key: cord-0008956-egbs1rto
authors: Leidinger, E.; Kramberger-Kaplan, E.; Gemeiner, Manfred
title: Optimization of culture conditions for feline × murine heterohybridomas
date: 2002-11-13
journal: Comp Immunol Microbiol Infect Dis
DOI: 10.1016/0147-9571(93)90158-2
sha: 1efc431c39a8f2eb00f2601e8acb4b879d965c6a
doc_id: 8956
cord_uid: egbs1rto

Feline splenocytes were fused to the murine myeloma lines NSO or Ag8. Autologous serum and taurine were used as media supplements for the cat × mouse heterohybridomas. The best results were obtained by the use of NSO as fusion line with taurine-supported media.

Feline Infectious Peritonitis (FIP) is a severe disease in cats caused by a coronavirus (FIPV) and characterized by the formation of FIP-associated immune complexes. Immunoglobulin G (IgG) is considered to be a major component of these complexes [1] . Unlike many other mammalian species, in cats only two IgG subclasses, designated IgG~ and IgG2, have been postulated so far [2, 3] . For a better understanding of the involvement of these complexes in the immunopathology of FIP we were interested in the two proposed IgG-subclasses.

For the subclass determination of human immunoglobulins the use of paraproteins, secreted by myelomas (plasmacytomas) in vivo or by cell lines adapted to in vitro growth, proved to be extremely useful in the past [4] . Compared to other species, the occurrence of feline myelomas is a very rare event. Most feline paraproteins which have been more closely characterized until now, have been determined as IgG without further discrimination of IgG-subclasses [5, 6] .

Another possibility to obtain antibodies belonging to one distinct subclass is offered by the hybridoma technology developed by Krhler and Milstein [7] for the production of monoclonal antibodies (mAb). This approach is not directly applicable in cats, because no *Author for correspondence. [8] for the characterization of bovine IgG subclasses by bovine x murine heterohybridomas, and by Galakhar et al. [9] for IgG subclass studies in minks using mink x mouse heterohybridomas.

In this preliminary study we tried to optimize the outgrowth efficiency of feline x murine mAb by heterohybridomas.

NSO were obtained from European Culture Collection (ECACC No. 85011432), P3X63-Ag8.653 (Ag8) were from ATCC (ATCC CRL 1580). The cells were propagated in a culture medium consisting of RPMI-1640 supplemented by 7% heat-inactivated foetal bovine serum (Boehringer), 2mM L-glutamine, l mM of sodium pyruvate, 10IU/ml penicillin, 10 #g/ml streptomycin and 5 x 10 2 mM 2-mercaptoethanol. Both cell lines do not synthesize antibodies and were checked for HAT-sensitivity regularly.

Lymphocytes were obtained from seven cats which had to be sacrificed due to severe non-infectious diseases. The spleens were removed immediately after euthanasia and immersed in 70% ethanol. The splenocytes were prepared as single cell suspensions from small pieces of the spleen, washed three times with cold PBS containing antibiotics and counted in a haemocytometer (Biirker-Tiirk ruling). The percentage of viable cells was estimated by 0.5% trypan blue dye exclusion test.

An aliquot of the isolated lymphocytes was frozen for further experiments in a medium containing 20% foetal bovine serum and 10% DMSO (Sigma), at a density of 1 x 10 7 cells/ml according to Schreier et al. [10] and stored in liquid nitrogen.

Cat serum was collected by venous puncture from healthy donors, incubated for 25 min at 56°C for complement-inactivation, filter sterilized twice and frozen in small aliquots at -30°C and used at a final concentration of 5% (v/v). Taurine (2-aminoethanesulphonic acid, Sigma) was used at a final concentration of 0.8 mM (0.1 mg/ml culture medium).

Fusions were done following standard protocols [11, 12] with 50% (v/v) polyethylene glycol 1500 (Boehringer), using the myeloma lines Ag8 or NSO, respectively, at the logarithmical phase of growth. Fused cells were seeded at a concentration of 5 X 106 lymphocytes/ml in 48-well culture plates (Costar). The culture medium described above was used, additionally supplemented by 1 × 10-4M hypoxanthine, 4 × 10 7M aminopterin and 1.6 x 10 -5 M thymidine (HAT, Boehringer) and 25% (v/v) macrophageconditioned medium [13] instead of feeder cells (=complete culture medium, CCM). In the initial phase of growth the heterohybridomas were cultured as described in Table 1. HAT was replaced by HT without macrophage-conditioned medium 12-15 days and 

The outgrowth efficiency (E) was used as a parameter for the comparison of fusion lines and media supplements. Culture plates were screened for clonal growth of the hybridomas 10-20 days after fusion and then about once a week. Wells which contained colonies consisting of at least four to eight large, contiguous cells were counted positive. The outgrowth efficiency was calculated by the formula: Growth rates and size of heterohybridomas and parental lines Numbers of parental lines and heterohybridomas were adjusted to 5 × 105 cells/ml and 1 ml of cell suspension was added in triplicate in a 24-well culture plate. Cell numbers were counted at 24, 48, 72 and 92 h by a haemocytometer and an automated counter (Cobas Minos Vet, Roche); the results were expressed as means of the three wells at each time point. The cell size was determined by microscope from native monolayers.

Culture supernatants were screened by ELISA for feline IgM and IgG production 1 week after the second medium replacement. High affinity 96-well assay plates (Greiner) were coated overnight at 4°C with 50/~1 culture supernatant diluted with the same volume of a carbonate-bicarbonate buffer (pH 8.9). Supernatants from control wells (NSO or Ag8) were taken as blanks, CCM containing 5% cat serum was used as positive control. Goat anti-cat-IgM (H-and L-chain specific, 1 : 10,000, Accurate Chemicals) or goat anti-cat-IgG (H-chain specific, 1:50,000, Bethyl) was used as first antibody. After each step the plates were washed three times with PBS containing 0.01% Tween 20 using an automatic ELISA-washer (SLT). The assay was developed with an affinity purified rabbit anti-goat-IgG (H-chain specific, 1: 20,000, Accurate Chemicals) conjugated to horseradish peroxidase with OPD as substrate.

Chromosome preparations were made following standard procedures. The metaphase spreads were examined after G-banding [14] . For each cell line to be karyotyped, the total number of chromosomes and the number of cat chromosomes were determined for 15 individual cells. Cat chromosomes were differentiated from mouse chromosomes on the basis of banding pattern and the position of the centromere [15] .

Twelve experiments resulted in 223 colonies (7.4% of wells seeded) which survived at least 15 days, 23 (0.8%) of them survived for several months and were stored in liquid nitrogen after propagation in HAT-medium for 2 weeks. Under the conditions described above, the fusion experiments did not result in more than one colony per well, i.e. there was one developing colony in 5 x 10 6 cells seeded. No heterohybridomas secreting feline IgM or IgG could be detected by ELISA.

The heterohybridoma cells were larger than their parental lines. Typically, one clone (37/5C5) was 15.13 _ 1.14#m dia, compared to 12.71 _ 0.69/tin for its parental line Ag8 (Fig. 1) . Depending on the parental line heterohybridomas generally multiplied slower [ Fig. 2(a), (b) ] and had a lower maximum cell density compared to NSO or Ag8.

Native lymphocytes showed > 95% viability, in frozen splenocytes viability was reduced to c. 40% as determined by 0.5% trypan blue dye exclusion test. The outgrowth efficiency of native and previously frozen splenocytes was compared for NSO-controls until day 15 after hybridization ( Table 2) .

Compared to the controls, autologous serum as well as taurine influenced the growth of some of the hybridomas during the first 12-15 days after fusion (Fig. 3 ).

The fusion of feline splenocytes with NSO or Ag8 caused an increase in the number of chromosomes of the resulting hybridomas, depending on the parental line (Table 3) . A variable number of feline chromosomes was found in different heterohybridoma clones (Fig. 4) .

Little is known about the specific needs of feline lymphoid cells in culture. Cat leukocytes and neutrophils, like human ones, contain a high intracellular concentration of taurine (22 and 26 mM respectively) [16] . Unlike other species, taurine is an essential amino acid in cats and the effects of feline taurine deficiency have been investigated during the last years [17] . It was shown, that a taurine-free diet has an impact on the development and function of the feline immune system in vivo [16] . Added to a lymphocyte transformation assay in vitro, taurine causes an increase in proliferation to Con A [18] . Human lymphoblastoid lymphocyte-derived ceils are also stimulated and proliferate in taurine-supplemented media [19] . In our experiments we found that the addition of taurine to culture media significantly improved the outgrowth efficiency for Ag8-derived hybridomas (Fig. 3) , but no marked effect was noticed on NSO-derived hybridomas.

Autologous serum was successfully used by Raybould and Takahashi [20] for the production and improvement of stability for rabbit x mouse heterohybridomas. Compared to controls, cat serum had a clear growth-supporting effect in Ag8-derived heterohybridomas, but when NSO was used as parental line, cat serum obviously had a rather growth-suppressive effect. The reasons for the different modes of action of cat serum and taurine on the two fusion lines are unknown so far, but the choice of the appropriate fusion line seems to be important for the production of interspecific hybridomas. Galakhar et al. [9] found the fusion lines Ag8 and NSO to be superior to Sp2/0-Agl4 in the yield of mink heterohybridomas. Other possible influences of Ag8 or NSO on heterohybridomas, such as antibody production could not be studied in the present experiments.

A number of HAT-resistant clones could be isolated from either fusion partners which contained cat chromosomes and were larger than their parental lines, proving that a fusion of feline and murine cells had taken place.

No clones secreting feline IgG (or IgM) could be detected for two possible reasons. (1) Splenocytes of unstimulated cats were used for our fusions experiments. According to literature some experiments using lymphocytes of unimmunized animals resulted in antibody-secreting heterohybridomas [21, 22] , others did not and showed reduced outgrowth efficiency [23] . (2) Early screening followed by rapid cloning is frequently recommended to stabilize secreting clones [22, 24] , but due to slow cellular growth ELISA screening could not be done earlier than 4 weeks after the fusion.

Experiments with in vivo stimulated (originated from FIP-infected animals) or in vitro stimulated cat splenocytes are in progress.

Somatic hybridization experiments (cat × mouse and cat x Chinese hamster) were reported by O'Brien and Nash [15] for the study of chromosomal linkage groups in cats, but no information about culture conditions was given. Therefore the chosen approach was useful in order to optimize the culture system and experiments to adapt some of the heterohybridoma clones described above for growth in 8-azaguanine are currently in progress. They should be valuable as HAT-sensitive fusion partners for the production of 2 ° heterohybridomas (hybridhybridomas). Such cell lines tend to be more stable than 1 ° heterohybridomas in the production of monoclonal antibodies [25] .

Virion polypeptide specificity of immune complexes and antibodies in cats inoculated with feline infectious peritonitis virus

Studies on Ig A immunoglobulins in man and animals. Thesis, Dept de medicine experimental

Chemical typing of immunoglobulins

Characterization of three feline paraproteins

The immunoglobulins of the cat

Continuous culture of fused cells secreting antibody of predefined specificity

Production and characterization of monoclonal bovine immunoglobulins G~, G 2 and M from bovine x murine hybridomas

Mink mouse hybridomas that secrete mink immunoglobulin G

Hybridoma techniques

Preparation of monoclonal antibodies: strategies and procedures

Antibody production by hybridomas

Macrophage-derived factor required by plasmazytomas for survival and proliferation in vitro

Giemsa staining patterns for identification of the pig mitotic chromosomes

Genetic mapping in mammals: chromosome map of domestic cat

Immunologic consequences of taurine deficiency in cats

Nutritional problems in cats: taurine deficiency and vitamin A excess

Evaluation of immunity in taurine-deficient cats

Taurine uptake by cultured human lymphoblastoid cells

Production of stable rabbit-mouse hybridomas that secrete rabbit mAb of defined specificity

Production and characterization of monoclonal antibodies to bovine immunoglobulin G 2

Rabbit-mouse hybridomas secreting intact rabbit immunoglobulin

Characterization of heteromyloma fusion partners which promote the outgrowth of porcine hybridomas

Bovine and ovine monoclonal antibodies to erythrocyte membrane determinants produced by interspecific (hetero-) myelomas

Murine/bovine hybridomas producing monoclonal antibodies to bovine red cell antigens