Monoclonal antibodies against murine gamma interferon

Monoclonal antibodies against murine immune interferon (IFN-gamma) were produced by fusing the murine nonsecreting myeloma cell line P3.X63.Ag8.653 with spleen cells from rats immunized with IFN-gamma-containing supernatants obtained by stimulating a T-cell lymphoma, L12-R4, with phorbol 12-myristate 13-acetate. Supernatants from a twice-cloned hybridoma (AN-18.17.24) were found to neutralize and to adsorb in depletion experiments up to 27 units of mouse IFN-gamma but not equivalent amounts of mouse leukocyte or fibroblast IFNs. The AN-18.17.24 monoclonal antibody neutralized to the same extent mouse IFN-gamma from different sources--namely, (i) concanavalin A-stimulated spleen cells, (ii) alloantigen-stimulated spleen cells, and (iii) monkey fibroblasts transfected with the cloned gene of murine IFN-gamma. Moreover, the monoclonal antibody displayed species specificity, since it did not neutralize IFN-gamma of human origin. Binding inhibition experiments with murine IFN-gamma preparations exposed to enzymatic or physicochemical degradation demonstrated that the protein moiety and not the carbohydrate residues were responsible for the binding to the AN-18.17.24 monoclonal antibody. Finally, this monoclonal antibody immunoprecipitated two molecular species of IFN-gamma of about 16.8 and 17.8 kilodaltons, respectively, from [35S]methionine- or [3H]glucosamine-labeled supernatants of stimulated L12-R4 cells.