Generation of hybridoma cell lines producing monoclonal antibodies against Toxoplasma gondii or rabies virus following fusion of in in vitro-immunized spleen cells with myeloma cells

Abstract The present experiments proved that specific antibody against Toxoplasma gondii or rabies virus (RV) could be induced in vitro in spleen cells from normal (i.e. not deliberately immunized) mice following seven days exposure to the relevant antigen presented in association with syngeneic macrophages. Using such in vitro -primed spleen cells as fusion partners with non-secretor myeloma cells, we were able to isolate hybridomas secreting anti-toxoplasma or anti-RV antibody with high frequency. A total of 12 cloned continuously propagable hybridoma lines were established, which secreted T. gondii -specific antibodies; 9 of the antibodies were directed against trophozoite membrane-associated antigen(s) and the remaining 3 could recognize soluble cytoplasmic antigen(s). Among the panel of 9 clonal antibodies reacting with trophozoite membrane antigen(s), only 4 showed reactivity in the Sabin-Feldmann dye test (complement-dependent cytotoxicity test). Fifteen stable cloned hybridomas obtained from fusions between myeloma cells and spleen cells primed in vitro against RV secreted antibodies which reacted with intact RV in radioimmunoassay. Since 10 of these 15 different hybridoma-derived antibodies could neutralize RV and also stained the membranes of RV-infected cells in the indirect immunofluorescence test, it can be postulated that they were directed against the coat glycoprotein (G-protein) of RV. The remaining 5 monoclonal antibodies which failed to neutralize RV stained the intracytoplasmic inclusions exclusively. This suggests that they may be recognizing the RV nucleocapsid determinant(s). Immunochemically, 17 monoclonal antibodies obtained in the present study (7 against T. gondii and 10 against RV) were of IgM class, 3 antibodies (2 against T. gondii and 1 against RV) were of IgG1 subclass and 7(3 against T. gondii and 4 against RV) were of IgG2a subclass. The continuously propagable hybridomas established in the present study provide a useful source of large quantities of homogeneous antibodies specific for T. gondii and RV.