIUM 124
CYTOTOXINS BY THE USE OF NUCLEOPROTEIDS 125
Opsonins 127
HISTORICAL 127
X CONTENTS
PAGE
BACTERIOTROPIC SUBSTANCES 129
OPSONINS DISTINCT ANTIBODIES. 130
STRUCTURE OF OPSONINS 130
THE OPSONIC INDEX 131
TECHNIQUE 131
VALUE OF THE OPSONIC MEASUREMENTS 133
vSnake Venoms and their Antisera 137
THE VENOMS 137
ANTIVENIN'S 139
Anaphylaxis 141
HISTORICAL 141
THE PHENOMENON 142
SERUM RASHES 143
THEORIES OF ANAPHYLAXIS 144
ALLERGY 147
SUPPOSED RELATION TO PRECIPITIN ACTION 148
PATHOLOGY OF ANAPHYLACTIC SHOCK 150
RELATION OF ANAPHYLAXIS TO SERUM THERAPY 151
Infection and Immunity 154
INFECTION 154
THE INFECTING AGENT 154
ANAPHYLATOXIN 157
RESISTANCE AGAINST INFECTION 158
NATURAL IMMUNITY 159
ACQUIRED IMMUNITY 160
MECHANISM OF IMMUNITY 162
RELATION OF ANAPHYLAXIS TO IMMUNITY 163
IMMUNITY REACTION OF THE PART OF BACTERIA 165
ATREPSY 166
Bacterial Vaccines 169
HISTORICAL. . . 169
METHODS OF ACTIVE IMMUNIZATION 171
TREATMENT WITH VACCINES 173
THE VACCINES 174
DOSES :....' 175
RESULTS 176
CONTENTS xi
PAGE
Leucocyte Extracts in the Treatment of Infec-
tions 177
THEORY 177
PREPARATION OF THE EXTRACTS 177
APPLICATION AND RESULTS 173
Principles Underlying Treatment of Syphilis with
Salvarsan 179
PARASITOTROPISM AND ORGANOTROPISM 179
CHEMORECEPTORS 181
Appendices 185
A. THE WASSERMANN TEST FOR SYPHILIS 185
B. NOGUCHI'S MODIFIED WASSERMANN REACTION 200
C. BLOOD EXAMINATION PREPARATORY TO TRANS-
FUSION 204
D. THE CONGLUTINATION REACTION 207
THE MUCH-HOLZMANN COBRA VENOM REACTION . .. 208
THE MEIOSTAGMIN REACTION 208
WEIL'S COBRA VENOM TEST IN SYPHILIS 210
ANTITRYPSIN DETERMINATIONS. .'.. . 212
IMMUNE SERA
ANTITOXINS
Historical. The researches of Buchner 1 in 1889
had shown that the serum of animals artificially
immunized against a certain bacterium possessed
marked bactericidal properties for that particular
organism. In studying immunity on animals which
had been successfully immunized against diphtheria
infection, Behring, 2 working in Koch's laboratory
was struck by the fact that in these animals
living virulent diphtheria bacilli were often demon-
strable in the scab at the site of. injection several
weeks after the infection, and furthermore that the
blood serum of the animals did not possess bacteri-
cidal properties. In a study published in 1890
Behring showed that the serum of rabbits arti-
ficially immunized against diphtheria was able to
confer a specific immunity against diphtheria infec-
tions in other animals. He also demonstrated that
such a serum could be used therapeutically to cure
an infection already in progress. Such a serum
1 Buchner, Centralblatt Bacteriologie, Vol. v, 1889. Archiv.
f. Hygiene, Vol. x. 1890.
2 Behring & Kitasato, Deutsche med. Wochenschrift, No.
49, 1890.
I
2 IMMUNE SERA
was not bactericidal, and retained its therapeutic
power for a considerable time. He believed that
the action of the serum was effected by a neu-
tralization of the bacterial toxin by an " antitoxic
serum constituent." The action was strictly specific,
an antitoxic serum obtained after a diphtheria
infection protected only against diphtheria; one
derived from a tetanus animal, only against tetanus.
Subsequently Behring and Knorr showed that im-
munization could be effected with bacterial-free
filtrates of tetanus cultures and that the serum thus
produced protected not only against tetanus
infection but against poisoning by the toxic prod-
ucts of the bacilli. After considerable experi-
mental work Behring and his collaborators devised
an effective method of immunizing Lheep and
certain other animals against diphtheria and against
tetanus and so produced antitoxic sera in con-
siderable amounts.
The following account taken from Park shows the
present methods of producing diphtheria antitoxin.
Production of the Diphtheria Toxin. A strong
diphtheria toxin should be obtained by taking a very
virulent culture and growing it in broth which is about
8 cc. normal soda solution per liter above the neutral
point to litmus. The culture fluid should be in com-
paratively thin layers and in large-necked Erlenmeyei
flasks, so as to allow of a free access of air; the tem-
perature should be about 35 to 36 C. The culture,
after a weeks growth, is removed from the incubator,
ANTITOXINS 3
and having been tested for purity by microscopic and
culture tests is rendered sterile by the addition of 10
per cent of a 5 per cent solution of carbolic acid. After
48 hours the dead bacilli have settled on the bottom of
the jar and the clear fluid is filtered through ordinary
sterile filter paper and stored in full bottles in a
cold place until needed. Its strength is then tested by
giving a series of guinea pigs carefully measured
amounts. Less than o.oi cc. when injected hypoder-
mically should kill a 250 gram guinea pig.
Immunizing the Animals. The horses used should
be young, vigorous, of fair size, and absolutely healthy.
Vicious habits, such as kicking, etc., make no difference,
except, of course, to those who handle the animals.
The horses are severally injected with an amount of
toxin sufficient to kill five thousand guinea pigs of 250
grams weight (about 20 cc. of strong toxin). After
from three to five days, so soon as the fever reaction
has subsided, a second subcutaneous injection of a
slightly larger dose' is given. With the first three
injections of toxin 10,000 units of antitoxin are given.
If antitoxin is not mixed with the first doses of toxin
only one-tenth of the doses advised is to be given.
At intervals of from five to eight days increasing injec-
tions of pure toxin are made until at the end of two
months from ten to twenty times the original amount
is given. There is absolutely no way of judging which
horses will produce the highest grades of antitoxin.
Very roughly those horses which are extremely sensi-
tive, and those which react hardly at all are the poorest,
but even here there are exceptions. The only way,
therefore, is at the end of six weeks or two months to
bleed the horses and test their serum. If only high
grade serum is wanted all the horses that give less
4 IMMUNE SERA
than 150 units per cc. are discarded. If moderate
grades only are desired, all that yield 100 units may be
retained. The retained horses receive steadily in-
creasing doses, the rapidity of the increase and the
interval of time between the doses (three days to one
week) depending somewhat on the reaction following
the injection, an elevation of temperature of more than
3 F. being undesirable. At the end of three months
the antitoxic serum of all the horses should contain
over 300 units and in about 10 per cent as much as 800
units per'cc. Very few horses ever give over 1000
units, and none so far has given as much as 2000 units
per cc. The very best horses, if pushed to their
limit continue to furnish blood of gradually decreasing
strength. If every nine months an interval of three
months' freedom from inoculations is given, the best
horses furnish high grade serum during their periods of
treatment for from two to four years.
Collecting the Serum. In order to obtain the serum
the blood is withdrawn from the jugular vein by means
of a sharp-pointed canula which is plunged through the
vein wall, a slit having been made in the skin. The
blood is carried by a sterile rubber tube attached to the
canula, into large Erlenmeyer flasks and allowed to
clot, the flasks, however being placed in a slanting
position before clotting has commenced. The serum
is drawn off after 4 days by means of sterile glass and
rubber tubing, and is stored in large flasks in a refrige-
rator. From this as needed small vials are filled.
The vials and their stoppers, as indeed all the utensils
used for holding the serum, must be absolutely sterile
and every possible precaution must be taken to avoid
contamination of the serum. An antiseptic may be
added as a preservative, but is not necessary. Diph-
ANTITOXINS 5
theria antitoxin, when stored in vials and kept in a cool
place away from light and air contains within 10 per
cent of its original strength for at least two months;
after that it can be used by allowing for a maximum
deterioration of 3 per cent for each month.
Testing the Strength of the Antitoxin. This is carried
out as follows: Six guinea pigs .are injected with mix-
tures of toxin and antitoxin. In each of the mixtures
there is 100 times the amount of a toxin (similar to
that adopted as the standard) which will kill a 250
grams on an average in 96 hours. In each of the
mixtures the amount of antitoxin varies; for instance,
No. i would contain 0.002 cc. serum; No. 2, 0.003 cc. ;
No. 3, 0.004 cc. ; No. 4, 0.005 cc -> etc - ^ at tne en d f
the fourth day Nos. i, 2 and 3 were dead and Nos. 4,
5 and 6 were alive we would consider the serum to
contain 200 units of antitoxin for each cubic centi-
meter. When we mix only ten fatal doses of toxin
with one-tenth of the amount of antitoxin used with
100 fatal doses, the guinea pig must remain well. The
mixed toxin and antitoxin must remain together for
fifteen minutes before injecting.
Behring's publication was followed in the next
two years by considerable work along these lines,
valuable contributions being made by Aronson, 1
Roux, and Martin, 2 Wernicke, 3 Knorr 4 and -others.
The statements of Behring as to the strict specifi-
city of the antitoxins were fully confirmed. Certain
1 Berliner med. Gesellschaft, Sitzung, Dec. 21, 1892. Also
Berliner Klin. Wochenschrift, 1893 and 1894.
3 Roux and Martin, Annal. Pasteur 1894.
8 Behring and Wernicke, Zeitsch. Hygiene, 1892. Vol. xi.
Behring and Knorr, Zeitsch. f. Hygiene, 1893. Vol. xii.
6 IMMUNE SERA
observations by Buchner 1 and by Roux and Martin
threw doubt, however, on the correctness of Beh-
rings view that the toxin was neutralized by the
specific serum just as a base was neutralized by an
acid. It was claimed, for example, that the specific
serum acted mainly on the body cells causing them
to become non-susceptible to the poison in question.
Various theories were formulated to account for the
production of the antitoxins, their specificity, etc.,
but of them all only one has at all maintained itself.
This, is the so-called side-chain theory, which was
formulated by Ehrlich 2 in 1897.
Ehrlich's Side-Chain Theory. Originally the
side-chain theory was applied by Ehrlich only to
the production of the specific antitoxins, i.e., sub-
stances in the blood, which act not only on the
living bacteria, but also and especially on their
dissolved toxins. Later on he extended it so as
to apply also to the formation of specific bacteri-
cidal and haemolytic substances in the serum of
animals treated with living bacteria or with animal
cells.
Toxins Toxoids Special Function of the Side
Chains. The basis of the theory is the fact that
poison and counter-poison, toxin and antitoxin,
combine directly in any given quantity. This
combination always occurs in definite proportions
1 Buchner, Miinchener med. Wochenschrift, 1894.
1 Ehrlich, Klinisches Jahrbuch, 1897.
ANTITOXINS 7
following the laws of chemical combination; and,
still following those laws, is slower at lower tem-
peratures than at higher, stronger in concentrate J.
than in dilute form. Ehrlich could further show
that each poison for which by the process of immun-
izing one can develop a counter-poison possesses
two groups which are concerned in the combina-
tion with the counter-poison or antitoxin. One of
these, the so-called haptophore group, is the combin-
ing group proper; the other, the toxophore group,
is the carrier of the poison. A poison molecule,
therefore, might lose the one, the toxophore, and
still be capable by means of its haptophore group
of combining with antitoxin. Such a modified
poison, which because of the loss of the toxophore
group can hardly be called a poison, but which still
possesses the power to combine with antitoxin,
Ehrlich calls a toxoid. Toxoids may be produced
spontaneously in old poisons through decomposi-
tion of the poison molecule, or they may be pro-
duced artificially by causing certain destructive
agents such as heat or chemicals to act on bacterial
poisons. The toxophore group is a very delicate
one and much more readily decomposed than the
combining (haptophore) group. Ehrlich reasoned
that in order for a poison to be toxic to an organ-
ism, i.e., in order that the toxophore group be able
to act destructively on a cell, it is necessary for the
haptophore group of the poison to combine with
8 IMMUNE SERA
the cell. IS In every living cell," Ehrlich says,
" there must exist a dominating body [Leistungs
Kern] and a number of other chemical groups or
side chains. These groups have the greatest variety
of function, but especially those of nutrition and
assimilation."
The side chains, then, according to this author,
toxophore group
, POISON MOLECULE
FlG. I
are able to combine with the greatest variety of
foreign substances and convert these into nourish-
ment suitable to the requirements of the active
central body. They are comparable to the pseudo-
podia of the lower animals, which engulf food par-
ticles and assimilate the same for the immediate
use of the organism. In order that any substance
ANTITOXINS 9
may combine with these side chains it is necessary
that certain very definite relations exist between
the combining group of the substance and that
of the side chain. Using the well-known simile of
Emil Fischer, the relation must be like that of lock
and key, i.e., the two groups must fit accurately.
Hence not every substance will fit all the side
chains of an organism. It will combine only with
those for which it possesses a fitting group.
Receptors Weigert's Overproduction Theory.
This doctrine of the chemistry of the organism's
metabolism Ehrlich applied to the action of toxins
and antitoxins. ' The toxin," he said, " can act
only when its haptophore group happens to fit to
one of the side chains," or receptors, as he now pre-
fers to call them. As a result of this combination,
the toxophore group is able to act on the cell and
injure it. If we take as an example tetanus, in
which all the symptoms are due to the central ner-
vous system, the side-chain theory assumes that
the haptophore group of the tetanus poison fits
exactly and is combined with the side chain or
receptors of the central nervous system. Other
experiments, which we will not reproduce here,
have shown us unquestionably that the action of
the antitoxins depends on the fact that this com-
bines with the haptophore group of the poison and
so satisfies the latter 's affinity. Ehrlich, therefore,
concluded that the antitoxin is nothing else than
10 IMMUNE SERA
the side chains or receptors which are given off by
the cells and thrust into the circulation. The way
in which these side chains or receptors are thrust
off as a result of the immunizing process, Ehrlich
explains by means of Weigert 1 s Overproduction
Theory.
At the meeting of German Naturalists and
Physicians held at Frankfurt in 1896, Weigert * in
discussing regeneration, advanced an hypothesis the
essential features of which are that physiological
structure and function depend upon the equilibrium
of the tissues maintained by virtue of mutual
restraint between their component cells ; that destruc-
tion of a single integer or group of integers of a
tissue or a cell removes a corresponding amount of
restraint at the point injured, and therefore destroys
equilibrium and permits of the abnormal exhibi-
tion of bioplastic energies on the part of the remain-
ing uninjured components, which activity may be
viewed as a compensating hyperplasia; that hyper-
plasia is not, therefore, the direct result of external
irritation, and cannot be, since the action of the
irritant is destructive and is confined to the cells
or integers of cells that it destroys, but occurs
rather indirectly as a function of the surrounding
uninjured tissues that have been excited to bio-
plastic activity through the removal of the restraint
1 Weigert, Verhandlungen der Ges. deutscher Naturforscher
iind Aerzte, 1896
ANTITOXINS II
hitherto exerted by the cells destroyed by the
irritant; and, finally, when such bioplastic activity
is called into play there is always hypercompen-
sation i.e. there is more plastic material gene-
rated than is necessary to compensate for the
loss.
Ehrlich points out that owing to the combination
of the toxin with the side chain of a cell, these
side chains are practically lost to the cell ; that the
latter or its fellows now produces new side chains to
replace this loss, but that this production always
goes so far as to make a surplus of side chains ; that
these side chains are thrown off by the cell as
unnecessary ballast, and then circulate in the blood
as antitoxin. The same substances, therefore, which
when part of the cell combine with the haptophore
group of the toxin, enabling that to act on the cell,
when circulating free in the blood combine with
and satisfy this haptophore group of the toxin,
and prevent the poison from combining with and
damaging the cells of the organism.
It does not follow from Ehrlich's theory that the
antitoxin is produced by the same set of cells whose
injury by the toxin gives rise to the particular
clinical symptoms. Thus we might believe that
although in tetanus the cells of the central nervous
s}^stem give rise to the characteristic symptoms, cells
entirely apart from these, e.g., in the bone marrow,
might be the main source of the antitoxin. The
12 IMMUNE SERA
fact that we appreciate symptoms from only one
organ is, obviously, no proof that other tissues
have been unaffected.
It may be well here to call attention to another
rather common misconception regarding the pro-
duction of antitoxin, namely that the body cells
have to become educated, so to speak, to produce
the antitoxin. This, it is believed, is effected by
giving gradually increasing doses of toxin. As a
matter of fact the reason for this gradual increase
in the dose injected is quite different. The object
in view is the administration of an enormously
large dose of toxin, one that will engage the recep-
tors of many cells, The previous injections have
brought about some production of antitoxin and
this partially neutralizes some of the toxin in-
jected, making it possible to give a larger dose than
before. If one gives at the outset a large amount of
toxin, partially neutralized by antitoxin, one will
produce an amount of antitoxin equal to that
ordinarily obtained in response to the same quan-
tity of unaltered toxin given as the tenth or
twentieth injection of a series. Park and Atkinson
for example, injected a fresh horse with one litre
of a toxin neutralized ij times for guinea pigs.
At the end of a week the horse had produced a serum
containing 60 units per cc. When the toxin was
neutralized 6 fold no antitoxin whatever was pro-
duced.
ANTITOXINS 13
Experimental Evidence for Ehrlictis Theory.
According to Ehrlich, then, the formation of specific
antibodies must proceed in three stages:
1. The binding of the haptophore group to the
receptor.
2. The increased production of the receptors
following this binding.
3. The thrusting-off of these increased receptors
into the blood.
So far as the first point is concerned Wassermann *
showed that with tetanus, in which, as is well
known, all the symptoms are referable to the cen-
tral nervous system, tetanus toxin was bound by
central nervous system substance in vitro. A
mixture of tetanus poison and normal central
nervous system was innocuous to animals, showing
that certain substances present in the central
nervous system combine with and thus satisfy the
affinity of the haptophore group of the poison.
This of course prevents the latter from combining
with any cells of the organism. Organs other than
the central nervous system do not possess this
property of combining with tetanus poison, just
as the central nervous system is, on the contrary,
incapable of combining with diphtheria poison,
which clinically does not show any pronounced
affinity for the central nervous system.
Wassermann 2 also believes recently to have given
1 Wassermann and Takaki, Berliner Klin. Wochenschr, 1898,
1 Wassermann, New York Medical Journal, 1904.
14 IMMUNE SERA
experimental proof of the second and third points,
the increased production of the receptors and their
thrusting off. For this purpose he employed a
tetanus poison which he had kept for about eight
years, and which was originally very poisonous.
In the course of years, however, owing to the
damaging action of light, of oxidation, etc., it had
become so weak that it was no longer toxic at all.
Injections of one cc. into a guinea pig produced
no tetanus. Nevertheless the haptophore group
remained intact, as could readily be proved, for
this non-poisonous tetanus toxin was still able to
bind tetanus antitoxin, i.e. thrust-off receptors. On
injecting rabbits with this nOn-poisonous tetanus
toxoid in increasing doses, and then examining the
blood serum of the animal he found not a trace of
tetanus antitoxin. This absence could have either
of two causes: It might be that the toxoid no
longer produced any physiological effect whatever
in the organism; or although it still caused an
increase in the receptors, these increased receptors
remained in the organs (sessile) and were not
thrust off into the blood. In order to decide this
question Wassermann first determined the exact
quantity of fresh tetanus toxin which constituted a
fatal dose for guinea pigs. He reasoned that if
he injected first the toxoid, and shortly after, say
in one or two hours, the fresh toxin, he should in
such an animal have to increase the fatal dose,
ANTITOXINS 1 5
i.e. more tetanus toxin should be required to kill
this animal than a normal one, because owing to
the previous toxoid injection part of the cells sus-
ceptible to tetanus toxin would already have been
occupied. Provided Ehrlich's theory were correct,
so that this binding of the toxoid really occurred,
the conditions should be entirely different when,
instead of injecting the toxin shortly after the
toxoid, he waited somewhat longer, one to three
days, and then injected the fresh tetanus toxin
In that case Weigert's law should come into play
and the receptors have commenced to increase
in number, i.e. the organ should now possess more
sensitive groups than before. This would manifest
itself in such fashion that in contrast to the first
experiment the fatal dose of fresh tetanus toxin
could now be decreased ; in other words a small dose
would now tetanize the animal in a shorter time.
As a matter of fact Wassermann's experiments
yielded exactly the results deduced theoretically.
He injected a guinea pig with some of the non--
poisonous toxoid and then, an hour later, with
tetanus toxin. He found that much more toxin
was required to kill this animal than a normal
guinea pig of equal size. When, on the contrary,
he waited one to three days, it was found that then
a dose of tetanus toxin which would not even
tetanize a normal guinea pig was sufficient to kill
this one.
16 IMMUNE SERA
It will be seen that in the above experiments
the completely non-poisonous toxoid, although it
effected an increased production of receptors, did
not cause their thrusting-off. The serum of the
rabbit treated with toxoid contained no antitoxin
whatever. Wassermann concludes from this and
other experiments that the thrusting-off cannot be
a function of the haptophore group, and that
something additional is required. This " some-
thing," he claims is a function of the toxophore
group. It may be stated that Von Dungern has
also published experiments (with majaplasm) point-
ing to the existence of the second stage, the stage of
sessile receptors.
Antigens or Haptins. It has been found that
it is impossible to produce any immunity against
all poisons, e.g. strychnine or morphine. Accord-
ing to Ehrlich these simpler chemical molecules do
not enter into a true chemical combination with
the tissues, but form rather a kind of solid solution,
a loose combination with the cells, so that they can
again be abstracted from these cells by all kinds of
solvents, e.g. by shaking out with ether or chloro-
form. The point can perhaps be likened to the
difference between saccharin and sugar. Both sub-
stances taste sweet, but despite this similarity in
their physiological action they behave very dif-
ferently toward the cells of the organism. Sac-
charin simply passes through the organism without
ANTITOXINS 17
entering into a firm combination, i.e. without being
assimilated, and is therefore no food. Its sweeten-
ing action is a mere contact effect on the cells
sensitive to taste. Sugar, on the contrary, is
actually bound by the cells, assimilated and burnt,
and so is a true food. Until recently it was believed
that the simpler chemical substances could not
excite the production of antibodies. Ford and
Abel 1 have however been able to show that toad
stool poison, a true toxin, against which an anti-
toxin can be produced is chemically a glucoside.
As we shall subsequently see it is possible to
immunize the animal body against a large number
of substances, including not only such cell products
as ferments, toxins and venoms, but also cells of
the greatest variety, bacteria, dissolved proteids, etc.
All these substances, therefore, must possess hapto-
phore groups able to combine with the side chains
or receptors in the animal body. Collectively,
we speak of such substances as antigens or haptins.
Nature of Antitoxins in General. But little is
known concerning the constitution of antitoxins,
for we do not know them apart from serum or
serum constituents. It seems probable that they
are proteid in character, but this has not been
positively decided. It has been found that like
the globulins they are quite resistant to the action
of trypsin, but are acted on by pepsin-hydrochloric
1 Ford and Abel, Journal of Biological Chemistry, Vol. ii, 1907
1 8 IMMUNE SERA
acid. In general they withstand a fair degree of
heat, certainly far more than the toxins. Anti-
toxins are to be regarded as inactive substances,
effecting merely a blocking of the haptophore
group of the corresponding toxin. They do not
act on the toxins destructively. This is indicated
by experiments of Wassermann on pyocyaneus toxin,
and of Calmette and Morgenroth 1 on snake venom,
which showed that in the toxin-antitoxin com-
bination, the toxin could again manifest itself after
the antitoxin had been destroyed. The antitoxins
therefore are not ferment-like substances. As far
back as 1897 attempts were made to determine the
chemical nature of the antitoxins. In that year
Belfanti and Carbone 2 found that the antitoxin
was precipitated with the globulins of the serum by
means of magnesium sulphate. Dieudonne 3 had
previously shown that the proteids thrown out of
solution by acetic and carbonic acids contained
none of the antitoxin. In 1901 Atkinson 4 showed
that the globulins increase markedly in the serum
of horses as the antitoxic strength increases. The
most recent work on this subject is that of Gibson, 5
who shows that if the ammonium sulphate precipi-
1 Morgenroth, Berlin, klin. Wochenschr. 1905.
2 Beifanti and Carbone, Centralblatt Bacteriologie (Ref.),
Vol. xxiii, 1898.
3 Dieudonne, Arbeiten a.d. kaiserl. Gesundheitsamte. VoL
xiii, 1897.
4 Atkinson, Jour. Exper. Medicine, Vol. i, 1901.
5 Gibson, Journ. Biological Chemistry, Vol. i, 1906.
ANTITOXINS 1 9
tate (globulins, nucleo-proteids, etc.) is treated with
saturated sodium chloride solution, practically all
the antitoxic fraction passes into solution. Gibson's
was the first really practicable method of concentrat-
ing the antitoxin. By means of it solutions of
antitoxic globulin could easily be made to contain
1500 units per cc. Continuing Gibson's work,
Banzhaf discovered that if the antitoxic serum or
plasma was heated to 57 for 18 hours, there was
a change of a considerable portion of the soluble
globulins (soluble in Nad solution) into insoluble
globulins. The antitoxin remained unchanged.
This procedure, therefore, permits of a still greater
elimination of the non-antitoxic proteids.
Gibson has recently studied the possibility of
differentiating other antibodies by means of their
precipitation characteristics. He believes that a
differentiation of the antibodies into those precip-
itated with the pseudo globulins and with the
euglobulin fractions, according to the Hofmeister
classification, is based on a misconception of the
application of ammonium sulphate in separating
proteids by their precipitation characters. While
there seem to be some differences in the dis-
tribution of the antibodies in individual specific
sera in comparative experiments, this is not so
absolute as maintained by Pick l and others. Gib-
son's work on the fractionating of poly agglutina-
1 Pick, Beitiage z. chem. PhysioL u- PathoL, VoL i, 1901.
20 IMMUNE SERA
tive serum shows that no separation of the several
antibodies developed in an individual serum is
possible. In the case of antitoxic sera both Gibson
and Ledingham find that in goat serum the antitoxin
is not invariably associated with the euglobulin
fraction as maintained by Pick, but shows the same
solubilities as that in horse serum.
Toxins and other Poisonous Cell Derivatives, in
General. Soon after bacteriology had demon-
strated the etiological connection between bacteria
and disease, the conviction gained ground that it
was less the actual destruction wrought by the
bacteria directly, than the injury produced by their
chemical products that gave rise to the lesions in
the infectious diseases. Brieger, especially, was
one of the first to direct attention to the probable
existence of specific poisons in the bacteria. He
isolated a number of well defined chemical sub-
stances called ptomaines, most of which were highly
toxic. Subsequent study, however, showed that
these were not the specific bacterial poisons. The
latter, the true toxins are something quite different
as we shall see in a moment. Still later other
substances were isolated from bacteria, and these
were termed toxalbumins. We now know that
some of these were identical with the true toxins,
but that others were entirely unrelated.
What then are the true toxins? A number of
pathogenic bacteria, when grown in pure culture,
ANTITOXINS 21
produce dissolved poisons in the culture fluid.
These poisons are neither ptomaines nor proteid
substances; their chemical nature is still absolutely
unknown. They are extremely sensitive to exter-
nal influences, especially against heat, and in many
ways are very analogous to ferments. Physio-
logically the toxins are extremely poisonous, far
beyond that of any of the ordinary' well known
poisons, and this poisonous action manifests itself
only after a certain latent period known as the
period of incubation. Finally one of the funda-
mental properties of the toxins is their ability
to excite, in the organism attacked, antitoxins
directed specifically against them, so that for every
true toxin there is a corresponding antitoxin.
In addition to these bacterial toxins we know
of other poisonous substances possessing similar
characteristics. Among these are the " zootoxins,"
- snake venoms, spider and toad poisons, the
toxin of eel blood, and the " phytotoxins,"
ricin, crotin, abrin, etc. It may be mentioned that
some of these are of somewhat more complex con-
stitution than the ordinary bacterial toxins. Ricin,
for example, appears to possess one haptophore
group but two ergophore groups, a toxic and an
agglutinating one. In the case of the snake
venoms it is not yet definitely known whether
they are haptins of the first order or of the
second. (See page 49.)
22 IMMUNE SERA
The Relations Existing between Toxin and Anti-
toxin. The exact nature of the toxin-antitoxin
reaction has long been the subject of study and has
given rise to considerable discussion. For obvious
reasons most of the work has been done with
diphtheria and tetanus toxins and their antitoxins.
In order to give the reader some conception of
the diverging views of various authorities we shall
devote a few pages to a brief study of ths diphtheria
toxin-antitoxin reaction.
During the earlier years of toxin-antitoxin in-
vestigations the filtered or sterilized bouillon, in
which the diphtheria bacillus had grown and pro-
duced its " toxin," was supposed to require for
its neutralization an amount of antitoxin directly
proportional to its toxicity as tested in guinea pigs.
Thus, if from one bouillon culture ten fatal doses
of " toxin" were required to neutralize a certain
quantity of antitoxin, it was believed that ten
fatal doses from every culture, without regard to
ths way in which it had been produced or preserved,
would also neutralize the same amount of antitoxin.
Upon this belief was founded the Behring-Ehrlich
definition of an antitoxin unit. 1
The results of tests by different experimenters
of the same antitoxic serum, but with different diph-
1 This unit was " ten times the amount of antitoxic serum
necessary to just protect a 250 gramme guinea pig against
ten fatal doses of the toxin "
ANTITOXINS
theria toxins, proved this opinion to be incorrect.
Ehrlich 1 deserves the credit for first clearly per-
ceiving and calling attention to this fact. He
obtained from various sources twelve toxins and
compared their neutralizing value upon antitoxin;
these tests gave interesting and important in-
formation. The following table gives the results
in four of his toxins and well illustrates the point in
question :
Smallest num-
Fatal doses re-
ber of fatal
doses of toxic
quired to
" completely
Estimated
bouillon re-
neutralize " one
L+ minus
Serial
minimal fatal
quired to kill a
antitoxin unit
Num-
ber.
dose for
250 gm.
guinea pigs.
2sogm. guinea
pig within
5 days when
mixed with one
as determined
by the health
of the guinea
pig remaining
L(> in
fatal
doses.
Remarks.
antitoxin unit.
unaffected.
("L t Ehrlich.")
(" LO Ehrlich.")
A
o . 009 cc.
39-4
33-4
6
Old; deterio-
rated from 0.003
;o o. 009.
B
0.0165 cc
76.3
54-4
22
Fresh toxin,
preserved with
:ricresol.
C
o. 039 cc.
123.
108.
15
A numbei of
"resh cultures,
grown at 37 C.
four and eight
days.
D
0.0025 cc.
100
5
5
Tested immedi-
ately after its
x
withdrawal.
It was natural to suppose, as the early investi-
gators did, that a just neutral mixture of toxin and
1 Ehrlich, Die Werthbemessung des Diphtherieheilserums.
Klinisches Jahrbuch, 1897.
24 IMMUNE SERA
antitoxin, would require the addition of but one
fatal dose of toxin in order to regularly kill the test
animal. In the above table, however, we see that
this difference ranges from six to fifty fatal doses.
Partial Saturation Method - - Toxons, Toxoids.
Ehrlich obtained considerable additional informa-
tion by means of his " partial saturation " method.
Certain experiments had led him to believe that the
original antitoxin on which he had based his " unit "
determinations, while able to neutralize 100 fatal
doses (per unit) really represented 200 " binding
units," and that the toxic bouillon really contained
several kinds of poisonous substances able to com-
bine with antitoxin.
He now believes that the diphtheria bacilli excrete
at least two such poisons, " toxins " and " toxons ; "
that these very quickly decompose to a greater or
less extent forming various " toxoids."
In the case of a hypothetically pure toxin Ehrlich
believes that one antitoxic unit would correspond
to 200 fatal doses or 200 binding units. If the
entire amount of antitoxin, i.e. | is added to
the amount of toxin in question, the result will be
just complete neutralization. If the toxin is entirely
pure, ^{jf of the antitoxin unit would neutralize all
but ?hu of the initial toxicity and M&, or ?$% or ^A,
etc. of the antitoxin added would permit correspond-
ing degrees of toxicity to be demonstrated through
animal inoculations. It was found, however, that
neutralization according to this simple scale did not
ANTITOXINS 2 5
take place. The results were complicated andEhrlich
found it convenient to express them graphically in
the form of the so-called "toxin spectra. " Without
10 20 30 40
Toxon
70 80 90 100
150
200
FlG. 2.
going much deeper into the subject the point maybe
illustrated by the appended diagrams or " spectra."
Fig. 2 shows the simplest conceivable diphtheria
poison. In this case the following values would
be obtained.
# cc poison (100 fatal doses) + f antitoxin
units = o, i.e. absolutely neutral.
# cc poison + iffft = Free toxon.
# cc poison + *{} = Free toxon.
That is to say, if the proportion of antitoxin added
was Mti of the amount required for complete
neutralization, it would be found that the poison
thus uncombined was much less, and differently
toxic than a corresponding amount of the original
toxin. It was found that these fractions possessed
a rather constant though low degree of toxicity
with characteristic action. This consisted in the
production of some local oedema, followed by a
long incubation period, and finally the develop-
ment of cachexia and paralysis. Ehrlich believes
26
IMMUNE SERA
that this action is due to a separate poison excreted
by the diphtheria bacillus which he calls a toxon.
If we continue with the above poison we shall
obtain these values:
# cc poison + ^ = Toxin action (i fatal dose).
# cc poison + sVu =30 fatal doses.
x cc poison + sVk = 90 fatal doses, etc.
That is to say, if we add only $fo units antitoxin,
i.e. sitf unit less than in the JH mixture, we find
that one fatal dose is set free. This relation would
exist right to the end. The fact that in this experi-
ment the toxins are liberated after the toxons,
shows that the toxons have less affinity for the anti-
toxin than have the toxins.
As a matter of fact, however, conditions are prob-
ably never as simple as this. In the process of
toxin formation a double action is always going
on that of toxin and toxon production, and that
of their decomposition. As was pointed out on
a previous page the poisons quickly change into
non-poisonous toxoids, and these substances are
still able to bind antitoxin.
This is shown in the following " spectrum."
Protoxoid
10 20 30 40 50 60
1GO
FIG. 3.
Toxon
150 160
ANTITOXINS
Here we would obtain the following figures:
2 7
# cc poison -f
lutely neutral.
# cc poison +
# cc poison +
# cc poison -f
oo poison +
oc cc poison +
antitoxin unit = o, i.e. abso-
= Toxon free.
= Toxon free.
= Toxin free (i fatal dose.)
- Toxin free (60 fatal doses. )
= Toxin free (100 fatal doses.)
Now we come to the non-poisonous "prototoxoids" :
# cc + inm = Toxin free(ioo fatal doses.)
# cc + *oo = Toxin free (100 fatal doses.)
x cc + sio = Toxin free (100 fatal doses.)
We see here that after we have reduced the
antitoxin to ^ no further increase of toxicity is
brought about by any further reductions. Ehrlich
calls these toxoids " prototoxoids " because they
have such a high affinity for the antitoxin. But
there are apparently still other toxoids, as is shown
by the following spectrum:
Protoxoid
Syntoxoid
Toxon
' 100 ' 10 ' '200
FIG. 4.
Here we would obtain values as follows:
x cc poison -f- f $% = o, i.e. absolutely neutral.
x ce poison -f iol = Toxon.
28 IMMUNE SERA
x cc poison 4- M<! = Toxin free (i fatal dose).
oc cc poison + Mf = Toxin free ( 2 fatal doses.)
x cc poison 4- ^B = Toxon free (30 fatal doses.)
Here we find that in the middle part of the
" spectrum " we encounter a zone in which each ^"o
antitoxin unit neutralizes one fatal dose. Ehrlich
believes that this part of the mixture consists of
equal parts of syntoxoid and toxin that is to
say, he believes there are also toxoids which
have the same degree of affinity for antitoxin
that this toxin has. He speaks of these as " syn-
toxoids."
By following out this conception of the toxin-
antitoxin combination, Ehrlich comes to the con-
clusion that diphtheria poison is a very complex
substance, containing toxin, toxon, and perhaps still
other primary secretion products in addition to the
various secondary modifications of these, toxoids,
toxonoids, etc. It is difficult to escape the feeling
that the existence of some of these hypothetical
substances is more apparent than real.
Views of Arrhenius, Bar del and Others. Bordet
and others refuse to accept Ehrlich 's views and
the whole matter is still unsettled. Thus the exist-
ence or non-existence of toxons has excited a great
deal of discussion among investigators.
The great Swedish chemist, Arrhenius, has given
much attention to the toxins; and has applied
the principles of physical chemistry to the toxin-
ANTITOXINS
29
antitoxin reaction. It is, of course, well known
that a solution of a compound such as sodium
chloride represents not only NaCl in solution,
but also sodium ions and chlorine ions. There is
a certain amount of dissociation going on hand in
hand with a combination of the two components.
The degree of this varies with the temperature and
the dilution of the substances. Arrhenius believes
that the same process goes on with the toxin-
antitoxin combination and that such more or less
dissociated compounds give rise 'to the effects
Ehrlich ascribes to the toxon. There is, however,
no direct evidence that the combination of toxin-
antitoxin is reversible. It is true that Morgenroth
has been able to dissociate the two components of
a neutral mixture of cobra venom and its antitoxin.
But even here we are not dealing with a reversible
reaction, for it requires certain manipulations to
disrupt the neutral combination. In their work
on the toxin of symptomatic anthrax, Grassberger
and Schattenfroh found that different mixtures
were obtained, depending on whether they mixed
the toxin and antitoxin after diluting them, or
diluted the toxin-antitoxin mixture. This fact
is not in favor of Arrhenius' theory, for according
to that, the same state of equilibrium should exist
in both instances owing to reversibility, and the
same fraction of the toxin of necessity remain
free.
30 IMMUNE SERA
Bordet * believes that the neutralization of toxin
by antitoxin is an adsorption phenomenon, and
compares it with the process of dyeing. The
molecules of the toxin would " stain " more or less
deeply by the antitoxin molecule, and the com-
plexes that result in the various instances would
be less toxic in proportion as they contained more
antitoxin and less toxin. If a large piece of filter
paper is placed in a certain volume of sufficiently
diluted dye, it takes a uniform shade of intensity;
if, on the other hand, the same sized piece of paper
is cut in pieces and added in fragments, the first
pieces are stained deeply, and the last find no color
left. In the same way, on adding toxin to antitoxin
in divided doses, the last portions of the poison
cannot be neutralized, as the first are supersatu-
rated with antitoxin. When the entire mixture
is made at once, on the contrary, the antitoxin is
spread all over the toxin molecules and a complex
is obtained which contains an even proportion of
the antidote, and which, consequently, is not as
fatal as even a small dose of free toxin. The action
which Ehrlich therefore ascribes to toxons, Bordet
refers to toxin which is partially saturated with
antitoxin. Bordet also cites the researches of
Grassberger and Schattenfroh on the toxin of
symptomatic anthrax. The toxic fluid which these
1 Bordet-Gay, Collected Studies in Immunity, Wiley & Sons,
1909.
ANTITOXINS 3I
authors employed contains only a single poison:
there is no reason for assuming the existence of
toxoids, inasmuch as the toxic power of the poison
is constantly parallel to its neutralizing power for
antitoxin. On mixing a certain dose of the toxin
either with little or with much antitoxin, complexes
of toxin-antitoxin were obtained which varied
in their reaction to heat. Moreover, these authors
found that their poison absorbs much more anti-
toxin than is necessary to destroy its entire toxicity,
and forms a stable compound with it. Bordet's con-
ception of the toxin-antitoxin reaction thus seems
to be very simple. The main difficulty which it
encounters is the strict specificity of the combi-
nation. However, recent investigations make it
probable that the affinity of adsorption is similar
to a true chemical affinity, in that both are elective.
It is possible, therefore, that the existence of strict
specificity may still be found entirely compatible
with the adsorption theory.
AGGLUTININS
The Agglutination Phenomenon. We have just
seen that pathogenic bacteria may be divided into
those which produce extracellular toxins in culture
media, and those which do not. Against the
former the organism defends itself by the production
of antitoxins ; against the latter it produces a variety
of antibodies : bacteriolysins, agglutinins, precipi-
tins, opsonins and possibly others.
The agglutinins can be observed either in a test-
tube or in a microscopical preparation. For example,
if typhoid or cholera immune sera are added respec-
tively to a 24-hour culture of typhoid or cholera
bacilli, and the mixture placed in a thermostat,
the following phenomenon will be noticed: The
bacteria which previously clouded the bouillon
uniformly, clump together into little masses, settle
to the sides of the test-tub 2 and gradually fall to
the bottom until the fluid is almost entirely clear.
In a control test, on the contrary, to which no active
serum is added, the fluid remains uniformly cloudy.
The reaction is completed in twenty-four hours
at the most. If the reaction is observed in a hang-
ing drop, it is seen that the addition of the active
serum first produces an increased motility of the
32
AGGLUTIN1NS 33
bacteria which lasts a short time and is followed
by a gradual formation of clumps. One gets the
impression that the bacteria are dying together.
Frequently one sees bacteria which have recently
joined a group make violent motions as though they
were attempting to tear themselves away; then
they gradually lose their motility completely. Even
the larger groups of bacteria may exhibit movement
as a whole. After not more than one or two hours
the reaction is completed; in place of the bacteria
moving quickly across the field, one sees one or
several groups of absolutely immobile bacilli. Now
and then in a number of preparations one sees
a few separate bacteria still moving about among
the groups. If the reaction is feeble, either because
the immune serum has been strongly diluted or
because it contains very little agglutinin, the groups
are small and one finds comparatively many iso-
lated and perhaps also moving bacteria. It is
essential each time to make a control test of the
same bacterial culture withoiit the addition of
serum. Under some circumstances the reaction
proceeds with extraordinary rapidity so that the
bacilli are clumped almost immediately. By the
time the microscopical slide has been prepared
and brought into view nothing is to be seen
of any moving or isolated bacteria, and only by
means of the control test is it t possible to tell
whether the culture possessed normal motility.
34 IMMUXE SERA
We are not yet informed as to the nature of these
phenomena. A number of theories have been ad-
vanced, into which, however, we cannot here enter.
In some cases the agglutinins are active even in
very high dilutions. Thus in typhoid patients
and typhoid convalescents a distinct agglutination
has been observed in dilutions of i : 5000, and this
action persisted for years, though not, of course,
in the same degree. Even normal blood-serum,
when undiluted, often produces agglutination. But
the above specific agglutinins, which do not exist
beforehand, being formed only in consequence of
an infection, are characterized by this, that the
agglutination occurs even when the serum is diluted
(at least i : 30 to i : 50), and, furthermore, that after
this dilution the action is still specific, i.e. cholera
immune serum agglutinates only cholera bacilli,
typhoid immune serum only typhoid bacilli, etc.
This specificity, however, as will be shown later,
is not always absolute.
Agglutinins can also be developed against red
blood cells and against certain protozoa (trypan-
osomes). We speak of iha former as hamag-
glutinins. Analogous to the hsemolytic action or
normal serum on the red cells of certain other
species, we find that normal serum is able to
agglutinate the red cells of many species and bac-
teria. For example, normal goat serum aggluti-
nates the red cells of man, pigeon, and rabbit;
AGGLUTININS 35
normal rabbit serum agglutinates typhoid and
cholera bacilli.
Of practical interest is the fact that normal serum
may agglutinate the red blood cells of another indi-
vidual of the same species. Following Ehrlich's nomen-
clature, we speak of this as is o agglutination. The
subject has been studied by a number of investigators,
and mostly in human blood. According to the extensive
investigations of Moss, isoagglutinins occur in the serum
of about 90% of adult human beings. Landsteiner
divided the individuals into three groups, namely :
Group i. The corpuscles are not agglutinated by
sera of the other two groups, while the sera agglu-
tinate the corpuscles of both groups.
Group 2. The corpuscles are agglutinated by the
sera of the other two groups, while the sera agglutinate
the corpuscles of Group 3, but not of Group i.
Group 3. The corpuscles are agglutinated by the
other two sera, and the sera agglutinate the corpuscles
of Group 2, but not of Group i.
An examination of this grouping shows that in no
case is there an agglutination of erythrocytes by their
own serum, in other words these are isoagglutinins but
not autoagglutinins. A somewhat different classification
was made by Jansky, and independently of him also by
Moss. Both these authors find it necessary to establish
four groups in order to embrace all the cases met with.
Gay calls attention to the fact that the clumping of
erythrocytes by serum is not necessarily due to the pres-
ence of an agglutinin at all, but may be due to variations
in the molecular concentration of the serum constituents
or of the constituents of the blood cells. Whatever finally
proves to be the correct explanation, it is obvious that
the occurrence of isoagglutination in human blood might
36 IMMUNE SERA
possibly prove disastrous in human homologous trans-
fusion. For this reason it is now common practice to
always precede homologous transfusion by an examina-
tion of the blood of both donor and recipient. A brief
outline of these tests is given in the appendix.
Purpose of Agglutination. It is not yet clear
what the purpose, if any, of ths agglutinating
function is. Gruber, the first to thoroughly study
and appreciate the bacterial agglutinins, assumes
that the process injures the affected cell, preparing
it for solution and destruction. After numerous
experiments I have not been able to convince
myself of any damaging influence of the agglutinins
on the affected cell, be this blood cell or bacterium,
and the observations of other authors confirm this
opinion. Agglutinated bacteria are capable of
living and of reproduction, and agglutinated red
blood cells are no more fragile or easier to destroy
than normal, non-agglutinated cells. Neither can
anything be discovered microscopically which would
indicate any injury to their structure.
One thing is certain: that the agglutinins are in
no way related to the lysins found in serum, and
so of course are not identical with these. The
simultaneous occurrence in a serum of immune
bodies, interbodies, complements, and agglutinins
is an entirely independent phenomenon which is
in no way regular. There are sera which dissolve
certain cells without agglutinating them, and others
which agglutinate cells without dissolving them.
AGGLUTININS 37
Historical Serum diagnosis by means of the
agglutinins was introduced chiefly through the
labors of Gruber and Widal. The studies under-
taken by Gruber and his pupil Durham began as
early as 1894. At the Congress for Internal Medi-
cine in 1896 l Gruber first announced that he had
discovered the reaction in typhoid convalescents,
and asked that his observations be verified if pos-
sible. Soon after this Pfeiffer and his co-workers
published a study which confirmed Gruber's results. 2
The significance of the reaction as a diagnostic
help was unquestionably first pointed out by Widal, 3
who showed that the reaction appears at a relatively
early period of the disease, and may therefore be
employed as a diagnostic measure. We must not
omit to state that Griinbaum 4 in March, 1896, several
months before Widal's publication, had also grasped
the significance of the reaction as a diagnostic
measure. Owing to insufficient clinical material
his publication did not appear until some time after
Widal's. Hence, in acknowledgment of the labors
of the two authors most concerned in the discovery
and introduction of this reaction, we now speak
1 Transactions of the Congress, edited by E. von Leyden and
R. Pfeiffer, Wiesbaden, 1896.
2 Pfeiffer and Kolle, Deutsche med. Wochenschrift, 1896,
No. 12.
3 Widal, Bulletin de la soc. mdd. des hop., June 26, 1896.
4 Grunbaum, Lancet, Sept. 19, 1896; Muench. med. Wochen-
schrift, 1897, No. 13; Blood and the identification of bacterial
species, Science Progress, Vol. I, No. 5, 1897.
38 IMMUNE SERA
of it as the "Gruber-Widal reaction," whereas in
the beginning only the term " Widal reaction "
was used.
The manner in which the reaction proceeds in
microscopical preparations as well as when mac-
roscopically observed has been described above
(page 32). Nowadays the microscopic method is
given the preference x because in many cases it is
distinct when the macroscopic reaction fails; and
further because the former yields distinct results
within an hour at the most, whereas in many cases
twenty-four hours are required for the macroscopic
test.
Pfaundler's Reaction (Thread Reaction). It
may be well at this point to call attention to a
peculiar reaction described by Pfaundler 2 in 1896.
This author showed that certain bacteria, though
they might not be agglutinated by a given serum,
would often, when they were grown therein, develop
in the form of long threads more or less interlaced.
This occurred only in the specific serum and wa
absent in the normal serum. Most authorities
regard the thread reaction as a manifestation of
agglutinins. According to Metchnikoff this reaction
sometimes gives more information concerning a
serum than does the ordinary agglutination test.
1 This applies to typhoid ; in other diseases the macroscopic
method is sometimes preferable,
'* Pfaundler, Centralblatt Bacteriologie, Vol. xix, 1896.
AGGLUTININS
39
Nature of the Agglutinins, and of the Agglutina-
tion Reaction. The agglutinins are fairly resistant
substances which withstand heating to 60 C., and
lose their power only on heating to 65 C. It is pos-
sible, therefore, to make a serum bacteriolytically in-
active by heating to 5 5 C., and still preserve its agglu-
tinating power. It has been found that agglutinins
when heated may keep the property of uniting
with bacteria, although they lose the property of
agglutinating them. To explain this fact, Ehrlich
supposes that agglutinins possess two groups, a
haptophore group, effecting the specific union with
the cell, and an ergophore group, which effects the
clumping. He supposes further that under the con-
ditions mentioned the agglutinin loses its aggluti-
nating group but keeps its combining group. Such
a modified agglutinin Ehrlich calls an agglutinoid,
just as toxins which have lost their toxophore
groups are called toxoids. The nature of agglutinoid,
however, is still very obscure. In fact, as we shall
presently see, the opponents of the Ehrlich school
refuse to believe in the existence of agglutinoids.
It has occasionally been observed that agglutination
is absent in concentrated serum, and present in dilute
serum. This zone of no agglutination, preceding that
of agglutination, is often spoken of as the pro zone
and was first described by Eisenberg and Volk. Ac-
cording to Ehrlich. it is due to the presence in the
serum of agglutinoids. These are assumed to possess
higher affinity for the bacteria than do the agglutinins
4 o IMMUNE SERA
and so prevent the latter from acting on the bacteria.
Since, however, the agglutinins are usually far more
abundant than the agglutinoids, dilution of the serum
dilutes the latter to practically nothing, thus allow-
ing the agglutinins to combine with the bacteria.
Ehrlich's conception of the structure of the agglu-
tinin molecule and his views on the nature of the
agglutination reaction have been sharply combated.
Elser very properly points out that not enough
attention has been paid to the effect of heat on serum,
and that alterations in the physical characters of the
serum may be sufficient to account for phenomena
heretofore ascribed to chemical changes. Among
other things he cites the effect of heat on horse serum ;
heating produces a marked increase in the viscosity
of the serum. It is obvious, therefore, when heated
sera are used in agglutination experiments, that this
purely physical characteristic exerts a profound in-
fluence on the result of the reaction. Differences in
the behavior of an agglutinating serum before and
after heating must therefore be interpreted with great
caution, and must not at once be taken to indicate
the chemical alteration of the agglutinin complex.
Bordet, for example, cites an interesting experi-
ment of Gengou. An aqueous solution of agar,
so diluted as to be only slightly viscous at room
temperature, agglutinates barium sulphate sus-
pended in water. Heating such a solution destroys
this property without affecting the adsorbing
property; under these conditions it produces the
AGGLUTININS 4 !
opposite effect, namely, disseminates the particles
of barium and gives a milky appearance to the
fluid. Can we, says Bordet, claim that by heating
this solution we have caused it to lose its agglu-
tinating group? Bordet agrees with Forges, who
believes that the hypothesis of such a group in the
antibody molecule has no foundation. Forges
found, on studying the effect of heat on the agglu-
tinating power of the albuminous substances of
serum for mastic emulsions, that he could obtain
results entirely similar to those that have been
noted for agglutinins. Bordet insists that we have
no right to localize the cause of agglutination in a
molecule of the antibody rather than in one of the
antigen. The hypothesis of a functional group in
the molecule of the .agglutinin, he says, is all the
more doubtful, inasmuch as it is not the only sub-
stance which can render bacteria sensitive to the
flocculating action of salts. Bacteria that have
adsorbed iron, uranium, or aluminium compounds
are subsequently flocculable by salts, and silicic
acid is similar in its action. According to Bordet,
the essential phenomenon with agglutination, as
with other active substances in sera, is its union
with the antigen; as far as the agglutination itself,
which follows this union, is concerned, it is only a
secondary phenomenon on which we cannot depend
in considering agglutinins as functionally different
in molecular structure from the other antibodies.
The influence of salts upon agglutination is in
4 2 IMMUNE SERA
a sense comparable to their action upon the pre-
cipitins. Joos found that antityphoid serum did
not agglutinate typhoid bacilli in the absence of
salts. For agglutination to take place he considers
it as necessary as the agglutinin and agglutinable
substance. He believes that salts play an active
part in the process. Bordet, on the other hand,
believes that the absence of salts offers only a
physical impediment to agglutination. Friedberger
does not consider that the salts act chemically, for he
found that agglutination took place in the presence
of grape sugar, asparigin, etc., in the place of salts.
In view of the fact that the protoplasm of the
body and the albuminous constituents of serum
have a close relationship to, or really are, colloids,
investigators have studied certain reactions which
occur among the colloids with the expectation
that these would throw some light on the reactions
of protoplasm and of serums. 1 Colloids diffuse
very slowly and exert little or no osmotic pressure,
supposedly because of the large size of the particles.
They do not conduct electricity, but the particles
react to the electric current by alterations in the
direction of their motion (i.e., toward the positive
or the negative pole) and, moreover, carry electric
charges themselves. The features of colloids which
bring them into relation with the subject in hand
are their coagulable nature in certain instances and
the fact that their particles may be agglutinated
1 This subject is well presented in : Pauli- Fischer, Physical
Chemisty in the Service of Medicine. Wiley & Sons, N. Y.
AGGLUTININS 43
or precipitated by the addition of minute amounts
of salts (electrolytes). This of course is entirely
analogous to the need of salts in the agglutination
of bacteria by sera. In the latter reaction the
agglutinins carry a positive, the bacteria a negative
charge. The resulting combination, therefore, does
not precipitate from the menstruum supposedly
because there is still sufficient difference in the
electric potential. When salts are present the
kations so alter the electric conditions, of the colloi-
dal particles, i.e., of the agglutinin-bacterium com-
bination, that their surface tension is increased.
In order to overcome this the particles get together,
presenting in a clump less surface tension than if
they remained as individual particles. Some experi-
ments by Field indicate that the pro zone may be
explained on the assumption that the bacteria and
agglutinins behave as colloids. It has already
been stated that the union of agglutinin and bac-
terium does not precipitate because, possibly, there
is still sufficient electric potential; the combination
carries a negative charge. Field believes that with
very large amounts of agglutinin (as in the pro
zone) the bacteria load themselves with so much
agglutinin that the combination now carries a con-
siderable positive charge. The surface tension there-
fore is not sufficient to cause a clumping to occur.
Naturally, the presence of salts does not alter the con-
dition, as the kations also carry a positive charge.
Group Agglutinins. For some time after their
44
IMMUNE SERA
discovery the agglutinins were regarded as strictly
specific, i.e., a serum derived, for example, from a
typhoid infection would agglutinate only typhoid
bacilli and no others. After a time, however, it was
found that such a serum would frequently aggluti-
nate somewhat related organisms, though not,
usually, to so high a degree. In other words, while
agglutinins may be nearly, if not quite, specific in
their action, a serum which produces agglutination
may be far from being so.
The following examples will illustrate the point.
In a case of infection with paratyphoid bacilli,
type B, the bacilli of the infecting type B were
agglutinated 1:5700; typhoid bacilli, however, only
1:120, while paratyphoid bacilli type A were not
agglutinated at all. In a case of typhoid infection
an agglutination with a dilution of i : 40 was obtained
for paratyphoid type B, while typhoid bacilli were
agglutinated in a dilution of i : 300 and over. As a
rule the agglutination with the infecting agent is by
far the strongest, i.e. it proceeds even in high dilu-
tions, whereas other bacteria require a stronger
concentration.
This phenomenon is known as group agglutina-
tion. The bacteria which are agglutinated by one
and the same serum need not necessarily be related,
although usually this is the case. Conversely,
microorganisms which, because of their morpho-
logical or other biological characteristics, are re-
garded as entirely identical or nearly so, are sharply
AGGLUTININS
45
differentiated by means of their agglutination.
Because of this lack of absolute specificity the serum
diagnosis of infection or the identification of bac-
teria by means of agglutination tests, has value
only when very carefully tested. We have said
above that while agglutinins are specific, a serum
which produces agglutination may be far from
being so. The reason for this is that the serum
may contain several agglutinins. In fact, when
immunizing an animal with a particular bacterium
both specific and group agglutinins are produced.
This will perhaps be made clearer by reference to
the following diagram. We assume that the typhoid
A B C B ft E F
Typhoid Bacillus
Colon Bacillus
Dysentery Bacillus
FIG. 5.
bacillus possesses considerable protoplasm A, which
is specific for the typhoid bacillus; that it possesses
also certain protoplasm B, which is common to it,
and to the colon bacillus; and some protoplasm
C, common perhaps to some other bacterium. In
the case of the colon bacillus, protoplasm D is
specific, i.e., possessed only by this bacillus, while
B is common to it and the typhoid bacillus, and E
common to colon and dysentery bacilli. By immu-
nization with the typhoid bacillus we would obtain
4 6 IMMUNE SERA
a serum containing agglutinins against protoplasm
A, B, and C. By virtue of this the serum \vould exert
some agglutinating power also on colon bacilli.
Absorption Method for Differentiating between
a Mixed and a Single Infection and for Identifying
Bacteria. In 1902 Castellani called attention to
a procedure which consists in saturating the diluted
immune serum with successive quantities of the
bacteria most strongly agglutinated until the agglu-
tinating power for these is zero. After centrifug-
ing, the clear fluid is tested on the second variety
of bacteria, and from this one learns whether mixed
or single infection was present. According to Castel-
lani, if the serum of an animal immunized against a
certain microorganism is saturated with that organ-
ism, the serum will lose its agglutinating power not
only for that organism, but also for all other varieties
that it formerly acted on. Saturated with the others,
its action upon the first is reduced little or none at all.
The serum of an animal immunized against two
microorganisms A and B. loses its agglutination
when saturated with A, only for A. Saturated with
A and B it loses agglutinating power for both.
The absorption test is extensively used in the
identification of bacteria, but it must be used with
caution, as its interpretation is open to error. Refer-
ring to the figure illustrating specific and group
agglutinins, let us assume we have obtained a
specific typhoid serum by immunization with typhoid
bacilli. By virtue of the common agglutinin, this
AGGLUTININS
47
serum will act also on colon bacilli. On extracting
such a serum with typhoid bacilli, all the aggluti-
nating power would be lost, that for typhoid bacilli
as well as that for colon. On extracting the serum
with colon bacilli, we would remove the aggluti-
nating power for these bacilli, but leave the specific
agglutinating power for typhoid bacilli. If we
extracted the serum with a culture suspected to be
typhoid bacilli, and found after extraction that
the serum no longer agglutinated known typhoid
bacilli, we could conclude that the suspected culture
was also one of typhoid bacilli.
Formation of the Agglutinins According to the
Side-Chain Theory Receptors of First, Second and
Third Order. Ehrlich's theory as outlined in the
preceding chapter offers a ready explanation for the
development of these bodies. Certain peculiarities
of the agglutinins require merely a slight elabora-
tion of detail in order to be clearly understood.
According to Ehrlich the prime function of the side
chains of a cell is to provide for the nutrition of the
cell. Obviously the simplest mechanism for this
purpose will be a side chain which merely anchors
the food molecule, leaving the digestion entirely to
the cell proper. This type of receptor suffices for
comparatively small molecules such as those of the
toxins, for these are, after all, but the products of
cellular activity. When the protoplasm of the
bacterial cell itself, however, is to serve as food for
the animal cell the latter needs more than a mere
4 g IMMUNE SERA
anchoring group, it needs also an active group
which can in some way act on the huge food par-
ticle and make it more readily assimilable. Such
receptors then possess two groups, a haptophore
group and another functional group acting on the
food particle thus anchored. Ehrlich calls these
his " receptors of the second order," and places in
this class the agglutinins and the precipitins. The
same action can perhaps be more economically
brought about by having these receptors, in addi-
tion to their specific haptophore group, possess the
means by which the action of a ferment-like sub-
stance can be brought to bear on the anchored
food particle. Such a receptor would then possess
two haptophore groups, one for the food particle,
the other for the ferment-like substance. These
are Ehrlich's " receptors of the third order " and
will be discussed in the next chapter. Confining
ourselves for the present to the agglutinins we find
that the existence of the two groups (haptophore
and agglutinating) has experimental confirma-
tion. We have seen that an agglutinin may be
changed by the action, for instance, of acids, so that
it will no longer possess any agglutinating action,
but will still combine with the bacteria. Once the
agglutinating power is lost it cannot be restored,
in which respect the agglutinins differ from the
bacteriolysins.
FIG. 6. THE VARIOUS TYPES OF RECEPTORS ACCORDING TO
EHRLICH.
I. Receptors of the First Order. This type is pictured in a. The
portion e represents the haptophore group, whilst b represents a
toxin molecule, which possesses a haptophore group c and a
toxophore group d. This represents the union of toxin and
antitoxin, or ferment and antiferment, the union between anti-
body and the toxin or ferment being direct.
II. Receptors of the Second Order are pictured in c. Here e represents
the haptophore group, and d the zymophore group of the
receptor, f being the food molecule with which this receptor
combines. Such receptors are possessed by agglutinins and
precipitins. It is to be noted that the zymophore group is an
integral part of the receptor.
III. Receptors of the Third Order are pictured in III, e being the
haptophore group and g the complementophile group of the
receptor. The complement k possesses a haptophore group h
and zymotoxic group z; whilst f represents the food molecule
which has become linked to the receptor. Such receptors are
found in hsemolysins, bacteriolysins, and other cytolysins, the
union with these cellular elements being effected by the ambo-
ceptor (a thrust-off receptor of this order). It is to be noted
that the digesting body, the complement, is distinct from the
receptor, a point in which these receptors therefore differ from
those of the preceding order.
BACTERIOLYSINS AND ILEMOLYSINS
Historical. As far back as 1874, Gscheidlen
and Traube l demonstrated that considerable quan-
tities of septic material could be injected into the
circulation of warm-blooded animals without
apparently any effect on the animal. Very little
was thought of this observation at the time, and it
is not until more than ten years later that we find
a similar observation made by Fodor. 2 In 1888
Nuttall 3 showed that normal blood serum possessed
marked germicidal properties, and his observations
stimulated a number of workers who undertook to
determine the conditions most favorable to the
exhibition of this phenomenon, and further to
decide upon the constituent of the serum to which
this property was due or whether it was a function
of the serum as a whole. In 1889 Buchner 4 pub-
lished a series of experiments and showed that an
exposure of 55 C. robs the serum of its bacteri-
cidal property. He concluded that the active
element in the process was a living albumen and
1 Gscheidlen and Traube. Schlez. Gesellschaft. f. Vater-
land. Cultur, Med. Sect., 1874.
2 Fodor, Deutsche med. Wochenschr, 1886.
3 Nuttall, Zeitschr. f. Hygiene, Vol. iv, 1888.
4 Buchner, Centralblatt Bacteriologie, Vol. v, 1889. Archiv.
f. Hygiene, Vol. x, 1890.
5
BACTERIOLYSINS AND HALMOLYSINS 51
suggested for it the name " alexin." He found that
it was possible to greatly increase the bactericidal
action, (i.e. the quantity of " alexin ") for a par-
ticular bacterium by immunizing an animal with
that bacterium.
Pfeiff er's Phenomenon. An enormous advance
in the study of immunity was made in the dis-
covery of Pfeiffer's phenomenon in 1894, and it is
to Pfeiffer's splendid observations 1 that we owe
the first and most important insight into the mode
of action . of the bacteriolytic immune sera. A
normal guinea pig is able to kill and dissolve a
number of living cholera spirilla if these are in-
jected intraperitoneally. If in such an animal we
gradually increase the dose injected, it will be pos-
sible after a time to inject at one dose an amount
of cholera spirilla that represents many times an
ordinary fatal dose. If from this animal we now
withdraw serum and inject it into another animal,
we find that this serum, even in such small amounts
as the fractional part of a centigram or even of a
milligram, is able to protect the second animal
against living cholera spirilla. Under the influence
of these small amounts of serum of the treated ani-
mal, the organism of the untreated animal is able
to dissolve large amounts of cholera spirilla, amounts
which would otherwise be invariably fatal. This
process, as R. Pfeiff er showed, is a specific one, i.e.,
1 R. Pfeiffer, Zeitschr. Hygiene, Vol. xviii, 1894.
5 2 IMMUNE SERA
the serum of the guinea pig treated with cholera
spirilla transmits an increased solvent power only
for cholera spirilla, but not for any other species of
bacteria. The active substance of such a bacterio-
lytic immune serum Pfeiffer called a specific bac-
tericide. If we allow some of this specific cholera
immune -serum to remain for some time outside of
the body, e.g. in a bottle, and then test it for
solvent properties against cholera spirilla, not in a
living body but in a test-tube, we shall find that its
power is almost nil. If we add to this serum in
the test-tube some fresh peritoneal exudate or
some other body fluid, suoh as serum of a normal,
untreated guinea pig, as Metchnikoff first did, we
find that this serum has now acquired the power
to rapidly dissolve cholera spirilla even in a test-
tube. Bordet, 1 in 1895, showed that in order for
the specific immune serum to dissolve spirilla in a
test-tube, it is unnecessary to add fresh normal
serum or peritoneal fluid; but that immune serum
freshly drawn from the vein is able even under
these circumstances to dissolve the spirilla.
Haemolysis. In his experiments with the bac-
teriolysis of cholora spirilla, Bordet used as an
immune serum the serum of a goat that had been
immunized against cholera spirilla, and as alexin,
fresh normal guinea-pig serum. It often happened
that the latter contained a certain number of red
1 Bordet, Annal. Inst. Pasteur, 1895.
BACTERIOLYSINS AND HsEMOLYSINS 53
blood cells and he found that these were agglutinated
and would be agglutinated even when mixed with
normal goat serum. Knowing, as he did, that
immunization against bacteria increases the agglu-
tinating property toward a given organism over
that in the normal animal, it was natural that he
should experiment to see whether similar results
could be obtained with red blood cells. Accord-
ingly he injected guinea pigs several times with
5 cc. defibrinated rabbit's blood, and found that
not only did this guinea-pig serum acquire agglu-
tinating properties, but also the property to dissolve
rapidly and intensely, in a test- tube, the red blood
cells of a rabbit. The serum of a normal guinea
pig was incapable of doing this, or did it in only a
slight degree. Bordet could further show that this
action is a specific one, i.e., the serum of animals
treated with rabbit blood acquires this dissolving
property only for the red cells of rabbits, not for
those of any other species of animal. ' For the
latter, such a serum is no more strongly solvent
than the serum of a normal animal. The same
property that Bordet had demonstrated in the serum
of guinea pigs treated with rabbit blood could now
be shown for the sera of all animals treated with
blood cells of a different species. We can formulate
this as follows: The serum of animals, species A,
after these have been injected either subcutaneously,
intraperitoneally, or intravenously with erythrocytes
54
IMMUNE SERA
of species B, acquires an increased solvent action
for erythrocytes of species B, and only for this
species. 1 It is therefore a specific action. We call
this hcemolysis, and the substances which effect
the solution of the red cells, hcemolysins or hczmo-
toxins.
At about the same time, and independently of
Bordet, similar experiments with similar results
were published by Landsteiner 2 and v. Dungern. 3
As a result of this work, the acquired toxicity of
horse serum, found by Belfanti and Carbone when
they treated horses with red cells of rabbits, was
explained. The serum of the horses so treated had
become h&molytic for rabbit blood, and therefore
caused a solution or destruction of the red cells
in the living body just as it did in a test-tube.
Nature of Hcemolytic Sera. In a subsequent
study Bordet 4 was able to show that the sol-
vent power of the specific haemolysins depended
on the combined action of two constituents of the
specific serum. When the fresh haemolytic serum
was heated for half an hour to 55 C., it lost its
power. If to this inactive serum a very small
amount of the serum of a normal guinea pig was
added (a serum which of course was not haemolytic
for rabbit red cells), the full haemolytic power was
1 We shall point out a few exceptions later on.
2 Landsteiner, Centralblatt Bacteriol., Vol. xxv, 1899.
3 Von Dungern, Munch, med. Wochenschrift, 1898.
4 Bordet, Annal. Inst. Pasteur, Vol. xii, 1898.
BACTERIOLYSIS S AND H&MOLYSINS 55
restored to this inactive serum. In other words,
it had been reactivated by this addition.
This experiment permits of only one conclusion,
namely, that the hDemolytic action of the specific
haemolytic serum depends on two substances. One
of these is able to withstand heating to 55C., and
is contained only in the specific serum. The other
is destroyed by heating to 55 C., and is contained
not only in the specific haemolytic serum, but also
in the serum of normal untreated animals.
Buchner, we have seen, applied the term alexins
to the constituents of normal serum which were
actively destructive to corpuscular elements, bac-
teria, and other cells with which they came in con-
tact. This term was retained by Bordet to desig-
nate that constituent of normal serum which did
not withstand heating to 55 C., and which was one
of the factors in the haemolytic process. The other
substance, which was found only in the specific
serum and which withstood heating to 55C., he
termed substance sensibilatrice.
According to Bordet, therefore, the substances
required for haemolysis are the substance sensibila-
trice of the specific haemolytic serurn and the
alexin which exists even in normal serum. The
action of these two substances Bordet explains by
assuming that the red cell is not vulnerable to the
alexin ; just as, for example, there are certain sub-
stances that will not take a dye without the previous
56 IMMUNE SERA
action of a mordant. The substance sensibilatrice
plays the role of mordant. It makes the blood
cells vulnerable to the alexin, so that the latter can
attack the cells and dissolve them. The alexin he
regards as a sort of ferment body with digestive
powers.
Bordet says further, that the substance sensi-
bilatrice sensitizes the blood cells not only for the
alexin derived from the serum of the same species
as that from which it (the substance sensibilatrice)
is derived, but sensitizes such cells also for the
alexins of normal sera of other species. For ex-
ample, in the foregoing experiment of Bordet, the
substance sensibilatrice derived from the guinea
pig by treatment with rabbit blood sensitizes the
red blood cells of rabbits not only for the alexin
of normal guinea pig blood, but also for the alsxins
of other normal sera. In another experiment
this author showed that rabbit red cells sensitized
with an inactive specific haemolytic serum derived
from a guinea pig would dissolve rapidly on the
addition of normal rabbit blood. Here, then, the
rabbit red cells, sensitized (according to Bordet) by
the substance sensibilatrice of the guinea pig, dissolve
on the addition of the alexin of their own serum.
Resume. Reviewing the important facts we have
learned, we find them to be as follows: By means
of the treatment of one species of animal with the
erythrocytes of a different one, the serum of the
B ACT ERIOLY SINS AND H&MOLYSINS $7
first species acquires an uncommonly increased
power to dissolve and to agglutinate the erythro-
cytes of the second species. This increased hsemo-
lytic power shows itself not only in vivo, so that an
animal so treated is able to cause red cells injected
into it to rapidly dissolve and disappear, but it
shows itself also in vitro when the serum of this
animal is used. The process consists in the com-
bined action of two substances, that which is excited
in response to the injection, the substance sensi-
bilatrice, and the alexin of normal serum.
Analogy between the Bacteriolytic and Haemolytic
Processes. If we now recall the main points in
cholera immunity the close analogy between this and
the subject of haemolysis is apparent. Just as, when
immunizing an organism against cholera bacilli
the organism responds with an increased solvent
power for those bacteria, so does, the organism
respond when it is treated, i.e. immunized, with
red cells of another species, by increasing the sol-
vent power of its serum for those particular cells.
Furthermore, just as the haemolytic process was
seen to depend on the combined action of two sub-
stances, one developed in the hagmolytic serum,
the other already present in normal serum, so also
in the bactericidal process just studied there are
two factors. It is easy to understand, therefore,
what formerly was not at all clear, why a specific
bactericidal serum against cholera, typhoid, or
58 IMMUNE SERA
other infectious disease should not act in a test-
tube unless there had first been added some normal
serum (according to Metchnikoff), or there had
been employed a perfectly fresh serum (according
to Bordet) : simply because in either of these
ways the alexin necessary to co-operate with the
substance sensibilatrice is introduced. This alexin
no longer exists in the immune serum, if this be
not perfectly fresh, for we have seen that it decom-
poses either on warming, or spontaneously on stand-
ing. A bactericidal serum, therefore, that has
stood for some time is incapable of dissolving
bacteria. It is possible, however, to make an old
inactive serum again capable of dissolving bacteria
in vitro by adding a little fresh alexin, according
to the suggestion of Metchnikoff. In other words,
it is thus reactivated. Another obscure point was
cleared up by these studies: why a specific bac-
tericidal serum which is inactive in vitro should
be intensely active in the living body. This is
because in the living body the serum finds the alexin
necessary for its working, which is not the case in
the test-tube unless fresh normal serum be added.
We see from all this that even the first experiments
in haemolysis have served to clear up a number of
practical points in an important branch of bacteri-
ology.
Ehrlich and Morgenroth on the Nature of Haemo-
lysis. In continuing the study of hsemolysins we
BACTERIOLYSINS AND H.EMOLYS1NS 59
must note particularly the researches of Ehrlich
and Morgenroth. 1 These authors asked themselves
the following questions: (i) What relation does
the haemolytic serum or its two active components
bear to the cell to be dissolved? (2) On what
does the specificity of this haemolytic process
depend ? Ehrlich was led to these researches partic-
ularly by his so-called Side-chain Theory, which
we shall examine in a moment.
He made his experiments with a hasmolytic
serum that had been derived from a goat treated
with the red cells of a sheep. This serum, there-
fore, was haemolytic specifically for sheep blood
cells; i.e., it had increased solvent properties exclu-
sively for sheep blood cells.
Basing his reasoning on his side-chain theory,
Ehrlich argued as follows: " If the haemolysin is
able to exert a specific solvent action on sheep
blood cells, then either of its two factors, the sub-
stance sensibilatrice of Bordet or the alexin of nor-
mal serum, must possess a specific affinity for these
red cells. It must be possible to show this experi-
mentally." Such in fact is the case, and the experi-
ments devised by him are as follows :
Experiment i . Ehrlich and Morgenroth, as
already said, experimented with a serum that was
specifically haemolytic for sheep blood cells. They
1 See the various papers in "Collected Studies on Immunity,"
Ehrlich- Bolduan, Wiley & Sons, New York, 1910.
60 IMMUNE SERA
made this inactive by heating to 55 C., so that then
it contained only the substance sensibilatrice.
Next they added a sufficient quantity of sheep
red cells, and after a time centrifuged the mixture.
They were now able to show that the red cells had
combined with all the substance sensibilatrice, and
that the supernatant clear liquid was free from the
same. In order to prove that such was the case
they proceeded thus: To some of the clear centri-
fuged fluid they added more sheep red cells; and,
in order to reactivate the serum, a sufficient amount
of alexin in the form of normal serum was also
added. The red cells, however, did not dissolve
there was no substance sensibilatrice. The next
point to prove was that this substance had actually
combined with the red cells. The red cells which
had been separated by the centrifuge were mixed
with a little normal salt solution after freeing them
as much as possible from fluid. Then a little alexin
in the form of normal serum was added. After
remaining thus for two hours at 3 7 C. these cells
had all dissolved.
In this experiment, therefore, the red cells had
combined with all the substance sensibilatrice,
entirely freeing the serum of the same. That the
action was a chemical one and not a mere absorp-
tion was shown by the fact that red blood cells of
other animals, rabbits or goats for example, exerted
no combining power at all when used instead of
BACTERIOLYSINS AND H&MOLYSINS 6l
the sheep cells in the above experiment. The
union of these cells, morever, is such a firm one
that repeated washing of the cells with normal salt
solution does not break it up.
So far as concerns the technique of the experi-
ments, I should like to observe that the addition
of red cells in this as well as in all the following
experiments was always in the form of a 5% mix-
ture or suspension in 0.85%, i.e., isotonic, salt solu-
tion.
The second important question solved by these
authors was this; What relation does the alexin
bear to the red cells ? They studied this by means
of a series of experiments similar to the preceding.
Experiment 2. Sheep blood was mixed with
normal, i.e. not haemolytic, goat serum. After a
time the mixture was centrifuged and the two por-
tions tested with substance sensibilatrice to deter-
mine the presence of alexin. It was found that in
this case the red cells acted quite differently. In
direct contrast to their behavior toward the sub-
stance sensibilatrice in the first experiment, they
now did not combine with even the smallest por-
tion of alexin, and remained absolutely unchanged.
Experiment 3. --The third series of experiments
was undertaken to show what relations existed
between the blood cells on the one hand, and the
substance sensibilatrice and the alexin on the
other, when both were present at the same time,
62 IMMUNE SERA
and not, as in the other experiments, when they
were present separately. This investigation was
complicated by the fact that the specific immune
serum very rapidly dissolves the red cells for which
it is specific, and that any prolonged contact be-
tween the cells and the serum, in order to effect
binding of the substance sensibilatrice, is out of
the question. Ehrlich and Morgenroth found that
at o C. no solution of the red cells by the haemo-
lytic serum takes place. They therefore mixed some
of their specific hsemolytic serum with sheep blood
cells, and kept this mixture at o-3 C. for sev-
eral hours. No solution took place. They now
centrifuged and tested both the sedimented red
cells and the clear supernatant serum. It was
found that at the temperature o-3 C. the red
cells had combined with all of the substance sen-
sibilatrice, but had left the alexin practically
untouched.
It still remained to show the relation of these
two substances to the red cells at higher temper-
atures. At 37-4o C., as already mentioned,
haemolysis occurs rapidly, beginning usually within
fifteen minutes. It was possible, therefore, to
leave the cells and serum in contact for not over
ten minutes. Then the mixture was centrifuged
-as before. The sedimented blood cells mixed with
normal salt solution showed haemolysis of a moder-
ate degree. The solution became complete when
'BACTERtOLYSINS AND HMMOLYSMS 63
a little normal serum was added. The supernatant
clear fluid separated by the centrifuge did not dis-
solve sheep red cells. On the addition, however,
of substance sensibilatrice it dissolved them com-
pletely.
From this experiment Ehrlich concludes that the
substance sensibilatrice possesses one combining
group with an intense affinity (active even at o C.),
for the red cell, and a second group possessing a
weaker affinity (one requiring a higher temperature)
for the alexin.
Nomenclature. In place of the name substance
sensibilatrice Ehrlich first introduced the term
immune body; later on he called it the amboceptor,
to express the idea that it served as a link between
alexin and cell. Other names proposed for this sub-
stance have been substance fixatrice by Metchnikoff,
copula, desmon, preparator by Muller. Instead of
the name alexin, Ehrlich now uses the term com-
plement in order to express the idea that this body
completes the action of the immune body.
According to Ehrlich the red blood cells possess
specific affinity for the immune body, but none
whatever for the alexin. The alexin, therefore,
possesses no combining group which can attach itself
directly to the red blood cell. It acts on these cells
only through an intermediary, the immune body,
which therefore must possess two binding groups one
of which attaches to the red blood cell and the other to
64 IMMUNE SERA
the alexin of normal serum. As already stated, the
group which attaches to the red blood cell possesses
a much stronger affinity than that which combines
with the alexin. This follows from the last two
experiments of Ehrlich before cited, in which he
showed that at the lower temperature, and with
both substances present with the blood cells, only
the immune body combined with the cells, while
the alexin remained uncombined. At the higher
temperature the alexin also exerted its affinity, foi
then the red cells combined with all the immune
body and with part of the alexin. We saw that
after a time the red cells partially dissolved, but
that complete solution occurred only after some
fresh alexin had been added. This showed that
although the red cells had combined with all the
immune body necessary for their solution, they had
been unable to bind all the alexin necessary. We
may say, therefore, that that group of the immune
body w r hich combines with the red cell has a
stronger affinity than that which combines with the
alexin.
Role of the Immune Body. According to Ehrlich,
then, the role of the immune body consists in this,
that it attaches itself to the red cell on the one hand,
and to the complement on the other, and in this way
brings the digestive powers of the latter to bear
upon the cell, the complement possessing no affinity
for the red cell. Immune body and complement
BACTERIOLYSINS AND H^EMOLYSINS 65
have no very great affinity for each other. At o C.
they may exist in serum side by side, and they
combine only at higher temperatures.
The amount of immune body which combines
with the red cells may vary greatly, as the experi-
ments of Bordet and of Ehrlich clearly show.
Some red cells combine with only just enough
immune body to effect their solution. Others are
able to so saturate themselves with immune body
that they may have a hundred times the amount
necessary for their solution.
On what the Specificity Depends. From the pre-
ceding it follows that the specific action of the
haemolytic sera, and, I may at once add, of the bac-
tericidal sera also, is due exclusively to the immune
body. This possesses a combining group which is
specific for the cells with which the animal was
treated; e.g., the combining group of an immune
body produced by treatment with rabbit blood
will fit only to a certain group in the blood cells of*
rabbits; an immune body produced by treatment
with chicken blood will fit only to parts of the red
cells of chickens; one produced by treating an ani-
mal with cholera bacilli will fit only to this species
of bacteria and combine only with the members of
it. Keeping to the well-known simile of Emil
Fischer, the relation is like that between lock and
key, each lock being fitted only by a particular
key.
66 IMMUNE SERA
To repeat for the point is of the greatest
importance the role of the immune body consists
in tying the complements of normal serum, which
have no affinity for the red cells or for the bacteria,
indirectly to these cells so that their solution and
digestion may be effected by the complements.
In other words, the immune body serves to con-
centrate on the corpuscular element to be dis-
solved all the widely distributed complement found
in normal serum.
Ehrlich's conception of the relation existing be-
tween complement, immune body (i.e., amboceptor)
and erythrocyte is shown in the accompanying
figure.
i. H.
symotoxic group
\
COMPLEMENT ^
haptophore group
complementophile gr.
IMMUNE BODY ~
:ytophile group
receptor
CEL1
FIG. 7
Difference between a Specific Serum and a Normal
One. The difference, then, between a specific
haemolytic or a specific bactericidal serum and a
normal one consists in this * that the specific serum
contains an immune body which is specific for a
BACTERIOLYSINS AND H&MOLYS1NS 67
certain cellular clement and by means of which the
complement present in all normal serum can be con-
centrated on this element to cause its solution. We
shall return to this subject later.
Diverging Views of Ehrlich and Bordet. Now if
we recall the first experiments of Bordet and his
conclusions respecting the manner in which the
factors concerned a^.ted, we shall at once see how
Ehrlich and Bordet differ. Bordet assumes that
the substance sensibilatrice (the immune body)
acts as a kind of mordant on the red cells or bac-
teria, sensitizing these to the action of the alexin
(complement). That is to say, neither the cell nor
the immune body has alone any manifest affinity
for the alexin, but they form by their union a
complex which can absorb alexin, in other words
which has particular properties of adhesion. Accord-
ing to Bordet, then, there is no such thing as an
amboceptor, and no complementophile group. He
cites the experiments of Muir as showing that the
hypothesis of a complementophile group is untenable.
This author found that blood corpuscles which had
fixed the sensitizer (immune body) and had been
saturated with alexin could subsequently, by dif-
fusion, lose a certain amount of their sensitizer,
although they retain the complement, and what is
more, in this instance they lose as much sensitizer
as if they had not absorbed complement. Con-
sequently, says. Bordet, it is in no way through the
68 IMMUNE SERA
mediation of the sensitizer that the alexin attaches
itself to the corpuscles; if this were the case the
removal of the sensitizer would necessarily imply
that of the alexin (complement) , which, as we have
just mentioned, does not leave the corpuscles.
According to Ehrlich, however, the process is not
analogous to a staining process, but follows definite
laws of chemical combination, there being, in fact,
no affinity whatever between the complement and
the blood cells or bacteria. Furthermore, according
to this authority, the complement always acts
through the mediation of the immune body, which
possesses two combining groups; one, the cytophile
group, combining with the cell, and another, the
complementophile group, combining with the com-
plement.
The Side-Chain Theory Applied to these Bodies. -
All of the specific relations which, in a previous
chapter, we saw existed between toxin and anti-
toxin, Ehrlich and Morgenroth in their experi-
ments above noted found existed also between
immune body and the specific blood cell. The
immune body must therefore possess a haptophore
group which fits exactly to certain receptors or
side chains of the red cells, just as the anti-body
according to the side-chain theory possesses a
group that fits exactly into the specific combining
group i.e., haptophore group of the toxin or
toxoid used for exciting the immunity.
BACTERIOLYSINS AND H&MOLYS1NS 69
If, for example, we produce a haemolytic serum
specific for red cells of a rabbit by injecting an
animal with these cells, the haptophore groups of
this serum, i.e., the free side chains thrust off, must
possess specific combining relations with the red
cells of rabbits. That such is the case in the haemo-
lytic immune serum we saw from the experiments
of Ehrlich and Morgenroth.
In consequence of all this, Ehrlich widened
the application of his side-chain theory so as to
include not only the production of antitoxin but
also the production of bactericidal, haemolytic,
and other immune bodies. He expressed this
somewhat as follows: // any substance, be it toxin,
ferment, constituent of a bacterial or animal cell, or
of animal fluid, possess the power by means of a
fitting haptophore group to combine with side chains
(receptors) of the living organism, the possibility for
the overproduction and throwing off of these recep-
tors is given, i.e., the possibility to produce a cor-
responding anti-body.
Specific anti-bodies in the serum as a result of
immunizing processes can only be produced, there-
fore, by substances which possess a haptophore
group l and which, in consequence, are able to form a
firm union with a definite part of the living or-
ganisms, the receptor. This is not the case with
1 Such substances Ehrlich terms ''haptins." See page
16.
70 IMMUNE SERA
alkaloids, e.g., morphine, strychnine, etc., which
according to Ehrlich enter into a loose union, a kind
of solid solution with the cells. It is for this reason
that we are unable to produce any anti-bodies in
the blood serum against these poisons. Ehrlich
says further that all of the substances taking part
in the production of immunity, including of course
complement and immune body, have certain
definite affinities for each other, and in order
to act they must fit stereochemically to each
other.
As we have already seen, we are able by means
of the injection of a variety of substances or cells
to produce a similar variety of immune bodies in
the serum. Thus we can immunize a rabbit so
that its serum will possess specific haemolytic
bodies against the red cells of guinea pigs, goats,
chickens, and oxen and specific bactericidal
bodies against cholera and typhoid bacilli, etc.,
and as we shall see, still other groups of anti-
bodies.
Multiplicity of Complements. Under these cir-
cumstances an important question presents itself:
Is there in normal serum one single complement
which completes the action of all these various
immune bodies, one, for example, which in the
above illustration will fit all the haemolytic immune
bodies as well as all the bactericidal ones, or
are there a great many different complements?
BACTERIOLYSINS AND HMMOLYSINS 71
Ehrlich, as a result of his experimental work
with Morgenroth, claims that the latter is the case ;
namely, that it takes a different complement to fit
the immune body specifically haemolytic for guinea
pig blood than it does to fit that specific for chicken
blood.
Bordet, on the other hand, assuming that the
immune body plays the role of mordant, believes
that there is but one single complement in the
serum. According to him, this complement is
able to dissolve blood cells as well as bacteria after
these have been sensitized by their specific immune
body. Each of these authors supports his claims
by means of ingenious experiments, for the details
of which, however, we must refer to the original
articles, as they require the knowledge of a specialist
for their comprehension. We shall, however, give
one of Bordet 's l experiments on this point in some
detail, since it has found extensive application in
another direction.
The Bordet-Gengou Phenomenon. Bordet sensi-
tized blood corpuscles with appropriate amboceptors,
and then exposed them to the action of a freshly drawn
normal serum. If now he waited for the occurrence
of haemolysis and then added sensitized cells (bacteria
or blood corpuscles of a different species), the latter re-
mained entirely unchanged, although the serum that had
been used as complement was capable in its original con-
1 Bordet and Gengou, Annal. Inst. Pasteur. Vol. xv, 1901.
72 IMMUNE SERA
dition of destroying these also. When fresh serum was
first brought into contact with sensitized bacteria, simi-
lar results were obtained. The blood corpuscles sub-
sequently added did not then undergo haemolysis.
// such an action on one of the sensitive substrata has
once taken place, the active sera, as a rule, are deprived
of all their complement functions, from which Bordet
concludes that the destruction of the most varied
elements by one and the same serum must be due to a
single complement.
It may be said in passing that Ehrlich admits the
correctness of the above experimental results, but
brings forward arguments to show that Bordet 's inter-
pretation as to the existence of only a single complement
cannot be accepted.
This experiment of Bordet is usually spoken of as
the " Bordet-Gengou phenomenon " and is now used
largely in determining whether or not a given serum
possesses certain amboceptors. The serum to be
tested is first heated and then mixed with a small
quantity of fresh normal serum (complement) and
with an emulsion of the bacterium whose amboceptors
it is desired to discover. After standing for six hours
at room temperature, red blood cells previously treated
with heated haemolytic serum are added. If there is
no hsemolysis it is held to mean that the complement
in the fresh serum which was suitable for lysis of
properly prepared blood corpuscles, has been absorbed
by the bacteria by reason of the presence of specific
amboceptors in the serum tested.
Wassermann 1 has attempted to apply this method
in measuring the amboceptor content of specific menin-
* Wassermann, Neisser and Brack, Deutsche med. Wochen-
schr., 1906; Wassermann and Plaut, Ibid.
BACTERIOLYSINS AND H&MOLYS1XS 73
gococcus sera and has successfully adapted the Bordet-
Gengou test to the diagnosis of syphilitic infections. 1
Neisser and Sachs 2 have recently described a procedure
for the forensic diagnosis of blood stains. The principle of
this is the same as in the preceding, although in so far as
a specific precipitin serum is made use of, the procedure is
really modelled after the "Gengou-Gay" phenomenon.
If human blood serum is mixed with a specific
human precipitin serum derived from rabbits, it will
be found that the mixture binds complement. Hae-
molysin subsequently added is unable to dissolve its
specific red blood cells, owing to this locking up of
the complement. Only the serum of monkeys has a
similar effect. The amount required is extremely
minute, T W^o to TTy <yV<y<T cc - human blood or monkey
blood sufficing. Extracts of human blood stains will also
produce the desired effect. The authors believe that
the immunization with human blood serum gives rise
not only to precipitins but also to amboceptors which
then are able to unite with their corresponding unformed
albuminous bodies and so bind complement. Others
are of the opinion that the complement is bound by
the precipitin-precipitum combination.
The test is extremely delicate and has been found
trustworthy by a number of investigators. In view
of the importance of such tests in medico-legal cases,
Neisser and Sachs suggest that it should always be
used in addition to the well known Wassermann-
Uhlenhuth precipitin test.
Normal Serum, its Haemolytic and Bacteriolytic
Action. Inquiring now into the essential differ-
1 See also page 185.
. 2 Neisser and Sachs, Berliner klin. Wochenschrift, 1905.
74
IMMUNE SERA
ence between a specific haemolytic or bactericidal
serum and a normal one, we must first of all study
the behavior of normal serum toward alien red
cells and bacteria. It has long been known to
physiologists that fresh normal serum of many
animals has the power to dissolve blood cells of
another species. This was studied especially by
Landois. One-half to one c.c. of normal goat serum,
for example, is able to dissolve 5 c.c. of a 5% mix-
ture (in normal salt solution) of rabbit or guinea
pig red cells. In the same way, these red cells
are dissolved by the sera of oxen, of dogs, etc.
This normal globulicidal property of the serum cor-
responds to another which fresh normal serum was
found to possess, namely, the property to dissolve
appreciable quantities of many species of bacteria.
This analogy was pointed out by Fodor, Nutall,
Nissen, and especially by Buchner. We call this
the bactericidal property of fresh normal serum.
This property is well illustrated by the following
protocol from Park.
No. of bacteria
in i cc. fluid.
Amount of
serum added.
Approximate number alive after being kept at 37 O
One hour.
Two hours.
Twenty-seven hrs.
30,000
100,000
1,000,000
. I CC.
. I CC.
0. I CC.
400
5,000
400,000
2
1,000
2,OOO,OOO
200,000
10,000,000
It is at once apparent that the number of bacteria
introduced is an important factor, the normal serum
being able to kill off only a certain number.
BACTERIOLYSINS AND H^MOLYSINS 7-
Buchner, as we have already seen, had studied
this bactericidal action carefully and ascribed the
action to a substance found in all normal serum,
which he called alexin. According to his experi-
ments, this is a very unstable substance, decom-
posing spontaneously on standing or on heating for
a few minutes to 55 C., or readily on the action
of chemicals. According to this author all the
globulicidal and bactericidal functions of normal
serum are performed by this one substance, the
alexin.
Active and Inactive Normal Serum. In taking
up the study of the haemolytic action of normal
serum Ehrlich and Morgenroth sought par-
ticularly to discover whether in normal serum
the haemolytic property depended on the action of
a single substance, the complement (Buchner's
alexin), or whether here as in the specific haemo-
lytic serum it depended on the combined action
of two substances. For this purpose they used
guinea-pig blood, which is dissolved by normal
dog serum. If this serum was heated to 55 C., it
lost its haemolytic power. It was necessary now
to show that in this inactive dog serum there
remained a second substance which could be reacti-
vated after the manner of reactivating an old
specific haemolytic serum. This had its difficulties,
for they could not add normal dog serum. This,
as we saw, is already haemolytic for guinea-pig
7 6 IMMUNE SERA
blood. " Possibly/' said they, " there exists a com-
plement of another animal which will fit the hypo-
thetical second substance of this dog serum."
This proved to be the case, the complement of
guinea-pig blood fulfilling the requirements. If
they added to the inactive normal dog serum about
2 c.c. normal guinea-pig serum the haemolytic prop-
erty was restored and the guinea-pig red cells
dissolved completely. According to Ehrlich, this can
only be explained by assuming that in guinea-pig
blood there exists a complement which happens to fit
the haptophore group of the second substance or
inter- body, of the normal dog serum. This com-
bination of guinea-pig blood, inactive normal dog
serum, and a reactivating normal guinea-pig serum
is well adapted to demonstrate the existence in
normal dog serum of an inter-body; for the guinea-
pig serum should be the best possible preservative
for the guinea-pig red cells. The haemolysis fol-
lowing the addition of this serum shows positively
the existence of a substance in the dog serum which
has acted with something in the guinea-pig serum. 1
1 Of such combinations, i.e., combinations in which a com-
plement derived from the same animal from which the red cells
are derived fits to the inter-body of other species of animals,
causing the solution of red cells of the latter, Ehrlich and
Morgenroth found still other examples. For instance, guinea-
pig blood, inactive calf serum, guinea-pig serum; goat blood,
inactive rabbit blood, goat serum; sheep blood, inactive rabbit
blood, sheep serum; guinea-pig blood, inactive sheep serum,
guinea-pig serum.
BACTERIOLYSINS AND H&MOLYSINS 77
Inter-body and Complement. We see, then, that
the haemolytic action of normal sera depends, just
as that of the specific haemolytic sera, on the com-
bined action of two bodies: one, the inter-body,
which corresponds to the immune body of the
specific sera, and a second or complement. In
speaking of the constituents of normal serum,
Ehrlich and Morgenroth prefer to use this term
inter-body to distinguish it from the immune bodies
of specific hasmolytic sera.
Action not Entirely Specific. It has also been
found that there frequently exist normal sera which
are haemolytic not only for one species of red cell,
but for several. We saw, for instance, that normal
goat serum dissolved the red cells of guinea pigs
and rabbits. The question now arises, Is this prop-
erty of normal goat serum due to two inter-bodies
existing in the serum side by side, one fitting the
red cells of the guinea pig, the other those of the
rabbit? Ehrlich and Morgenroth answered this
in the affirmative, for in the following experi-
ment they succeeded in having each of the two
inter-bodies combine with its respective cell. To
some inactive normal goat serum they added rab-
bit blood and centrifuged the mixture. To the
separated clear fluid they again added some rab-
bit red cells as well as normal horse serum to reac-
tivate the mixture. Horse serum is not haemo-
lytic for rabbit red cells. The mixture remained
7 8 IMMUNE SERA
unchanged, no haemolysis taking place. If, how-
ever, they added some of this normal horse serum
to the centrifuged red cells, the latter immediately
dissolved. Now, to the clear centrifuged fluid,
which as we have seen would not dissolve rabbit
red cells, they added guinea-pig red cells and again
some normal horse serum to reactivate the mixture.
The guinea-pig red cells all dissolved. This proved
conclusively that in the normal goat serum there
had existed two specific inter-bodies. One, for
rabbit red cells, had been tied by these cells and
carried down with them in centrifuging ; the other,
specific for guinea-pig red cells, had remained
behind.
Multiplicity of the Active Substances. Further
investigation led these authors to assume a still
greater multiplicity in the substances in normal
serum which are concerned in haemolysis. In
addition to the two interbodies just mentioned, they
demonstrated the existence in goat serum of two
specific complements, one for each interbody, and
they were able by means of Pukall filters to separate
these two. In this filtration the complement fit-
ting the inter-body for rabbit blood remained
behind for the greater part, while that fitting
the inter-body for guinea-pig blood mostly passed
through.
Whereas then, according to Buchner, only one
substance, the alexin, is concerned in the haemo-
B ACT ERIOLY SINS AND H JEM OLY SINS 79
lytic action of this normal goat serum, these
experiments of Ehrlich and Morgenroth show
us four substances, viz., two inter-bodies and
two complements. This at once makes clear the
opposing views of these authorities. According to
Ehrlich, however, the number of active substances
in normal serum is still greater, for it often hap-
pens that a specific inter-body shows itself to be
made up of several inter-bodies, all, to be sure,
fitting the same specific red cell, but differing
from each other by their behavior toward dif-
ferent complements. Ehrlich, therefore, regards
the substances concerned in haemolysis which
occur in normal serum to be of great number and
variety.
Difference between a Normal and a Specific
Immune Serum. Practical Application. Return-
ing now to the question of the difference between a
specific immune serum and a normal one, we find
this to be as follows : Normal serum contains a great
variety of inter-bodies, in very small amounts, and a
considerable amount of complements. In immune
serum, on the other hand, the amount of a specific
inter-body, the one which fits the haptophore group
of a certain cell, is enormously increased. This
specifically increased inter-body, it will be remem-
bered, is called the immune body. The comple-
ment, as shown by v. Dungern, Bordet, Ehrlich
and Morgenroth and Wassermann, is in no way
80 IMMUNE SERA
increased by the immunizing process. The increase
affects solely the immune body. It is therefore
possible to have a serum which contains more
immune body than complement to satisfy it, and if
we withdraw such a serum from an animal we shall
find that it contains some free immune body. This
serum can only then exert its 'full power when the
full amount of complement is present, i.e., when
some normal serum is added. If we treat a rabbit
with the red cells of an ox, as v. Dungern did, we
shall obtain a serum which is haemolytic for ox
blood. Of this freshly drawn serum 0.05 c.c. suf-
fice to dissolve 5.0 c.c. of a 5% mixture of ox
blood. If now we add to this haemolytic serum
a little normal rabbit serum, we shall find that
only one-tenth of the amount of serum is required;
i.e., only 0.005 c - c - to dissolve the same quantity
of ox blood. This means that through the addi-
tion of the rabbit serum, which, of course, is not
haemolytic for ox blood, a sufficient amount of
complement was added to enable all the immune
body of the specific serum to act. This specifically
increased power of the immune serum to act on
certain definite cells depends on the fact that the
immune body resulting from the immunizing
process concentrates the action of the comple-
ment scattered through the serum, on cells for
which it has definite affinities. If 2 c.c. of normal
guinea-pig serum are able to dissolve, we will say
BACTERIOLYSINS AND H&MOLYSlNS 8l
5 c.c. of a 5% defibrinated rabbit-blood mixture,
and if we find that after the immunizing process
0.05 c.c. of the guinea-pig serum suffice to dissolve
the same amount of rabbit blood, we conclude
that through this process the inter-body, i.e. the
immune body, has been increased forty times. We
know that the complement has not been increased,
but this is now able to act by means of forty times
increased combining facilities. This increase, how-
ever, is exclusively for rabbit-blood cells. In a
bactericidal immune serum this specific increase is
sometimes as much as 100,000 times that of normal
serum.
The practical idea to be gained from this for
the therapy of infectious diseases is this: that
with the injection of an immune serum we supply
only one of the necessary constituents to kill
and dissolve the bacteria, and that is the immune
body.
We do not, however, supply the second, i.e. the
complement, for this we have seen is not increased
by the immunizing process. As matters stand,
then, the use of a specific immune serum for
therapeutic purposes assumes that the complement
which is essential for the action of the immune
body will be found in the organism to be treated.
Since in certain infectious diseases the required
complement is present in too small amounts in
the organism, Wassermann suggested that the
82 IMMUNE SERA
curative power of many bactericidal sera might be
increased by the simultaneous injection of the sera
of certain normal animals in order thus to gain
an increased amount of complement; but we
shall soon see that this procedure, while of great
value in animal experiments, presents certain dif-
ficulties.
Nature of the Immune Body Partial Immune
Bodies of Ehrlich Turning now to a closer study of
the nature of the immune body, we again find a dif-
ference of opinion. Whereas Bordet, MetchnikofI,
and Besredka assume each immune body to be a
single definite substance, Ehrlich and Morgenroth
as a result of their experiments hold to a plurality
of bodies.
These authors say that each immune body
is built up of a number of partial-immune bodies,
a point to which we have already alluded. In
support of this view they offer the following ex-
periment. On immunizing a rabbit with ox blood,
they obtained a serum haemolytic not only for
ox blood but also for goat blood; on immunizing
a rabbit with goat blood they obtained a serum
haemolytic for goat blood and ox blood. 1
The conditions present can be readily under-
stood by reference to Fig. 7, which represents
schematically three portions of the combining groups
1 We have already called attention to these exceptions to the
rule of specific action.
BACTERIOLYSINS AND HAZMOLYSINS 83
of the blood cells. Of these a is present only in the
ox-blood cells, ^ only in the goat-blood cells, and ft
in both. If a rabbit is injected with ox blood, the
immune bodies corresponding to groups a and ft
will be formed. On subjecting such a serum to
absorption with ox-blood cells we shall find that
these, by means of their a and ft groups will be able to
absorb all the immune bodies, whereas goat-blood
cells will in a similar test absorb only the immune
FIG. 7
body of portion ft, leaving the immune body of
portion a in solution.
According to Ehrlich's theory, then, the red cells
of the ox possess certain receptors which are identi-
cal with receptors possessed by the goat red cells.
From this it follows that in a single red cell there
are several or many groups each of which is able,
when it finds a fitting receptor, to take hold of a
84 IMMUNE SERA
single immune body. Ehrlich and Morgenroth,
therefore, claim that the immune body of a haemo-
lytic serum is composed of the sum of the partial
immune bodies which correspond to the individual
receptors used to excite the immunity. It may be
assumed, then, that not all of the combining groups
of a cell, be this a blood cell or a bacterium, will
find fitting receptors in every animal organism,
and that therefore not all the possible partial im-
mune bodies will be equally developed. In one
animal there may be receptors which are not pres-
ent in another, and in this way there might be a dif-
ferent variety of partial immune bodies in the two
animals. This would lead to the possibility of the
occurrence of immune bodies, for the same species
of blood cell or bacterium, differing from each other
in the partial immune bodies composing them,
according to the variety of animals used in prepar-
ing the serum.
Metchnikoff's Views Practical Importance of
the Point. This view is directly opposed to that of
MetchnikofT and Besredka, who believe that a cer-
tain immune body, e.g. one specific for ox blood,
is always the same no matter from what animal it
is derived. The point is not merely theoretical,
but under certain circumstances of great practical
importance. If we believe, as Ehrlich does, that
the immune body differs according to the species of
animal from which it is derived, i.e., that it is made
BACTERIOLYSINS AND H&MOLYSINS 85
up of different partial-immune bodies, then we must
admit that we have better chances for finding fit-
ting complements if we make use of immune bodies
derived from a variety of animals. We would, for
instance, be likely to achieve better results in treat-
ing a typhoid patient with a mixture of specific
bactericidal typhoid sera derived from a variety of
animals than if we used a serum derived only from
a horse. For in such a mixture of immune bodies
the variety of partial-immune bodies must be very
great and the chances that the complements of the
human body will find fitting immune bodies, and so
lead to the destruction of the typhoid bacilli, are
greatly increased. Ehrlich and his pupils have
actually proposed such a procedure in the use of
bactericidal sera for therapeutic purposes. 1
Support for Ehrlich' s View. Besides the above
experiments we possess others which support the
theory that the immune body is not a simple but
a compound substance, v. Dungern had already
shown that following the treatment of an animal
with ciliated epithelium from the trachea of an ox,
there were developed immune bodies which acted
not only on the ciliated epithelium but also on the
red cells of oxen. We must assume, therefore, that
1 Reasoning along similar lines, namely, that the human
complement must fit the immune body of the therapeutic
serum, Ehrlich has also proposed that these bactericidal sera
be derived from animals very closely related to man, e.g.,
apes, etc.
86 IMMUNE SERA
the ciliated epithelium and the red cells of the ox
possess common receptors. Analogous to this is
the action of the immune body resulting from the
injection of spermatozoa, as was pointed out by
Metchnikoff and Moxter.
We see, then, that the specific action of immune
bodies is not so limited as to apply only to the cells
used in the immunizing process, but extends to
other cells which have receptors in common with
these."
So far as concerns the site in the organism where
the substances used in immunizing find their
receptors, this is not known for the hsemolytic
immune body. For the bactericidal immune bodies
of cholera and typhoid the researches of Pfeiffer,
Marx, and others show that the chief site of pro-
duction is in the bone-marrow, spleen, and lymph
bodies. Wassermann's experiments on local immu-
nity indicate that the site of infection determines
largely the site of the development of the immune
bodies.
Antihaemolysins : their Nature Anti-complement
or Anti-immune Body? A further step in the
study of haemolysins is one discovered independ-
1 The same holds good for the agglutinins and the pre-
cipitins still to be studied. In these the action extends also
to closely related cells and bacteria, or in the case of the precipi-
tins to closely related albumins, as these possess a number of
receptors which are common to them and to the cells or sub-
stances used for immunizing.
BACTERIOLYSINS AND H^EMOLYSINS 87
ently by Ehrlich and Morgenroth on the one hand,
and Bordet on the other. These authors succeeded
in producing an antih&molysin. The procedure is
closely related to the results gained by immuniza-
tion against bacterial poisons. A specific haemoly-
sin, one, for example, specific for rabbit blood,
derived by treating a guinea pig with rabbit red
cells, is highly toxic to rabbits. Injected into the
animals intravenously in doses of 5 c.c. it kills the
animals acutely, causing intra vitam a solution of
the red cells. Such a haemolytic serum, then, acts
the same as a bacterial poison, and it is possible to
immunize against this just as well as against a bac-
terial poison. For example, to keep to our illustra-
tion, rabbits are injected first with very small doses
of this specific haemolytic serum. The dose is
gradually increased until it is found that the animal
tolerates amounts that would be absolutely fatal to
animals not so treated. If some of the serum of
this animal is now abstracted and added to the
specific haemolytic serum, it is found that the power
of the latter will be inhibited. According to
Ehrlich an antihanwlysin has been formed. As
we know that the action of the haemolysin depends
on the combined action of two substances, the
immune body and the complement, the question
arises to which of these two the antihasmolysin is
related. Is it an anti-immune body or an anti-
complement? A study of this question shows that
88 IMMUNE SERA
both these substances are apparently present. In
the serum of the rabbit treated with specific haemo-
lysin, both an anti-immune body and an anti-
complement have been found. Ehrlich and Mor-
genroth were further able to show that the action
of the anti-complement depended on a haptophore
group which it possessed, enabling it to combine
with the haptophore group of the complement,
thus satisfying this and hindering its combination
with the complementophile group of the immune
body.
" Anti- complement." l *Since the complements are
constituents of normal serum, it should be possible
to produce anti-complements by injecting animals
merely with normal serum; and they can, in fact,
be so produced. If rabbits are treated by inject-
ing them several times with normal guinea pig
serum, a serum may be obtained from these rabbits
which contains anti-complements against the com-
plements of normal guinea-pig serum. A serum
obtained in this way of course contains only one of
the antihaemolytic bodies, the anticomplement,
and not the antiimmune body. This is because
normal serum is too poor in immune body (inter-
body) to excite the production of any antiimmune
body.
1 The existence of anti-complements is denied by Bordet,
Gay and others. For a statement of their views see pa<je
96.
BACTERIOLYSINS AND IL-EMOLYSINS 89
If to a hsemolytic serum derived from guinea pigs
we add an anticomplement serum derived, as just
stated, from rabbits, and containing an anticom-
* I
ii.
f
COMPLEMENT
COMPLEMENT
ANTICOMPLEMENT
IMMUNE BODY J| f IMMUNEBODY
CELL MB m m CELL
FIG. 8. (After Levaditi.)
plement specific for guinea-pig complement, the
haemolytic action of the former will be inhibited, for
the reason that the complement necessary for the
haemolysis to take place has been bound by the
anticomplement. (See Fig. 8.) One must, how-
ever, observe the precaution to heat the anticom-
plement serum of the rabbit to 55C. before so
mixing it, in order to destroy the complement which
it contains and which would otherwise reactivate the
guinea-pig immune body.
From the foregoing we see that either anti-
immune body alone, or anticomplement alone, is
able to inhibit the hxmolytic action. Haemoly-
sis cannot take place when either of the two
9
IMMUNE SERA
necessary factors is bound and prevented from
acting. l
The action of anticomplements is specific, i.e., an
anticomplement combines only with its specific
complement. Thus an anticomplement serum
derived from rabbits by treatment with guinea-
pig serum combines only with the complement of
normal guinea-pig serum, not, however, with the
complements of other animals. Exceptions to this
are those cases in which the complement of the
other species possess receptors identical with those
of the first.
In order that a normal serum of species A,
injected into species B, produce anticomplements
there, the side-chain theory demands that the com-
plements of A find fitting receptors in species B.
According to Ehrlich, however, normal serum con-
tains many different complements and not merely a
single one. Under the circumstances, it is easily
possible that only a few of the complements in the
serum of A find fitting receptors in species B. We
shall then obtain an anticomplement serum which
inhibits the action of some, but not of all the com-
plements of species A. Thus it might inhibit the
1 By treating animals with normal sera of certain other
species, it is possible to produce not only anti-complements,
but also specific anti-bodies against certain other constituents
of normal serum. These are, for example, anti-agglutinins,
which inhibit the action of the haemagglutinins of normal serum,
and anti-precipitins, which we shall discuss later.
BACTERIOLYSINS AND HsEMOLYSINS 91
action of a complement fitting to a certain bacteri-
cidal immune body and not of one contained in the
same serum which fitted a certain haemolytic im-
mune body, etc.
Fluctuations in the Amount of Complement and
other Active Substances in the Blood We have
come to know certain conditions under which there
may be a decrease of certain complements in
normal serum. Ehrlich and Morgenroth showed
that in rabbits poisoned with phosphorus and in
whom, therefore, the liver was badly damaged^
the serum on the second day (the height of the
disease) had lost its power to dissolve guinea-pig
blood, and that this was due to a disappearance of
the complement. Metchnikoff also reported that in
an animal suffering from a continuing suppurating
process, the complement had fallen considerably in
amount. Especially interesting are the experi-
ments of v. Dungern, who showed that animal cells,
hence emulsions of fresh organs, are able to attract
and combine with complements.
Even more important than the question of a
decrease in complements, or an inhibition of their
action, is that of the possibility to artificially in-
crease them. A number of authors, among them
Nolf and Miiller, have answered this question in the
affirmative. They believe they have noticed such
an increase following the injection of an animal with
92 IMMUNE SERA
all sorts of substances, such as normal serum of
another animal, sterile bouillon, etc. v. Dungern,
Wassermann and others, have not been able to con-
vince themselves of the possibility of such a definite
increase. Wassermann tried to excite the increased
production of complement by injecting guinea pigs
for some time with anticomplement. This being
the opposite of the complement, he hoped to be
able by immunizing to excite an increase of the
complements. In this he was unsuccessful, though
of course it maybe possible with another species of
animal.
Despite all this, we must believe that the amount
of complement, as well as the amount of other active
substances of the blood, inter-bodies, agglutinins,
antitoxins, ferments, antiferments, etc., is subject
to great fluctuations even in the same individual,
a constant change going on within the organism.
Ehrlich, in particular, has pointed out these indi-
vidual and periodic variations and has insisted on
their importance. Very likely, under circumstances
of which we now know very little, these substances
are at certain times produced in greater amounts,
at other times in lesser; sometimes they may be
absent entirely in an individual in whom they were
previously present. For example, the serum of a
dog will at times dissolve the red cells of cats, rab-
bits, and guinea pigs, at other times not. Further-
more, the serum of one and the same animal may
B ACT ERIOLY SINS AND H.EMOLYSINS p*
possess specific haemolytic properties for certain
cells, and later on may lose this property entirely.
In human serum these same individual and periodic
variations may be demonstrated, as Wassermann
was able to prove experimentally. However, the
circumstances on which these variations depend are
as yet entirely unknown to us. Possibly we are
dealing here with subtle pathological changes.
Source of the Complements Leucocytes as a
Source Other Sources. Where do the comple-
ments or alexins originate? This question has been
studied particularly by Metchnikoff and by Buch-
ner; also by Bail, Hahn, Schattenfroh, and others.
These investigators believe that the leucocytes are
the source of the complements or alexins. There
is, however, this difference between the views
of Metchnikoff and Buchner: whereas Buchner
believes the alexins to be true secretory products,
Metchnikoff believes that they originate on the
breaking up of the leucocytes, i.e., that they are de-
composition products. Metchnikoff bases his belief
chiefly on the work of his pupil, Gengou, who showed
that although the serum was rich in alexin (i.e., com-
plement) the plasma contained none at all.
Other authors, as Pfeiffer and Moxter, as a result
of their experiments, are not willing to assume the
existence of any relationship between the alexins
and the leucocytes. Gruber as well as Schatten-
froh are ready to believe the leucocytes to be the
94 IMMUNE SERA
source of an alexin, but claim that this is different
from that found in serum. Wassermann believes
that the leucocytes are a source of complements
(alexins), for he succeeded in producing anticom-
plement by means of injections of pure leucocytes
which had been washed free from all traces of serum,
and which had been obtained by injections of aleu-
ronat. In view of the plurality of the comple-
ments, Wassermann expressed the view that the
leucocytes are probably one source, but not the
only one, for the complements of the serum. Land-
steiner and Donath have confirmed this experi-
mentally. They succeeded in producing anticom-
plement by the injection, not only of leucocytes,
but of other animal cells. Furthermore, the experi-
ments of Ehrlich and Morgenroth already mentioned,
in which the complements disappeared after the
destruction of the liver function, show that the liver
cells are concerned in the formation of complements.
Structure of Complements Haptophore and Zy-
motoxic Groups Complementoids. Ehrlich 's con-
ception of the structure of complements is based
principally on the fact that when complement is
heated to 55 C., its complementing power is lost.
If animals are injected with such a heated serum,
anticomplement will be produced, showing, accord-
ing to Ehrlich, that heating has not destroyed the
entire complement body, but only that part which
effects the digesting, solvent action. The hapto-
BACTERIOLYSINS AND HMMOLYSINS
95
phore group must have remained intact. Ehrlich
therefore concludes that the complements consist
of a combining haptophore group which withstands
heating to 55 C., and another, more fragile group,
which possesses the actual solvent properties and
which Ehrlich calls the zymotoxic group. The
complements, according to this conception, are
entirely analogous to the toxins already studied.
zymotoxic group
COMPLEMENT
haptophore group
IMMUNE BODY
FIG. 9.
And just as those toxins which had lost their
toxophore group were called toxoids, so Ehrlich
terms complements which have lost their zymo-
toxic group, complementoids .
Bordet, it will be recalled, does not believe in
any complementophile group in the amboceptor.
So also in the case of the complement, or alexin
as he prefers to call it, he refuses to accept Ehrlich 's
view that this contains a zymotoxic and a hapto-
96 IMMUNE SERA
phore group. The fact that heated complement
produces an anticomplementary serum is readily
explained by Bordet as due to the absorption of
complement by precipitates. An illustration will
bring out this point more clearly.
Goat serum heated to 55 C., and therefore con-
taining no active complement, is injected into a
rabbit. According to Ehrlich it excites the pro-
duction of an " anticomplement " in response to
the " complementoid " which it contains. As we
shall see in the next chapter, the injection of this
heated goat serum in addition to whatever else it may
do does actually excite the production of a specific
precipitin in the serum of the rabbit, so that when
such a rabbit serum is mixed with goat serum, a pre-
cipitate will be "produced. Ehrlich 's demonstration
of the " anticomplement " is somewhat as follows:
Ox blood corpuscles, plus suitable amboceptor (serum
of a rabbit injected with ox blood corpuscles), plus
fresh, normal goat serum as complement, undergo
haemolysis. When, however, the fresh, normal
goat serum is mixed with the serum of a rabbit
previously injected with goat serum, and then the
above combination carried out, no haemolysis
occurs. The rabbit serum contains an " anti-
complement," says Ehrlich. On the other hand,
Bordet and Gay believe that the anticomplement-
ary action is due to the absorption of goat com-
plement by the precipitate produced by the mix-
BACTERIOLYSINS AND H.&MOLYSINS
97
ture of goat serum and its precipitin, the rabbit
serum. Moreschi comes to the same conclusion.
Isolysins Autolysins Anti-isolysins. All of
the preceding studies in haemolysis have concerned
themselves with the results obtained by injecting
animals of one species with blood cells of another.
Ehrlich and Morgenroth now sought to discover
what the results would be if they injected an animal
with blood cells of its own species. They injected
goats with goat blood, and found that when the
amount injected at one time was large the serum
of the goat injected acquired hasmolytic properties
for the blood of many other goats, but not for all.
The substances thus formed the authors called
isolysins. These, then, are substances which will
dissolve the blood of other individuals of the same
species. Substances which dissolve the blood cells
of the same individual are called autolysins. But
autolysins have so far been demonstrated experi-
mentally only once (by Ehrlich and Morgenroth).
If one tests the properties of an isolysin of a goat on
the blood of a great many other goats, it will be
found that this will be strongly solvent for the
blood of some, slightly for the blood of others, and
not at all for still others.
By using a blood that was readily dissolved by
the isolysin, and proceeding in the same series of
experiments which we have already studied under
haemolysis, Ehrlich and Morgenroth showed that
98 IMMUNE SERA
the isolysins, like the hsemolysins, consist of an
immune body and a complement of the normal
serum. The experiments undertaken by these
authors were made on thirteen goats, and the sur-
prising fact developed that the thirteen resulting
isolysins were all different. For example, the iso-
haemolytic serum of one goat dissolved the red cells
of goats A and B\ that of a second goat those of
C and D ; of- a third those of A and D, but not of C,
and so on. If now they produced antiisolysins by
injecting animals with these isolysins, they found
that these antiisolysins were specific; i.e., the anti-
isolysin of A would inhibit the action only of iso-
lysin of A, but not of C, etc. These results are of
the highest clinical interest, for they show a differ-
ence in similar cells of the same species, something
that had never before been suspected. In the above,
the blood cells of species A must have a different bi-
ological constitution than those of species C, etc.
Moss finds that isolysins occur in about 2 5 % of adult
human individuals, and that the relative frequency is
the same in health and disease, so that the presence
of isolysins has no diagnostic significance. The sub-
ject has recently acquired importance because of the
revival of homologous transfusion, and it is customary
now to always test the blood of both donor and re-
cipient prior to carrying out such a transfusion. The
technique of the test is described in the appendix.
The fact that after injections of large amounts of
BACTER10LYSINS AND HMMOLYSINS
99
cells of the same species isolysins develop, but that
autolysins are almost never formed, caused Ehr-
lich and Morgenroth to assume that the body pos-
sesses distinct regulating functions which naturally
prevent the formation of the highly destructive
autolytic substance. It is obvious that if there
were no such regulating facilities, the absorption of
large bloody effusions and hemorrhages might lead
to the formation by the organism of autolysins
against its own blood cells. Gengou, a pupil of
Metchnikoff, believes to have shown experimen-
tally that the destructive action of these auto-
lysins is hindered by the simultaneous production
of an auto-antiimmune body which immediately
inhibits their action.
In order that isolysins may be formed, it seems
necessary to overwhelm the organism once or sev-
eral times with large amounts of cells or cell prod-
ucts of the same species ; to produce, as Ehrlich says,
an ictus immunisatorius. Wassermann tried, by
using various blood poisons, such as haemolytic sera,
toluylenediamine, etc., for a continued length of
time, to cause the formation of these isolysins, but
without success, although in these experiments
each injection was followed by an appreciable
destruction of red cells and absorption of their
decomposition products. The gradual and even
repeated absorption of not too large quantities of
decomposed red cells does not therefore lead to the
100 IMMUNE SERA
formation of isolysins; but, as already said, a sudden
overwhelming of the organism by large amounts
of the cells or their products is necessary.
Deflection of Complement. In the use of the
antitoxic sera, experience has shown that the em-
ployment of a large dose is of paramount importance.
So far as the antitoxic action is concerned l one
cannot do harm by giving a large excess. Con-
cerning the action of bactericidal sera, however,
the literature contains a number of examples which
indicate that here an excess of immune serum is
occasionally injurious. Perhaps the earliest proto-
col of this kind is that published by Loffler and
Abel 2 on their experiments with bacillus coli and a
corresponding immune serum. Out of nineteen
guinea pigs which had been inoculated with the
same amount of culture and had received varying
amounts of immune serum, only six animals were
protected, those which had received doses of 0.25
c.c. to 0.02 c.c. Eight animals with larger doses,
as well as five with smaller doses of serum died.
Neisser and Wechsberg 3 encountered the same
phenomenon in bactericidal test-tube experiments,
and concluded as a result of their experiments
1 We shall discuss the rash production, or " serum sickness,"
page 141.
3 F. Loffler and R. Abel, Centralblatt Bacteriol., 1896, Vol.
xix, p 51.
8 M. Neisser and F. Wechsberg, Munch, med. Wochen-
schrift. 1901. No. 1 8.
BACTERIOLYSINS AND H&MOLYS1NS IOI
102 IMMUNE SERA
that the only satisfactory explanation was one based
on the views of Ehrlich and Morgenroth. In Fig.
10, A II represents schematically a bacterium a
with a number of receptors; for there are many
reasons for assuming that each bacterium possesses a
number of receptors of the same kind. According
to the side-chain theory, if we inject this bacterium
into an animal an over-production of the corres-
ponding group will occur, resulting in a serum
which is rich in body b. This body b, however, is
not able by itself to injure the bacteria, and a
bacterium all of whose receptors are laden with b
need not at all be injured in its vitality. Body b
normally possesses a peculiar function, namely,
to serve as a coupling member or link, and hence it
possesses two groups (amboceptor). As has already
been discussed, one of these groups fit the receptors
of the bacterium on the one hand and the com-
plement on the other. When, therefore, to a normal
serum which contains suitable complement, we add
equivalent amounts of immune serum, the con-
dition pictured in A I will result. On adding the
corresponding bacterium to this we get the con-
dition shown in A II, in which all the bacterial
receptors are occupied with immune bodies, or
more accurately, with immune bodies which on
their part are loaded with bacteriolytic comple-
ment c. in the case here presented let us say that
it requires the occupation of all of the receptors
BACTER10LYSINS AND HJEMOLYSINS IO3
with complemented interbodies to cause the death
of the bacterium.
If now to an equivalent mixture of comple-
ment and inter-body we add an excess of inter-body,
it will be possible for only a part of the inter-body to
be loaded with complement, leaving a portion of
the inter-body uncomplemented. On adding the
corresponding bacteria a number of conditions may
result; the affinity of the inter-body for the bac-
terial receptor may, as a result of the loading with
complement, (i) remain unchanged, (2) it may
thereby be increased, or (3) be diminished.
In the figure, B II shows the condition of in-
creased affinity. Of the six inter-bodies only those
combine with the bacterium which have become
laden with complement. In this case, therefore,
the excess of inter-bodies will have no influence on
the bactericidal effect. The condition is really the
same as A II, except that free inter-body is also
present.
C II shows the condition of unchanged affinity.
In this case, if we add the bacterium to the mixture
of complement and excess of inter-body, all the
receptors of the bacterium will, to be sure, be occu-
pied by inter-bodies, but this will be entirely with-
out regard to the fact that these inter-bodies are or
are not loaded with complement. It may there-
fore happen that only a few of the bacterial receptors
will be occupied by complemented (i.e., active)
104 IMMUNE SERA
inter-bodies, while the rest of the bacterial receptors
are occupied by uncomplemented (hence inactive)
inter-bodies. As already stated, however, the
vitality of such a bacterium is not necessarily
destroyed.
D II represents the last conceivable case. It i^
assumed that the " completion " of the inter-body
has resulted in a diminution of the latter 's affinity
for the bacterial receptor. In this case primarily
only the uncomplemented inter-bodies will com-
bine with the bacterial receptors, while the free
fluid will contain complemented inter-bodies.
In cases C II and D II, therefore, the excess of
inter-body exerts a deflecting action on ike complement,
thus diminishing the end results.
It is difficult to say to what extent " deflection of
complement " really occurs in the experiments
referred to above. Studies by Buxton, Gay, and
others show that deflection of complement will not
always explain the phenomenon, and that in these
instances other factors must be responsible for the
paradoxical results.
Practical Value of Injections of Anti-Bacterial
Sera. We use the term " antibacterial" advisedly,
because, after all, when we immunize an animal
against a certain bacterium we do not produce
merely a bactericidal serum, but one which con-
tains agglutinins, precipitins, opsonins, and per-
haps still other antibodies as well. The use of
BACTERIOLYSINS AND H.EMOLYSINS
specific antibacterial sera has been tried in man both
to cure existing infections and as a preventive of
infection. The therapeutic use has in most instances
been rather disappointing, though in dysentery,
plague, gonococcus and meningococcus infections
the results have been somewhat better. Recently
also, fairly good reports are heard from the admin-
istration of large doses of antistreptococcus serum.
In susceptible animals injections of some of the
very virulent bacteria, as pneumococci, strepto-
cocci, typhoid bacilli and cholera spirilla, can be
robbed of all danger if small doses of their respective
sera are given before the bacteria have increased to
any great extent in the body. If given later they
are ineffective. Conditions in man are probably
very similar.
The reasons for the failure of these antibacterial
sera when used therapeutically demand a moment's
consideration. It is apparent from all that has
gone before that a deeper insight into the mechan-
ism of immunity discloses many difficulties to be
overcome before we can hope for much in a practical
way. In the case of the bacteria sera, for example,
we have as yet found no method of increasing the
complements, and these are apparently highly im-
portant in destroying the invading bacteria. Nor
have we any way to determine the proper dose so
as to avoid the phenomenon termed "deflection of
complement." Possibly, also, as Ehrlich has sug-
106 IMMUNE SERA
gested, the complements present in human serum
may not be able to reactivate immune bodies
derived from the horse, sheep, or other animal
furnishing the therapeutic serum. Probably the
most important cause of the failure of these sera
is that they do not reach the bacteria in the body.
In the case of cholera, for example, it is hardly to
be expected that the serum injected would affect
the spirilla, for most of these are in the intestinal
contents, and therefore really outside of the body.
In many of the bacterial infections the organisms
accumulate in the lymph glands and other sites
where they cannot readily be reached by anti-
bodies circulating in the blood. Instructive in this
connection are the good results achieved by intra-
spinal injections of antimeningococcus serum, when
exactly the same serum had proven valueless when
given subcutaneously.
PRECIPITINS
Definition. All ol the foregoing experiments
have concerned themselves with the results obtained
by injection of cellular material of one animal into
another. In the further study of this subject,
experiments were made to discover what happens
when dissolved albuminous bodies of one species are
injected into animals of another species. This line
of investigation was first pursued by Tchistowitsch, 1
who injected rabbits with the serum of horses and
of eels. On withdrawing serum from such rabbits
and mixing it with horse or eel serum, the mix-
ture became cloudy, owing to the precipitation of
part of the albumin of the horse or eel serum by
that of the rabbit. Normal rabbit serum does not
possess this property. Bordet was able to demon-
strate that the same thing takes place if rabbits are
treated with chicken blood. On mixing such a
serum with chicken serum, a precipitate formed.
The substances which develop in the serum by
treating an anima 1 with albuminous bodies of
another animal, and which precipitate these a'bumins
when the sera of the two animals are mixed, are
1 Tchistowitsch, Annal. Pasteur. Vol. xiii, 1899.
107
io8 IMMUNE SERA
called precipitins.' 1 This power of the organism to
react to the injection of foreign dissolved albuminous
substances has been found to be very extensive.
Bacterial Precipitins. In 1897, R. Kraus showed
that the serum of a rabbit immunized against
typhoid often produces a precipitate in the bac-
terial-free nitrate of a bouillon culture of typhoid
bacilli. This fact has been verified by subsequent
investigators and the reaction found to be specific.
In general, the best results are obtained with old
bouillon cultures which contain a larger proportion
of the autolytic products. It was natural that this
reaction should at once be applied to the diagnosis
of typhoid and other diseases. Numerous experi-
ments however have shown that Kraus' phenomenon
is not nearly so constantly observed as that of
agglutination, and the reaction is therefore but
little used. Whether the bacterial precipitins are
identical in character with those obtained by
injecting an animal with an unrelated serum (zoopre-
cipitins), is still undecided. Rostoski, as well as
Nuttall, believes that they are probably different.
So much for bacterial precipitins.
Lactoserum Other Specific Precipitins. Bordet,
by injecting cows' milk into rabbits', was able to
produce a serum which precipitates the casein of
1 It will be recalled that, besides the production of pre-
cipitins, the above procedure causes the formation of other
anti-bodies such as anti-complements, anti-agglutinins, etc.
PREC1PITINS
log
cows' milk. He called this lactoserum. Ehrlich,
Morgenroth, Wassermann, Schtitze, Myers, and
Uhlenhuth showed that by treating a rabbit with
chicken albumin a precipit'n is formed which pre-
cipitates chicken albumin. Myers, by treating ani-
mals with Witte's pep ton and globulin, produced a
serum that contained specific antipeptons and anti-
globulins. Pick and Spiro, by using albumose,
produced antialbumoses. Leclainche and Vallee,
Stern, Mertens, and Ziilzer treated animals with
human albuminous urine and produced a serum
which contained a precipitin specific for this sub-
stance. Schutze, by treating rabbits with a vege-
table albumin, as well as with human myoalbumin,
produced a precipitin specific for these albumins.
This does not exhaust the recital of the work done
in this field, and there is a host of other albuminous
bodies which, when injected into an animal, are
able to excite the production of precipitins.
Specificity of the Precipitins. It was soon recog-
nized that the specificity is not absolute. Above all,
this depends upon the strength of the serum, i.e.,
its degree of activity. This is measured by the
dilution in which it will still react. Thus a highly
active serum, one, for example, which will still
give a distinct reaction when diluted i : 1000 or over,
will produce a marked precipitate with the serum
used to excite its production ; whereas, in the serum
of other animal species it will produce slighter pre-
HO IMMUNE SERA
cipitates, or only cloudings. A less highly active
serum will likewise cause a marked precipitate in
the homologous blood solution, and a slight pre-
cipitate, or only a clouding, at the most, in a closely
related species. For example, the serum of a rabbit
which has been treated with sheep blood produces
a marked precipitate in a solution of sheep blood ; a
slight precipitate in a goat -blood solution ; and a still
fainter one in an ox-blood solution. In some in-
stances th2 two latter will show only a clouding. If
we employ a very weak serum, even the cloudings
will be absent, and a precipitate is formed only in
the sheep-blood solution. If human blood or
blood serum has been injected, the clouding and
precipitation will occur most readily (aside, of
course, from human-blood solution) in that of apes.
In the precipitin reaction, therefore, the relationship
of the single animal species is an important factor.
This peculiar behavior has first been thoroughly
studied by Nuttall * who made observations on
five hundred different animals. As a result of these
we know that a weak human-blood antiserum,
besides reacting on human blood, causes a clouding
only in the blood of anthropoid apes (chimpanzee,
gorilla, orang-outang) ; a stronger serum causes a
clouding also in the blood of other monkeys ; finally
1 British Medical Journal, 1901, Vol. ii, and 1902, Vol. i.
See also Nuttall, Blood Immunity and Blood Relationship,
1904. The Macmillan Co., N. Y.
PRECIPITINS III
a very highly active serum reacts with the blood of
all the mammalia. In that case, of course, only a
faint clouding is produced even after considerable
time. Nuttall also obtained antisera, each of which
was specific for one of the large animal classes
(birds, reptiles, amphibia). Here, too, the same
quantitative differences were noted.
Nature of the Precipitins. The precipitins are
fairly resistant bodies, whose power gradually
declines at a temperature of 60 C., but is not lost
until 70 C. is reached. Once their action is lost,
it cannot be restored by the addition of normal sera,
showing according to Ehrlich, that the precipitins
are receptors of the second order and are not ambo-
ceptors. The resulting precipitate is soluble in
weak acids and alkalies. Peptic digestion destroys
the substances which effect the precipitation.
Leblanc found that the precipitins were precipitated
from the serum in that fraction which Hofmeister
calls the pseudo globulins. Eisenberg, on the other
hand, in his experiments found them in the eu-
globulm fraction. The latter result was also obtained
by Obermayer and Pick in precipitins obtained
from goats and rabbits. The discordant results
are comprehensible in view of recent publications
concerning the unreliability of ammonium sulphate
fractionation of serum globulins. The nature of
the resulting precipitate has also been studied by
Leblanc. He finds that it is a combination of the
112 IMMUNE SERA
precipitated albumin with the antibody of the
specific serum. In this combination the properties
of the pseudo globulin predominate showing that
it is the specific serum which furnishes the greater
part of the precipitate. The presence of salts seems
to be necessary for the precipitin reaction. A tem-
perature of 37 C. hastens, while a low temperature
markedly retards the reaction. In either case, the
amount of precipitum is uninfluenced. The pres-
ence of even small quantities of acids or alkalies
markedly reduces the amount of precipitum formed,
but an increase of salt (NaCl) has little effect.
Practical Application. These precipitins have
very recently found a practical application. Fish,
Ehrlich, Morgenroth, Wassermann, and Schutze
investigated the specific action of lactoserum. They
found that a serum derived by treating an animal
with cows' milk contained a precipitin which reacted
only on the casein of cows' milk, but not on that of
human milk or goats' milk. The serum of an ani-
mal treated with human milk was specific for the
casein of human milk, etc. Ehrlich, Morgenroth,
and Wassermann also experimented with the serum
resulting from treatment with chicken egg albumin,
and found that this, while not strictly specific so far
as closely related species are concerned, is yet so
against other species. The precipitins, therefore,
react on closely related albumins, but are specific
against those of unrelated species.
PRECIP1TINS 113
The Wassermann- Uhlenhuth Blood Test. As a
result of these researches Wassermann proposed,
at the Congress for Internal Medicine, 1900, to use
these sera as a means of differentiating albumins,
i.e., to distinguish the different albumins from one
another, and particularly to distinguish those
derived from man from those of other animals.
This proposal thus to use the Tchistowitsch-Bordet
precipitins had important practical and theoretical
results. Uhlenhuth, Wassermann, Schutze, Stern,
Dieudonne, and others showed that a serum could
be produced from rabbits by injecting them with
human serum, by means of which it is possible to
tell positively whether a given old, dried blood stain
is human blood or not.
Uhlenhuth * tested nineteen kinds of blood and
only obtained a reaction with human blood upon
adding antihuman serum to the series of dilutions.
He, moreover, found that human blood which had
been dried four weeks on a board could be readily
distinguished by means of antihuman serum from
the blood of the horse and ox. On the following
day Wassermann 2 demonstrated experiments simi-
lar to Uhlenhuth 's at the meeting of the Physiologi-
cal Society, Berlin. Outside of human blood only
that of a monkey gave the reaction with anti-
human serum.
1 Uhlenhuth, Deutsche med. Wochenschrift, 1901. xxvii.
* Wassermann A. and Schutze, Berliner klin. Wochenschr.
1901. No. xxviii.
114 IMMUNE SERA
The reliability of this reaction in medico-legal
questions has been abundantly established. In the
forensic blood diagnosis the subjects of the test
are usually blood stains on clothing, and on wood
and metal objects. After such a doubtful stain
has been dissolved in physiological salt solution,
one first proceeds to determine that it is really
blood. For this purpose Teichmann's test (the
production of haemin crystals), the guaiac test, and
the spectroscopic examination are undertaken. This
is of considerable importance, for not merely blood
but other albuminous solutions derived from the
same animal react with an antiserum obtained by
injecting an animal with blood or serum, ^Having
found that the stain is that of blood, we next deter-
mine the special kind of blood.
Immunizing the Animals. For the production of
the antisera, we make use of rabbits. These can be
injected either with sterile, freshly-defibrinated blood
or with sterile serum, the latter being preferable
for intravenous inoculation. It is well to begin
with small doses and gradually increase; thus for
intravenous inoculations the first injection should
be about one c.c. and increased up to three or
four c.c. With intraperitoneal injections about
double these doses can be given. Ordinarily, the
interval between injections is three or four days,
and the entire duration of treatment from two
weeks to a month. Long-continued treatment
PRECIPITINS 115
leads to a disappearance of precipitins from the
blood.
Collecting the Serum. When the animals have
received five to six injections, and some days have
elapsed it is well to draw off samples of the blood
and to test for precipitins. This is easily done by
shaving the ear and cleansing the skin with alcohol
and sterile water. An incision is then made into
the marginal vein and a few drops of blood collected
in a small test-tube. This is then set aside to allow
the blood to coagulate. After the serum has sepa-
rated it can be tested and if it prove insufficiently
powerful, treatment may be continued, otherwise
the animal may be killed, preferably a week or ten
days after the last injection. The animals may be
killed in a variety of ways. Uhlenhuth chloroforms
them, opens the thoracic cavity under aseptic
precautions, and, cutting through the beating heart,
the blood is allowed to flow into the thoracic cavity,
whence it is removed by means of sterile pipettes
to suitable vessels. Nuttall's method is to shave
the neck and disinfect the skin with lysol solution;
bend the animal's head backward to put the skin
of the neck on the stretch, and have an assistant
make a clean sweep with a sterilized knife through
the tense skin to and through the vessels. The
blood spurts into a large sterile dish which is
immediately covered when the main flow has ceased.
The dishes are placed horizontally until a clot has
Il6 IMMUNE SERA
formed ; they are then slightly tilted, and as soon as
serum enough has been expressed, this is pipetted
off into sterile test containers which are stored in a
cool place. It is well not to add any preservative
to the serum, as such an addition may occasionally
lead to pseudo reactions.
The Test. In carrying out the test the sus-
pected clot is mixed with a small quantity of normal
salt solution and then filtered. Whether or not the
blood specimen has gone into solution can best be
judged by the foam test. Air is blown gently through
the pipette which is used for transferring the solu-
tion into the test-tubes. Solutions of blood or
serum of i : 1000 and over, still foam well. The color
of the fluid is not so reliable an index of solution.
To some of this solution in a test-tube, about double
the amount of the specific serum (derived as above)
is added. As a control test, we place a little blood
of another species, e.g., of an ox, in a second test-
tube together with some of the specific serum and a
little normal salt solution. In a third tube we
place some of the suspected blood solution, and 'n a
fourth some of the specific serum mixed with the
normal salt solution. All four tubes are placed in
the incubator at 37C. for one hour, or are left at
room temperature for several hours. If the sus-
pected clot was one of human blood, the first tube
will show distinct evidence of precipitation, while
all the control tubes will have remained clear. It
PRECIPITINS
117
is desirable to dilute the suspected blood as far as
possible when testing, for when concentrated sera
are brought together reactions may occur which
will lead to erroneous conclusions. In medico-
legal work it will be well to progressively dilute a
suspected blood sample and to reach a conclusion
upon the highest (within limits) which reacts to a
given antiserum. In routine work one can com-
mence with dilutions of the suspected blood of
i : 100 or i : 200. We must not omit to say that it
is necessary to test to litmus all solutions to be
examined, and to neutralize any that are found
decidedly acid or alkaline.
Appearance of the Reaction. When antiserum
is added to blood dilution it sinks to the bottom of
the tube, forming a milky white zone at the point of
contact. The milkiness gradually extends upward
until the whole fluid is clouded. Where the fluids
have been mixed by shaking this diffuse cloudiness
undergoes a change; after ten to twenty minutes,
or later, very fine granules of precipitum begin to
appear, and the upper layers of the fluid begin to
clear, due to sedimentation of the precipitum.
The fine particles soon become aggregated into
coarser ones, and these into flocculi which, gradually
sinking to the bottom of the tube, give rise to more
or less deposit of a whitish appearance. With blood
dilutions of, say i : 40 to i : 200 and over, the deposit
formed is usually sharply defined ; where more con-
Ii8 IMMUNE SERA
centrated dilutions are used, the deposit may form
an irregular mass at the bottom of the tube.
The reaction may be followed microscopically by
means of the hanging-drop method. By this method
a reaction can be observed within ten to fifteen
minutes, which macroscopically becomes visible
only after two hours.
Delicacy of the Precipitin Test. Whereas the
ordinary chemical tests for blood cease to give reac-
tions in dilutions of about i : 1000, powerful antisera
greatly exceed this limit, as the reported results of
independent observers have shown. Working with
an antihuman serum, Strube reports a re.action
with a blood diluted 20,000 times, and Stern one
with a blood diluted 50,000 times. Ascoli obtained
a reaction with a specific serum with egg albumin
diluted 1,000,000 times.
Other Applications of the Precipitin Test. It can
be readily understood that this test finds ready
application in the detection of horse, dog, or cat
meat in sausage.
The principle and the method are the same in all
these various applications. We treat animals with
the albumins which we wish to differentiate, and so
obtain sera specific, each for its particular kind of
albumin. These sera, then, produce precipitates
only in solutions of their respective albumins. For
example, if we wish to determine whether a given
PREC1P1T1NS
Iig
sample of meat is horse-flesh or not we must inject
an animal with horse serum, or, if we prefer, with
an extract of horse-flesh. The serum derived from
this animal will then produce a precipitate in the
aqueous extract of the meat if this be horse-flesh,
but not if it be beef. Animals treated with dog
serum yield a serum which precipitates an aqueous
extract of dog-flesh, etc. The method of examina-
tion consists in scraping the meat and extracting it
with water or normal salt solution. It takes a long
time to extract the meat in some cases. An extract
is suitable for testing when it foams on being shaken.
If the extract is very cloudy it should be cleared by
filtration through a Berkfeld filter. In testing, add
ten to fifteen drops of antiserum to 3 cc. of the
saline meat extract.
Antiprecipitins Iso-precipitins. Biologically,
the precipitins are found to behave -like the sub-
stances already studied. It is possible, for example,
by injecting an animal with a precipltin, say
lactoserum, to obtain an antiprecipiiin, an anti-
lactoserum, which counteracts or inhibits the
action of the precipitin. This is entirely analo-
gous to the antihaemolysins, the an tispermo toxin,
etc.
If rabbits are treated with rabbit serum, a serum
is obtained which will, in certain cases, precipitate
the serum of other rabbits. This was done by
120 IMMUNE SERA
Schutze, and he called this serum iso-precipitin.
Whether or not iso-precipitins ever occur in normal
serum has not yet been definitely established.
Their occurrence in human serum might be of
importance in homologous transfusion.
II. CYTOTOXINS
Cytotoxins Definition Leucotoxin Nature of
the Cytotoxin Anticytotoxin. After it had been
found that the injection of an animal with red blood
cells of another animal was followed by the produc-
tion of definite, specific reaction substances, investi-
gators experimented to see whether this was also
the case if other animal cells were used. Injections
were made with white blood cells, spermatozoa of
other animals, etc., and there resulted a series of
reaction substances, entirely analogous to the
haemolysins, which were specific for the cells used for
injection. These sera Metchnikoff calls cytotoxins.
After Delezenne had published a short article on a
serum haemolytic for white blood cells, Metchnikoff
undertook a study of the substances produced in
sera of animals treated with leucocytes of another
species. He injected guinea pigs with the mesen-
teric glands and bone marrow of a rabbit. He
also injected for several weeks half an Aselli's pan-
creas at a time, at intervals of four days. If he
withdrew serum from such a guinea pig he found
this to be intensely solvent for white blood cells of
a rabbit. He called this serum leucotoxin. This
leucotoxin is very poisonous for these animals, and
121
122 IMMUNE SERA
kills them within a few hours. Non-fatal doses at
first excite a marked hypoleucocytosis, which is
followed after a few days by a compensatory hyper-
leucocytosis. Leucotoxin destroys the mononu-
clear as well as the polynuclear leucocytes of the
animal, as was shown by Funk. Leucotoxin which
had been derived by injection of the leucocytes of
horses, oxen, sheep, goats, or dogs acted only on
the leucocytes of that species, not on the leucocytes
of man. So far as the mechanism of the cy to toxic
action is concerned, it has been found that this is
the same as that of the hasmolysins. The action of
the specific cytotoxic serum is always due to the
combined action of two substances in the serum, a
specific immune body, and an alexin or comple-
ment present also in normal serum. The cyto-
toxic sera, like the haemolytic sera, are rendered
inactive by heating to 55 C. In other respects
also the cytotoxic sera maintain the analogy to
the haemolytic sera. Thus it is possible by immu-
nizing with a cy to toxin to obtain an anticytotoxin.
MetchnikofT, for example, was able to produce' an
antileucotoxin by injecting animals with leuco-
toxin. This antibody inhibited the action of the
leucotoxin.
Neurotoxin. Delezenne and Madame Metchni-
koff have injected animals with central-nervous-
system substance, and so produced a specific neuro-
ioxin. They injected ducks intraperitoneally, giving
CYTOTOX1NS 123
them five or six injections of ten to twenty grammes
of dog brain and spinal cord mixed with normal
salt solution. The serum of these ducks injected
intracerebrally into dogs in doses of 0.5 c.c.
caused the dogs to die almost at once in complete
paralysis, whereas if normal duck serum was in-
jected in the same way no effects of any kind were
produced. If smaller doses of the specific neuro-
toxic serum were administered, say o.i to 0.2 c.c.,
various paralyses and epileptiform convulsions set
in, from which the animals sometimes recovered.
The action of this serum is specific, i.e., the serum
of ducks treated with dog brain causes these symp-
toms only in dogs, while on rabbits it acts no
differently than normal duck serum.
Spermatoxin. Another specific cell-dissolving
serum was produced by Landsteiner, Metchnikoff,
and Moxter, by injecting animals with the sperma-
tozoa of other animals. Such a serum rapidly
destroys the spermatozoa of the animals whose
product was injected. This cytotoxin was named
spermatoxin. If animals are treated with spermato-
zoa there is produced a serum which is not only a
spermatoxin, but which is also hasmolytic for the
red cells of that animal. This was demonstrated
by Metchnikoff and Moxter, and has already been
referred to in discussing hsemolysins. If, for ex-
ample, we inject the spermatozoa of sheep into
rabbits, we shall obtain a serum that is sperma-
124 IMMUNE SERA
toxic for sheep, as well as haemolytic for sheep
red cells.
Common Receptors. At first it was thought that
the haemolysin so produced was due to the presence
of small quantities of blood injected with the sper-
matozoa. The same result however was obtained
when all traces of blood could be excluded ;* further-
more a number of investigators produced haemoly-
sins by the injection of fluids entirely free from red
corpuscles, such as serum and urine. The produc-
tion of this haemolysin is not hard to explain if we
hold fast to the side-chain theory. We have
merely to assume that the spermatozoa or these
other substances possess certain receptors in com-
mon with the red blood cells of the same animal.
Ehrlich and Morgenroth 2 have repeatedly pointed
out that specificity is a matter not of cells, but of
receptors. Despite these very conclusive demon-
strations later investigators, who attempted to
produce antisera for the cells of various organs,
continued to use emulsions of unwashed organs, in
utter disregard of the presence of free receptors in
the organ juices and also without consideration of
the antibodies certain to be produced by the red
cells normally present.
Cytotoxin for Epithelium. As far back as 1899,
1 Von Dungern. See "Collected Studies on Immunity, " Ehr-
lich-Bolduan, p. 47. Wiley & Sons, New York, 1910.
2 Ehrlich and Morgenroth. Ibid., p. 100.
CYTOTOX1NS 125
von Dungern showed that it was possible to produce
an antiepithelial serum by treating animals with
the ciliated tracheal epithelium of oxen. This
serum was rapidly destructive for this particular
kind of epithelium, but it contained also a specific
haemolytic body just as was the case in the sper-
motoxic serum, and for the same reasons. This
antiepithelial serum aroused considerable interest
since it indicated the possibility of producing sera
which were cytotoxic for certain varieties of epi-
thelial cells, especially those of pathological origin,
as carcinoma. The numerous experiments made
in this direction failed however to produce the
desired results. Owing to the extensive distribu-
tion of common receptors the antisera were found
to exhibit quite general properties and to lack
that degree of cell specificity, essential for practical
purposes.
Cytotoxins by the Use of Nucleo-Proteids. In
order to prevent the adventitious formation of
those bodies resulting from impure methods of
immunization, and also in the hope of obtaining
greater specificity, a few investigators have utilized
the nucleo-proteids of the cell for immunization.
This method seems to have been tried first by
Marrassini in 1903, but with indifferent results.
In 1905 Beebe 1 published an extensive study along
1 S. P. Beebe, Cytotoxic Serum Produced by the Injection
of Nucleo-Proteids. Journ. Exper. Medicine, Vol vii, 1905.
126 IMMUNE SERA
these lines and described the formation of a nephro-
toxic serum which caused albuminuria and acute
degeneration of the kidney without changes in
the other organs. Albuminuria appeared gene-
rally on the fourth or fifth day, increased rapidly
in amount, and was accompanied by the excretion
of hyaline and granular casts. Subsequently Pearce
and Jackson, 1 after a careful experimental study on
the production of cytotoxic sera by the injection of
nucleo-proteids, conclude " that the results do not
support the theory that specific cytotox ! c sera may
be developed in this way, but indicate, rather, that
such sera have certain mildly toxic properties acting
in a general way and affecting especially the principal
excretory organ, the kidney."
1 R. M. Pearce and Holmes Jackson, Journal of Infectious
Diseases, Vol. iii, 1906.
OPSONINS OR BACTERIOTROPIC
SUBSTANCES
Historical. - - The early work of Nuttall and
others on the bactericidal action of normal serum,
and Pfeiffer's demonstration of the bacteriolysis
of cholera and typhoid bacilli by immune sera in the
absence of cells, formed the chief basis on which
rested the humoral theory, which attributed the
protection in such cases to the destructive action
of the serum on the microbes. It was found, how-
ever, that cases of protection resulting from the
use of immune serum occurred where no such
bacteriolytic action could be demonstrated; infec-
tion with plague or streptococcus may be men-
tioned as examples. It is now pretty generally
accepted that immunity in these cases is due largely
to the phagocytic action of the leucocytes. As far
back as 1858 Haeckel had observed that particles
of indigo injected into the veins of certain molluscs
could shortly afterwards be found in the blood
cells of the animal. However, the significance of
this and other observations was not appreciated
until Metchnikoff * in 1883 called attention to their
bearing on infection and immunity. The outcome
1 Arbeiten des Zoo log. Institutes in Wien, 1883, Vol. v.
127
128 IMMUNE SERA
of his investigations was the establishment of the
well-known doctrine of phagocytosis, the principle
of which is that the wandering cells of the animal
organism, the leucocytes, possess the property of
taking up, rendering inert, and digesting micro-
organisms which they may encounter in the tissues.
Metchnikoff believes that susceptibility to or
immunity from infection is essentially a matter
between the invading bacteria on the one hand
and the leucocytes of the tissues on the other. He
realizes that the serum constituents play an im-
portant role, but this role consists in their stimulat-
ing the leucocyte to take up the bacteria.
Thus if a highly virulent organism is injected
into a susceptible animal, the leucocytes appear to
be repelled, and to be unable to deal with the
microbe, which multiplies and causes the death of
the animal. If, however, the suitable immune
serum is injected into the animal before inoculation,
the phagocytes attack and devour the invading
micro-organisms. Admitting that the phagocyte
plays an important part in certain infections the
question must still be considered whether the
immune serum has acted on the injected microbes
or on the phagocytes. Metchnikoff, we have seen,
takes the latter view.
In 1903 A. E. Wright 1 called attention to certain
substances present in serum which acted on bacteria
1 Wright and Douglas, Proc. Royal Society, Vol. 72, 1903.
OPSONINS 129
and rendered them mere easily taken up by the
phagocytic cells. He called this substance opsonin
and showed that it is present in normal as well as
immune sera. By means of absorption tests
modelled after those of Ehrlich and Morgenroth, he
showed that the opsonin has a specific affinity for
the bacteria and none for the leucocytes. The
opsonins for staphylococcus prepare only staphy-
lococci for the leucocytes, those for tubercle bacilli
only these bacteria, etc. As a result of his obser-
vations Wright supposes that the phagocytes play
only a passive r6le, which depends on the pre-
liminary action of the opsonin.
Bacteriotropic Substances. - - Independently of
Wright, though somewhat later, Neufeld and Rim-
pau l of Berlin published experiments on the pha-
gocytic effect of immune sera. They also found
that in these sera there exists a substance which has
no direct action on the phagocytes, but which can
fix itself on the corresponding bacteria and so modify
these that they are more readily devoured by the
phagocytes. They call this constituent a " bacte-
riotropic substance." There is little doubt that this
bacteriotropic substance and Wright's opsonin are
identical. Certain differences in the effect of heat
are probably to be explained by the differences in
the quantities of these sensitizing substances in
normal and immune sera.
1 Neufeld and Rimpau, Deutsche med. Wochenschrift, 1904.
IMMUNE SERA
Opsonins Distinct Antibodies. It was natural to
question whether these " opsonins " were really dis-
tinct from other antibodies, or whether they were
perhaps identical with the immune body (or sub-
stance sensibilatrice). In a series of papers on this
subject Hektoen l shows that the former is the case,
opsonins are distinct substances. This is not only
indicated by the results of absorption tests, but by
the fact that, by immunization, a serum can in cer-
tain cases be obtained which is opsonic but not lytic,
or in other cases one which is lytic but not opsonic.
Similar experiments have differentiated opsonins
from agglutinins.
Structure of Opsonins. In structure the opso-
nins are like the agglutinins. Following Ehrlich's
conceptions they possess two groups, opsoniferous
end haptophore. On heating an opsonic serum
the former group is destroyed, but the haptophore
group remains intact, as can be seen from suitable
combining experiments. There is still consider-
able difference of opinion as to the degree of heat
necessary to inactivate the opsonins. Once the
opsoniferous group has been destroyed it is impos-
sible to restore the opsonic action by the addition
of a complementing substance. Hence the opsonins
are to be regarded as receptors of the second order
and similar in structure to the agglutinins and
precipitins. In this connection it will be we.ll to
1 Hektoen, L., Journal Infect. Diseases, 1905 and 1906.
OPSONJNS I3 i
remember Bordet's objections to the assumption
of two groups in the agglutinin molecule. These
have already been considered oh page 40.
The Opsonic Index. In the study of these opso-
nins Wright developed the idea that they were
highly important in combating a number of bacterial
infections, such as staphylococcus and tubercle.
His observations showed that inoculations of the
corresponding bacteria produced marked changes in
the opsonic contents of the infected individual and
that it was possible to estimate accurately the im-
munizing effect of such inoculations.
Technique. Wright's technique of measuring the
opsonic power is a slight modification of the Leish-
man l method and is as follows : An emulsion of
fresh human leucocytes is made by dropping twenty
drops of blood from a finger prick into 20 c.c.
normal salt solution containing one per cent sodium
citrate. The mixture is centrifuged, the supernatant
clear fluid, removed and the upper layers of the sedi-
mented blood cells transferred by means of a fine
pipette to 10 c.c. normal salt solution. After cen-
trifuging this second mixture the supernatant fluid
is pipetted off and the remaining suspension used
for the opsonic tests. Such a " leucocyte emulsion, '
of course, contains an enormous number of red
blood cells; the proportion of leucocytes, however,
is greater than in the original blood.
1 Leishman, British Medical Journal, Jan., 1902.
132 IMMUNE SERA
One volume of this emulsion is mixed with one
volume of the bacterial suspension to be tested and
with one volume of the serum. This is best accom-
plished by means of a pipette whose end has been
drawn out into a capillary tube several inches in
length. With a mark made about three-quarters
of an inch from the end it is easy to suck up one such
volume of each of the fluids, allowing a imall air
bubble to intervene between each volume. All
three are now expelled on a slide and thoroughly
mixed by drawing back and forth into the pipette.
Then the mixture is sucked into the pipette, the end
sealed and the whole put into the incubator at 37 C.
The identical test is made using a normal serum in
place of the serum to be tested. Both tubes are
allowed to incubate fifteen minutes and then ex-
amined by means of smear preparations on slides
spread and stained in the usual way. The degree of
phagocytosis is then determined in each by count-
ing a consecutive series of fifty leucocytes and find-
ing the average number of bacteria ingested per
leucocyte. This number for the serum to be tested
is divided by the number obtained with the normal
serum and the result regarded as the opsonic index
of the serum in question. The presence of a high
opsonic index Wright regards as indicative of in-
creased resistance. He further states that the fluc-
tuation of the opsonic index in normal healthy
individuals is not more than from .8 to 1.2, and that
OPSONINS
133
an index below .8 is therefore almost diagnostic of
the presence of an infection with the organism tested.
Application of the Opsonic Measurements. At
the present time Wright has correlated all his obser-
vations and built up a system of treating bacterial
infections by means of active immunization con-
trolled by opsonic measurements. The principles
underlying his method may be briefly summarized
as follows: In localized bacterial infections the
infected body absorbs but small amounts of bacterial
substances or antigens. In consequence of this the
amount of active immunity developed is but slight.
Localized infections therefore tend to run a chronic
course. The logical method of effecting a cure in
these cases is to actively immunize the body with
the invading organism. In a number of infections,
notably those of staphylococcus, streptococcus, and
tubercle, the degree of immunity is measured accu-
rately by the opsonic index. Following an inocu-
lation with the infecting bacteria (dead cultures in
salt solution) there is first a drop in the opsonic
index, the " negative phase," then, depending on
the size of the dose and the reacting power of the
individual, there comes a rise of the index, the
" positive phase," or a continuation of the negative
phase. The former is obtained with proper dosage ;
the latter with doses too large or too small. In
estimating the size of the dose given, Wright counts
the number of bacteria per c.c. of emulsion injected.
134 IMMUXE SERA
Thus in the case of localized staphylococcus infec-
tions the doses for adult humans range from 100
million to 500 million bacteria. In the case of strep-
tococcus the doses are smaller, averaging about 50
to 100 million. The bacterial suspensions are heated
to 60 C. for twenty minutes, 0.5% carbolic acid
is added, and tests are made to insure sterility.
The time for inoculation is governed by the opsonic
index. If the first inoculation has been properly
gauged there is a brief negative phase, followed by
a positive phase of some days' duration. As this
positive phase gradually drops, one gives another
inoculation and watches the effect on .the opsonic
index. If the index drops markedly and rises but
little, the dose has been too large. Or if the nega-
tive phase is slight, and the positive phase slight and
transitory, the dose has been too small. With
proper dosage the negative phases are small, and the
opsonic index is kept fairly well above normal.
Hand in hand with this goes an improvement in the
clinical symptoms.
Wright and his pupils have published accounts of
a large number of cases successfully treated accord-
ing to this method. The results are reported as espe-
cially good in cases of severe acne, multiple boils,
lupus, tubercular glands, and bone tuberculosis.
In judging of the value of Wright's method we
must bear clearly in mind that the essential feature
of it is the control by opsonic measurements; treat-
OPSONINS 135
ment of bacterial infections by the inoculation of
dead cultures has long been known.
The results obtained by most workers in this coun-
try fail to bear out Wright's claims for the method.
Thus the author 1 finds that the variation in the
opsonic indices of several normal persons is often
considerable; that opsonic counts based on fifty
leucocytes may occasionally vary by more than
50% and that it is therefore necessary to count from
150 to 200 leucocytes for each test; that duplicate,
triplicate and more tests made of the same serum,
at the same time, and under identical conditions so
far as one can tell, frequently give widely divergent
results; that the opsonic index and the clinical
course of the disease do not always run parallel.
Cases may do very well and have the index remain
low; other cases may do poorly with an increased
opsonic index. It is to be noted, furthermore, that
some of these variations in results are unavoidable,
at least with the present technique.
To one who has followed the progress of immunity
studies, it is not at all surprising to find that the
opsonic index is not necessarily a measure of the
patient's immunity. When Gruber and Durham
published their observations on agglutinins the
phenomenon was at once Hailed and interpreted by
many as measuring the degree of immunity possessed
by the patient. The same error was made when
1 Bolduan, Long Island Med. Journal, Vol. i, 1907.
IMMUNE SERA
some time later the bacteriolytic substances were
discovered. In both cases it was soon found that
these were but accompaniments of greater or less
significance to the complex phenomenon of immun-
ity. When we consider how manifold are the defen-
sive agencies which the animal organism possesses,
and how very complex they become the more they
are studied, we shall not marvel at the absence of
parallelism between the clinical course of the disease
and the opsonic index. There is little doubt that
the opsonic indices do measure a certain fraction or
phase of the immunity reaction; we do not believe
that they replace clinical observations in measuring
the effect of immunizing injections.
VII. SNAKE VENOMS AND THEIR ANTISERA
Despite the fact that venomous serpents have
excited the fear and interest of mankind for centuries
it is only very recently that we have come to know
anything definite about their poisons. This is
perhaps in part due to the fact that Europe possesses
but few poisonous snakes, and so offered little
material for study. Some idea of the importance
of the subject for certain countries, however, can be
seen when it is stated that in India more than
20,000 persons annually die from the bite of the
hooded cobra. It was quite natural, therefore, that
one of the earliest modern researches into the
nature of snake venom, that of Calmette, 1 should
have come from that country. This author also
found that he could produce an antitoxic serum by
injecting animals with the snake venom.
The Venoms. Our present knowledge of snake
venoms and their antisera is due largely to the
researches of Flexner and Noguchi 2 and of Kyes
and Sachs. 3 The venoms of different snakes vary
1 Calmette, Annal. Inst. Pasteur, Vol. vi, 1892; Comptes
rend. Soc. Biol., 1894.
2 Flexner and Noguchi, Journal Exp. Medicine, 1902, et seq.
s Kyes and Sachs. See in Collected Studies on Immunity,
Ehrlich-Bolduan, New York, 1910.
138 IMMUNE SERA
a great deal in their toxic properties, and this is
due to their relative contents of different consti-
tuents, as follows:' haemagglutinins, haemolysin,
hsemorrhagin, and neurotoxin. The first two act
exclusively on the blood cells, the haemorrhagin on
the endothelium of the blood vessels, and the
neurotoxin on the cells of the central nervous
system. The last named causes death by paralysis
of the cardiac and respiratory centers. The ven-
oms of the cobra, water-moccasin, daboia and
some poisonous sea snakes are essentially neuro-
toxic, although they have strong dissolving powers
for the erythrocytes of some animals. In study-
ing the haemolytic powers of the venoms of cobra,
copperhead, and rattlesnake, Flexner and Noguchi
found cobra venom to be the most haemolytic and
that of rattlesnake the least. They attribute the
toxicity of rattlesnake poison chiefly to the action
of haemorrhagin. The venoms of the water mocca-
sin and the copperhead also contain haemorrhagin.
Unlike the bacterial toxins the action of the snake
venoms is preceded by no appreciable incubation
period. In addition to this the poisons are very
rapidly absorbed. Thus Calmette found that a rat
inoculated into the tip of the tail could not be saved
by amputating the tail orie minute later. Such
animals died within about five minutes of the time
required for control animals.
The haemolysin and neurotoxin and perhaps also
SNAKE VENOMS AND THEIR ANTISERA 139
the other cytotoxic substances of venom consist of
amboceptors which find a complement in the body
of the poisoned animal. Not only does ordinary
serum-complement serve for activation, but, accord-
ing to Noguchi, 1 the fatty acids contained in the red
blood cells also act as complement. Lecithin is
also able to reactivate the haemolysins of cobra
venom, forming, according to Kyes, a "cobra-
lecithid." Recent experiments by Manwaring, 2
however, show that the product obtained by Kyes
was really a venom-free lecithin derivative and not
a "lecithid."
Antivenins. Calmette was the first to produce
an antiserum against snake venom, utilizing for
this purpose rabbits. He began with injections of
Jv of a fatal dose, and injected gradually increasing
doses until at the end of four or five weeks the
animals tolerated double a fatal dose. By con-
tinuing the treatment he finally got the animals
to stand 80 fatal doses (40 mg.) without any
reaction whatever. Five drops of the serum of such
an animal neutralized i mg. cobra poison. It
has been found that anticobra serum protects
against the neurotoxic " components of other snake
venoms, furthermore against scorpion poison and
the poison of eel blood. The serum also contains
1 Noguchi, Journ. Exper. Medicine, Vol. ix, 1907.
2 Manwaring, Johns Hopkins Hospital Bulletin, September,
1910,
140 IMMUNE SERA
an antihaemolysin, but no antibody against haemor-
rhagin (of the rattlesnake). It is therefore without
effect on rattlesnake venom. Antivenin for the
latter may be prepared by immunizing goats with
corresponding venoms which have been attenuated
by weak acids. Such a serum, of course, possesses
no antineurotoxin and is therefore useless against
cobra and viper venoms.
VIII. ANAPHYLAXIS
Historical. In 1898 Richet and Hericourt showed
that when dogs were injected with eel serum they
not only failed to develop an immunity against this
poison, but actually became more susceptible.
Subsequently they made similar observations with
a toxin, mytilo-congestin, isolated from mussels.
Richet applied the term " anaphylaxis " to this
phenomenon to distinguish it from immunization
or prophylaxis. Arthus, in 1903, reported that
similar effects could be obtained with substances
ordinarily not poisonous. Thus he found that if
rabbits were injected with horse serum they were
rendered very susceptible to a second injection
made after an interval of six to eight days. The
second injection produced severe symptoms, and
sometimes led to death in these animals. Little
or no attention was paid to these observations. Fol-
lowing a statement made to him by Theobald Smith
in 1904, Ehrlich caused his pupil, Otto, to study
why guinea pigs which had been injected with
toxin-antitoxin mixtures in the course of standard-
ization of diphtheria antitoxin, should so often be
killed by a subsequent injection of horse serum.
Independently of this the subject was being investi-
141
1 42 IMMUNE SERA
gated by Rosenau and Anderson in the Hygienic
Laboratory. Almost simultaneously with the ap-
pearance of these studies came a comprehensive
monograph on the serum rashes by v. Pirquet and
Schick, and this fitted in so well with the labora-
tory studies of Otto and of Rosenau and Anderson
that a great deal of interest was aroused in this
subject.
The Phenomenon. As a result of all the work
that has been done we now know that when an
animal is injected with an alien proteid, there
develops, after a time, a specific hypersusceptibility
for this proteid. After a definite interval if the
animal is given a second injection of the same
proteid, violent symptoms appear, often leading
to the death of the animal. The reaction is specific,
so that animals sensitized, for example, to horse
serum, manifest little of no hypersusceptibility
to other sera. It is possible, however, to sensitize
an animal to several proteids simultaneously. The
sensitizing dose may be very small -even as little
as one millionth cubic centimeter of horse serum
has sufficed to render guinea pigs sensitive. A
varying length of time must elapse after the sen-
sitizing injection before the animal becomes fully
sensitized. In guinea pigs injected with small doses
of horse serum, from twelve to fourteen days
suffices. With larger doses, however, the time
required is much longer, and may extend over
ANAPH YLAXIS 143
weeks or even months. The hypersusceptibility
is transmitted from mother to offspring, and may
also be passively transferred to other animals by
transferring some of the serum of the sensitized
animal to normal animals. Animals recovering
from the symptoms induced by the second injection
are thereafter no longer hypersensitive to the pro-
teid employed, but are immune. This immunity
is spoken of as " antianaphylaxis." This condi-
tion can also be brought about artifically by inject-
ing the animals after they have received their
sensitizing injection and just before the end of
the anaphylactic incubation time, with compara-
tively large quantities of the same proteid. Rosenau
and Anderson have shown that animals may be
sensitized by feeding them with the proteid.
Whether this has any practical application to the
clinical use of specific immune sera derived from
horses in persons habitually eating horse flesh is not
known.
Serum Rashes. Turning our attention for a
moment to the serum rashes, we find that in 1874
Dallera reported that urticarial eruptions might
follow the transfusion of blood. Neudorfer as well
as Landois also refer to this complication. In the
year 1894 the use of diphtheria antitoxin introduced
the widespread practice of injecting human beings
with horse serum. In the same year several cases
were reported in which these injections were fol-
I 4 4 IMMUNE SERA
lowed by various skin manifestations, mostly of
an urticarial character. Following these came
a great mass of evidence which made it clear that
following the injection of antidiphtheric serum
these sequelae were usually comparatively harmless.
Heubner in 1894 and von Bokay somewhat later
expressed the opinion that these manifestations
were due to other properties than the antitoxin
in the serum, and this has proved to be the case.
Johannessen produced the same effects by injecting
normal horse serum. It has been shown that the
skin eruptions and other symptoms follow in direct
proportion to the amount of serum injected, a fact
which has led to the concentration of the sera by
getting rid of the non-antitoxic proteid constituents.
In their exhaustive study, already mentioned,
v. Pirquet and Schick described the various clinical
manifestations following the injection of horse serum
into man, and gave the name " serum disease " to
the symptom complex. The principal symptoms
of this disease are a period of incubation varying
in length from eight to thirteen days, fever, skin
eruptions, swelling of the lymph glands, leucopenia
joint symptoms, oedema, and albuminuria.
Theories of Anaphylaxis. It was difficult tc
account for the long period of incubation in the pro-
duction of these serum rashes. With poisons
capable of self -multiplication (bacteria, etc.), this
period was usually referred to the time necessary
ANAPHYLAXIS
for them to accumulate in sufficient number
and virulence to produce symptoms. But serum
is not a poison capable of multiplication.
Pfeiffer's work on the endo toxins led to the view
that the antibodies played an important part in
bringing on the symptoms by setting free the endo-
toxins. The results of these observations are very
closely related to von Pirquet and Schick's explan-
ation of the production of serum disease. The
endotoxin theory, in the sense of bacteriolysis,
naturally cannot be applied to albuminous sub-
stances in solution. We can only accept it in the
sense that by means of the reaction between the
antibodies and the antigen the poisonous substance
is formed. The period of incubation, both in serum
rashes and in bacterial infections, is thus readily
understood, for it is at once apparent that the
formation of antibodies requires time. The
general idea underlying von Pirquet and Schick's
theory of serum disease is that the injection of the
horse serum into man causes the development of
specific reaction products which are able to act upon
the antigens introduced. These antibodies encoun-
ter the antigens, i.e., some of the serum still present
in the body, and so give rise to a poisonous sub-
stance. This accounts also for the cases of " imme-
diate reaction" described by von Pirquet and Schick
in which the second injection of a serum produces
an attack of serum disease without any period of
146 IMMUNE SERA
incubation. Here the second injection comes at a
time when the accumulation of antibodies is at its
height. It has been claimed that this explains the
cases of sudden death in humans following injec-
tions of serum, but investigation shows that most
of these deaths occurred after but a single injection
of serum. Moreover in most of them such conditions
as status lymphaticus sufficed to explain the fatal
ending.
This theory has found some experimental con-
firmation from the work of Vaughan and Wheeler,
who have been able to prepare a number of split
products from the proteid molecule, some of which
in animals give- rise to a symptom complex not
unlike that of typical anaphylaxis.
Gay and Southard hold a somewhat different
view. According to them the " horse serum con-
tains a substance, anaphylactin, which is not neu-
tralized, and is eliminated from the body with great
slowness. When a normal guinea pig is injected
with a small amount of horse serum, the greater
part of its elements are rapidly eliminated; the
anaphylactin, however, remains and acts as a
constant irritant to the body cells, so that their
avidity for the other assimilable elements of horse
serum which have accompanied the anaphylactin
becomes enormously increased. At the end of two
weeks of constant stimulation on the part of the
anaphylactin, and of increasing avidity on the part
ANAPHYLAXIS
of the somatic cells, a condition has arrived when
the cells, if suddenly presented with a large
amount of horse serum, are overwhelmed in the
exercise of their assimilating functions, and func-
tional equilibrium is so disturbed that local or
general death may follow." According to this view
the intoxication caused by the second injection
depends upon constituents of the serum eliminable
by the animal body.
Allergy. It is apparent that what has been said
concerning the production of anaphylaxis in re-
sponse to serum injections will apply also to bac-
terial infections, for in these the body is injected,
as it were, with bacterial proteids. The phenomena
of anaphylaxis are therefore of general application in
immunity. This is well expressed by von Pirquet, 1
who calls attention to the fact that the main differ-
ence between a normal and an immune individual
is one relating to the alteration in the latter's re-
activity. He speaks of this alteration as "allergy" :
from ergeia, reactivity, and allos, altered, meaning
thereby a changed reactivity as a clinical conception
unprejudiced by bacteriological, pathological or
biological findings. This alteration may relate to
the quality and quantity of the symptoms and to
their rate of development. Allergy seems to be
associated more with some infections than with
others. Experimentally it can best be studied by
1 C. E. von Pirquet, Archives of Internal Medicine, Feb., 191 r.
148 IMMUNE SERA
observing the effect of cow-pox inoculation in
primary and subsequent vaccinations. The re-
vaccinated overcomes the whole process with a very
slight local reaction a few millimeters in size, while
the person vaccinated the first time shows extensive
local inflammation, fever, and other general symp-
toms. If the reaction is studied on the day follow-
ing the vaccination, we shall find that the re-
vaccinated is really hypersensitive, because at this
time the first vaccinated does not show any reaction,
while the revaccinated responds with a local inflam-
matory process. In tuberculosis, glanders, and
other infections the injection of extracts of the
infecting bacterium (tuberculin, mallein, etc.) pro-
duces characteristic local and general symptoms,
because of the specific hypersensitive condition
present in such infections. These reactions can
therefore be employed in the diagnosis of such in-
fections. The symptoms of hay fever, and of urti-
caria appear to be merely examples of proteid
hypersensi ti ven ess .
Supposed Relation to Precipitin Action.
Attempts have also been made to associate the
phenomena of anaphylaxis with the action of pre-
cipitins. Hamburger and Moro were the first (1903)
who found that man forms precipitins after the
injection of horse serum. Precipitin was present
after the appearance of serum rashes ; therefore they
suggested a connection between serum exanthem
ANAPHYLAXIS
149
and precipitin formation, without looking on the
precipitation itself as the cause of the rash. More
recently Doerr anci Russ, as the result of experi-
ments, hold that the phenomena of anaphylaxis are
due to a reaction between precipitins attached to
the tissue cells, and the precipitable antigen. The
anaphylactic shock is looked upon as an intracellu-
lar precipitin reaction. In quantitative investiga-
tions these authors showed that the amount of
anaphylactic antibody in the serum of rabbits
was always parallel to its precipitin content. It
has also been found that animals which do not form
precipitins, like white mice, are incapable also of
forming the anaphylactic antibody. Against the
view that precipitins have anything to do with
anaphylaxis in man is the fact that the symptoms
of serum disease appear within eight to thirteen
days following the first injection of horse serum,
whereas it requires about three weeks for precipi-
tins to appear in the blood in children after the
injection of horse serum. Furthermore, the forma-
tion of precipitins does not take place as readily
in man following the injection of horse serum as
it does in rabbits. In fact von Pirquet found that
sometimes even after the injection of 200 cc. there
was no production of precipitins. Finally it may
be remembered that there is no evidence that the
precipitin action is other than a test-tube phenom-
enon, or that it ever occurs in vivo. Friedemann
1 50 IMMUNE SERA
has shown that the precipitates produced in vitro
will, when injected intravenously into animals, pass
through the capillaries without harmful effects.
Pathology of Anaphylactic Shock. Acute ana-
phylactic death in guinea-pigs was originally attrib-
uted to asphyxia of central origin. Auer and
Lewis, 1 however, showed that the asphyxia is due
to a tetanic contraction of the bronchial muscle,
the contraction being so pronounced that the lumina
of the smaller bronchi are occluded, thus preventing
both the entrance and the escape of air. In a
recent study of the subject, Schultz and Jordan 2
show that in guinea-pigs the point of occlusion is
usually just beyond the place where the secondary
bronchi leave the primary, and in all cases at points
commanding large areas of lung tissue. At this
point there is the greatest relative (to diameter of
lumen) amount of smooth muscle, and there is also
normally a thicker mucosa and greater degree of
folding of the same relative to the lumen. The
fatal asphyxia observed in guinea-pigs is therefore
due to the peculiar anatomical condition of the
bronchial tree in these animals. In white mice
the anaphylactic reaction shows itself by increased
peristalsis, contractions of the bladder, increased
irritability of the skin, etc. The respiratory symp-
1 Auer and Lewis, Journal Exp. Medicine, Vol. xli, 1910.
2 Schultz and Jordan, Journal Pharmacol. and Exp. Thera-
peutics, Vol. ii, March, 1911.
ANAPHYLAXIS
toms are absent. This is clearly because the mucosa
of the bronchial tree is nowhere sufficiently thick
or folded, relative to the amount of muscle and to
the diameter of lumen, to produce occlusion under the
amount of constriction produced by the contracting
musculature. The recent work of Schultz shows
that serum anaphylaxis is essentially a hyper-
sensitization of smooth muscle generally.
It is possible that the occasional occurrence of
severe symptoms and even of death in man follow-
ing the injection of serum is sometimes due to an
abnormal development or condition of the mucous
membrane and smooth muscle of the bronchi.
Some support is given to this view by the more
frequent occurrence of these disturbances in asth-
matic individuals.
Relation of Anaphylaxis to Serum Therapy.
Returning now to the relation of the experimental
work in anaphylaxis to serum therapy, attention
should be called to the work of Steinhardt and Banz-
haf, who show that the anaphylactic reaction in
rabbits differs considerably in character from that
observed in guinea-pigs. These authors, therefore,
warn against utilizing the results of experiments on
guinea-pigs without reservation for the interpreta-
tion of phenomena observed in human beings. It
is probable that man cannot be sensitized in the
same way as guinea pigs, the most susceptible of
the laboratory animals. Children have in numerous
152 IMMUNE SERA
instances been injected with antidiphtheric horse
serum at short and long intervals, without, so far
as we are aware, causing death. Certain serums,
for example, the anti tubercle serum of Maragliano
and the antirheumatic serum of Menzer, are habit-
ually used by giving injections at intervals of days
or weeks. It may, of course, be pbjected that
possibly these injections are so spaced as to produce
antianaphylaxis. If a person had once before had
an injection of horse serum, would it be safe, say
some months, or a year, or several years later, to
give him another injection of horse serum? Or if
a child had been immunized against diphtheria
would it be safe to repeat the injection a year
later if the child were again exposed? The exper-
ience of clinicians is practically unanimous in show-
ing that such second injections need not be feared.
Even if the results obtained in guinea pigs were
applicable to man, a subcutaneous injection in man
comparable to the amount required to produce
sickness in a guinea pig would be over 200 cc.
To date about twenty cases of sudden death follow-
ing the injection of horse serum have been recorded
in the literature, and while this undoubtedly does
not represent all the cases that have occurred, the
total number is insignificant when compared to the
enormous number of such injections already made.
In New York City, in over 50,000 persons injected,
but two deaths attributed to the serum injection
ANAPHYLAXIS ! 53
have occurred. A number of fatal cases have been
reported in asthmatic individuals, and this may be
borne in mind when about to make serum injec-
tions. It is also of interest to know that Banzhaf
and Famulener have shown that chloral in large
doses will prevent the anaphylactic reaction in
sensitized guinea pigs. Such animals after the
second injection are immune to further injections.
IX. INFECTION AND IMMUNITY
Infection. In the preceding chapters we have
studied the formation and mode of action of the
various antibodies. Let us now summarize briefly
our knowledge concerning the factors involved
in infection and immunity. An infectious disease
is one caused by a living organism wiiich has gained
access to the tissues of the body. A study of
infection and immunity, therefore, embraces a
study of the pathogenesis of these organisms on the
one hand and of the defensive agencies of the body
on the other. So far as the invading organism
is concerned, we know that this may remain localized
or be widespread through the body. The absorption
of chemical products from a local infection may
produce general symptoms. This is known as an
intoxication, and is observed in cholera, diphtheria,
tetanus, local abscess, etc. In general we apply
the term ' pathogenic ' to organisms capable of
producing disease, but it must be borne in mind
that this is a relative term, for an organism patho-
genic for one species of animal need not necessarily
be pathogenic for another species.
The Infecting Agent. In studying the patho-
genicity of various bacteria, it is apparent that
154
* INFECTION AND IMMUNITY 155
we can distinguish several classes of organisms.
One class is characterized by the secretion of
highly toxic soluble substances, both in the living
body and in the culture fluid. The type of this
class is the diphtheria bacillus. Another class pro-
duces highly toxic substances, which instead of
being given off, remain within the body of the
bacterium. These poisons may be demonstrated
in old cultures in which a certain amount of dis-
solution (" autolysis ") has taken place, or they
may be obtained by mechanically breaking up the
bacteria by pressure and grinding. These sub-
stances are spoken of as endotoxins, and are liber-
ated in the body when the bacteria are disintegrated
by the bacteriolytic agencies. The type of this
class is the spirillum of cholera, an organism which
produces a powerful endotoxin and which very
readily undergoes bacteriolysis. In addition to
these two classes we know of a large number of
bacteria which neither secrete a highly toxic soluble
substance as do diphtheria bacilli nor disintegrate
as readily as the cholera spirilla, and which never-
theless are extremely pathogenic. Hiss has sug-
gested that many organisms, if not all, secrete sub-
stances which are not soluble in their condition
at secretion, but which are susceptible to digestion
in the animal body. These substances thus become
soluble and assimilable, and when toxic act harm-
fully on the body cells. Under ordinary circum-
156 IMMUNE SERA
stances these substances are broken up within the
leucocytes and the poisons . thus set free at once
neutralized by neutralizing bodies present within
the cells. According to this, conception the leu-
cocytes exercise a double function, one bactericidal
and bacteriolytic, the other a poison-neutralizing
one. The bactericidal and bacteriolytic bodies
appear to escape from the leucocytes quite readily,
and can be demonstrated in the blood plasma; the
neutralizing bodies, on the other hand, do not appear
to be given off from the cell. It is obvious, there-
fore, that the bacterial substances may be broken
up in the blood plasma, and from them may thus
be liberated a poisonous body. When this poison-
ous body is assimilated in sufficient quantity by the
higher cells of the animal organism, death ensues,
and ensues the more quickly the more rapid the
process of liberation.
In discussing allergy it was pointed out that the
phenomena of anaphylaxis should also be applied to
bacterial infections, because in these the body was
treated with small doses of bacterial proteid. As a
result of his studies, Friedberger concludes that it is
unnecessary to assume the existence of specific endo-
toxins in bacteria to account for the various symp-
toms seen in bacterial infections. By repeatedly
injecting sensitized animals with minute doses of
sheep or horse serum, he found it possible to produce
all manner of fever curves at will, merely by varying
INFECTION AND IMMUNITY 157
the size of the dose and the interval between injec-
tions. From this he concludes that the diversity
of clinical symptoms of various infectious diseases
can readily be explained on the assumption of but
a single poison. He speaks of it as anaphylatoxin,
and regards it as a cleavage product of proteid of
whatever origin introduced parenterally. Just as in
enteral digestion uniform cleavage products are
formed from most diverse proteids, so he believes
that in the parenteral proteid decomposition lead-
ing to the formation of anaphylatoxin, a uniform
poison is produced. Whether or not in addition to
the anaphylatoxin there are other specific poisons for
the various infectious diseases is entirely immaterial ;
their existence has not been proved 1 and the as-
sumption of their existence is unnecessary. In con-
sidering the diversity of the clinical symptoms of
various infectious diseases, it must be remembered
that the various species of bacteria differ in their
virulence and in their rate of multiplication, and
the invaded organisms also differ considerably in
their antibody production. All these factors serve
to modify the clinical picture. According to Fried-
berger the assumption of a common "anaphyla-
toxin" is only apparently in contradiction to the
well-known law of specificity of the infectious dis-
eases. In the infectious diseases it is not the poison
1 This applies only to the infectious bacteria, not to those pro-
ducing extra-cellular toxins.
158 IMMUNE SERA
which is specific, but only the mode of its produc-
tion. The production of anaphylatoxin requires the
action of antibodies; the mere solution or disinte-
gration of bacteria by other means does not suffice.
In other words, a definite cleavage of the proteid
molecule is necessary. The anaphylatoxin, there-
fore, is not identical with Pfeiffer's "endotoxins,"
though perhaps the latter may be the mother sub-
stance from which the anaphylatoxin is derived.
Another important factor in pathogenesis, accord-
ing to Bail, is the ability of many bacteria to pro-
duce certain neutralizing substances, not directly
injurious, but able to inhibit or neutralize the anti-
bacterial activities of the body. These substances
Bail calls aggressins. There is still some doubt
whether they are a distinct class of bacterial
products. Wassermann and Citron, Doerr, and
others regard them as consisting of dissolved
bacterial substances, extracted endotoxins and
toxins.
Resistance Against Infection. The ability of
an animal to resist the effects of a pathogenic
organism is spoken of as immunity, and may be
either natural or acquired. For example, it is well
known that the lower animals are immune against
syphilis and gonorrhoea, that dogs and goats are
rarely affected with tuberculosis, and that man is
naturally immune against chicken cholera and
rinderpest. These are instances of natural im-
INFECTION AND IMMUNITY 159
muiiity. Furthermore, it is well established that
with certain diseases one attack usually protects
the individual for life. This is well seen in
small-pox, scarlet fever, and measles. Inasmuch
as the individual was previously susceptible,
this form of immunity is spoken of as acquired
immunity.
Natural Immunity. It is seldom that natural
resistance is absolute. Young animals are often
susceptible to an infection against which adults are
resistant. Thus young pigeons are readily infected
with anthrax while older pigeons are usually
refractory. Moreover, the resistance of animals
toward infections against which they are relatively
immune can often be lowered by artificial means.
Frogs can be infected with anthrax if they are kept
in water at a temperature of 35 C. Conversely,
chickens, which also are relatively immune to
anthrax, can be infected if they are chilled. White
rats, which are ordinarily resistant to anthrax
infection, become susceptible after fatigue or when
fed on an exclusively vegetable diet.
Ehrlich believes that natural immunity is some-
times due to the absence, in the body of the invaded
animal, of suitable receptors for the virus. After
what has been said in connection with the side
chain theory, it is obvious that the virus cannot
exert its pathogenic action if there are no receptors
whereby it is anchored to the body cells. More-
160 IMMUNE SERA
over, as Ehrlich points out, even if such receptors
are present, it is possible for the animal to be immune
provided the receptors are situated only in indif-
ferent, vitally unimportant tissues. In some of
the lower animals there is reason to believe that the
toxin of tetanus does combine with such tissue
(Metchnikoff).
Acquired Immunity, This may be either active
or passive, and either form may be acquired naturally
or artificially. As examples of naturally acquired
active immunity w r e may mention the immunity
developed by one attack of small-pox, scarlet
fever, etc. The immunity against small-pox con-
ferred by vaccination is an example of artificially
acquired active immunity; so is the preventive
inoculation with bacterial vaccine against typhoid
fever. The best illustration of artificially acquired
passive immunity is the injection of diphtheria
antitoxin into humans, while the transmission of
antitoxic immunity from mother to offspring is
an example of naturally acquired passive im-
munity.
So far as maternal transmission of immunity is
concerned, a number of writers, among whom may
be mentioned Ehrlich, 1 Anderson, 2 and Theobald
1 See Morgenroth's article in Kolle and Wasserman's Hand-
buch, Vol. iv, p. 784.
2 Anderson, Bull. Hyg. Lab. U. S. Pub. Health and Mar.
Hosp. Serv., No. 30.
INFECTION AND IMMUNITY .16 1
Smith, 1 noted that an actively immunized female
parent may transmit antibodies to the immediate
young, who, receiving the immunity passively, soon
lose it again. The male parent is unable to trans-
mit any immunity. In his classical studies with
ricin and abrin, Ehrlich showed that lactation
played an important part in the transmission of
immunity from female mice to their immediate off-
spring. By immunizing a nursing mother mouse
(after the birth of the litter) he was able to demon-
strate the transmission of immunity to swine plague
to the nursing young. Smith, on the other hand,
in his experiments with guinea pigs, immunized
against diphtheria toxin, found that lactation played
no appreciable part in the passive immunity of the
young. Salge nursed infants with the milk of
goats which had been immunized against diphtheria
and against typhoid, and was unable to'demonstrate
the passage of antibodies to the infants.
In contrasting active with passive immunization
we may say that the former is usually more effect-
ive, more lasting, and productive of a general
immunity and not merely of one particular kind.
It is, however, sometimes difficult to carry out,
may involve some risk to the patient, and takes
time. Passive immunization, on the other hand,
is usually productive of only a limited kind of
immunity, i.e., antitoxic, bactericidal, opsonic, etc.,
1 Smith, Jour. Exper. Med., Vol. xi, 1909.
1 62 IMMUNE SERA
and therefore is often ineffective. Consisting, as
it usually does, in the injection of an alien serum,
passive immunization produces an immunity of but
short duration, the body apparently getting rid of
the alien proteid as rapidly as possible. The great
advantage of this form of immunization, however,
is its convenience, freedom from risk to the patient,
and above all, the fact that the immunity is pro-
duced instantaneously.
Mechanism of Immunity. Infection, whether
natural or artificial, is usually followed by a
remarkable series of alterations in the tissues of
the infected host. Representing, as it does, all the
tissues of the body, it is natural that these changes
are most strikingly exhibited in the blood. The
alterations vary, however, both with the kind
of bacterium, and with the animal species involved.
Against the true toxins, including probably the
leucocidins and hasmolysins, the body produces anti-
toxins; against the bacterial bodies it directs the
action of the leucocytes and the lytic combinations
formed by the union of amboceptor and comple-
ment; against the so-called aggressins it directs
the opsonins and perhaps also the bacteriolysins.
Before leaving the consideration of the reaction
of the body to infection, attention should be called
to the comprehensive investigations of Opie. This
observer showed that the cells which accumulate in
response to an irritant contain enzymes, the enzyme
of the polynuclear leucocytes resembling trypsin
INFECTION AND IMMUNITY I 6 2 a
and the enzyme of the macrophages resembling
pepsin in its action. The blood serum, on the
other hand, contains an antienzyme. The varying
relation existing between these enzymes and the
antienzymes serves to explain how the same irritant
in the same quantity may cause two different types
of inflammation. This is well illustrated by the
following experiment made by Opie: 1 If a small
quantity of turpentine is injected into the sub-
cutaneous tissue of dog, a large fluctuating abscess
filled with creamy pus is formed within four days;
there is a widespread undermining of the skin.
The same quantity of turpentine injected into the
pleural cavity causes a serofibrinous inflammation
which undergoes resolution so that the pleural
cavity is restored to its normal condition after about
ten days; there is no destruction of tissue and a
scar is not formed. In the subcutaneous tissue
only a small amount of oedematous exudate can
accumulate; the undiluted irritant causes active
migration of leucocytes so that the antibody of the
exuded serum is soon overbalanced by the enzyme
set free by disintegrated pus cells. In the pleural
cavity, on the contrary, a large quantity of serum
quickly accumulates and the exudate is sero-
fibrinous instead of purulent; the antienzyme it
contains is capable of holding in check the enzyme
1 E. L. Opie, Lecture before the Harvey Society, New York,
Feb. 1910. The Harvey Lectures, J. B. Lippincott Co. 1910.
l62b IMMUNE SERA
of the accumulated leucocytes. If a bit of the
fibrinous exudate is suspended in the exuded
serum, it is preserved intact. Nevertheless, by
repeated injection of turpentine at short intervals
into the pleural cavity, accumulation of leucocytes
can be prolonged so that finally a condition is
produced in which antienzyme can no longer
restrain the enzyme. The softened fibrin of such
an exudate quickly disintegrates in the serum of
the exudate. These observations, as Opie points
out, help to explain how the typhoid bacillus pro-
duces abscesses in certain situations such as the
kidney and bone; how the pneumococcus, which
rarely causes abscess of the lung, in which condi-
tions are somewhat similar to those within the pleural
cavity, may cause suppuration in other localities,
such as the middle ear, or in the subdural space, etc.
In addition to the antibodies already mentioned,
the animal body produces agglutinins and precip-
itins directed against the invading bacteria, but
the relation of these antibodies to immunity is not
at all clear. So far as the action of the agglutinins
is concerned, we have already pointed out (on page
36) that this appears to have no destructive effect
on the agglutinated organisms. Moreover, while ag-
glutination is often observed to precede lysis, there is
no reason to believe it a necessary factor in the lytic
process, nor even an aid thereto. Whether this list
exhausts the number of serum antibodies is doubtful.
INFECTION AND IMMUNITY 163
The antibody content of the serum, ^wever, is
not always the same as that of the blood plasma.
Thus Gengou, by collecting the plasma in vaselined
tubes, found it often to be almost devoid of bac-
tericidal power, while the corresponding serum was
capable of destroying large numbers of microor-
ganisms. In these cases, it is evident, we cannot
regard plasma destruction of bacteria as the impor-
tant factor in immunity. Moreover, in the case of
bacteria containing considerable quantities of endo-
toxin, it is conceivable that plasma destruction of
bacteria may even do considerable harm by
causing an enormous liberation of endotoxin. This
point is perhaps of practical importance as contra-
indicating the use of bacteriolytic sera in the
curative treatment of certain infections.
From what has been said it is evident that the
exact mechanism of immunity, at least so far as
most infections are concerned, is still very obscure.
Like most biological phenomena, the deeper we
analyze the problem, the more complex and more
marvelous it becomes. Enough has, however, been
presented to show some of the difficulties to be
overcome and the method of attacking the sub-
ject.
Relation of Anaphylaxis to Immunity. We have
already discussed the relation of anaphylaxis to
infection and may now take up briefly its relation
to immunity. We know that the subcutaneous,
164 IMMUNE SERA
intraperitoneal, or intravenous introduction of alien
proteid is followed by the formation of antibodies;
at the same time it can readily be shown that no
antibodies develop after the oral introduction of
milk, eggs, or even of raw meat. In other words,
there is a marked contrast in the behavior of the
body between the enteral and the parenteral intro-
duction of proteid. In the former the proteid is
acted on by the gastric and intestinal juices (pepsin,
trypsin, and enterokinase) . These so break down
the proteid molecule that it loses its species identity.
After this, absorption takes place, and with it there
is a synthesis, or rearrangement, of the molecule
whereby it is built up into the specific proteid of
the body. Under normal conditions it is im-
possible to produce specific antibodies by feeding
alien proteid. Precipitins have, however, been pro-
duced by overfeeding animals with large quantities
of alien blood. When proteid is introduced paren-
terally it gives rise to the formation of specific
antibodies, and thus to the state of anaphylaxis.
The term anaphylaxis is unfortunate, for the con-
dition is not always opposed to immunity. Von
Pirquet, it will be remembered, called attention to
the altered reactivity during the anaphy lactic state.
We must not lose sight of the fact that the symp-
toms of anaphylaxis are brought on when sensitized
animals are subsequently injected 'with relatively
large quantities of the same proteid. Following
INFECTION AND IMMUNITY 165
such an injection there is a sudden liberation of
large amounts of toxic material. The parent eral
introduction of large quantities of alien proteid
must, however, be very exceptional under natural
conditions. The number of bacteria primarily
involved in an infection certainly represents but a
very small amount of alien proteid. If the body is
in the condition of allergy (anaphylaxis) at the
time of infection it will be able to respond more
quickly than otherwise and perhaps destroy the
invaders. Under these circumstances it is con-
ceivable that the condition is really an immunity
reaction. Looking at the entire question broadly
we may regard the mechanism which lies at the
bottom of the phenomenon of anaphylaxis as a
useful contrivance which enables the organism to
rid itself of alien proteid, both organized and un-
organized, which has been introduced parent erally.
Immunity Reaction on the Part of Bacteria. It
may be well at this point to call attention to a view
advanced by Welch some years ago. According to
this it is reasonable to suppose that just as the
animal body produces antibodies against an invad-
ing organism, so does the latter, owing to the action
of the body fluids, produce antibodies directed
against the tissues of the invaded body. In this
way the infecting organism would be adapting itself
to unfavorable surroundings, and this we know it
often does. It is certain that the animal body often
1 66 IMMUNE SERA
successfully overcomes an infectious disease without
entirely overcoming the infecting bacteria. This is
well shown by what we call chronic germ carriers.
Deutsch regards the increase in virulence brought
about by successive passage of a bacterium through
a susceptible animal as representing an immunity
developed by the bacterium against the anti-
bacterial agencies of the body.
Atrepsy. Ehrlich has investigated this phe-
nomenon in the case of trypanosomes. He found
that a monkey which had been infected with a
particular strain of trypanosome and then cured
by means of chemo- therapeutic agents, when
tested with the original strain was not immune,
the disease reappearing after a long incubation.
If mice were inoculated with blood from the diseased
animal, i.e., with blood containing trypanosomes,
they became infected and died. Curiously, how-
ever, if the trypanosomes were first removed from
this monkey blood, it was found that the serum
was able to kill the original strain of trypanosomes.
This showed that the trypanosomes had undergone
some change in the body of the monkey; they
differed from the original strain in their behavior
toward the serum; they had become " serum-fast."
Similar observations were made at the same time
by Kleine, and recently also by Mesnil.
In explanation of this adaptation, Ehrlich sug-
gests that certain particular receptors of the para-
INFECTION AND IMMUNITY i6f
site are concerned entirely with the parasite's nutri-
tion. Owing to the destruction brought about by
the chemical agent, some of these receptors pass
into the monkey's body, and, acting as antigens,
excite the production of antibodies directed against
these particular receptors. When living parasites
are brought into contact with this antibody, either
in vitro or in vivo, the antibody is anchored by
the parasites. As a result of this occupation of
its receptors, the parasite undergoes a biological
alteration which consists in the disappearance of
the original receptor group and its replacement
by a new group. Ehrlich's researches lead him to
believe that the antibody has merely an anti-
nutritive action, blocking the nutrireceptor of the
parasite and so bringing about starvation. The
parasite thus develops immunity by getting rid
of certain of its nutrireceptors, and replacing them
with different ones. This form of immunity Ehrlich
speaks of as " atrepsy," while the antibodies de-
veloped against the nutrireceptors he terms " atrep-
sins." A somewhat different example of atrepsy
is the following: Bird-pox, virulent for both fowl
and pigeon, if passed through the pigeon becomas
completely avirulent for the fowl. Ehrlich believes
that the parasite in passing through the pigeon has
to assimilate substances different from those assim-
ilated in its passage through the fowl. There-
fore that part of the receptors which deals with the
1 68 IMMUNE SERA
nutritive substances in the fowl's organism is not
in use during the passage through the pigeon and
may become atrophied, so that on the parasite
being transferred back to the fowl, supposing one
of the specific constituents of fowls to be neces-
sary for its proliferation, it would no more be
able to grow. We have, therefore, a loss of cer-
tain receptors which are absolutely necessary for
nutrition.
Ehrlich suggests that probably the majority of
so-called non-pathogenic micro-organisms, if intro-
duced into an animal's body, perish by this mechan-
ism. It is not necessary to assume the presence
of special poisons in the body, it suffices to suppose
that the bacteria in question do not find the needful
means of existence in the body and therefore cannot
multiply. They thus fall a prey to the phagocytes
which destroy the invaders in a non-specific
manner.
X. BACTERIAL VACCINES
Historical. Early in the eighteenth century
attention was called to the fact that in Oriental
countries individuals were immunized against small-
pox by inoculating them with a little small-pox
virus under the skin. In 1796 Jenner showed that
similar immunity could be produced by inoculating
the virus of cow-pox, and this procedure was free
from the dangers that attended small-pox inocu-
lations. Following the discovery of the specific
microbe of anthrax, attention was directed to the
problem of combating this disease. Pasteur, who
had been greatly impressed with Jenner's work
with cow-pox, felt that attempts should be made
to produce a mild attack of the disease, and that
this would then protect against a virulent infection.
After considerable experimental labor he devised
the plan of inoculating animals with cultures of
anthrax which had been attenuated by being grown
at high temperatures, 43 C. These animals had
a mild attack of the disease from which they soon
recovered, and then were resistant to infection with
virulent virus. Soon after this, inspired by Pas-
169
1 70 IMMUNE SERA
teur's work, successful vaccines * were prepared
against chicken cholera and swine plague.
The discovery of diphtheria antitoxin in 1893 by
v. Behring marked the beginning of the search for
specific sera, and it was not long before a number
of such were produced and employed clinically.
The use of sera for therapeutic purposes was very
attractive, because it was possible to have some
animal, like the horse, manufacture the antibodies,
and one needed then merely to transfer the animal's
immunity to the patient by injecting some of the
animal's serum. Clinical trials, however, soon
showed that most of these sera had little thera-
peutic value, and subsequently laboratory experi-
ments disclosed a large number of difficulties in
their practical application. After what has been
said under haemolysins and bacteriolysins it will
be unnecessary to dwell on these difficulties. Among
them is the problem of providing sufficient com-
plement, the determination of the optimum dose
so as to avoid the parodoxical results known as
the Neisser-Wechsberg phenomenon, the ability
of producing really effective antibodies, and finally
the question whether immunity in a given case is
really directly due to the presence of these anti-
bodies in the serum.
1 The French have long used the term "vaccin" to denote
any virus which is used for immunization, and that is the
sense in which the term is used here. There is, of course,
nothing of the cow, vacca, about them.
BACTERIAL VACCINES 171
In the past few years it has become more and
more apparent that the limitations of serum therapy,
at least in the great majority of infectious diseases,
are at present almost insuperable. Attention was
therefore again turned to treatment by active
immunization. It was perhaps only natural, in
view of his discoveries in fermentation, that Pasteur
should have believed that the production of immu-
nity required the action of the living virus. He
therefore vigorously combated the idea that im-
munity could be brought about by means of dead
virus, or of lifeless products of growth of the virus.
Touissant, as far back as 1880, had held out for
the latter possibility, but the imperfections of
his technique were such that his views were not
accepted. To Salmon and Smith of this country
belongs the honor of first clearly demonstrating
the possibility of immunization w r ith dead cul-
tures.
Methods of Active Immunization. Active im-
munization can be carried out in several ways:
(i) By means of living cultures of the virus.
Usually the cultures are attenuated, but there are
some exceptions.
A number of different procedures may be employed
to attenuate the virus. Thus, by drying, as is done
with rabies virus in the Pasteur treatment; or by
growing the virus at a temperature unsuited for the
development of virulence, as is done in the case of
172 IMMUNE SERA
anthrax; or by passing the virus through a less sus-
ceptible animal, as is done in vaccination against small-
pox; or by means of chemicals such as the addition
of iodine solution to diphtheria toxin, as was formerly
done by Behring; or by means of heat, as was also
formerly done with diphtheria toxin.
(2) By means of dead cultures of the virus. The
cultures can be killed either by heat or by the use
of chemicals.
(3) By the so-called " combined method," i.e.,
by first administering a dose of the specific immune
serum and subsequently the virus. This method
has been used in typhoid fever, cholera, and plague.
(4) By means of the products of autolysis of the
cultures. This has also been used in typhoid fever,
and seems to possess certain advantages over the
use of native cultures.
(5) By means of various combinations of the
preceding methods.
The choice of these various methods of immuni-
zation depends on the nature of the infecting virus.
With some infections dead cultures apparently are
able to cause the production of full protective pow-
ers, while in other infections the body seems to
require a greater stimulus. In these, the use of
attenuated living cultures may bring about the
desired immunity. Finally there are infections
in which nothing short of fully virulent cultures
seems to bring about the development of sufficient
BACTERIAL VACCINES 173
immunity. In these cases it is necessary to first
prepare the way by the use of dead or of attenuated
cultures.
Treatment with Vaccines. The treatment of
infections by means of active immunization has
been greatly stimulated by the work of Wright,
who has published favorable results in a large
number of infections. Already several hundred
thousand persons have been actively immunized
against cholera, and large bodies of troops have
been immunized against typhoid fever. Until
recently the method found application particu-
larly in the prophylactic immunization of persons
liable to be exposed to infection. At the present
time, however, owing largely to the efforts of
Wright, the method has come to be used for cura-
tive purposes, i.e., for infections already in progress.
This author has clearly formulated the conditions
in which he thinks this form of treatment is indi-
cated, and he has also devised methods for the
more exact determination of doses than were
formerly in use.
In the employment of bacterial vaccines, one
must constantly keep in mind the nature of the
bacterium with which one is working, and the kind
of immunity one wishes to bring about. Every-
thing depends on the way in which the vaccine is
prepared. With bacteria making considerable
quantities of a toxin, it will be necessary, if we
174 IMMUNE SERA
wish to immunize against this toxin, to grow the
culture for the requisite length of time and under
the proper conditions for producing the toxin.
In the case of bacteria possessing certain endo-
toxins, it may be necessary to let the cultures
autolyze, so as to set these substances free, or the
bacteria may be crushed and ground for the same
purpose. On the other hand, we may wish to use
these bacteria for producing a specific agglutinat-
ing serum. In that case we often try to avoid
injecting these toxic substances. Our entire pro-
cedure might then have to be quite the reverse of
what has just been indicated.
The Vaccines. Wright's method of preparing a
staphylococcus, typhoid, streptococcus, or gono-
coccus vaccine, is as follows:
Several streak slant agar cultures are planted and
incubated for twenty to twenty-four hours. The
cultures are then washed off with normal salt solu-
tion, using from one to several cc. for each culture.
These suspensions are next heated to 55 C. in
order to kill the bacteria, and are then standard-
ized. By this is meant determining the number of
organisms per cc., for Wright always used definite
numbers of bacteria in his inoculations. This
standardization is readily accomplished by means
of the method devised by Wright, which is as fol-
lows : From a finger prick a drop of blood is sucked
up in a capillary tube to a mark made at any con-
BACTERIAL VACCINES 175
venient point with a wax pencil. Next an equal
amount of the bacterial suspension is drawn into
the tube, allowing a tiny air-bubble to intervene.
The two fluids are then expelled on a glass slide,
and thoroughly mixed by sucking back and forth
a number of times. After this has been done the
mixture is spread in the ordinary way of making
blood smears. If these blood smears, after stain-
ing, are examined with a microscope having a ruled
eye-piece, it is a simple matter to determine the
ratio of bacteria to blood cells. Taking the red
blood cells as 5,000 million per cc., one calculates
the number of bacteria per cc. In practice it is
advisable to so dilute the bacterial suspension that
the dose to be injected is contained in about one
cc. of fluid. Finally \ per cent, of carbolic acid
is added as a preservative. Such a suspension is a
" bacterial vaccine." It goes without saying that
the vaccines should be tested by means of cultures
to insure sterility, and that contaminations should
be excluded by means of microscopical exami-
nation.
Doses. So far as doses are concerned, these vary
with different bacteria, and also according to the
indications, opsonic or clinical. The ordinary dose
for the staphylococcus vaccine is from 200,000,000
to 1,000,000,000 organisms; for the streptococcus
it is from 50 to 75 or 100,000,000, and for typhoid
from 750,000,000 to 1,000,000,000 bacteria. All
T y6 IMMUNE SERA
the injections are given subcutaneously, and it is
well to repeat the injections every three or four
days.
Results. The clinical results obtained by means
of bacterial vaccines have varied. There seems
considerable agreement on the part of most observers
that certain localized infections, such as acne, mul-
tiple boils, etc., usually respond remarkably well
with this method of treatment. In the treatment of
bone tuberculosis the results are not so harmonious,
and in the treatment of general infections many
failures have been reported. There is no doubt,
however, that treatment by means of bacterial
vaccines is a valuable addition to our therapeutic
armamentarium .
XL LEUCOCYTE EXTRACTS IN THE TREAT-
MENT OF INFECTIONS
Theory. Attention has already been called to
Hiss's view concerning the role of leucocytes in
combating infections. Believing that the phagocytic
power of leucocytes of persons suffering from infec-
tions to be less than that of leucocytes of the normal
individual, Hiss was led to extract these cells with
a view to utilizing their neutralizing and other pro-
tective substances in readily diffusible form. By
this means it was thought possible to furnish to the
infected organism such assistance as would enable
its phagocytic cells to properly protect the various
tissues from poisons elaborated from the invading
bacteria.
Preparation of the Extracts. In the preparation of
the extract, double pleural inoculations of aleuronat l
suspensions are made into rabbits. After 24 hours
the rabbits are killed and the turbid fluid collected
from both pleural cavities. The quantity obtained
varies from about 30 to 60 cc. The fluid is quickly
centrifuged and the serum decanted. The cells are
1 Aleuronat, a vegetable product similar to gluten, is pre-
pared by Hundhausen, in Hamm, Westphalia, Germany, and
is supplied in packages containing 100 grams. The suspensions
are prepared with thin starch paste and boiled.
177
1 78 IMMUNE SERA
then thoroughly emulsified in distilled water, using
about as much water as the volume of serum orig-
inally poured off, and the mixtures allowed to
stand for a few hours at 37 C. This more or less
autolyzed fluid is used for the injections, and the
dose employed varies from 5 to 15 cc., repeated
several times.
Application and Results. In their work, clinical
and experimental, Hiss and Zinsser 1 thought they
saw little indication of immediate bactericidal
power possessed by the leucocyte extract, but that
the results pointed rather to a marked power on
the part of the extract to reduce the purely tox-
semic manifestations in infected subjects. Favorable
clinical results have been reported by these authors
in cerebrospinal meningitis, lobar pneumonia, 2 and
other infections, and while the data are still too
scanty to justify definite conclusions as to the
value of this treatment, enough has been done to
warrant further careful clinical investigations along
this line.
1 Hiss and Zinsser, Journal Med. Research, Vol. xix, Nov.,
1908.
2 See also Floyd and Lucas, Journ. Med. Research, VoL
xxi, Sept., 1909.
XII. THE PRINCIPLES UNDERLYING THE
TREATMENT OF SYPHILIS WITH
SALVARSAN (" 606 ")
As a result of some of his earliest researches and
in entire accordance with his views as already set
forth under "Antitoxins," Ehrlich has always held
that the action of a chemical substance on a given
cell denotes the existence of definite chemical affini-
ties between the substance and the cell. Applying
this conception to the germicidal action of chem-
icals, he maintains that the latter must have a
certain chemical affinity for the parasites in order
to kill them. Substances having such affinities
he terms " parasitotropic." It is clear, however,
that substances which can destroy parasites will
also be poisonous for the animal body, i.e., they
will have chemical affinity for the vital organs of
the host. They are, therefore, also " organotropic."
In the employment of chemical substances in com-
bating infectious diseases it follows that success
can only be attained if their chemical affinity for
the infecting parasite bears certain relations to their
affinity for the infected body.
179
i8o IMMUNE SERA
In his researches on trypanosomes Ehrlich found
that if the dose of germicide administered to an
infected animal was too small to kill all the para-
sites, there developed after a time a strain of organ-
isms which were resistant to the further action of
the germicide. It was usually futile to repeat the
dose, for the resistant parasites would survive.
At the same time the interesting observation was
made that this resistance manifested itself only
in the animal body; in vitro the parasites could
still be filled by the germicide in question. It was
also found that the various chemical substances
which exerted therapeutic effects in animals infected
with trypanosomes could be grouped into three
classes, namely i, various arsenicals (arsenious
acid, atoxyl, arsacetin, arsenophenylglycin, and
finally "606,"); 2, certain azo dyes (among them
trypan red, trypan blue, and trypan violet) ; and 3,
certain basic triphenylmethane dyes (among them
parafuchsine, methyl violet, pyronine, etc.). Against
each of these classes it was possible to produce speci-
fically resistant strains of trypanosomes, so that a
strain which had been made resistant to fuchsin was
also resistant to related basic dyes, but vulnerable
to the azo dye and to the arsenicals. Moreover,
by appropriate treatment it was found possible
to produce strains resistant against all three classes
of trypanocidal agents.
According to Ehrlich the union of the chemical
TREATMENT OF SYPHILIS WITH SALVARSAN 181
with the parasites is brought about by certain
" chemoreceptors " of the parasites. The arsenic
compounds, for example, are anchored by arseno-
receptors. When a strain therefore becomes re-
sistant to the arsenicals one might suppose that
this was due to the parasite ridding itself of its
arseno-receptors ; similarly also with the other
classes of trypanocidal substances. But this appears
not to be the case, for, as we have already said, the
resistance is manifested only in the animal body,
and not in vitro. Ehrlich explains this by assuming
that in the resistant strain the affinity of that par-
ticular chemo-receptor has been reduced, so that
when the germicidal agent is introduced into an
animal infected with the resistant strain, the pro-
portion of distribution of the germicide is altered
in favor of the chemo-receptors of the organism.
In other words the organotropic affinity is greater
than the parasitotropic affinity. Had the parasite's
chemo-receptors quite disappeared there should
have been no difference in the resistance as mani-
fested in the animal and in vitro.
In searching for germicidal substances whose
parasitotropic affinity should be great in comparison
to their organotropic affinity, Ehrlich made careful
pharmacological studies with each of the three
classes of trypanocidal substances already mentioned.
After testing homologues and substitution products
of almost every variety he finally concluded that
ig2 IMMUNE SERA
dioxydiamidoarsenobenzol fulfilled the require-
ments. As each of the substances was tested it
received a laboratory number for identification;
dioxydiamidoarsenobenzol bore the serial number
606, whence the designation by which this substance
is still commonly known. Its trade name is " Sal-
varsan."
It is not our purpose, in these pages, to enter into
the chemistry of " 606," or to discuss the treatment
of syphilis by this drug. Suffice it to say that
Ehrlich lays considerable stress on the fact that
in 606 the arsenic is in the trivalent unsaturated
form. Pentavalent arsenic compounds, he believes,
are less efficient in their trypanocidal action. As
supplied in the market, Salvarsan is a bright yellow
powder containing theoretically 34.15 per cent
arsenic. It is the hydrochloride of dioxydiami-
doarsenobenzol, and is administered by suspending
or dissolving it in water with the addition of NaOH
to neutralize, thus forming dioxydiamidoarseno-
benzol plus NaCl and H 2 O. Subcutaneous, intra-
muscular, and intravenous injections have been
employed, as also solutions and suspensions of the
drug. The results in the treatment of syphilis
have been encouraging, but the time has not yet
come to express a definite opinion concerning the
ultimate value of this drug.
The principles here outlined, however, deserve
to be carefully studied and tested experimentally,
TREATMENT OF SYPHILIS WITH SALVARSAN 183
for if correct they point the way for devising an
effective therapy for many infections at present
quite beyond our control.
LITERATURE
EHRLICH-BOLDUAN. Collected Studies in Immunity,
(Chapter XXXIV) 1910. Wiley & Sons, New
York.
EHRLICH. Experimental Researches on Specific Thera-
peutics. 1909. Hoeber, New York.
EHRLICH and HATA. Die Experimented Chemothera-
pie der Spirillosen. 1910. Springer, Berlin.
MARTINDALE and WESTCOTT. " Salvarsan " (" 606 ").
1911. Hoeber, New York.
WECHSELMANN. The Treatment of Syphilis with Di-
oxydiamidoarsenobenzol. (English translation by
A. L. Wolbarst). 1911. Rebman Company, New
York.
APPENDIX A
THE WASSERMANN TEST FOR SYPHILIS.
As has already been pointed out on page 72,
Wassermann applied the principle of the Bordet-
Gengou phenomenon to the detection of syphilis
antibodies in the serum and cerebrospinal fluid of
persons infected with syphilis. In the few years
which have elapsed since Wassermann's first pub-
lication, the reliability of this method of diagnos-
ing syphilis has been confirmed by a large number
of investigators, and it has already proven of con-
siderable value in several departments of medicine.
In response to numerous requests, the writer has
undertaken to give a clear description of the test,
together with a brief review of the results thus far
achieved by its use.
When an animal is repeatedly injected with red
blood cells of another species, it reacts to such in-
jections by producing substances in its serum which
have the power to dissolve these foreign blood cells.
Examined by means of a test tube experiment, it
is found that the serum exerts this power only
while it is fresh. Serum several days old is unable
to dissolve the recj cells. The fresh serum also
'85
l86 APPENDIX
loses its solvent power by exposure to heat, say to
55 C. Investigations showed that the solvent
action could be restored to these sera by the addi-
tion of small quantities of a fresh normal serum,
i.e. of a serum which by itself had no solvent power
whatever. The inactive serum had thus been
reactivated. The original specific dissolving serum
therefore contained two substances, one of which
is very labile and the other stable. The stable
substance is specific for the blood cells against
which it is directed, i.e. against the cells used for
immunizing the animal. It is called the " immune
body," or the " amboceptor." The labile sub-
stance, as we have seen, is present in all fresh sera,
and is spoken of as the " complement." The action
of the immune body seems to consist in bringing
the solvent action of the complement to bear on
the given cells. We must conceive that the com-
plement possesses the solvent power, but has no
way of laying hold of the cell to be dissolved. The
immune body merely effects this combination.
Ehrlich's diagram on page 66 will serve to make
this conception clear.
All that has been said regarding immune bodies
and complement for the solution of blood cells,
holds for the substances which effect destruction of
bacteria when bacteria are used for immunization.
In fact, the process is the same, no matter what
cells are injected into the animal. The immune
body is always directed specifically against the
cells injected, and against no others.
APPENDIX
As can be seen from Ehrlich's diagrams, the
bacteria or blood cells combine directly only with
the immune body. The complement, as already
said, has no way of laying hold of the cells. As
soon as the bacteria or cells have anchored the
immune body, however, conditions change. The
combination at once attracts and unites with the
complement. If the amount of complement is not
too large, the combination may unite with all of it,
i.e. may abstract the complement from the serum.
Just let us examine this by means of an illustra-
tion: Let us suppose we have immunized an ani-
mal with typhoid bacilli, and have obtained a
specific serum directed against these bacilli. This
serum has been inactivated by heating it to 55 C.,
so that now it will act on typhoid bacilli only when
some fresh normal serum is added to complement
the immune body. For this purpose we have
provided ourselves with some freshly drawn serum
from a guinea pig. The guinea pig serum, there-
fore, is the "complement." On mixing typhoid
bacilli with the specific immune serum and then
with the complement, these three factors enter into
combination, and this results in the destruction of
the typhoid bacilli. The quantities can easily be
so arranged that this combination uses up all of the
complement, so that the fluid contains not a trace
of free complement after the substances have com-
bined.
Suppose, now, that we also had a specific serum
obtained by injecting an animal with red blood
188 APPENDIX
cells, for example, by injecting a rabbit with sheep
blood cells. This rabbit serum would then be
specifically directed against sheep blood cells. Let
us inactivate this serum, by heating it to 55 C., so
that now it requires the addition of a fresh normal
serum to exert its solvent effect. For this purpose
we can again use fresh, normal guinea-pig serum.
When, then, we mix sheep blood cells with our
specific immune serum (against sheep blood cells)
and with the complement, i.e., with fresh normal
guinea-pig serum, all three factors unite, and bring
about destruction of the red blood cells. This is
manifested by the blood cells dissolving and
giving off their haemoglobin to the rest of the
fluid.
Let us now suppose we have carried out the first
part of this experiment, that with the typhoid bacilli,
and have left typhoid bacilli, specific typhoid serum
and complement in contact for several hours in a
warm place in order to cause the three factors to
combine. At the end of this time let us add sheep
blood cells and the specific serum directed against
sheep cells, but let us add no further complement,
because the fresh guinea-pig serum was able, as we
saw, to serve as as complement also for the blood
combination. The mixture is again placed in a
warm place for several hours, and then for twenty-
four hours in the refrigerator, after which it is ex-
amined. We shall find that no haemolysis has
occurred, from which we conclude that the previous
combination (typhoid bacilli, immune serum and
APPENDIX 189
complement), had used up all the complement, and
left none for the blood combination.
If we were to repeat the whole experiment, but
leave out, in the first part of the test, say the spe-
cific typhoid serum, we should find that the- blood
cells would be dissolved. This is readily under-
stood when it is remembered that then we would
have only typhoid bacilli and complement, two
factors which cannot ccmbine directly. The com-
plement would therefore be left free to act in the
blood combination.
If hasmolysis occurs we may therefore conclude
that one of the factors in the first combination was
absent, and conversely, if hasmolysis does not occur,
we know that the first combination must have
been perfect, i.e. all three factors must have been
present.
It is at once apparent that in adapting this test
to the detection of syphilis antibodies, pure cultures
of the causative organism, i.e. of the "antigen,"
were not available. Wassermann therefore made
use of extracts of syphilitic organs rich in spiro-
chastes in place of the typhoid bacilli, and used
either the serum or the cerebrospinal fluid of the
suspected case in place of the typhoid antiserum.
The rest of the test was similar to that described
above. When haemolysis of the sheep cells occured,
Wassermann said it showed that the first combina-
tion was incomplete; when haemolysis was com-
pletely inhibited, it showed, he said, that the first
combination was perfect, i.e. that the serum or
190 APPENDIX
spinal fluid contained syphilis antibody. As in
most such tests, only a positive result determines ;
a negative result does not necessarily exclude the
presence of syphilitic infection.
While the above exposition will serve to fix the
general plan of the test in the mind of the reader,
we must at once say that the mode of action is not
as simple as Wassermann first believed. Before
going into this phase of the subject, it will be
advisable to present a description of the technique
of the test.
For carrying out the test the following materials
are required:
(i)" Antigen," i.e. fluid containing syphilis ma-
terial. This is comparable to the pure culture of
typhoid in the test described above.
(2) Serum or cerebrospinal fluid from the patient
to be examined.
(3) Sheep blood cells.
(4) Haemolytic antibody, i.e. inactivated serum
of a rabbit immunized against sheep blood cells.
(5) Complement, i.e. fresh normal serum from a
guinea-pig.
For the syphilis antigen it is best to use the
organs of a syphilitic foetus, i.e. one dead of heredi-
tary syphilis, as these tissues are particularly rich
in spirochaetes. The organs are chopped up and
macerated in a clean vessel in a mixture composed
of water, 1000; NaCl, 8.5; carbolic acid, 5.0; one
part of the tissue to four of the fluid. The mixture
is shaken in a shaking apparatus for twenty hours ;
APPENDIX
the supernatant fluid poured off and centrifuged so
as to be perfectly clear.*
The serum for the test is collected from the
patient in the usual way by drawing from 5 to
10 cc. of blood from a vein at the elbow, placing
the blood in a sterile test tube and allowing it to
clot. Cerebrospinal fluid, obtained by lumbar
puncture, is preserved with 0.5% carbolic acid, and
then strongly centrifuged so as to make it perfectly
clear.
The sheep blood cells are obtained by defibrinat-
ing sheep blood, centrifuging and washing the blood
cells repeatedly with normal salt solution to remove
traces of adherent serum. A 5% suspension in salt
solution is used.
The haemolytic antibody consists of the serum of
a highly immunized (against sheep blood cells)
rabbit, the serum being inactivated by heating to
56 C. In the tests cited by Wassermann, one cc.
of a 1/1500 dilution of serum dissolved one cc. of
5% suspension of sheep blood cells at 37 C. in
two hours.
The complement consists of freshly drawn guinea-
pig serum. The test is carried out as follows :
To constant quantities of spinal fluid (e.g. i cc.
of the i/io dilution) decreasing amounts of the
extract of organs are added, thus 0.2, o.i, 0.05 cc.
Then i cc. of a i/io dilution of fresh normal guinea-
* In a very recent article, Wassermann states that more uni-
formly active extracts can be obtained by using 96% alcohol in
place of the water in the above formula.
1.9 2 APPENDIX
pig serum is added, and the mixtures allowed to
remain in contact at 37 C. for one hour in order to
bind the complement.
In this mixture we have antigen; we may or may
not have antibody; we have complement.
If the antibody is present, the complement will
be anchored by the combination, and so be unavail-
able for the haemolytic test next in order. If no
antibody is present, the complement will still be
free to act in the haemolytic test.
At the end of the hour, we add to the above
mixtures: one cc. of a 5% suspension of sheep
blood cells, and one cc. of the amboceptor dilution
containing double the solvent dose for that amount
of sheep blood cells. Thus, if the titer of the
haemolytic serum is 1/1800, we take one cc. of a
dilution 1/900.
All the tubes are made up to the same volume
with normal salt solution, namely, to 5 cc.,and are
then placed in the incubator at 37 C. and kept
there for two hours. Then they are placed on ice
until the next day, when the results are noted-
The whole procedure is clearly shown by the pro-
tocol from Wassermann and Plaut reproduced on
page 193.
Few experiments in immunity require such care-
ful technique, or are open to so many sources of
error as this serum test for syphilis. In view,
too, of the enormous responsibility assumed in
making a positive diagnosis of syphilis, it is appa-
rent that the test should only be undertaken by
APPENDIX
193
J
Haemoly-
tic Ambo-
Syphilit.
Foetus
Extract.
(0.2 gm.)
One c.c.
of I Dilu-
tion.
Spinal Fluid
of Patient
M. 0.2, i.e..
One c.c. of
the Dilu-
tion.
Normal
Guinea-
pig Serum
O.I CC.,
i.e. i cc.
of a A
Dilution.
ceptor
I CC.
equals
Double
the
Solvent
dose for
Sheep
Blood
Cells i cc.
of a 5%
Suspen-
sion.
Results.
i cc. of a
5% Sus-
pension.
0.2
0.2
I.O
I.O
i .0
Complete inhibition
of haemolysis
O.I
0.2
I.O
I.O
I.O
Compl. inhibition
0.2
0. I
I.O
I.O
i .0
Marked inhibition
O.I
O. I
I.O
I.O
I.O
Marked inhibition
0.2
I.O
I.O
I.O
Complete solution
O.I
I.O
I.O
I.O
Complete solution
0.2
I.O
I.O
i .0
Complete solution
O.I
I.O
I.O
I.O
Complete solution
Spinal Fluid
of
Non-syphil.
Person.
0.2
0. 2
1 .0
I.O
I.O
Complete solution
0. I
0. 2
I.O
I.O
I.O
Complete solution
O. 2
0. I
I.O
I.O
I.O
Complete solution
O.I
O.I
1 .0
1 .0
I.O
Complete solution
0.2
I.O
I.O
I.O
Complete solution
O. I
I.O
I.O
I.O
Complete solution
highly trained laboratory workers. On the other
hand, most who have busied themselves with the
test agree that suitable controls always lead to a
detection of possible sources of error, and that
IQ4 APPENDIX
therefore the reaction, when properly performed,
can be relied upon.
When the test was first published Wassermann
regarded the reaction which occurred as one between
mutually specific bodies, i.e. between antigen and
antibody, the resulting combination having the
power to anchor the complement. Through the
work of Marie and Levaditi, of Landsteiner, Miiller
and Potzl, of Weil and Braun, and still other in-
vestigators, it soon became apparent that the test
could also be carried out by using extracts of non-
syphilitic tissue, i.e. of other pathological tissue or
normal tissue. That, of course, meant that the
view of a reciprocal specific relation between anti-
body and organ extract, in the sense that typhoid
antibody and typhoid bacilli are reciprocally re-
lated, had to be abandoned. This does not, how-
ever, effect the reliability of the reaction for diag-
nostic purposes, for it has been found that positive
results are still only obtained when the serum or
spinal fluid is of syphilitic origin.*
Working under Wassermann's direction, Forges
and Meier studied the nature of the substances
concerned in the reaction, and began by precipi-
tating the organ extracts with alcohol and testing
*It may be well to state that according to Landsteiner, Muller
and Potzl the serum of animals infected with dourine (trypanoso-
miasis) also gives rise to inhibition of haemolysis when tested
according to the above method. This has been confirmed by
Hartoch and Yakimoff. Whether this will affect the value of the
Wassermann test in humans can only be decided by further
clinical tests, especially in cases of human trypanosomiasis.
APPENDIX 195
the resulting precipitate and clear fluid separately.
It was found that the substance concerned in the
reaction was soluble in alcohol, and the authors
thereupon made alcoholic extracts of the syphilitic
organs. These worked satisfactorily in making the
test. It was natural to think that the substance
which effected the reaction might be related to the
lipoids, and so the authors next studied the be-
havior of alcoholic extracts of normal human and
animal organs. While these extracts also sufficed
to produce the reaction, the authors felt that they
were not as active as extracts from syphilitic
organs. After it had been found that alcoholic ex-
tracts could be used for the test, a number of
authors almost simultaneously published favorable
results with chemically defined substances. Forges
and Meier used lecithin, Levaditi glycocholate of
soda, Sachs and Altmann oleate of soda, and
Fleischmann even used vaseline. The last-named
also used cholesterin with favorable results, but
Forges and Meier obtained only negative results
with this substance.* On the whole, however, it
seems that the extracts, especially of syphilitic
organs, give the most uniform results.
At the present time, therefore, Wassermann be-
lieves that the really active principle in the antigen
may be a combination of lipoids with certain protein-
like substances, and that the latter component,
* See especially Noguchi, The Relation of Protein, Lipoids and
Salts to the Wassermann Reaction, Journ Exp Medicine, vol.
xi, 1909.
196 APPENDIX
when it is derived from syphilitic material, has
something of a specific character. In this connec-
tion Wassermann refers to the researches of
Noguchi, Landsteiner, and others which show that
minute quantities of proteid mixed with lipoids
may cause, extensive alterations in the physico-
chemical behavior of the latter. He thinks that
under certain circumstances this proteid component
may play an important role in determining the
reliability of the reaction, a view which is borne out
by the investigations of Neisser and Bruck.
While Forges and Meier were engaged in the
studies just mentioned, Fornet and also Michaelis
showed that when the serum of individuals infected
with syphilis was mixed with certain antigens a
zone of precipitation might at times be observed at
the point of contact of the two fluids. The antigen
employed by Fornet was serum from individuals in
the florid stage of syphilis ; Michaelis used extracts
of organs from a syphilitic foetus. This of course
agrees with what was already known from the work
of Bordet, Gengou, and Gay. In fact, according
to Gay, the deflection or absorption of complement,
on which the Bordet-Gengou test depends, may be
due to the precipitate formed in the combination.
Forges and Meier thereupon tested the alcoholic
extracts, and solutions of lecithin and of glycocho-
late of soda to see whether this zone of precipita-
tion was at all constant, and whether it might not
be possible to substitute such a simple precipitation
test for the complicated Wassermann reaction.
APPENDIX 197
While it was found that the test was roughly spe-
cific, it was soon realized that a precipitate might
at times be produced with the serum of surely
non-syphilitic individuals, and similar unfavorable
results, have since been published by other authors.
At the present time, therefore, the only reliable
serum diagnosis of syphilis is that based on the
absorption of complement.
The results obtained with the Wassermann test are
well reflected in the findings of Fleischmann, as
follows :
The total number of persons tested was 230, of
which 38 were controls. None of the latter gave a
positive reaction. The other cases can be arranged
into four groups thus :
1 i ) Cases surely syphilitic, with clinically manifest
signs of syphilis at the time of the test. Of 89 such
cases tested, 83 gave a positive reaction (93%).
(2) Cases surely syphilitic but without clinical
symptoms at the time of the test. Of 64 such
cases tested, 33 gave a positive reaction (52%), and
31 gave a negative reaction (48%).
(3) Cases with symptoms suggestive of syphilis,
and with an indefinite history of infection. Of 32
such cases, 16 gave a positive reaction (50%), and
the rest a negative reaction.
(4) Surely syphilitic individuals showing cutaneous
lesions which the dermatologists diagnosed as very
probably not syphilitic. Of 7 such cases, i gave a
positive reaction and the rest a negative reaction.
Bruck and Stern tested 378 cases suspected to be
198 APPENDIX
syphilitic, and obtained a positive reaction in 204.
They also tested 157 surely non-syphilitic individuals
as controls, and found all but two negative. These
two gave a doubtful reaction.
In a recent paper Wassermann has collected data
on about 3000 tests, as follows: There were 1010
tests on cases surely non-syphilitic (controls), and
not one of these gave a positive reaction. Of the
1982 surely syphilitic cases tested, those examined
at the time when they had manifest symptoms
reacted in about 90% of the cases. When the cases
tested were without manifest symptoms, so-called
"latent syphilitics," about 50% reacted.
As a matter of interest it may be mentioned that
Blumenthal and Wile tested the urine of syphilitic
individuals, and found that this too would give the
reaction.
Marie and Levaditi examined the cerebrospinal
fluid of 30 cases of general paresis. All but two of
the cases gave a positive reaction. When the serum
was tested in place of the cerebrospinal fluid, the
percentage of positive findings dropped to 59%.
Michaelis examined 20 cases of general paresis
and obtained a positive reaction in 1 8 of them.
Citron examined 43 tabetics and paretics, and ob-
tained a positive reaction in 34 cases (79%). He
also tested the serum of 108 persons surely infected
with syphilis, or suspected to be infected, and ob-
tained a positive reaction in 80 (74%). None of the
sera from 156 surely non-syphilitic individuals gave
a positive reaction.
APPENDIX
199
Favorable reports have also been published con-
cerning the reliability of the test in ophthalmology,
dermatology, and other departments of medicine.
From the researches of Noguchi and others it
appears that with the progress of the cure of syphi-
litic infection not only do the symptoms of the
disease abate, but the blood reaction also grows
weaker and weaker, until ultimately, when cure
has been established, the reaction can no longer
be obtained. This is of clinical interest, for
it enables one, by means of the serum test, to
control the duration and efficacy of anti-syphilitic
treatment. At the same time it must be remem-
bered that the disappearance of the reaction need
not necessarily be permanent. Its reappearance, of
course, signifies that the virus is still present in the
body.
APPENDIX B
NOGUCHI'S MODIFIED WASSERMANN REACTION.*
A MODIFICATION of the Wassermann test recently
devised by Noguchi has been found both simple
and reliable. In principle, the test is the same as
Wassermann's, the only difference being in the
hasmolytic system employed. By making use of an
antihuman amboceptor, Noguchi avoids having con-
stantly to obtain fresh sheep blood cells. In addi-
tion there is some gain in accuracy owing to the
fact that human blood serum, in the quantities
ordinarily employed in the Wassermann test, often
contains normal amboceptors for sheep blood which
interfere with the reaction.
The reagents employed by Noguchi are as follows :
1. Antihuman haemolytic amboceptor. This con-
sists of a serum obtained from rabbits immunized
with washed human erythrocytes.
2. Complement. This consists of fresh guinea-
pig serum.
3. " Antigen." Alcoholic extracts of organs, or
crude preparations of lecithin. The preparation of
the organ extracts has been described on page 152.
* Noguchi, A New and Simple Method for the Serum Diagonosis
of Syphilis, Journ. Exp. Medicine, Vol. XI, 1909.
200
APPENDIX 201
In using lecithin, 0.3 gms. lecithin are dissolved in
50 cc. absolute alcohol, shaken with 50 cc. physio-
logical salt solution and filtered. The filtrate must
be clear.
4. Suspension of human blood corpuscles. One
drop of blood from a normal individual is mixed
with 4 cc. physiological salt solution.
5. Serum to be tested. About 10 or 15 drops of
blood are collected in a small tube and allowed to
clot. The clear serum is used for the tests.
Method. Take six clean test tubes, size 10 cm.
by i cm. Into the first two of these place one drop
from a capillary pipette of the patient's serum to be
tested. Into each of the second two tubes (which
serve as controls) put one drop of serum of a syphi-
litic case known to give a positive reaction. Into
each of the third pair of tubes put one drop of
serum of a normal person. Now to each of the six
tubes add i cc. of the suspension of human blood
corpuscles and 0.04 cc. fresh guinea-pig serum as
complement. Lastly, into one tube of each pair
put one drop of the " antigen " solution from a
capillary pipette. The second tube of each pair
receives no antigen.
After being well mixed by shaking, the six tubes
are incubated at 3 7 C. for one hour, after which each
tube receives two units * of antihuman amboceptor.
The tubes are returned to the incubator for two
* The term "amboceptor unit" is used to designate the amount of
ambcceptor which, on the addition of the optimum amount of comple-
ment, just suffices to produce complete haemolysis.
202
APPENDIX
hc'irs longer, and then kept at room temperature,
the reaction being read from time to time. The
interpretation of the result is identical with that of
the Wassermann reaction.
The following scheme accompanying Noguchi's
article will serve to elucidate the technique.
First Central Set.
Second Control Set.
Test for Diagnosis.
Test with a known
svphilitic -erum.
Test with a normal
serum.
(Positive reaction.)
(Negative reaction.)
a. Patient's
a 1 '. Posi-
a". Normal
serum
tive
serum
luetic
Control tube
serum
without
Q b. Human
Q b. Human
Q b. Human
"antigen"
blood
blood
blood
in each test
suspen-
suspen-
suspen-
sion
sion
sion
c. Comple-
c. Comple-
c. Comple-
ment
ment
ment
Determina-
a. 1
a'. I
a "'l
tive tube,
^ &. [ Ditto
^ b. \ Ditto
r ^ b. i Ditto
contains
U c. J
U c. J
U c. J
"antigen"
d. Antigen
d. Antigen
d. Antigen
Incubation at 37 C. for one hour.
Antihuman haemolytic amboceptor to all tubes.
Incubation at 37 C. for two hours longer, then at
room temp.
Noguchi has very cleverly adapted his method
for clinical purposes by drying the various reagents
on paper and standardizing them. He prepares
three kinds of slips, as follows:
APPENDIX 203
Antihuman amboceptor slips, each containing two
amboceptor units.
Complement slips, each containing sufficient com-
plement for one tube.
Antigen slips, each containing sufficient for one
tube.
These papers can be kept indefinitely at room
temperature in a dry place. Prior to using the slips
for a blood test it is necessary to make preliminary
tests to prove their activity and strength.
In employing the filter paper slips they are
dropped by means of a forceps into the test tubes
containing the human blood suspension and patient's
serum in the order and at the intervals already
stated for the respective reagents. If an incubator
is not at hand, the tubes can be kept warm by
carrying them in the vest pocket.
APPENDIX C
BLOOD EXAMINATION PREPARATORY TO TRANSFUSION
Reasons for Making the Examination. We have
already called attention to the occurrence of iso-
agglutinins, isohaemolysins, and isoprecipitins and
their bearing on homologous transfusion. The
mere occurrence of these substances in blood serum,
to be sure, does not at all prove that isoagglutina-
tion or isohaemolysis or isoprecipitation occur when
such transfusions are done. In fact we do not even
know whether these substances exist at all in the
blood plasma. Nevertheless, until we learn other-
wise, it will be well to bear in mind the possible
danger from this source, and to undertake no
transfusions in which examination shows the exist-
ence of homologous antibodies.
Technique of the Tests. It is evident that our
tests must be reciprocal, i.e., we must test the
serum of both donor and recipient against the
blood corpuscles of the other. To do this we col-
lect part of the blood from each individual, part
in citrated salt solution and part in a plain test-
204
APPENDIX 205
tube. The latter is allowed to clot and furnishes
the serum; the former is prevented from clotting
by the sodium citrate and serves to supply the
blood corpuscles. Instead of using sodium citrate,
Crile, 1 defibrinates the blood by shaking it in a
test-tube with a glass bead, and suspending the
blood corpuscles in physiological salt solution.
Either method may be used, though with the
sodium citrate it is necessary to centrifuge, wash
the blood corpuscles, and then resuspend them in
salt solution. The suspensions are usually 5%
strength.
In carrying out the test equal parts of serum and
blood suspension are mixed in a small test-tube, or,
as Epstein 2 has suggested, in small pipettes such
as Wright uses for his opsonic tests. After mixing,
the tubes are placed in the thermostat for two hours.
At the end of this time most of the cells have
usually settled to the bottom and pronounced
haemolysis can be seen. For finer grades of haemo-
lysis it is usually necessary to allow the tubes to
stand over night in the refrigerator.
Agglutination, when it occurs, is rather prompt,
and can be readily observed in the gross by the
clumping and sedimentation of the blood corpuscles.
1 Crile, Hemorrhage and Transfusion, 1909, Appleton and
Co., New York.
2 Epstein and Ottenburg, Archives of Internal Medecine,
Vol. iii, page 286, 1909.
206 APPENDIX
It is important in the haemolytic tests that all
the glassware be absolutely clean and dry, though
it need not be sterile.
In testing for the presence of isoprecipitins, equal
parts of the two sera are mixed in a small test-tube,
the mixtures kept in the incubator for two hours
and then examined.
APPENDIX D
OTHER REACTIONS
The Conglutination Reaction
IN 1906 Bordet and Gay described the presence
in bovine serum of a substance having the property
of producing a characteristic clumping of red blood-
cells and of accelerating their lysis provided the
cells had first been treated with both sensitizer
(amboceptor) and alexin (complement) ; its action
was possible under no other circumstances. Subse-
quently it was shown that the same phenomenon
could be produced with bacteria treated with a
specific sensitizer and alexin. This substance is
spoken of as "conglutinin." From the work of
Bordet, Streng, Gay, and others it would appear
that the conglutination reaction can be employed
to discover the presence of specific sensitizers
(amboceptors) , and thus be applied to the diagnosis
of bacterial infections. The experimental work
thus far done on the subject is still too meagre to
warrant any definite statements as to the diag-
nostic value of the reaction. In some infections
studied by Gay the conglutination reaction appeared
earlier than the agglutination reaction.
207
20$ APPENDIX
The Meiostagmin Reaction
Weichardt called attention to the fact that the
union of antigen with its antibody in certain dilu-
tions caused an increase in the rate of diffusion,
i.e., gave rise to changes in the osmotic pressure
and of the surface tension. Ascoli showed that the
decrease in the surface tension arising when bac-
terial substances combined with their specific antigen
could be measured by counting the number of drops
per given time interval delivered from a Traube
stalagmometer. Thus where a mixture of normal
serum with extract of typhoid bacilli showed 56
drops, a similar mixture of serum from a typhoid
fever patient with the extract showed 58 drops.
Attempts have been made to utilize the meiostag-
min reaction in the diagnosis of various infectious
diseases, and while the results on the whole have
shown the correctness of the underlying principles,
they have also demonstrated that other reactions
are far more convenient and decisive.
The Much-Holzmann Cobra Venom Reaction
It has long been known that cobra venom haemo-
lyzes red blood corpuscles, and that certain cor-
puscles, such as those of man, dog, pig, horse,
rabbit, and guinea-pig haemolyze directly on mixing
them with cobra v-enom, while others require the
APPENDIX 209
intervention of an activating substance. To the
latter class belong the blood corpuscles of ox,
sheep, and goat. As already pointed out in dis-
cussing snake venoms, the activating substance is
present in blood serum; it is also present in com-
mercial lecithin. Haemolysis of either group of
blood corpuscles can be inhibited by means of
cholesterin, though just how this substance acts is
not clear. Much and Holzmann showed that the
blood serum of patients suffering from various
mental disorders, especially dementia praecox, and
manic-depressive insanity frequently inhibits hae-
molysis of human red blood corpuscles, and they
suggested that the reaction could be used for
diagnostic purposes. While it appears to be true
that the psychoses yield the largest proportion of
positive reactions, the value of the reaction for
diagnostic purposes is practically nil. At the same
time it is interesting to note that diseases of the
nervous system accompanied by demonstrable
lesions of the nerve tissue give the same reaction as
psychoses in which such lesions have not yet been
demonstrated. Much 1 therefore concludes that in
both cases the same substance circulates in the
blood, and that, moreover, in both this substance
is derived from a degeneration of the nerve tissue.
1 Much, Die Immunitatswissenschaft. C. Kabitzsch, Wiirz-
burg, 1911. i
210 APPENDIX
Weil's Cobra Venom Test in Syphilis
In studying the varying resistance of red blood
corpuscles to haemolytic agents, Weil 1 noted that
the corpuscles of syphilitics were regularly more
resistant to the action of cobra venom than those
of normal individuals. Just what is the cause of
this increased resistance is not entirely clear. It
is known that syphilis attacks the lipoids of the
body, and that the amount of lecithin which can
be extracted from the tissues is less in syphilitic
conditions than in normal individuals. The in-
creased resistance has therefore been thought to
be due to a decrease in the lecithin content of the
red blood corpuscles.
In testing the corpuscles the blood is collected
in citrated salt solution, and then washed by
repeated centrifugalization in order to thoroughly
remove the serum. Even slight traces of
serum interfere decidedly with the reaction. The
washed cells are made up into a four per cent
suspension in 0.9 per cent common salt solution.
Centrifugalization is done in very accurately grad-
uated centrifuge tubes, as the accurate dilution of
the cells is a matter of very great importance.
The cobra venom, which is kept in the dried con-
1 Weil, Richard, Journal of Infectious Diseases, Vol. vi, Nov.,
1909. Proceedings Society Exp. Biology and Medicine, Vol. vi
and Vol. vii.
APPENDIX 211
dition, is made up very accurately in small quan-
tities into a 0.05 per cent solution in 0.9 per cent
solution of common salt. From this stock solution,
higher dilutions are prepared as required, and are
kept at a constant low temperature. In carrying
out the test four solutions are prepared, namely,
i in 10,000, 20,000, 30,000, and 40,000. One cc.
of the corpuscle suspension is added to one cc. of
the venom solution. The mixtures are incubated
for one hour at 40 C., after which a preliminary
inspection will almost invariably reveal the final
results of the tests. At this stage, cells showing
no haemolysis with i to 10,000 venom are strongly
positive, cells showing no haemolysis with i to
20,000 are positive, cells showing very slight hsemol-
ysis at i to 30,000 are weakly positive; cells show-
ing moderate destruction at i to 30,000 are negative,
and cells showing the least appreciable trace of
haemolysis at i to 40,000 are strongly negative.
After the preliminary reading the mixtures are
again shaken and then kept in the ice-box over
night, the final readings being made on the follow-
ing morning. The figures here given are merely
those of a particular specimen of cobra venom;
it is possible that other samples might differ in
strength. At all events a preliminary titration
against corpuscles derived from cases of known
syphilis and controls suffices to determine the
values for any given sample. Inasmuch as the
212 APPENDIX
strength of the dried venom does not vary, this
preliminary titration gives a constant standard,
which is practically permanent, since one gram of
venom suffices for about 5000 complete tests. The
standardization is different for infants as compared
with adults, for the red corpuscles of infants and
young children haemolyze much more readily than
those of adults. The cells, however, acquire the
same degree of resistance as do those of adults in
disease.
Weil states that the cobra venom reaction appears
to have certain advantages over the Wassermann
reaction. It is much simpler and the labor much
less. Less blood is required, so that a few drops
from the lobe of the ear suffice for a reaction; this
is of some importance in infants. Cases of scarlet
fever and of leprosy do not offer a source of con-
fusion. The reaction is more marked in old,
apparently dormant cases. The reaction persists
much longer after mercurialization, thus offering a
further diagnostic and therapeutic test. The reac-
tion is possible in cases of jaundice, whereas the
Wassermann is not.
Antitrypsin Determinations.
We have already pointed out that the animal
body responds to the injection of ferments by
the production of antiferments. Considerable in-
APPENDIX 21-
vy
terest has been aroused by the discovery that in
certain diseases, especially cancer, the antitrypsin
content of the patient's serum is markedly in-
creased. In cancer this increase is noted in about
90 per cent of the cases. The antiferment action
is not entirely specific, but extends to other pro-
teolytic ferments, and particularly to the ferment
of leucocytes. At the present time, therefore, a
marked increase in the antitryptic power of a
patient's serum is taken to indicate an increased
parenteral * destruction of proteid in the body. 2
The original method of demonstrating the presence
of this antitrypsin was by placing drops of pro-
teolytic ferment (trypsin) on the surface of a plate
of Loeffler's serum, and causing the development of
small concavities owing to the digestion of the
medium. The addition of inhibiting serum to the
drops was able to prevent the formation of - the
concavities. A more convenient and accurate
method is the one developed by v. Bergmann and
Meyer. This depends on the digestion of a per-
fectly clear solution of casein. If all the casein has
been digested, the addition of acid is obviously
unable to precipitate any casein from solution.
On the other hand, if the acid causes clouding or
precipitation, it follows that all the casein was not
1 Other than intestinal.
2 See the excellent digest of the work on this subject in Jahres-
bericht der Immunitatsforschung, Bd. V, 1909, Abteilung I, page
58.
APPENDIX
digested. It is evident that the quantity of trypsin
required to digest a given amount of casein can be
exactly determined, and that by employing grad-
uated amounts of the inhibiting serum accurate
determinations of the antitryptic content can be
made.
INDEX OF AUTHORS
Abel, 17, 100
Anderson, 142, 160
Altmann, 195
Aronson, 5
Arrhenius, 28
Arthus, 141
Ascoli, 208
Atkinson, 18
Auer, 150
Bail, 93, 158
Banzhaf, 19, 151, 153
Beebe, 125
von Behring, i, 5, 170
Belfanti, 18
von Bergmann, 013
Besredka, 82, 84
Blumenthal, 198
von Bokay, 144
Bolduan, 135
Bordet, 28, 40, 52, 56, 67, 87
96, 108, 185, 207
Braun, 194
Bruck, 72, 196
Buchner, i, 6, 50, 75, 78, 93
Buxton, 104
Calmette, 18, 137
Carbone, 18
Castellani, 46
Citron, 158, 198
Crile, 205
Dallera, 143
Delezenne, 121
Deutsch, 1 66
Dieudonne, 18, 113
Doerr, 149, 158
Donath, 94
Douglas, 128
von Dungern, 16, 54, 79, 85,
Qi, I2 5
Durham, 37
Ehrlich, 6, u, 13, 16, 23, 39,
47, 58, 67, 84, 87, 92, 97,
112, 159, 166, 179
Eisenberg, 39, in
Epstein, 205
Famulener, 153
Field, 43
Fischer, 9
Fleischmann, 195, 197
Flexner, 137
Floyd, 178
Fodor, 50
Ford, 17
Fornet, 196
Friedberger, 42, 156
216
INDEX OF A UTHORS
Gay, 73, 88, 96, 104, 146, 196,
207
Gengon, 40, 71, 99, 163, 185,
196
Gibson, 18
Grassberger, 29
Gruber, 36, 93
Griinbaum, 37
Gscheidlen, 50
Haeckel, 127
Hahn, 93
Hamburger, 148
Hektoen, 130
Hericourt, 141
Heubner, 144
Hiss, 177
Holzmann, 208
Jackson, 126
Jansky, 35
Jenner, 169
Jordan, 150
Kitasato, i
Kleine, 166
Knorr, 2, 5
Kraus, 108
Kyes, 137
Landois, 74, 143
Landsteiner, 35, 54, 94, 123,
194, 196
Leblanc, in
Leclainche, 109
Ledingham, 20
Leishman, 131
Levaditi, 194, 198
Lewis, 150
Loffler, 100
Lucas, 178
Manwaring, 139
Marie, 194, 198
Marrassini, 125
Martin, 5
Marx, 86
Meier, 194
Mertens, 109
Mesnil, 166
Metchnikoff, 38, 52, 58, 63,
82, 84, 91, 93, 99, 121, i27 ;
1 60
Meyer, 213
Michaelis, 196
Moreschi, 97
Morgenroth, 18, 29, 71, 109
Moro, 148
Moss, 35, 98
Moxter, 86, 93, 123
Much, 208
Muir, 67
Muller, 63, 91, 194
Myers, 109
Neisser, 72, 100, 196
Neudorfer, 143
Neufeld, 129
Nissen, 74
Noguchi, 137, 195, 199, 200
Nolf, 91
Nuttall, 50, 74, no, 115
Obermayer, in
Ottenburg, 205
Otto, 141
Opie, 162
Park, 2, 74
Pasteur, 169, 171
Pearce, 126
Pfaundler, 38
INDEX OF A UTHORS
217
Pfeiffer, 37, 51, 86, 93
Pick, 19, 109, in
von Pirquet, 142, 147, 164
Plant, 192
Porges, 194
Potzl, 194
Richet, 141
Rimpau, 129
Rosenau, 142
Roux, 5
Russ, 149
Sachs, 73, 137, 195
Salge, 161
Salmon, 171
Schattenfroh, 29, 93
Schick, 142
Schultz, 150
Schiitze, 109, 113, 120
Smith, 141, 161, 171
Southard, 146
Spiro, 109
Steinhardt, 151
Stern, 109, 113, 197
Streng, 207
Tchistowitch, 107
Touissant, 171
Traube, 50
Uhlenhuth, 109, 113
Vallee, 109
Volk. 39
von Wassermann, 13, 1 8, 72, 7-
86, 92, 93, 99, 109, 113, 15;
185, 192
Wechsberg, 100
Wechselmann, 183
Weichardt, 208
Weigert, 10
Weil, 194
Weil, R., 210
Welch, 165
Wernicke, 5
Widal, 37
Wile, 198
Wright, 128, 131, 173
Zinsser, 178
Ziilzer, 109
SUBJECT INDEX
PA.GE
Abrin 21
Absorption, in study of agglutinins 46
of complement, see Bordet-Gengou 71
and Neisser-Wechsberg 100
Active immunization, methods 171
Adsorption, as applied to toxin-antitoxin 30
elective character 31
Agglutination, in typhoid fever 37
influence of dilution on 34
phenomenon of 32
purpose of 36
specificity of 44
Agglutinin 32
against trypanosomes 34
effect of heat on 39
nature of 39
Agglutinoid 39
Aleuronat, in production of Icucocytic exudates 177
Alexin, see also complement 56
Allergy 147
Amboceptor, definition 63
unit 20 1
Antibacterial sera, practical value of 104
Antibodies, cells concerned in production cf n
Anticomplement 86, 88
views concerning '. . . . 96
Anticomplementary serum, specificity of 90
Anticy totoxin 122
Antigen, definition of 16
for syphilis test 190
Antihaemolysins, their nature 86
219
220 SUBJECT INDEX
PAGE
Antiprecipitin 119
Antiserum, for snake venoms 137
Antitoxic globulins 1 8
Antitoxin, action on toxins 18
diphtheria, production of 2
diphtheria, testing of 5
historical i
nature of r. 17
quantitative relation to toxin 31
relation to toxin 22
unit 22
Antitrypsin, determination of 212
Antivenins 139
Anaphylactic shock 142
pathology of 150
prevention by chloral hydrate 153
Anaphylatoxin 157
Aggressins 158
Anaphylaxis, historical 141
relation to precipitins 148
relation to serum therapy , 151
theories of 144
Anthrax, symptomatic, toxin of 29
Antianaphylaxis 1.^3
Asthma, relation to anaphylaxis 151
Atrepsy 166
Autolysins 97
Bacterial precipitins 108
Bactericidal serum, clinically 105
Bacteriolysins, historical 50
Bacteriolysis, of cholera spirilla 51
Bacteriotropic substances 127
Blood, biological reaction for 73, 1 13
examination for transfusion 204
its germicidal power 50, 74
cells, agglutination of 34
relationship, by means of precipitin test no
test, Neisser-Sachs 73
SUBJECT INDEX 2 2i
Bordet-Gengou phenomenon 71
Brain tissue, effect on tetanus toxin 13
Cancer, immunization against 125
Casein, precipitin against 108
Chemoreceptors 181
Chloral hydrate, to prevent anaphylactic shock 153
Cholera spirilla, Pfeiffer's test 51
Clumping, see Agglutination . 32
Cobra-lecithid 139
Cobra venom 137
as test in syphilis 210
in haemolysis 208
Colloids, coagulability of 42
in the study of agglutination 42
Common receptors 83
Complement 64
absorption by precipitates 96
absorption of, see Bordet-Gengou 71
artificial increase 92
definition of . 63
deflection of i oo
effect of heat on 94
effect of phosphorus poisoning on 91
multiplicity of 70
source of 93
structure of 94
Complementoid 94
Complementophile group, Bordet's views concerning 67
Conglutination 207
Copula, definition of 63
Crotin 21
Cytotoxins, definition 121
produced by nucleo-proteid injections 125
specificity of 124
Desmon, definition of 63
Deviation of complement 100
Dissociation, in toxin-antitoxin action 29
222 SUBJECT INDEX
Dosage, of lytic sera ..." 100
Dyeing, analogous to antitoxin reaction. 30
Electric charge, of agglutinins 43
Electrolytes, in relation to agglutination 41
Endotoxins, relation to infection 158
Endotoxin theory, applied to anaphylaxis 145
Epithelium, cytotoxin for 124
serum against 85
Fractional neutralization, in study of antitoxins 30
Ferments, in leucocytes 162
Globulin, antitoxic 18
Group agglutination 44
Gruber- Widal reaction 37
Haemagglutinins 34
Haemolysin, discovery of . . 52
Haemorrhagin, of snake venom 138
Haptins, definition of 16
Haptophore group, nature of 7
Heat, effect on serum 40, 96
Hypersensitization, to serum 142
Immediate reaction 145
Immune bodies, site of production 86
Immune body , . . . 64
nature of 82
Immunity, acquired 160
hereditary transmission 160
mechanism of 162
natural, variations in 159
relation of anaphylaxis to 163
varieties of 158
Immunization, active contrasted with passive 161
active, methods of 171
against diphtheria toxin. . .- 3
with partially neutralized toxins 12
SUBJECT INDEX 223
PAGE
Inactivated serum . . . . : 54
Index, opsonic 132
Infection, nature of 154
relation of anaphylaxis to 156
relation of virus to 154
Inflammation, Opie's researches on 162
Interbody of normal serum 77
Isoagglutinin, occurrence of in normal human sera 35
relation to homologous transfusion 36
Isolysins, occurrence of in man 98
nature of 97
Isoprecipitin 120
Lactoserum. . . i 108
Lecithid, of cobra venom 139
Leucocytes, as source of complement 93
ferments in 162
phagocytic action of 127
Leucocyte extract, in treatment of infections 177
Leucotoxin 121
Liver, role in production of complement 91
L and L| 23
Lysins, relation to agglutinins 36
Mastic, agglutination of 41
Meiostagmin reaction 208
Milk, human and bovine tested biologically 112
Much-Holzmann reaction 208
Mussels, toxin of 141
Negative phase 133
Neisser-Sachs blood test 73
Neisser-Wechsberg phenomenon 100
Nephrotoxin 126
Neurotoxin 122
of snake venom 138
Noguchi's test for syphilis 200
Normal serum, agglutinating poweY of 34
contrasted with specific immune serum. 79
224 SUBJECT INDEX
PAGE
Normal serum, its haemolytic and bacteriolytic action. . ; 73
Nucleo-proteid, for immunization 125
Opsonic index, clinical value cf 135
nature of 131
Opsonin, affinity of, for bacteria 129
histoi ical 127
structure of 130
Organotropism 179
Osmotic pressure, of serum, in infections 208
Parasitotropism 179
Parenteral digestion, of proteid 164
Partial immune bodies 82
Partial saturation method 24
Pfaundler's thread reaction 38
Pfeiffer's phenomenon 51
Phagocytosis 127
Phosphorus poisoning, effect on complement production. . . 91
Physical chemistry, applied to toxin-antitoxin reaction. ... 28
Precipitates, relation to anti-complement action 96
Precipitins, definition of , 107
in relation to animal species no
method of immunization 114
nature of in
specificity of 109
Precipitin test, for blood 116
various applications of 1 18
Preparator, definition of 63
Pro zone, in agglutination 39
relation of colloids to , 43
Proteid, cleavage of 157
parenteral digestion of 164
Protoxoid 26
Psychoses, serum diagnosis in 209
Ptomaines 20
Rash, from serum injections 143
Receptors, common 83
SUBJECT INDEX 22 ' 5
Receptors, nature of g
various orders of 47
Reversible reactions in toxin-antitoxin combination 29
Ricin 21
Calts, their relation to agglutination 41
Salvarsan, chemistry of 182
principles governing treatment by 1 79
Serum, active and inactive 54
against epithelium 125
antibody content of compared to plasma 163
effect of heat on 40
mode of collection 4, 115
Serum diagnosis, in typhoid fever 37
Serum-fast trypanosomes 166
Serum rashes 143
Sessile receptors 14
Side-chain theory, applied to antitoxins. . . ; . 6
applied to bacteriolysins and haemolysins 68
Snake venom 137
Specific therapy, principles of 179
Specificity, cause of 65
nature of 65, 124
of agglutination 45
Spectrum, Ehrlich's so-called. 25
Spermatoxin 123
Split products, in anaphylaxis 146
Stimulins 128
Substance fixatrice 63
sensibilatrice 55
Syntoxoid 27
Syphilis, cobra venom test in 210
serum diagonsis of 185
specific treatment of .* 179
tests for 196
Tetanus toxin, affinity for brain tissue 13
Thread reaction 38
Toxin -antitoxin reaction 22-31
226 SUBJECT INDEX
PAGE
Toxin, absorption of antitoxin by 31
diphtheria, preparation of 2
relation to antitoxin 22
Toxins, characteristics of 20
Toxoids, nature of 6
various kinds of 24
Toxons 24
Toxophore group, its influence in immunization 1 6
nature of 7
Transfusion, blood tests in 204
relation of isoagglutinins to 36
Trypanosomes, agglutinins against 34
serum-fast 1 66
Typhoid fever, Gruber-Widal reaction in 37
Uhlenhuth blood test 1 16
Vaccines, bacterial 169
bacterial, doses 175
Vaccine therapy 173
Venoms, of snakes 137
Viscosity of blood, relation to serum reactions 40
Wassermann test, for syphilis 185
Weigert's overproduction theory 9
Welch's hypothesis 165
Widal reaction 37
Zootoxins 21
Zymotoxic group 94
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