Monoclonal antibodies directed against human alpha-thrombin and the thrombin-antithrombin III complex.
Human alpha-thrombin was poorly immunogenic in Balb/c mice. Nevertheless, following fusion of spleen cells from a responding mouse with NS-1 cells, 8 mouse monoclonal antibodies against alpha-thrombin were isolated, and 6 were characterised. Five of these were isotype IgG2a, and one was IgG1. One, EST 1, bound thrombin only minimally, and was directed against a neoantigen on the thrombin-ATIII (T-AT) complex. This antibody also recognised a site on prothrombin, though with much lower affinity. Its binding was markedly temperature-dependent, indicating a requirement for molecular mobility. A second antibody, EST 4, would not bind the T-AT complex. It inhibited both the clotting and amidase activities of thrombin, and modification of the active site histidine, but not the active site serine, reduced the affinity constant of binding to EST 4. This antibody appears to be directed against an epitope in the vicinity of the enzyme active site. The epitopes for EST 1 and EST 4 were both remote from those of the other monoclonal antibodies, EST 2, 6, 7 and 8. These four competed with each other for binding to thrombin, and all inhibited clotting but not amidase activity. Thrombin binding was not affected by modification of the active site, though formation of the T-AT complex reduced the affinity of binding to EST 6 and EST 8. These monoclonals recognise epitopes in the region of the fibrinogen binding site.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D000915:Antibody Affinity; D000990:Antithrombin III / Q000276:immunology*; D005260:Female; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D011516:Prothrombin / Q000276:immunology; D011863:Radioimmunoassay; D013917:Thrombin / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000990:Antithrombin III; D011516:Prothrombin; D013917:Thrombin