LIBRARY 
 
 OF THE 
 
 UNIVERSITY OF CALIFORNIA. 
 
 RECEIVED BY EXCHANGE 
 
 Class 
 
Ube Tftniverstts ot Cbtcago 
 
 FOUNDED BY JOHN D. ROCKEFELLER 
 
 A STUDY OF THE CONCENTRATION 
 
 OF THE ANTIBODIES IN THE 
 
 BODY FLUIDS OF NORMAL 
 
 AND IMMUNE 
 
 ANIMALS 
 
 A DISSERTATION 
 
 SUBMITTED TO THE FACULTY OF THE OGDEN GRADUATE SCHOOL 
 
 OF SCIENCE IN CANDIDACY FOR THE DEGREE OF 
 
 DOCTOR OF PHILOSOPHY 
 
 (DEPARTMENT OF PHYSIOLOGY) 
 
 BY 
 
 FRANK C. BECHT and JAMES R. GREER 
 
 CHICAGO 
 1910 
 
mniperstts of Gbicago 
 
 FOUNDED BY JOHN D. ROCKEFELLER 
 
 A STUDY OF THE CONCENTRATION 
 
 OF THE ANTIBODIES IN THE 
 
 BODY FLUIDS OF NORMAL 
 
 AND IMMUNE 
 
 ANIMALS 
 
 A DISSERTATION 
 
 SUBMITTED TO THE FACULTY OF THE OGDEN GRADUATE SCHOOL 
 
 OF SCIENCE IN CANDIDACY FOR THE DEGREE OF 
 
 DOCTOR OF PHILOSOPHY 
 
 (DEPARTMENT OF PHYSIOLOGY) 
 
 BY 
 FRANK C. BECHT and JAMES R. GREER 
 
 CHICAGO 
 1910 
 
A STUDY OF THE CONCENTRATION OF THE ANTI- 
 BODIES IN THE BODY FLUIDS OF NORMAL 
 AND IMMUNE ANIMALS.* 
 
 FRANK C. BECHT AND JAMES R. GREEK. 
 (From the Hull Physiological Laboratory, The University of Chicago.) 
 
 THE presence of antibodies of various kinds in serum has long 
 been known, and the concentration in that fluid has been carefully 
 studied. The presence of antibodies in the various other body 
 fluids has not been so carefully investigated, nor has sufficient allow- 
 ance been made for individual variations in animals of the same 
 species. While the authors were associated with Dr. Carlson in his 
 work on lymph formation, he suggested that a careful comparison 
 between the concentration of the antibodies in the various body fluids 
 of the same animal might be of considerable importance in determin- 
 ing the differences between the lymph and serum, and in that way 
 throw light on some of the problems of lymph formation, and possibly 
 also on the point of origin of antibodies. When the work was begun, 
 we intended to collect lymph from the different organs, but the 
 practical difficulties encountered in introducing cannulae into the 
 delicate lymphatics of such organs as the spleen was so great, that 
 the project was temporarily abandoned, and the work has been con- 
 fined to a comparison between serum, lymph from the cervical lym- 
 phatics, lymph from the thoracic duct, pericardial fluid, cerebrospinal 
 fluid, and aqueous humor. Thus far work has been done on the 
 hemolysins, hemagglutinins, agglutinins for the typhoid bacillus, 
 the protein precipitins, and the opsonins, bacterial and erythrocytic. 
 No work has yet been undertaken on the bacteriolysins. We have 
 not enough data to enable us to draw any broad conclusions, and we 
 will content ourselves with presenting what we believe to be the facts 
 under the various conditions studied. 
 
 Literature. The first studies that we were able to find on the relative concentra- 
 tion of antibodies in the various body fluids were those of Pegano,36 w ho found the 
 concentration of hemolysins of the thoracic lymph in dogs lower than that of the serum. 
 Falloise ll and later Batelli* confirmed the work of Pegano. Hughes and Carlson- 8 
 working on normal dogs, horses, and cats found the concentration of hemolysins for 
 
 * Received for publication November 9, 1909. 
 
 127 
 
 218508 
 
BECHT AND JAMES R. GREER 
 
 rabbit corpuscles in the body fluids to form a descending series: serum, thoracic lymph, 
 neck lymph, pericardial fluid, aqueous humor. No lysins were found in the cere- 
 brospinal fluid. Straus and Wolf4 studied the hemolytic power against rabbit cor- 
 puscles of the cerebrospinal fluid, edema fluid, pleural and pericardial transudates, 
 and blister fluid, and attempted to correlate the hemolytic strength with the protein 
 content. Marshall and Morgenroth 2 s found anti-complement and anti-amboceptor 
 in a pathological exudate an ascites fluid. Hedinger j s studied the hemolytic power 
 of non-inflammatory exudates like those arising from cirrhosis of the liver and heart 
 failure, and found that they were not so hemolytic as the serum. The inflammatory 
 exudates arising from cases of tuberculosis and carcinoma were not so strongly hemolytic 
 as non-inflammatory exudates. He failed to find hemolysins in the fluid from an 
 ovarian cyst, or in the cerebrospinal fluid in two cases of tuberculosis. Marshall 2 ^ 
 found that pleural and ascites fluids were more strongly hemolytic than the serum 
 from an infant. But no conclusions can be drawn from this comparison in regard to the 
 comparative hemolytic power of serum and other body fluids in the same individual. 
 He found a multiplicity of amboceptors and complements in the fluids that he studied. 
 Grollo 4 could find no amboceptors for rabbit corpuscles in transudates, but found 
 them in exudates, altho in the latter complement is often lacking. He suggests this 
 method as a means of diagnosis between transudates and exudates. Liidke 22 con- 
 firmed the findings of Marshall in regard to the hemolytic strength of transudates and 
 exudates. Granstronvs found wide variations in the hemolytic content of transu- 
 dates and exudates, and could establish no characteristics essential for either. The 
 hemolysins did not run parallel with those of the blood. Isolysins are found less 
 frequently in transudates and exudates than in the blood. Hemolysins were not 
 found in the cerebrospinal fluid. Isolysins and heterolysins were found independent 
 of the albumen content, number of the leukocytes, and the osmotic pressure of the 
 fluids tested. Tedeschi4; found precipitins in both transudates and exudates, less 
 frequently in the latter than in the former. Mioni3 found amboceptor but no com- 
 plement for guinea-pig corpuscles in the pericardial fluid of the ox. Bard 2 claims to 
 have found hemolysins in the cerebrospinal fluid of patients, and found that they were 
 increased during various diseases. Massaglia 26 could not confirm the work of Bard. 
 His results in both healthy and diseased individuals were negative. The presence of 
 antibodies for syphilitic material in the cerebrospinal fluid has been shown by various 
 investigators, among them Morgenroth and Stertz,3i and Wassermann and Plaut.4 2 
 Gatti 2 could demonstrate no hemolysins in the aqueous humor of the ox. Levaditi 2 
 showed that there is normally no opsonin in the aqueous humor; but if the fluid of the 
 anterior chamber of the eye of an immune animal is withdrawn, the newly formed 
 aqueous humor will contain opsonin. Bohme 6 investigated the opsonin content of 
 pleural, peritoneal, and abscess fluids He found that usually in such cases the opsonin 
 content of the fluid was reduced for the infecting organism, but remained unchanged 
 for other bacteria. He could find no opsonin in normal cerebrospinal fluid, but found 
 them there after an inflammation had been set up in the dura. He could not develop 
 opsonins in the cerebrospinal fluid by repeated puncture as Levaditi had done by 
 drawing off the aqueous humor. He believes that there is a relation between the 
 protein content and the opsonin action of a fluid. 
 
 Methods. The plan of study adopted was to determine first the concentration 
 of the antibodies in the body fluids of normal cats and dogs; then the concentration in 
 actively immunized animals; and, finally, to study the passage of the antibodies from 
 
CONCENTRATION OF ANTIBODIES 129 
 
 the blood into the other body fluids in animals passively immunized by the with- 
 drawal of large quantities of blood, and the injection of a corresponding amount of 
 warm, defibrinated blood from an actively immunized animal. 
 
 The body fluids were secured under as nearly aseptic conditions as possible. The 
 animal was anesthetized with ether, and kept in a state of complete anesthesia, by the 
 administration of the vapor through a trachea cannula or through a tube introduced 
 through the larynx. The neck lymphatics were then isolated, and small, sterile, glass 
 cannulae provided with sterile, rubber tubing were inserted. If there was no free flow 
 of lymph, the neck was gently massaged. The lymph was never allowed to come in 
 contact with the air of the room, for as soon as it filled the cannula and a part of the 
 rubber tubing, it was drawn off by means of a fine, sterile Pasteur pipette, and placed 
 in a dry, sterile test tube plugged as for bacteriological work. The lymph was allowed 
 to coagulate spontaneously in the test tube, and was then defibrinated, and the delicate 
 coagulum removed. 
 
 The thoracic duct was tied off at the same time as the isolation of the neck lym- 
 phatics so that the lymph formed during the experiment was retained in the duct. Usually 
 the lymph from this duct was not collected until the animal had been bled to death, 
 altho sometimes it was collected simultaneously with the neck lymph. The routine 
 method was to draw the lymph by means of a Pasteur pipette provided with a bulb, so 
 that the fluid never came in contact with the air at all. This fluid was also defibrinated. 
 
 The pericardial fluid was never collected until after the death of the animal by very 
 complete bleeding from the arteries and veins of the neck. The thorax was opened 
 by removing the sternum, a small hole was cut into the pericardium, and the fluid was 
 removed by means of a sterile Pasteur pipette. 
 
 We found it a good plan in our experiments to suspend the animal by the jaws for 
 a few minutes before attempting to withdraw the cerebrospinal fluid. This drained 
 away the blood from the head and made admixture of this fluid with blood less likely. 
 Our method was to open the dura between the first and the second cervical vertebrae, 
 and then remove the fluid by means of the Pasteur pipette. The end of the pipette 
 must be well rounded in the flame, otherwise rupture of the delicate blood vessels of 
 the meninges is likely to follow. 
 
 The method of collecting the aqueous humor was simple and easy. It consisted 
 in thrusting a sharp pointed Pasteur pipette into the anterior chamber of the eye 
 through the cornea, and allowing the aqueous humor to flow into it, largely by the 
 tension within the eyeball. A little suction sufficed to remove the last drop of the fluid. 
 
 The serum was secured from blood drawn when the animal was bled to death, 
 and in most cases was freed from the corpuscles at once. 
 
 Careful notes were made in regard to the condition of the fluids, and in most 
 cases where there was any admixture of blood, the fluid was discarded. 
 
 HEMOLYSIS AND HEMAGGLUTININS. 
 
 Methods. The study of the hemolysins and hemagglutinins in normal* animals 
 was made on dogs only. The animals utilized were brought in from various parts of 
 the city. Most of the tests were made with rabbit corpuscles in 5 per cent suspension 
 in 0.9 per cent NaCl solution. In some cases rat and horse corpuscles were used. 
 
 *The term "normal" means animals which had not previously been immunized by us. We had 
 no way of knowing what their history had been previous to coming to the laboratory. 
 
130 FRANK C. BECHT AND JAMES R. GREEK 
 
 Our methods were the following: Quantities of the various body fluids of the animal 
 to be tested varying between o. i c.c. and o.oooi c.c. were placed in a series of eight 
 dry, sterile test tubes plugged as for bacteriological work. In order to make the 
 necessary measurements with a pipette graded to T fa of a c.c., dilutions of the body 
 fluids T V and ^ were made. To the fluid in each tube enough sterile 0.9 per cent 
 NaCl solution was added to make the total volume up to 0.4 c.c. To this was then 
 added o. 2 c.c. of a 5 per cent suspension of the corpuscles to be tested. In this way we 
 got dilutions of the fluid varying between i : 6 and i : 6, 144. All of the fluids from the 
 same animal were prepared, the tubes were placed in a block containing a suitable 
 number of holes, and adjusted to the sliding platform of a shaker in an incubator 
 warmed to 37 C. The shaker was run by water power, and the motion was rapid 
 enough to secure constant, thorough agitation, but not violent enough to injure the 
 corpuscles. The routine technic was to keep the tubes in the shaker for an hour and 
 in the ice-box from 12 to 20 hours to permit sedimentation of the corpuscles before the 
 final reading. 
 
 In determining the amount of hemolysis in the final reading, the following method 
 was employed: A measured sample of the corpuscle suspension in the test was sedi- 
 mented in the centrifuge, and the supernatant liquid drawn off with a pipette. The 
 corpuscles were then laked by adding distilled water to restore the original volume. 
 This sample contained three times as much hemoglobin as the hemolytic tests, because 
 0.2 c.c. of the corpuscles were added to 0.4 c.c. of the fluid tested. Therefore, the 
 above sample was diluted to three times its volume with water. This, then, would give 
 exactly the same concentration of hemoglobin as in any tube in the test, provided that 
 the hemolysis was complete, and is termed 100 per cent for this sample of corpuscles. 
 By further dilution tubes containing 90 per cent, 80 per cent, etc., were prepared. 
 No attempt was made to estimate closer than 10 per cent. A new scale was made for 
 each sample of corpuscles. 
 
 The agglutinins were read from the same tubes as the hemolysins. The method 
 employed to determine whether or not agglutination had occurred, was inspection of 
 the rim of sedimented corpuscles. When the corpuscles, after sedimentation by stand- 
 ing in the ice-box, show a perfectly smooth, knife-edged border, no agglutination has 
 occurred. If the border is slightly or decidedly roughened, agglutination has occurred 
 At first this method was carefully supplemented by microscopic examination, but it 
 was soon found so accurate that in the later experiments we depended entirely upon 
 the observation of the rim of the corpuscles, and dispensed with the use of the micro- 
 scope. 
 
 A. Normal animals. The concentration of lysins and hemag- 
 glutinins in the body fluids of normal animals varies within rather 
 narrow limits. This variation is great enough, however, to make it 
 necessary that the comparison be made between the body fluids of 
 the same animal. The following experiment shows the behavior 
 of the body fluids of the normal dog. 
 
 Table I shows that the concentration of hemolysins is greater 
 in the serum than in the other body fluids; thoracic lymph is next, 
 at least in the case of rabbit corpuscles; and neck lymph is third. 
 
CONCENTRATION OF ANTIBODIES 
 
 This difference in the hemolytic power of serum or other body fluid 
 against the corpuscles of different species of animals has been 
 explained by Ehrlich and his coworkers on the basis of a multiplicity 
 of amboceptors and complements, some of which are specific, some 
 
 TABLE i. 
 
 COMPARATIVE HEMOLYTIC AND AGGLUTINATING POWER OF THE BODY FLUIDS OF A NORMAL DOG ON 
 RABBIT AND RAT CORPUSCLES. 
 
 DILUTION 
 
 SERUM 
 
 NECK LYMPH 
 
 THORACIC LYMPH 
 
 Rabbit 
 
 Rat 
 
 Rabbit 
 
 Rat 
 
 Rabbit 
 
 Rat 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 :6 
 
 IOO 
 
 40 
 o 
 
 
 
 
 
 + 
 + 
 
 
 
 
 o 
 
 10 
 
 
 
 o 
 
 
 
 sp* 
 
 
 
 + 
 
 
 
 
 
 
 
 5 
 
 
 
 
 
 
 
 + 
 
 + 
 
 
 
 
 
 o 
 
 
 
 
 
 
 sp 
 o 
 
 
 
 
 
 
 o 
 
 40 
 
 
 
 
 
 
 o 
 
 + 
 + 
 + 
 + 
 
 
 
 o 
 
 
 
 
 
 sp 
 
 
 
 o 
 
 + 
 + 
 
 + 
 
 
 
 
 :24 
 = 48 
 
 :g6 
 :38 4 
 
 DILUTION 
 
 PERICARDIAL FLUID 
 
 CEREBROSPINAL FLUID 
 
 AQUEOUS HUMOR 
 
 Rabbit 
 
 Rat 
 
 Rabbit 
 
 Rat 
 
 Rabbit 
 
 Rat 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 :6 
 :i2 
 '.24 
 : 4 8 
 96 
 :34 
 
 
 
 o 
 
 
 
 
 
 
 + 
 + 
 
 
 
 o 
 
 
 
 
 
 
 
 sp 
 o 
 
 
 
 o 
 
 
 
 
 o 
 
 
 
 o 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 sp 
 
 
 
 o 
 sp 
 o 
 
 
 
 
 
 
 o 
 
 
 
 
 
 
 
 o 
 
 
 
 
 
 
 
 o 
 
 sp 
 sp 
 
 
 
 sp 
 
 
 
 o 
 
 o 
 
 
 
 
 *It will be noted that in this table several tubes are marked "sp." By that symbol is meant 
 hemolysis not due to the ordinary hemolysins. The appearance of the partially laked corpuscles is entirely 
 different from that in the ordinary hemolytic test. The hemoglobin can be seen diffusing from the sedi- 
 mented corpuscles, while the supernatant fluid remains perfectly clear. The hemoglobin has the peculiar 
 reddish purple tint of reduced hemoglobin, instead of the clear red of oxyhemoglobin. Furthermore, 
 laking may appear anywhere in the series, frequently, where no hemolysis is to be expected, and is met 
 more often in fluids like the cerebrospinal, or aqueous humor, which are normally not hemolytic, than in 
 the other fluids. Rat corpuscles seem more susceptible to this form of hemolysis than rabbit corpuscles. 
 Complement seems to inhibit this form of hemolysis. 
 
 non-specific. In most cases the thoracic lymph of normal dogs is 
 hemolytic for rat corpuscles, altho in the experiment cited above, such 
 was not the case. 
 
 As may be seen from the table above, the concentration of agglu- 
 tinins may be higher in the thoracic lymph than in the serum. Such, 
 however, is not the usual finding. In 10 experiments on normal 
 dogs we found in seven the concentration of agglutinins highest in 
 the serum; in two it was highest in the thoracic lymph; and in one 
 
132 FRANK C. BECHT AND JAMES R. GREER 
 
 the concentration was the same in both. The fact that the concen- 
 tration of agglutinins may be greater in the thoracic lymph than in 
 the serum, renders it hard to see how these antibodies can come from 
 the blood by pure nitration, for in that case, we should expect the 
 hemolysins to run a parallel course a thing which they do not do 
 or else we must assume that the agglutinins pass through membranes 
 more readily than the hemolysins. It would be necessary, also, on 
 the basis of filtration, to assume sudden great changes in the concen- 
 tration of the agglutinins in the blood, for on no other basis could we 
 explain the fact that the concentration of agglutinins would be so 
 much lower in the serum by the time the lymph reached the upper 
 end of the thoracic duct, than it was at the time the lymph was 
 formed. Of course other explanations are possible: there may be 
 an active secretion of the agglutinins into the lymph from the blood, 
 or the agglutinins, after being formed in the area drained by the 
 thoracic duct, are thrown into the lymph, reaching the blood by that 
 route. Much more investigation must be made before any conclusion 
 can be reached on this point. 
 
 The pericardial fluid when collected under the best conditions 
 never shows hemolysins for rabbit corpuscles. Agglutinins may or 
 may not be present. In four of our ten supposedly normal dogs 
 hemolysis was noted, in only one case amounting to more than 10 per 
 cent. Of these four animals, two were in poor condition, emaciated, 
 and generally run down, and both these dogs yielded excessive 
 amounts of pericardial fluid; in the other two cases, the pericardial 
 was found to contain a few erythrocytes. Agglutinins were found 
 in all four of these cases and in three others, making a total of 
 seven in ten. From these experiments we are inclined to believe that 
 hemolysins are not found in the pericardial fluid of normal dogs. The 
 fact that some animals showed hemolysins in the pericardial fluid 
 we would explain as a pericardial transudate in two cases, and to 
 admixture with blood in two cases. We did not test whether it was 
 amboceptor, or complement, or both which was absent from the 
 fluid, altho we have evidence on this point in immune animals. 
 Agglutinins for rabbit corpuscles may or may not be present in the 
 pericardial fluid of normal dogs. 
 
 As will be seen from Table i the cerebrospinal fluid and aqueous 
 
< x r 
 
 CONCENTRATION or ANTIBO^ES^ 133 
 
 humor of normal animals contain no lysins or agglutinins for rat or 
 rabbit corpuscles. In our 10 experiments on normal animals there 
 were no traces of hemolysis or agglutination in a single case where 
 admixture of blood was eliminated. Our results with cerebrospinal 
 fluid confirm those of Massaglia, who could find no lysins in that 
 fluid, and are contrary to those of Bard, who claims to have demon- 
 strated them there. 
 
 Conclusions. i. In the normal dog hemolysins for rabbit cor- 
 puscles are found in the serum, neck lymph, and thoracic lymph, 
 but are absent from the pericardial fluid, cerebrospinal fluid, and 
 aqueous humor. They are most concentrated in the serum, less 
 concentrated in the thoracic lymph, and are found only in traces in 
 the neck lymph. 
 
 2. Agglutinins are found in the serum, neck lymph, and thoracic 
 lymph of normal dogs. They may or may not be present in the 
 pericardial fluid, and are always absent from cerebrospinal fluid 
 and aqueous humor. In most cases the concentration descends in 
 the following order: serum, thoracic lymph, neck lymph, pericardial 
 fluid; altho in some cases, the order is thoracic lymph, serum, neck 
 lymph, pericardial fluid. 
 
 3. Serum and thoracic lymph show a weaker hemolysis and 
 agglutination toward rat than toward rabbit corpuscles. Neck 
 lymph lakes and agglutinates rabbit but not rat corpuscles. Peri- 
 cardial fluid agglutinates rabbit but not rat corpuscles. Cerebro- 
 spinal fluid and aqueous humor neither lake nor agglutinate rat or 
 rabbit corpuscles. 
 
 B. Immunized animals. Various methods of producing active 
 immunity were employed with good success. It is of interest to 
 ascertain what methods of immunizing yield the best results. We 
 employed the following: (i) Immunization of dogs with rabbit blood : 
 (a) intraperitoneally by a single large injection of from 80 to 150 c.c. 
 of blood, (b) intraperitoneally by repeated small injections, (c) sub- 
 cutaneously by repeated small injections. (2) Immunization of dogs 
 with horse serum:* (a) by a single large intraperitoneal injection of 
 100-150 c.c., (b) by repeated, small intraperitoneal injections, (c) by 
 
 * This serum was secured aseptically, November, 1908, by drawing the blood from the carotid of 
 a horse into jars. It was allowed to coagulate and stand in the ice-box until the serum came out. The 
 serum was then sealed into bulbs and kept in the ice-box until used. 
 
134 
 
 FRANK C. BECHT AND JAMES R. GREER 
 
 repeated, small subcutaneous injections. A comparison of the 
 results secured by using the fluids directly from the animal, so far as 
 the lysins are concerned, is not conclusive, for, as will be seen, com- 
 plement is not increased, at least not in proportion to the ambocep- 
 tors, if at all. This fact necessitates the use of sufficient complement 
 to supply all the amboceptors present to demonstrate the true state 
 of affairs. So far as the agglutinins are concerned, apparently a 
 single large dose of the serum or blood may develop them more 
 markedly than the other methods tried. The repeated, small intra- 
 peritoneal injections yielded the most uniform results. 
 
 As has been noted by numerous investigators, the increase in 
 complement does not keep pace with the increase in amboceptors. 
 The apparent increase in the hemolysins and in the agglutinins for 
 rabbit corpuscles is shown in Table 2. 
 
 TABLE 2. 
 
 LYTIC AND AGGLUTINATING ACTION OF THE BODY FLUIDS OF A DOG IMMUNIZED WITH RABBIT BLOOD. 
 
 (November 25, December 15, 10 c.c. rabbit blood intraperitoneally; December 15, 
 
 15 c.c. Fluids collected December 23.) 
 
 DILUTION 
 
 SERUM 
 
 NECK 
 LYMPH 
 
 THOKACIC 
 
 LYMPH 
 
 PERICAR- 
 DIAL FLUID 
 
 CEREBRO- 
 
 SPINAL 
 
 FLUID 
 
 AQUEOUS 
 HUMOR 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 :i* 
 '3 
 6 
 
 100 
 100 
 
 50 
 o 
 
 
 
 o 
 
 
 
 o 
 
 + 
 + 
 + 
 + 
 + 
 + 
 + 
 
 
 
 60 
 
 
 
 o 
 
 
 
 o 
 
 
 
 o 
 
 
 
 + 
 + 
 + 
 + 
 + 
 + 
 o 
 
 
 
 IOO 
 
 60 
 o 
 
 
 
 o 
 
 
 
 o 
 
 
 
 + 
 + 
 + 
 + 
 + 
 + 
 
 
 
 
 
 
 
 
 
 + 
 + 
 + 
 + 
 
 
 
 
 
 
 o 
 
 
 
 o 
 
 
 
 
 
 
 
 
 o 
 
 
 
 o 
 
 
 
 :i2 
 = 24 
 :48 
 :p6 
 :34 
 '.1,536 
 :6,i44 
 
 From Table 2 it can be seen by comparison with Table i that the 
 repeated injections of rabbit blood does not appear to increase to any 
 very marked extent the hemolytic power of the body fluids over that 
 of a normal animal. 
 
 There is, however, a marked increase in the power of the body 
 fluids to agglutinate rabbit corpuscles in those fluids which had the 
 power to agglutinate them previous to the injection; there is no 
 development, except in a few cases, of agglutinins in the cerebrospinal 
 fluid and the aqueous humor. Altho Table 2 does not show this 
 point, a careful comparison of this table with the succeeding ones 
 
CONCENTRATION OF ANTIBODIES 
 
 will show that the relative concentration of the agglutinins, in the 
 body fluids, remain the same during the process of immunization. 
 
 That there is, however, a marked increase in the amboceptor 
 content of the body fluids, normally containing them, during the 
 process of immunization is shown by Table 3. 
 
 TABLE 3 . 
 
 LYTIC AND AGGLUTINATING POWER ON RABBIT CORPUSCLES OF THE BODY FLUIDS OF DOG IMMUNIZED 
 
 WITH RABBIT BLOOD AS AFFECTED BY COMPLEMENT (0.2 c.c. FRESH GUINEA-PIG SERUM). 
 
 (Intraperitoneal injections of rabbit blood as follows: December i, 5 c.c.; December 5, 7^ c.c.; 
 
 December 10, 10 c.c.; December 15, 15 c.c.; December 21, 16 c.c.; January 16, 20 c.c. 
 
 Fluids collected February 8.) 
 
 DILUTION 
 
 SERUM 
 
 NECK LYMPH 
 
 THORACIC LYMPH 
 
 No Comple- 
 ment 
 
 Complement 
 
 No Comple- 
 ment 
 
 Complement 
 
 No Comple- 
 ment 
 
 Complement 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 :6 
 
 100 
 100 
 
 90 
 
 10 
 
 
 o 
 
 
 
 o 
 
 + 
 + 
 + 
 + 
 
 
 
 o 
 
 100 
 100 
 100 
 100 
 
 So 
 30 
 
 10 
 
 o 
 
 + 
 + 
 
 
 
 o 
 
 60 
 
 10 
 
 
 
 
 
 
 
 + 
 + 
 + 
 4- 
 
 
 
 
 
 o 
 
 E 
 
 40 
 
 20 
 
 
 
 
 o 
 
 + 
 + 
 + 
 + 
 
 
 
 
 
 
 100 
 
 7o 
 20 
 
 
 
 o 
 
 
 
 
 
 + 
 + 
 
 + 
 
 
 
 o 
 
 
 
 oooooooo 
 
 + 
 + 
 
 + 
 
 
 
 
 o 
 
 :24 
 = 48 
 
 :g6 
 
 : 3 84 
 
 : 1,336........ 
 
 
 DILUTION 
 
 PERICARDIAL FLUID 
 
 CEREBROSPTNAL FLUID 
 
 AQUEOUS HUMOR 
 
 No Comple- 
 ment 
 
 Complement 
 
 No Comple- 
 ment 
 
 Complement 
 
 No Comple- 
 ment 
 
 Complement 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 :6 
 :i2 
 = 24 
 = 48 
 : 9 6 
 
 
 
 o 
 
 
 
 
 
 + 
 + 
 + 
 
 + 
 
 
 
 20 
 10 
 
 O 
 
 
 + 
 + 
 + 
 + 
 
 
 
 
 
 
 
 
 
 
 
 
 
 o 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 o 
 
 
 
 
 o 
 
 
 
 
 o 
 
 1384 
 : 1,536 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 :6,i44 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 This experiment shows very clearly that in the immunized animal 
 the serum, neck lymph, thoracic lymph, and pericardial fluid do not 
 contain complement in sufficient quantity to activate all of the ambo- 
 ceptor present in the fluid, because the addition of guinea-pig com- 
 plement,* in doses of itself not ly tic, is able to produce stronger 
 
 * We experienced considerable difficulty in securing a complement which was effective and at the 
 same time did not of itself produce hemolysis. Rabbit serum, guinea-pig serum, and dog serum from 
 which the amboceptor had been separated in the cold were tried. The guinea-pig serum proved the only 
 effective one. 
 
136 FRANK C. BECHT AND JAMES R. GREER 
 
 hemolysis where only traces had appeared, and to produce hemolysis 
 in. other cases where there were no traces with exactly the same 
 amount of the fluid without complement. 
 
 The addition of c6mplement in a non-hemolytic dose is able to 
 cause hemolysis in the pericardial fluid. In 8 of 13 experiments on 
 blood-immune dogs traces of hemolysis occurred in the lowest dilu- 
 tions of that fluid. In these experiments we could detect no contami- 
 nation with blood, neither did we note that the fluid was present in 
 excessive amounts. Apparently, then, in dogs immune to a foreign 
 blood, amboceptor is always, and complement usually present in the 
 pericardial fluid. This agrees in part with the findings of Mioni 
 who found amboceptor but no complement for guinea-pig corpuscles 
 in the pericardial fluid of the ox. No amboceptors are found in the 
 cerebrospinal fluid or aqueous humor, for no hemolysis occurs when 
 effective complement is added, at least a complement which proved 
 effective in the case of the other body fluids. 
 
 The agglutinins in the various body fluids are seen from Table 5 
 to run practically the same course as in the normal animal, except 
 that the^concentration is highest in the serum, a little lower in the 
 thoracic lymph, still lower in the neck lymph, and lowest, but always 
 present, in the pericardial fluid. Sometimes, as in the normal animal, 
 the concentration of agglutinins in the thoracic lymph is equal to or 
 greater than that of the serum. In the 16 immune animals in which 
 we compared serum and thoracic lymph, in n cases the serum was 
 higher in the concentration of the agglutinins than the thoracic 
 lymph, in two cases the thoracic lymph was higher than the serum, 
 and in three cases the two fluids showed equal concentration. The 
 fact that the thoracic lymph may contain these antibodies in equal or 
 even considerably higher concentration than the corresponding serum 
 may be of significance as bearing upon the source of these substances. 
 
 In 15 experiments with cerebrospinal fluid from immune dogs, 
 a positive agglutination was secured in two. In 16 experiments with 
 the aqueous humor, positive results were secured in five. Thus it 
 will be seen that agglutinins may be found in the cerebrospinal fluid 
 and the aqueous humor, but their presence is the exception and not 
 the rule. 
 
 A point of considerable interest is the determination whether the 
 
CONCENTRATION OF ANTIBODIES 
 
 fluids of an animal immune to one kind of blood show an increased 
 hemolytic and agglutinating power toward the corpuscles of another 
 species. An experiment of this kind is shown in Table 4. 
 
 TABLE 4 . 
 
 LYTIC AND AGGLUTINATING ACTION ON HORSE AND RABBIT CORPUSCLES OF BODY FLUIDS OF A DOG 
 
 INJECTED WITH HORSE SERUM. 
 (100 c.c. of serum injected intraperitoneally January 16; fluids removed January 29.) 
 
 DILUTION 
 
 SERUM 
 
 NECK LYMPH 
 
 THORACIC LYMPH 
 
 Horse 
 Corpuscles 
 
 Rabbit 
 Corpuscles 
 
 Horse 
 Corpuscles 
 
 Rabbit 
 Corpuscles 
 
 Horse 
 Corpuscles 
 
 Rabbit 
 Corpuscles 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 6 
 
 100 
 100 
 100 
 
 70 
 30 
 
 
 
 
 o 
 
 + 
 + 
 + 
 + 
 + 
 
 100 
 100 
 
 30 
 
 IO 
 
 
 o 
 
 
 
 
 + 
 + 
 
 4- 
 
 
 
 
 
 100 
 
 90 
 60 
 
 IO 
 
 
 o 
 
 
 
 
 4- 
 4- 
 + 
 
 + 
 4- 
 
 
 
 
 So 
 
 IO 
 
 
 
 
 
 
 
 + 
 + 
 + 
 
 
 
 
 
 o 
 
 
 
 100 
 100 
 
 5 
 
 20 
 
 o 
 
 
 
 o 
 
 
 
 1 1 4-44 + + o 
 
 100 
 
 40 
 o 
 
 
 
 o 
 
 
 
 o 
 
 
 
 + 
 + 
 
 
 
 o 
 
 
 
 o 
 
 
 
 :i2 
 
 124 
 
 J'.'. 
 96 
 : 3 84 
 
 :i,536 
 :6,i44 
 
 DILUTION 
 
 PERICARDIAL FLUID 
 
 CEREBROSPINAL FLUID 
 
 AQUEOUS HUMOR 
 
 Horse 
 Corpuscles 
 
 Rabbit 
 Corpuscles 
 
 Horse 
 Corpuscles 
 
 Rabbit 
 Corpuscles 
 
 Horse 
 Corpuscles 
 
 Rabbit 
 Corpuscles 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 :6 
 
 48.".!'.".;".". 
 
 = 96 
 ;384 6 - 
 
 80 
 30 
 o 
 
 
 
 o 
 
 
 
 + 
 + 
 + 
 + 
 + 
 
 
 
 o 
 
 
 
 o 
 
 
 
 o 
 
 
 
 4- 
 
 
 
 
 o 
 
 
 
 
 
 
 o 
 sp 
 
 
 
 o 
 
 
 
 o 
 o 
 
 o 
 
 
 
 o 
 
 
 
 
 o 
 
 
 
 o 
 
 
 
 
 o 
 
 
 
 o 
 
 
 
 o 
 
 o 
 
 
 
 
 
 
 
 
 
 o 
 
 
 
 o 
 
 
 
 
 o 
 
 
 
 o 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Horse corpuscle control =o. Rabbit corpuscle control =o. 
 
 The animal tested here, while showing a marked immunity to 
 horse corpuscles, does not show immunity toward rabbit corpuscles 
 much higher than the normal. Indeed, in the 10 normal animals 
 studied, in two cases the lysins in the neck lymph were as concentrated 
 as here; and in five cases the lysins in the thoracic lymph were as 
 concentrated as here. Therefore, the lysins for rabbit corpuscles 
 are apparently little more concentrated as may be seen by comparing 
 Table 4 with Table i. The agglutinins are somewhat higher than 
 normal. Apparently the immunity is not entirely specific. 
 
 Muir and Browning 32 have advanced some evidence that a com- 
 
138 
 
 FRANK C. BECHT AND JAMES R. GREER 
 
 plement-like body plays a role in agglutination. They used ox 
 corpuscles, rabbit serum immune to ox blood, and guinea-pig serum 
 as complement. They do not make the claim that the complement 
 and the agglutinin are identical, merely that this form of complement 
 behaves like hemolytic complement, is thermostable, and acts only 
 when suitable amboceptor is present. They are not sure whether or 
 not this is the same complement concerned in hemolysis. In the 
 course of our experiments we found some evidence which points in the 
 opposite direction; viz., addition of complement or at least of rabbit 
 serum, as in one experiment, inhibits the agglutination of rabbit 
 corpuscles by the fluids of an immune dog. 
 
 TABLE 5. 
 THE INHIBITION OF THE AGGLUTINATION OF RABBIT CORPUSCLES BY THE BODY FLUIDS OF AN IMMUNE 
 
 DOG BY THE ADDITION OF RABBIT SERUM. 
 
 (150 c.c. rabbit blood intraperitoneally, February 16. Fluids drawn February 26. 
 o.i c.c. rabbit serum as complement.) 
 
 DILUTION 
 
 SERUM 
 
 NECK LYMPH 
 
 THORACIC LYMPH 
 
 No Comple- 
 ment 
 
 Complement 
 
 No Comple- 
 ment 
 
 Complement 
 
 No Comple- 
 ment 
 
 Complement 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 :6 
 
 100 
 100 
 
 80 
 
 10 
 
 
 
 
 
 + 
 + 
 + 
 + 
 + 
 + 
 
 100 
 IOO 
 100 
 
 70 
 
 10 
 
 o 
 
 
 
 o 
 
 + 
 + 
 + 
 
 
 
 o 
 
 IOO 
 10 
 
 
 o 
 
 
 
 
 
 
 + 
 
 + 
 + 
 + 
 + 
 + 
 
 
 
 IOO 
 
 30 
 
 
 
 
 
 
 
 
 000 + + + + 1 
 
 IOO 
 IOO 
 
 40 
 
 
 
 o 
 
 
 
 
 
 + 
 + 
 + 
 + 
 + 
 + 
 
 IOO 
 IOO 
 IOO 
 10 
 
 
 
 
 
 o o + + + | | | 
 
 :i2 
 124 
 
 = 48 
 : 9 6 
 
 :384 
 11,536 
 
 : 6,144 
 
 DILUTION 
 
 PERICARDIAL FLUID 
 
 CEREBROSPINAL FLUID 
 
 AQUEOUS HUMOR 
 
 No Comple- 
 ment 
 
 Complement 
 
 No Comple- 
 ment 
 
 Complement 
 
 No Comple- 
 ment 
 
 Complement 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 Lysis 
 
 Aggl. 
 
 :6 
 :i2 
 :24 
 
 20 
 
 
 
 sp 
 
 
 
 sp 
 
 + 
 + 
 + 
 
 + 
 
 20 
 10 
 
 
 o 
 
 
 
 o 
 
 + 
 + 
 + 
 + 
 
 
 
 
 o 
 
 
 
 o 
 
 
 
 + 
 
 + 
 + 
 + 
 
 o 
 
 
 
 o 
 
 
 
 
 
 
 o 
 
 
 
 sp 
 
 
 
 o 
 
 
 
 
 
 o 
 
 
 
 
 
 o 
 
 
 
 
 
 
 o 
 
 
 
 o 
 
 : 4 s :..: 
 
 : 9 6 
 
 '384 
 : i,536 
 : 6,144 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Corpuscle control=o. Complement control=o. 
 
 This experiment shows that the addition of rabbit serum as com- 
 plement instead of favoring agglutination as in the case; observed by 
 
CONCENTRATION OF ANTIBODIES 139 
 
 Muir and Browning actually inhibited it in every series in the experi- 
 ment by at least two dilutions. In this case we have a cerebrospinal 
 fluid and an aqueous humor which contained agglutinins for rabbit 
 corpuscles but in these fluids the addition of rabbit serum completely 
 inhibited the action. We would not give the impression that this 
 occurred normally in our work, or even frequently, for in the nine 
 cases where complement was used with body fluids of immune dogs, 
 this phenomenon was noted in only this one experiment. It appeared 
 only once in the fluids of normal dogs, and in that case the action was 
 less marked. 
 
 Conclusions. i . In the blood of dogs immunized with alien blood 
 hemolysins are faund in the serum, thoracic lymph, and neck lymph, 
 and usually in the pericardial fluid. They are not found in the cere- 
 brospinal fluid or aqueous humor. The comparative concentration 
 is the same as in the normal animal. 
 
 2. -The addition of guinea-pig serum as complement in non- 
 hemolytic doses increases greatly the hemolytic power of the serum, 
 neck lymph, thoracic lymph, and pericardial fluid; therefore, in the 
 course of immunization the amboceptors are increased in the fluids, 
 while complement is not. Cerebrospinal fluid and aqueous humor 
 do not become hemolytic on the addition of complement; therefore, 
 they do not contain amboceptors. 
 
 3. In the immunized dog the agglutinins are more concentrated 
 than in the same fluids of the normal animal. The usual order of 
 descending concentration is: serum, thoracic lymph, neck lymph, 
 pericardial fluid; but the order may be thoracic lymph, serum, neck 
 lymph, pericardial fluid. Cerebrospinal fluid and aqueous humor 
 may have agglutinins present but usually do not. If agglutinins 
 are present in these two fluids, the concentration is about equal and 
 lower than the pericardial fluid. 
 
 4. Immunization of a dog to horse serum increases the hemolytic 
 power of the body fluids for horse corpuscles, but little if at all for 
 rabbit corpuscle. The hemagglutinins are increased to a slight 
 extent. 
 
 5. Occasionally the addition of rabbit serum will inhibit the 
 agglutination of washed rabbit corpuscles by the fluids of a dog 
 immune to rabbit blood. 
 
140 FRANK C. BECHT AND JAMES R. GREER 
 
 PROTEIN PRECIPITINS. 
 
 Since most investigators who have worked with precipitins are 
 agreed as to the delicacy and specificity of the reaction, we chose 
 them as one of the antibodies best suited for study in our work on the 
 body fluids of normal and immune animals. In several cases these 
 were the same animals used in the work on hemolysins and hemag- 
 glutinins. 
 
 Our method was the same as is usually employed, namely, a 
 dilution method. Doses of the immune fluid varying between o . 2 c.c. 
 and o.oi c.c. were placed in a series of test tubes and made up to 
 2 c.c. with sterile 0.9 per cent Nad solution. To these tubes were 
 then added 0.15 c.c. of the same serum as used for immunization. 
 Control experiments were made in case of each of the fluids, and of 
 the serum, to eliminate any possibilities of a sediment from the protein 
 solutions confusing the results. The tubes were incubated for two 
 hours at 37 C., and then left in the ice-box 12 to 20 hours before the 
 final reading was made. 
 
 Our results were as follows: In the fluids of three normal dogs 
 tested with the fresh serum of the rabbit not a trace of precipitate 
 appeared in any tube. 
 
 In experiments with the fluids of seven dogs immune to rabbit 
 blood, three gave positive and four negative results. One dog gave 
 a precipitate only in the first dilution of the serum (i : 10) ; the other 
 mixtures and the control mixtures remained perfectly clear. This 
 animal had been immunized by repeated injections of rabbit blood 
 intraperitoneally, receiving in all 57 c.c. between November 14 
 and December 5, 1908. 
 
 A dog which 10 days earlier had received an intraperitoneal 
 injection of 150 c.c. of rabbit blood gave a positive reaction in 
 both serum and thoracic lymph. In this case the precipitation 
 occurred in a much higher dilution than in the former, tho in the 
 latter, neither the neck lymph, pericardial fluid, cerebrospinal fluid, 
 nor the aqueous humor nor any of the controls showed any pre- 
 cipitate. 
 
 A dog immunized by the intraperitoneal injection of 80 c.c. of 
 rabbit blood gave the best results of all, the serum and thoracic 
 lymph giving precipitation in dilutions of i : 40 and the <neck lymph 
 
CONCENTRATION OF ANTIBODIES 141 
 
 in i : 20 ; the pericardial and cerebrospinal fluids and the aqueous 
 humor, however, gave no reaction in i : 10. 
 
 Of the five attempts to produce precipitins in dogs by immuniza- 
 tion with horse serum, four were entirely negative, and even in the one 
 positive result only the serum, thoracic lymph, and the neck lymph 
 contained precipitin. The precipitation occurred in a higher dilution 
 in the thoracic lymph than in the serum, but only in the lower dilu- 
 tions of the neck lymph, which was considerably weaker than the 
 serum. 
 
 Our data are not uniform enough nor extensive enough to warrant 
 us in drawing conclusions. It is apparent that dogs develop pre- 
 cipitins with extreme difficulty, and that a good method of immuniza- 
 tion is by single, large, intraperitoneal injections. The results that 
 we have seem to show that the precipitins follow closely the hemag- 
 glutinins and the hemolysins in their distribution in the various body 
 fluids of immune animals, altho we have not as yet been able to 
 demonstrate any in the pericardial fluid. 
 
 BACTERIAL AGGLUTININS. 
 
 We also made a study of the concentration of the agglutinins for 
 the typhoid bacillus in the various body fluids of normal and immu- 
 nized cats and dogs, and intend later to extend the work to cover 
 the bacteriolysins. 
 
 Nuttall,34 employing both the hanging drop method and the plate method, found 
 bacteriolysins for the anthrax bacillus in the aqueous humor and the pleuritic exudate 
 of dogs and rabbits. Prudden38 found bacteriolysins in the amniotic, hydrocele, and 
 acites fluids. Meltzer and Norris 28 found the thoracic lymph nearly as bacteriolytic as 
 the serum for the typhoid bacillus. Widal43 found agglutinins for the typhoid bacillus 
 active in a dilution of i : 60 in the pericardial fluid, while the serum of the same patient 
 was active at 1:350. Edema fluid was found strongly agglutinating also, but the 
 result with cerebrospinal fluid was negative. Picks? found that cerebrospinal fluid 
 agglutinated at a dilution of i : i and i : 2. Kohler 20 found agglutinins in the cerebro- 
 spinal fluid in one case in ten examined of non-typhoid patients, and but three times 
 out of 19 cases of typhoid fever, and these in dilutions of i : i, 1:5, and i : 10. Braude 
 and Carlson? made a study of the concentration of the agglutinins for the typhoid 
 bacillus in the body fluids of normal and immunized cats and dogs. Inasmuch as they 
 used for comparison the action of the fluids of the same animal upon the same bacterial 
 suspension, their results give more nearly the true conditions of the body fluids in an 
 animal; they avoided the error which a comparison of the body fluids of different 
 animals is sure to introduce on account of the wide individual variations. The hanging 
 drop method which they employed, however, is not considered as accurate as the pre- 
 
142 
 
 FRANK C. BECHT AND JAMES R GREER 
 
 cipitation method which we used. They found bacterial precipitins in the cerebrospinal 
 fluid of immunized dogs and cats. 
 
 The fluids used in these experiments were collected as described 
 at the beginning of this paper. The test tubes were the same as 
 those used in the hemolytic work. The bacteria were secured from 
 2o-24-hour slant agar cultures, made up with sterile 0.9 per cent 
 NaCl solution to a distinctly cloudy suspension, and then filtered 
 through filter paper to remove all of the clumps. A series of tubes 
 was arranged for each fluid, then after the proper amount of fluid had 
 been measured into each, the bacterial suspension was added. The 
 dilutions used were i : 10, i : 50, i : 100, i : 500, i : 2,000, i : 6,000. Our 
 lowest dilution was, perhaps, too high to detect the traces of agglutinins 
 reported by Pick and Kohler. The tubes were all incubated together 
 at 37 C. for two hours and then kept in the ice-box for from 12-20 
 hours before the final observations. 
 
 In our study of agglutinins for the typhoid bacillus we used normal 
 cats and dogs, animals actively immunized by the repeated injection 
 of typhoid cultures, and animals rendered passively immune by the 
 withdrawal of large quantities of blood from the normal animal, and 
 the subsequent injection of an equal amount of warm, defibrinated 
 blood from an actively immunized animal of the same kind. 
 
 A. Normal animals. We studied first the concentration of 
 agglutinins in the body fluids of normal cats and found agglutinins 
 for the typhoid bacillus active in a dilution from i : 10 in the serum 
 and the thoracic lymph; the neck lymph and the pericardial fluid 
 usually contain them in the same concentration but the cerebrospinal 
 fluid and the aqueous humor do not contain them in such dilution. 
 
 DOGS. A study of the concentration of the typhoid agglutinins 
 in normal dogs gave the results shown in the following table : 
 
 TABLE 6. 
 
 THE COMPARATIVE AGGLUTINATING POWER ON THE TYPHOID BACILLUS OF THE BODY FLUIDS OF A 
 
 NORMAL DOG. 
 
 Dilution 
 
 Serum 
 
 Neck 
 Lymph 
 
 Thoracic 
 Lymph 
 
 Pericardial 
 Fluid 
 
 Cerebro- 
 spinal Fluid 
 
 Aqueous 
 Humor 
 
 i ; 10 
 
 + + 
 
 + + 
 
 + + 
 
 
 
 o 
 
 o 
 
 1:50 
 
 + + 
 + 
 
 
 
 
 
 o 
 
 o 
 o 
 
 
 
 o 
 
 
 
 
 
 o 
 
 o 
 
 o 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CONCENTRATION OF ANTIBODIES 
 
 This experiment is one of four performed on normal animals, and 
 gives a fair idea of the concentration of the agglutinins in the body 
 fluids. There are, of course, variations between animals even in 
 the same species. In all of our experiments the concentration of 
 agglutinins was highest in the serum, less in the thoracic and the 
 neck lymph, and the pericardial fluid contained agglutinins in only 
 one case. The cerebrospinal fluid and the aqueous humor did not 
 contain any in any case. 
 
 B. Actively immunized animals. We made a study of the fluids 
 of five immunized cats. Three had received repeated subcutaneous 
 injections of typhoid bacilli. For the first few injections killed cul- 
 tures, but later live cultures were used. The doses injected were 
 increased gradually and careful record of the weight and general 
 condition kept, to guard against pushing the process too rapidly. 
 The remaining two animals received a single large injection of six 
 living 24-hour cultures. The results given in Table 7 may be taken 
 as fairly typical. 
 
 TABLE 7. 
 THE COMPARATIVE AGGLUTINATING POWER ON THE TYPHOID BACILLUS OF THE BODY FLUIDS OF AN 
 
 IMMUNIZED CAT. 
 
 (Immunized by repeated subcutaneous injections. Fluids collected five 
 days after the last injection.) 
 
 Dilution 
 
 Serum 
 
 Neck 
 Lymph 
 
 Thoracic 
 Lymph 
 
 Pericardial 
 Fluid 
 
 Cerebro- 
 spinal Fluid 
 
 Aqueous 
 Humor 
 
 :io.. 
 : 50 
 
 + + 
 + + 
 
 + 4- 
 + + 
 
 + + 
 
 + + 
 
 + 
 o 
 
 ? 
 o 
 
 ? 
 o 
 
 :ioo 
 : 500 
 
 + + 
 
 + + 
 o 
 
 + + 
 + 
 
 o 
 o 
 
 o 
 o 
 
 
 
 o 
 
 -.2,000 
 : 6,000 
 
 + 
 + 
 
 
 
 
 o 
 
 
 
 
 
 
 
 
 
 
 
 o 
 
 Control=o. 
 
 The other experiments showed some variations. In two cases in 
 five the agglutinins were as concentrated in the thoracic lymph as in 
 the serum, in the remaining three of five the serum was by far the 
 more concentrated of the two. In one case the neck lymph showed 
 the same concentration of agglutinins as the thoracic lymph, but both 
 were considerably lower than the serum; in the four remaining cases 
 the concentration in the neck lymph was lower than in the thoracic 
 lymph. Agglutinins were found in four of five cases in the peri- 
 cardial fluid, but in no case in a dilution higher than i : 10. Agglu- 
 tination in the fifth case was questioned. The cerebrospinal fluid 
 
144 
 
 FRANK C. BECHT AND JAMES R. GREER 
 
 was negative in all dilutions used in four of five cases, and agglutina- 
 tion in the fifth case at i : 10 was questioned. The aqueous humor 
 gave negative results in three of five cases, and the agglutination was 
 questioned in the remaining two at a dilution of i : 10. 
 
 The development of agglutinins for the typhoid bacillus in dogs 
 runs a course which is strictly comparable to that in the cats under 
 similar circumstances. 
 
 TABLE 8. 
 THE COMPARATIVE AGGLUTINATING POWER FOR THE TYPHOID BACILLUS OF THE BODY FLUIDS OF AN 
 
 IMMUNE DOG. 
 (Immunized by repeated subcutaneous injections.) 
 
 Dilution 
 
 Serum 
 
 Neck 
 Lymph 
 
 Thoracic 
 Lymph 
 
 Pericardia! 
 Fluid 
 
 Cerebro- 
 spinal 
 Fluid 
 
 Aqueous 
 Humor 
 
 Control 
 
 : 10 
 .50 
 
 + + 
 + + 
 
 + + 
 
 + + 
 
 + + 
 + + 
 
 + + 
 + + 
 
 
 
 
 
 
 
 : 100 
 
 + + 
 
 + + 
 
 + + 
 
 + 
 
 
 
 
 : 500 . . . 
 
 + + 
 
 4. 
 
 + + 
 
 
 
 
 
 : 2,000 
 : 6,000. 
 
 + 
 
 
 
 + 
 
 o 
 
 
 
 
 
 
 
 
 
 
 
 
 From Table 8 it is evident that the concentration of the agglutinins 
 in the body fluids of immune dogs runs a course parallel to that in the 
 immune cats. The concentration in the neck lymph and the peri- 
 cardial fluid is considerably lower than that in the thoracic lymph 
 and serum, and of the two the concentration in the neck lymph is the 
 higher. In our seven experiments on typhoid immune dogs, thoracic 
 lymph and serum showed the same concentration of bacterial agglu- 
 tinins in four cases; in the remaining three cases the concentration is 
 greater in the serum. The pericardial fluid contained agglutinins 
 in six of seven cases. The highest dilution at which agglutination 
 occurred was i : 100 (three cases) . In no case were agglutinins 
 found in the cerebrospinal fluid in the dilutions used. Traces of 
 agglutinins were observed in three of six cases in the aqueous humor 
 in a dilution of i : 10. 
 
 We considered it of interest to determine whether immunity to 
 rabbit blood affected in any way the agglutinins for the typhoid 
 bacillus in the body fluids of a dog. With this in mind we tested the 
 usual six fluids of one of our immune dogs. An increased agglutinat- 
 ing power could be expected, if, in terms of Ehrlich's hypothesis, 
 there were any cell receptors common both to blood cells and the 
 typhoid bacilli. 
 
CONCENTRATION OF ANTIBODIES 
 
 TABLE 9. 
 THE COMPARATIVE AGGLUTINATING POWER ON THE TYPHOID BACILLUS OF THE BODY FLUIDS OF A 
 
 DOG IMMUNIZED WITH RABBIT BLOOD. 
 
 (Intraperitoneal injections as follows: October 10, ioc.c.; October 19, 8 c.c.; October 24, 8 c.c.; 
 October 29, 9 c.c.; November 3, 10 c.c.; November 9, 10 c.c.; November 14, 10 c.c.; fluids drawn 
 November 24.) 
 
 Dilution 
 
 Serum 
 
 Neck 
 Lymph 
 
 Thoracic 
 Lymph 
 
 Pericardial 
 Fluid 
 
 Cerebro- 
 spinal Fluid 
 
 Aqueous 
 Humor 
 
 
 + + 
 
 + 
 
 4- 
 
 o 
 
 o 
 
 o 
 
 I'-So 
 i : 100 
 i : 500 
 
 + 
 + 
 o 
 
 o 
 
 
 
 
 + 
 o 
 
 
 
 
 
 o 
 
 
 
 
 
 o 
 
 
 
 
 
 o 
 
 
 
 
 
 
 
 
 
 
 A comparison of this table with Table 7 shows that the agglutinins 
 for the typhoid bacillus were little if any higher than those of the 
 normal animal, altho this was one of our most highly immunized 
 animals and agglutinated rabbit corpuscles strongly in a dilution of 
 i : 1,536 in the serum and in a dilution of 1 1384 in the neck and the 
 thoracic lymph. The relative concentration in the body fluids is 
 strictly comparable to those in the normal animal cited. We tested 
 also the blood serum of two other dogs immune to rabbit blood with 
 exactly similar results. 
 
 C. Passive immunity. Evidence that antibodies of various kinds 
 are able to pass through membranes is not lacking in the literature. 
 Ehrlich 10 found that the young from a mouse immune to abrin, ricin, 
 or robin possess an immunity to these poisons which persists for two 
 months. Ascoli 1 found that the antibodies of the new born child 
 come from the maternal circulation and are not formed in the fetus 
 itself. This being true, the antibodies of the fetus have penetrated 
 the walls of at least a double membrane. Merkle 29 found that the 
 same was true for rabbits, since the young born of a mother immune 
 to human blood contain antibodies for human blood, altho they suckle 
 a normal mother from the very first. Liidke 23 confirmed the work 
 of Merkle. Ricketts 39 has shown that the young born to a guinea- 
 pig mother immune to Rocky Mountain spotted fever possess an 
 immunity to that disease, altho they suckled a normal mother. 
 DeBlasi 8 has shown that if a cat is immunized post par turn to B. 
 dysenteriae the young develop an immunity from the milk. This is 
 even a more striking example than the others of the ability of anti- 
 bodies to penetrate membranes, for in this case the antibodies after 
 reaching the alimentary canal of the young animal must penetrate 
 
146 
 
 FRANK C. BECHT AND JAMES R. GREER 
 
 the intestinal mucosa, and later the capillary or lymphatic wall, 
 depending upon whether the path of absorption was the blood or 
 the lymph. 
 
 It was hoped that a careful study of the passage of these antibodies 
 from the blood into the various body fluids in a passively immune 
 animal would throw some light upon the problem of lymph formation. 
 We have been disappointed thus far, for our results have not been 
 decisive enough to warrant any general conclusions. 
 
 Passive immunity was established by the withdrawal under light 
 ether anesthesia in most cases of a large amount of blood from a 
 normal animal, and the subsequent injection of an equal amount of 
 warm, defibrinated blood from an actively immune animal of the same 
 kind. 
 
 CATS. To show the concentration of agglutinins in the body fluids 
 of a passively immune cat we give Table 10 as typical of our results. 
 It will also show the concentration of agglutinins of the actively 
 immune cat from which the blood was taken. 
 
 TABLE io. 
 THE COMPARATIVE AGGLUTINATING POWER ON THE TYPHOID BACILLUS OF THE BODY FLUIDS OF CATS 
 
 ACTIVELY AND PASSIVELY IMMUNE TO THE TYPHOID BACILLUS. 
 
 (Cat n, immunized June 29 by the injection subcutaneously of six live 24-hour slant agar cultures 
 of the typhoid bacillus, operated July 9. Cat 12, passively immunized by the withdrawal on July 9 of 
 100 c.c. of blood and the injection of 100 c.c. of blood from Cat n. Operated July io.) 
 
 DILUTION 
 
 SERUM 
 
 NECK 
 LYMPH 
 
 THORACIC 
 LYMPH 
 
 PERICAR- 
 
 DIAL 
 
 FLUID 
 
 CEREBRO- 
 
 SPINAL 
 
 FLUID 
 
 AQUEOUS 
 HUMOR 
 
 ii 
 
 12* 
 
 12 
 
 ii 
 
 12 
 
 n 
 
 12 
 
 ii 
 
 12 
 
 ii 
 
 12 
 
 ii 
 
 12 
 
 :io 
 150 
 
 + + 
 + + 
 + + 
 + 
 + 
 
 
 
 + 
 + 
 
 
 
 
 
 + + 
 + 
 
 + 
 + 
 
 
 
 + + 
 + + 
 + 
 + 
 + 
 
 
 
 + + 
 + 
 
 
 
 
 
 + + 
 + + 
 + 
 + 
 + 
 
 
 
 + + 
 + 
 + 
 
 
 o 
 
 
 
 + 
 + 
 
 
 
 
 o 
 
 
 
 O 
 
 
 
 
 
 
 o 
 
 
 
 o 
 
 
 
 o 
 
 
 
 O 
 O 
 
 o 
 
 
 
 o 
 
 
 
 o 
 
 
 
 o 
 
 
 
 
 
 O 
 
 
 
 
 
 
 :ioo 
 
 12,000 
 
 : 6,000 
 
 * Normal serum before injection of immune serum 1 1 . 
 
 From Table io it is evident that it is possible to increase the agglu- 
 tinins not only in the serum but in both of the lymphs by a process of 
 passive immunization. The relative concentration is maintained 
 in the fluids which we find in the actively immunized animals of the 
 same grade of immunity. In no case in the passively immunized 
 animal did the pericardial fluid show any increase over the normal 
 animal. The cerebrospinal fluid and the aqueous humor^ showed no 
 
CONCENTRATION or ANTIBODIES 147 
 
 increase, and, indeed, one would not expect that they would, since 
 in the actively immune animal the presence of antibodies is the excep- 
 tion and not the rule. The results here are typical of all of our results. 
 In four passively immune cats this same increase in the concentration 
 of the agglutinins in serum, neck lymph, and the thoracic lymph was 
 noted. In two of the four cases the concentration in the neck lymph 
 and the thoracic lymph was equal, in the remaining two of the four 
 experiments the concentration in the thoracic lymph was greater 
 than that in the neck lymph. There appears on the whole to be a 
 tendency for the concentration of the agglutinins in the thoracic and 
 the neck lymph in the passively immune animals to run more nearly 
 parallel than in the actively immunized animals. 
 
 DOGS. Passive immunity produced in dogs in the manner 
 described yields exactly similar results; namely, the concentration in 
 agglutinins of the serum, neck lymph, and thoracic lymph can be 
 greatly increased. The concentration of the agglutinins in the peri- 
 cardial fluid, cerebrospinal fluid, and the aqueous humor is no higher 
 than in the normal animal. 
 
 In one of three passively immunized dogs the concentration in the 
 neck lymph and thoracic lymph was equal; in the other two the 
 thoracic lymph was much higher. The fact that 50 per cent of our 
 passively immunized cats and 33 per cent of the passively immune 
 dogs showed an equal concentration of the agglutinins in the thoracic 
 and the neck lymph may be significant, for the percentage of 
 such findings in the actively immune animals is 14 per cent for 
 the dogs and 16 per cent for the cats; but much more extensive 
 experimentation is necessary before any reliance can be placed on 
 these results. 
 
 In order to determine the relative rapidity with which these anti- 
 bodies pass from the blood into the lymph the following experiment 
 was made. A dog was anesthetized and rendered passively immune 
 by the withdrawal of 300 c.c. of blood from the femoral artery and 
 the injection of 300 c.c. of blood and serum from a typhoid immune 
 dog. Small samples of blood were drawn at stated intervals, as 
 were also samples of neck lymph. Thoracic lymph, pericardial 
 fluid, cerebrospinal fluid, and aqueous humor were collected at the 
 end of the experiment. All of the fluids except the thoracic lymph 
 
148 FRANK C. BECHT AND JAMES R. GREER 
 
 which was tinged with blood were in good condition and were tested 
 in the usual manner. The results are shown in Table 1 1 . 
 
 TABLE ii. 
 
 THE RATE OF PASSAGE OF ANTIBODIES FROM THE BLOOD INTO THE BODY FLUIDS IN PASSIVELY 
 
 IMMUNE ANIMALS. 
 
 Highest Dilu- 
 
 Fluid tion Showing 
 
 Agglutination 
 
 Normal serum 
 
 Immune serum injected into passively immune dog. . 
 
 Equal parts of normal and immune sera 
 
 Serum 25 min. after transfusion 
 
 " i hr. 25 min. after transfusion 
 
 " 2 " " " " " 
 
 14 3 " " " " " 
 
 " 4 " " " " " 
 
 Neck lymph 3 hr. " " 
 
 4 " 30 min. after transfusion. . 
 
 : 100 
 : 2,000 
 : 2,000 
 
 : 5 
 .500 
 : 500 
 = 500 
 
 '5 
 
 Thoracic lymph 4 hr. 30 min. after transfusion i : 100 
 
 Pericardial fluid 4 " 30 " " o 
 
 Cerebrospinal fluid o 
 
 Aqueous humor o 
 
 It may be noted that the results of this experiment are practically 
 identical with those secured from our 24-hour experiments on the 
 passively immunized dogs. The concentration of the agglutinins of 
 the serum remain practically the same during the 4^ hours of this 
 experiment. The neck lymph had the same concentration of agglu- 
 tinins three hours after the transfusion that it had at the close of the 
 experiment. The thoracic lymph in this case showed considerably 
 higher than the neck lymph, part of which may have been due to the 
 serum in the lymph, which came either through the increased permea- 
 bility of the capillary walls due to the action of the anesthetic or to 
 trauma, or to the natural anastomoses between the lymphatics and 
 the blood vessels of the splanchnic area. 
 
 The above experiment seems to indicate that the passage of such 
 substances as bacterial agglutinins from the blood to the lymphs is 
 a relatively rapid process, for the concentration of these bodies was 
 the same in the body fluids in 4^ hours as in 24 hours after passive 
 immunization. 
 
 From the results obtained the following conclusions seem justified: 
 
 i. Agglutinins for the typhoid bacillus are found in the serum and 
 thoracic lymph of normal cats in approximately equal amounts. 
 
W*a 4 
 
 i: 
 
 CONCENTRATION OF ANTIBODIES 149 
 
 They may or may not be present in the neck lymph and the pericardial 
 fluid. They are not found in the cerebrospinal fluid nor the aqueous 
 humor. 
 
 2. Agglutinins for the typhoid bacillus are found in the serum, 
 neck lymph, and the thoracic lymph in normal dogs in relatively 
 higher concentrations than in the same fluids in normal cats. They 
 are most concentrated in the serum, are in nearly equal concentration 
 in the two lymphs. They may or may not be found in the pericardial 
 fluid. They are found neither in the cerebrospinal fluid nor the 
 aqueous humor. 
 
 3. Agglutinins for typhoid bacilli are found in actively immune 
 cats in the serum, thoracic lymph, neck lymph, and pericardial 
 fluid in decreasing concentration in the order mentioned. If found 
 in the cerebrospinal fluid and aqueous humor there are only traces. 
 
 4. Agglutinins for typhoid bacilli are found in actively immune 
 dogs in the serum, thoracic lymph, neck lymph, and pericardial 
 fluid, usually in decreasing concentration in the order named. Serum 
 and thoracic lymph may show an equal concentration. Cerebrospinal 
 fluid shows no agglutinins in a dilution of i : 10. Aqueous humor 
 may or may not show agglutinins in a dilution of i : 10. 
 
 5. Immunization of a dog to rabbit blood does not increase the 
 concentration of agglutinins for typhoid bacilli in the body fluids 
 above the normal. 
 
 6. In the passively immunized animal the agglutinins for typhoid 
 bacilli pass readily from the blood stream into the lymphs, usually in 
 greater concentration into the thoracic lymph than into the neck 
 lymph, but the concentration may be equal in the two, showing the 
 permeability of the two systems to be the same. They do not pass 
 into the pericardial fluid, cerebrospinal fluid, nor the aqueous humor. 
 
 7. The passage of the agglutinins from the serum to the lymphs 
 in the passively immunized animal is a relatively rapid process, and 
 no difference is shown between the concentration at the end of 4^ 
 hours and 24 hours. 
 
 OPSONINS. 
 
 We made a quantitative study of the bacterial opsonins and the 
 hemopsonins in the body fluids of normal and immunized dogs. 
 
150 FRANK C. BECHT AND JAMES R. GREER 
 
 Since the discovery of the opsonins by Wright and Douglas44 there has been a 
 great deal of work done with these bodies, and as a result there has arisen an extensive 
 and conflicting literature. It is beyond the scope of this paper to undertake a general 
 discussion of this subject. We will confine ourselves, therefore, to those results which 
 most nearly relate to our problem. So far as we have been able to determine, there has 
 been up to this time no study of the relative concentration of the opsonins in the different 
 body fluids of the same animal. 
 
 Wright and Reid45 found that exudates may contain little or no opsonin. Opiess 
 found that the exudates following the injection of bacteria or turpentine were practically 
 opsonin free if unmixt with blood. Bohme 6 reports 15 cases of pleural and peritoneal 
 exudates showing a concentration of the opsonins ranging from less than half to even a 
 greater concentration than that of the serum. He also examined the edema fluid in 
 seven persons. In two of these cases there was very little opsonin while the remaining 
 five showed a considerable amount, but in no case was it equal to that of the serum. 
 This same investigator examined the cerebrospinal fluid in 16 cases and obtained opsonic 
 indices varying from 8 per cent to 76 per cent. He notes that these fluids were free from 
 blood. Bohme tries to correlate the concentration of the opsonin in these cases with 
 the amount of the protein in the fluid. 
 
 Levaditi and Inman 21 and others have shown that while the aqueous humor 
 normally contains little or no opsonin, yet that secured after a previous withdrawal 
 contains the opsonin. 
 
 Methods. The method of securing the fluids was the same as that described in 
 the foregoing. 
 
 The leukocytes were from the pleural exudate of young, healthy dogs which 24 
 hours previously had been given intraplural injection of a suspension of aleuronaut 
 in sterile 0.9 per cent NaCl solution. The exudate was secured after bleeding the 
 animal to death, and was drawn into a warm solution of i per cent sodium citrate in 
 0.9 per cent NaCl. The leukocytes were centrifugated out, and washed a second time 
 in warm, sterile salt solution. 
 
 The bacterial emulsion was obtained by suspending in 0.9 per cent NaCl solution 
 24-hour slant agar cultures of Staph. aureus. This emulsion was filtered through 
 absorbent cotton in order to remove the clumps and was then made up to the desired 
 opalescence. In all of the experiments a fairly rich suspension of the bacteria was 
 used, but no attempt was made to standardize this emulsion, and comparison of 
 individual experiments, except where the same suspension of leukocytes and bacteria 
 were used, would not be warranted. 
 
 The technic was essentially that described by Walker with slight modification. 
 This method in our hands gave the most satisfactory results. Dilutions of the various 
 body fluids were used in the same way as the whole fluid. The usual time for incuba- 
 tion was 20 minutes. Care was taken to make the smear from the incubated mixture 
 as soon after the period of incubation as possible, so that the time factor was as nearly 
 equal as it could be kept. We here incur a slight source of error in the difference in 
 the age of the material, for BeattieS has shown that the older citrated leukocytes are 
 up to 24 hours, the greater the phagocytosis, but the maximum difference in time 
 between the first and last smear was never greater than one hour. Great care was 
 taken to keep the leukocytic suspension of the same consistency throughout the experi- 
 ment. The leukocytes were used as fresh as possible, usually in about two hours or 
 less after removal from the animal's body. The number of leukocytes varied from 
 
CONCENTRATION OF ANTIBODIES 
 
 60 to 100 for each dilution, but the number was kept constant for each experiment. 
 The stains used were carbol-thionin and Giemsa's blood stain, but the same stain was 
 used throughout the same series of fluids. 
 
 Inasmuch as there has been so much criticism of the opsonic methods we made 
 the following tests of the accuracy of our observations. Using the same fluids, leuko- 
 cytes, and bacterial suspension, duplicate tests were made. The slides were labeled 
 in such a way that the person making the count had no way of knowing what the slide 
 contained, thus eliminating the personal equation. The result of this test was that 
 while most of the duplicates agreed fairly closely there were cases that varied as much 
 as 25 per cent, but the majority of the counts showed a much closer agreement. 
 
 Bacteriopsonins. We give the results of an experiment which we 
 consider as typical so far as concerns opsonin for Staph. aureus in 
 normal dogs. 
 
 TABLE 12. 
 COMPARATIVE STUDY OF THE OPSONIN FOR STAPH. AUREUS IN THE BODY FLUIDS OF A NORMAL DOG . 
 
 Dilution 
 
 Serum 
 
 Neck 
 Lymph 
 
 Thoracic 
 Lymph 
 
 Pericardia! 
 Fluid 
 
 Cerebro- 
 spinal Fluid 
 
 Aqueous 
 Humor 
 
 Whole 
 
 3 4 
 
 3 5 
 
 
 o 81 
 
 
 i 08 
 
 1:10 
 
 0.30 
 
 0.16 
 
 0.58 
 
 0.32 
 
 
 
 Control = 0.36. 
 
 It will be noted that in this experiment the opsonin had practically 
 disappeared in the dilution of i : 10 in all of the fluids. In the undi- 
 luted fluids the concentration of the opsonin in the serum, neck, and 
 thoracic lymph was nearly the same, but there was a slight excess in 
 favor of the thoracic lymph. The pericardial fluid, cerebrospinal 
 fluid, and the aqueous humor contained opsonin in a very much lower 
 concentration. 
 
 In four of nine normal animals the concentration of the opsonin 
 in the serum was considerably higher than in the thoracic and the 
 neck lymph. In three of nine cases the concentration was practically 
 equal in the serum and the two lymphs. In five of eight experiments 
 the amount of opsonin in the neck lymph was considerably less than 
 in the serum and thoracic lymph. It is thus evident, that, as far as 
 these three fluids are concerned, the concentration is greatest, on the 
 average, in the serum, least in the neck lymph, while the thoracic 
 lymph occupies an intermediate position. 
 
 Opsonin was found in the cerebrospinal fluid of four of seven dogs, 
 but in every case it was in decidedly smaller amounts than in the 
 serum and lymphs of the same animal. In the other three animals 
 there was no opsonin in this fluid. 
 
FRANK C. BECHT AND JAMES R. GREEK 
 
 The aqueous humor contained opsonin in five of eight cases, but 
 in only one of these did the concentration approach that of the serum 
 of the same animal. The remaining four showed very much less 
 phagocytosis, while three experiments gave negative results. 
 
 Opsonin was found in the pericardial fluid in two of seven dogs 
 in considerable amounts, in one it was practically equal to the neck 
 lymph of the same animal. In one of the remaining animals there 
 was a trace of opsonin. The other four animals contained no opsonin 
 in this fluid. 
 
 It is difficult to increase to any marked extent the concentration 
 of the opsonin for Staph. aureus in a dog by immunization. 
 
 We submit below two experiments in detail which we consider 
 quite typical of our results. 
 
 TABLE 13. 
 COMPARATIVE OPSONIC POWER FOR STAPH. AUREUS OF THE BODY FLUIDS OF IMMUNIZED DOGS. 
 
 (Both of these dogs received subcutaneously six slant agar cultures of Staph. aureus suspended in 
 0.9 per cent NaCl solution. Ten days later the fluids were all secured in apparently perfect condition. 
 Fluids kept over night in ice-box. Bacteria from a 24-hour slant agar growth. Incubation 20 minutes. 
 Carbol-thionin. Same bacterial suspension and leukocytes in both experiments.) 
 
 
 Dilution 
 
 Serum 
 
 Neck 
 Lymph 
 
 Thoracic 
 Lymph 
 
 Pericar- 
 dial 
 Fluid 
 
 Cerebro- 
 spinal 
 Fluid 
 
 Aqueous 
 Humor 
 
 Dog I 
 
 Whole 
 
 
 
 
 
 i 8s 
 
 
 
 i : 10 
 
 4-Si 
 
 i-7S 
 
 3-Si 
 
 i-33 
 
 0.88 
 
 0.50 
 
 
 1:50 
 
 1-75 
 
 0.86 
 
 i .01 
 
 0.22 
 
 0.28 
 
 0.28 
 
 Dog II 
 
 Whole 
 
 10.07 
 
 6.67 
 
 8-'3 
 
 4.18 
 
 0.92 
 
 i-33 
 
 
 1:10 
 
 4.21 
 
 1.50 
 
 2.88 
 
 1-47 
 
 0.22 
 
 * 0.30 
 
 
 1:50 
 
 O.po 
 
 0.38 
 
 0.52 
 
 0.17 
 
 0.22 
 
 0-35 
 
 Control for both=o.22. 
 
 It will be noted that the results of these experiments are com- 
 parable with those with respect to the other antibodies studied. In 
 the two experiments cited above the concentration of the opsonin is 
 considerably higher in the serum than in the other fluids. Of the 
 two lymphs there is a slight excess in that from the thoracic duct over 
 that from the cervical lymphatics. In both of these experiments 
 while there is considerable opsonin in the pericardial fluid yet it is 
 less than in the serum and the lymphs. In both of these experiments 
 the cerebrospinal fluid and the aqueous humor contained opsonin, 
 but in very much smaller amounts than in the corresponding serum 
 and lymphs. 
 
 Conclusions. There is a considerable amount of opsonin for 
 
CONCENTRATION OF ANTIBODIES 153 
 
 Staph. aureus in the body fluids of normal dogs. The amount found 
 in immune dogs is quite comparable with this, as regards both the 
 amount and the relative distribution. 
 
 Opsonin may be present in considerable concentration in all of 
 the body fluids studied by us, but in the serum, thoracic, and neck 
 lymphs they are always found in greater concentration than in the 
 other fluids studied. Of these fluids the serum usually contains the 
 greatest amount, and the thoracic lymph usually more than the cor- 
 responding neck lymph. The pericardial fluid, cerebrospinal 
 fluid, and the aqueous humor may or may not contain opsonin, but 
 rarely in amounts comparable to those of the serum and lymphs. 
 
 HEMOPSONINS. 
 
 It has long been noted that red corpuscles were taken up by phagocytes under 
 certain conditions but it was not until opsonins were discovered by Wright and Douglas 
 that the role of the serum in phagocytosis ^vvas understood. Neufeld and Topfer33 
 showed that there was something termed by them "hemotropic substance" in the 
 serum of a rabbit immune to goat blood, which caused the phagocytosis of goat erythro- 
 cytes by guinea-pig leukocytes in vitro. Barrats found the same antibody in the serum 
 of doves immune to hen blood. He proved these bodies thermostable. Hektoen 16 
 was the first to point out the similarity of these substances to bacterial opsonins and 
 suggested the name "hemopsonins." Neufeld and Topfer, Barrat, Keith, and Hektoen 
 all cite evidence to show that these substances are distinct from the amboceptors of the 
 hemolysins. The most extensive recent work on the hemopsonins is by Hektoen. ? 
 He shows that normal serum may contain hemopsonins for heterologous or even 
 homologous erythrocytes; that immune hemopsonins are highly resistant to heat; and 
 are in part specific and in part non-specific. 
 
 Methods. The body fluids of the dogs were secured as described earlier in this 
 paper, and inactivated by heat at 55 C. for 30 minutes. Rat corpuscles were used 
 throughout the work. They were made up to 5 per cent suspension after careful 
 washing in 0.9 per cent NaCl solution. The technic followed was to measure into 
 each of a series of small test tubes quantities of fluids ranging between 0.2 c.c. and 
 0.002 c.c. and then adding enough salt solution to make 0.2 c.c. To this was then 
 added 0.4 c.c. of a mixture of equal quantities of rat corpuscles and leukocytic suspen- 
 sion. In this way dilutions varying between 1:3 and 1:300 were secured. The tubes 
 were incubated in a shaker for one hour. Then the contents were mixt thoroughly, 
 smears made, and fixt in absolute alcohol for one hour, and then stained with Giemsa's 
 stain for 30-60 minutes. The percentage was figured from the number of the leuko- 
 cytes actively phagocytic. No attempt was made to count the number of erythrocytes 
 engulfed per leukocyte. In every case 500 leukocytes were counted. Our figures will 
 represent, then, the percentage of leukocytes phagocytic, and shows only the activity of 
 thermostable hemopsonins. 
 
 Early in the work an experiment was performed to test the accuracy of the method. 
 Two. complete sets of tests were made with the fluids of a normal dog, smears were 
 
FRANK C. BECHT AND JAMES R. GREER 
 
 made, fixt, and stained in the ordinary way. Labels were then attached to the slides 
 and one of us numbered the slides in a haphazard manner and kept a record of the 
 numbering used. The slides were all mixt together and then counted by the other. 
 In this way the subjective factor would not enter at all. The results were highly 
 satisfactory, for by counting 500 leukocytes and estimating the percentage from that 
 number, the corresponding slides agreed almost exactly. In one case there was a 
 difference amounting to over 2 per cent. 
 
 NORMAL DOGS. The first point was to determine the concentra- 
 tion of the hemopsonins in the body fluids of normal animals. As 
 a typical experiment we cite Table 14. 
 
 TABLE 14. 
 COMPARATIVE HEMOPSONIC POWER FOR RAT CORPUSCLES OF THE BODY FLUIDS OF A NORMAL DOG. 
 
 Dilution 
 
 Serum 
 
 Neck 
 Lymph 
 
 Thoracic 
 Lymph 
 
 Pericardial 
 Fluid 
 
 Cerebro- 
 spinal Fluid 
 
 Aqueous 
 Humor 
 
 1:3... 
 
 3 20 
 
 i . 20 
 
 0.60 
 
 o.oo 
 
 0.40 
 
 o. 20 
 
 i-6 
 
 O.2O 
 
 o oo 
 
 o 20 
 
 o oo 
 
 
 
 1:12 
 
 0.20 
 
 0.00 
 
 0.00 
 
 0.00 
 
 0.00 
 
 0.00 
 
 Control =0. 
 
 This table shows that the concentration of hemopsonins in the 
 various body fluids runs practically parallel with that of the other 
 antibodies. It is highest in the serum. The two lymphs are lower 
 but practically equal. There are also traces in the other fluids in this 
 case. In another normal animal the same conditions were found. 
 
 ACTIVELY IMMUNIZED DOGS. In establishing immunity for the 
 work on hemopsonins we followed a uniform technic. Each animal 
 received intravenously 0.5 c.c. of a 5 per cent solution of washed rat 
 corpuscles per kilo of body weight. The animal was operated the 
 loth day after the injection. We cite the experiment in Table 15 
 as typical. 
 
 TABLE 15. 
 COMPARATIVE HEMOPSONIC POWER FOR RAT CORPUSCLES OF THE BODY FLUIDS OF AN IMMUNE DOG. 
 
 Dilution 
 
 Serum 
 (Normal) 
 
 Serum 
 
 Neck 
 Lymph 
 
 Thoracic 
 Lymph 
 
 Pericar- 
 dial Fluid 
 
 Cerebro- 
 spinal 
 Fluid 
 
 Aqueous 
 Humor 
 
 3 
 6 
 
 24.4 
 
 45-o 
 3 8 4 
 
 43-4 
 28 o 
 
 39-4 
 28 " 
 
 25- 8 
 15 o 
 
 10.8 
 
 2.6 
 
 4.8 
 i .0 
 
 :i2 
 
 1.8 
 
 24.6 
 
 5 8 
 
 5-0 
 3 
 
 14.6 
 
 2 2 
 
 7.0 
 
 2 O 
 
 I . 2 
 O. 2 
 
 0.0 
 O.O 
 
 :6o 
 
 0.0 
 
 2.6 
 
 2.0 
 
 o 8 
 
 0.0 
 O O 
 
 0.0 
 O O 
 
 O.O 
 O.O 
 
 0.0 
 O.O 
 
 = 300 
 
 
 0.2 
 
 0.0 
 
 0.0 
 
 
 
 
 Table 15 shows that a considerable degree of immunity was 
 reached, the immune serum producing a much higher degree of 
 
CONCENTRATION OF ANTIBODIES 
 
 phagocytosis than the normal. The concentration was highest in 
 the serum; the two lymphs are practically parallel with a slight balance 
 in favor of the neck lymph, because it not only produces a higher 
 percentage of phagocytosis in the lowest dilution, but also causes 
 phagocytosis in a much higher dilution. The pericardial fluid shows 
 a somewhat lower concentration than the lymphs; the cerebrospinal 
 fluid and the aqueous humor contain the opsonin in lower concentra- 
 tion than the pericardial fluid. 
 
 In six experiments with immune dogs the concentration of hemop- 
 sonins ran the course shown in this experiment. We find some 
 variations in the cerebrospinal fluid and the aqueous humor. In 
 three out of five cases where the cerebrospinal fluid and aqueous 
 humor were tested the concentration of the hemopsonins was higher 
 in the former than in the latter. The fourth case is the one cited in 
 Table 15, and in the fifth case neither showed hemopsonins. In 
 one of six cases the hemopsonins of the pericardial fluid and aqueous 
 humor were practically equal in concentration. 
 
 PASSIVELY IMMUNE DOGS. Passive immunity was produced in 
 the same way described under bacterial agglutinins. We cite here 
 as an example the results obtained in the case of a dog immunized 
 by injecting intravenously 300 c.c. of the blood of an immune dog 
 after first removing 260 c.c. by bleeding. 
 
 TABLE 16. 
 
 COMPARATIVE HEMOPSONIC POWER FOR RAT CORPUSCLES OF THE BODY FLUIDS OF A DOG PASSIVELY 
 
 IMMUNIZED TO RAT BLOOD. 
 
 Dilution 
 
 Serum 
 (Normal) 
 
 Serum 
 
 Neck 
 Lymph 
 
 Thoracic 
 Lymph 
 
 Pericar- 
 dial Fluid 
 
 Cerebro- 
 spinal 
 Fluid 
 
 Aqueous 
 Humor 
 
 3 
 
 
 ,5 A 
 
 T r 5 
 
 
 , f. 
 
 _ Q 
 
 
 :6 
 
 
 
 7 8 
 
 
 
 
 
 : 12 
 
 4 6 
 
 !? fi 
 
 i 6 
 
 2 6 
 
 
 
 
 :24 
 
 
 2 6 
 
 
 i 6 
 
 o .0 
 
 O.O 
 
 
 :6o 
 
 
 i 8 
 
 
 
 o.o 
 
 
 
 : 120 
 
 
 o 6 
 
 
 
 
 
 
 300 
 
 
 
 
 
 
 
 
 The results in our experiment in passive immunity show first, an 
 increase in the concentration of the hemopsonins in the serum, and 
 second, a marked increase in the concentration of the antibodies in 
 the thoracic lymph, bringing it up to a concentration equal to that of 
 the serum, while the concentration of the hemopsonins in the neck 
 
156 FRANK C. BECHT AND JAMES R GREER 
 
 lymph remains low and from a comparison with other animals we 
 would say that the injection of the highly immune serum had had no 
 effect upon the concentration of the hemopsonins in the neck lymph. 
 The same is true of the pericardial fluid, the cerebrospinal fluid, and 
 the aqueous humor, where the concentration is no higher than in the 
 normal dog. It must be remembered that this dog showed a relatively 
 high hemopsonic power before immunization by injection of the 
 immune blood. We have other experiments which show the same 
 phenomenon. 
 
 From these experiments we draw the following conclusions : 
 
 1. Hemopsonins are found in the body fluids of normal dogs. 
 The concentration is highest in the serum, lower in the thoracic and 
 neck lymph, which run almost parallel, and are found in the other 
 body fluids only in traces. 
 
 2. By a process of immunization the hemopsonins of the body 
 fluids can all be increased. The order of descending concentration 
 is the serum, neck lymph, thoracic lymph, pericardial fluid, aqueous 
 humor, and the cerebrospinal fluid. Sometimes the arrangement in 
 the scale of the last two is reversed. 
 
 3. Hemopsonins can pass from the blood into the thoracic lymph, 
 but apparently they do not pass into the lymph of the head region. 
 The concentration of the hemopsonins in the pericardial fluid, cere- 
 brospinal fluid, and aqueous humor is not increased in 24 hours over 
 the normal by passive immunization as in our experiments. 
 
 GENERAL CONCLUSIONS. 
 
 In the normal animal the concentration of hemolysins, hemag- 
 glutinins, bacterial agglutinins, bacterial opsonins, and hemopsonins 
 decrease in the body fluids in the following order: serum, thoracic 
 lymph, neck lymph. Traces of hemagglutinins, bacterial agglutinins, 
 and opsonins are found in the pericardial fluid. Traces of opsonins 
 are found in the cerebrospinal fluid and aqueous humor. No pre- 
 cipitins for rabbit serum were found in any of the body fluids. 
 
 Immunization increases the concentration of the antibodies named 
 above in the fluids in which they are found in the normal animal. 
 Hemolysins are sometimes found in the pericardial fluid, and in a 
 few cases traces of bacterial agglutinins in the cerebrospinal fluid and 
 
CONCENTRATION OF ANTIBODIES 157 
 
 aqueous humor. Protein precipitins are found in the serum, thoracic 
 lymph, and neck lymph, the concentration being lower in the last than 
 in the first two. 
 
 The immunity is specific in the other body fluids as in the serum. 
 
 The introduction of an immune blood into a normal animal 
 increases the concentration of the bacterial agglutinins in both the 
 lymphs above the normal. The introduction of an immune blood 
 does not increase the concentration of hemopsonins in the neck lymph, 
 altho the concentration in the thoracic lymph is markedly increased. 
 The concentration of bacterial agglutinins and hemopsonins in the 
 cerebrospinal fluid is not modified by passive immunization. 
 
 The passage of antibodies from blood to the lymph is a relatively A 
 rapid process. 
 
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158 FRANK C. BECHT AND JAMES R. GREER 
 
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 36. PAGANO, Arch. Ital. di Biol., 1894, 20, p. no. 
 
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