title: Monoclonal antibodies to the chemotherapeutic agent methotrexate: production, properties and comparison with polyclonal antibodies.
abstract: Hybridomas that secrete monoclonal antibodies to the chemotherapeutic agent methotrexate (MTX) were produced by fusion of FO myeloma cells with splenocytes obtained from BALB/c mice immunized with L-MTX covalently bound to keyhole limpet hemocyanin (KLH). When the MTX/KLH molar ratio was 430/1 or 530/1 (high MTX series), 238/412 (58%) or 157/405 (39%), respectively, of antibody producing hybridomas secreted only antibodies to MTX. In contrast, only 15/412 (4%) or 54/405 (13%) hybridomas secreted only antibodies to KLH. When the molar ratio was 50/1 (low MTX series), only 14/343 (4%) of the wells contained anti-MTX and 93/343 (27%) contained antibodies to KLH. The predominance of anti-MTX over anti-KLH secretors in the 2 high MTX series, and the reverse predominance of anti-KLH secretors over anti-MTX hybridomas in the low MTX series was highly significant (P less than 0.001). The anti-MTX antibodies were mainly IgG1 with fewer IgG2a and IgG2b subclasses. All were kappa chain isotypes. No IgM or IgA antibodies were detected. Titers and binding constants of monoclonal anti-MTX antibodies in ascites fluid obtained from mice injected with selected cell lines were higher than those of conventional rabbit, mouse, or goat antibodies. Based on results of competitive inhibition radioimmunoassays, the ascites monoclonal antibodies were at least as specific as the conventional antibodies for MTX compared to structurally related compounds. IgG2a and IgG2b antibodies had high titers in complement mediated hemolysis of sheep erythrocytes that contained MTX on the cell surface (E-MTX) and also bound 125I-labeled protein A when complexed to MTX on these cells. IgG1 antibodies were relatively weak in the hemolysis assay and failed to bind 125I-labeled protein A directly. Hemolytic activity and 125I-labeled protein A binding were markedly enhanced when E-MTX cells were treated sequentially with IgG1 anti-MTX then rabbit IgG antibodies to mouse IgG1.
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000032:analysis / Q000096:biosynthesis*; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology; D001202:Ascitic Fluid / Q000276:immunology; D001667:Binding, Competitive; D005260:Female; D006041:Goats; D006433:Hemocyanins; D006461:Hemolysis; D006825:Hybridomas / Q000276:immunology; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000145:classification; D008727:Methotrexate / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D011863:Radioimmunoassay; D013205:Staphylococcal Protein A / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin; D008727:Methotrexate