PRODUCTION AND CHARACTERIZATION OF DOMAIN-SPECIFIC MONOCLONAL-ANTIBODIES AGAINST TISSUE-TYPE PLASMINOGEN-ACTIVATOR

Two mouse hybridoma lines (3E6 and WA3) have been isolated in order to generate specific antibody probes for fibrinolysis in bovine gonadal cells. Mice were immunized with human recombinant tissue plasminogen a ctivator (tPA) and the resultant spleen cells fused with SP2/0 myeloma s. Both the resultant monoclonal antibodies recognized human recombina nt tPA and bovine Sertoli cell tPA in a dose-dependent manner, and wer e identified as class IgG2b. The precise epitope recognized by each an tibody was characterized using modified enzyme-linked immunosorbent as says, immunoblotting analysis and protease assays. A synthetic inhibit or that irreversibly modifies the active site histidine of tPA (phe-pr o-arg-chloromethylketone) was used to identify antibodies which bind t o the tPA protease domain. Antibody 3E6 recognized this domain on the light chain of tPA, and will be useful for monitoring secreted tPA tha t is not complexed with its natural inhibitors. Antibody WA3 recognize d an epitope which is remote from the active centre, on the heavy chai n of tPA. Its binding to tPA obstructed the subsequent binding of fibr in, which indicates that WA3 binds to either the 'finger' or 'kringle 2' domain of tPA. This antibody has potential for monitoring tPA inter actions with fibrin or extracellular matrix proteins. Both antibodies have been used in the accompanying paper to investigate the hormonal r egulation of bovine Sertoli cell and granulosa cell fibrinolysis.