An Antibody that Binds to Primary Specific Pocket-Associated Structure in the Active Site of Bovine Thrombin

Abstract

We attempted to produce a monoclonal antibody (MAb) against the active site of native thrombin. Bovine thrombin was treated with diisopropyl fluorophosphate, and prepared diisopropylphosphoryl-thrombin was used for the immunization to BALB/c mice. Spleen cells of immunized mice were hybridized with mouse myeloma cells P3U1, and a hybridoma clone CC2, which produced a MAb against bovine thrombin was established. The MAb produced by hybridoma clone CC2 (MAb_CC2), consisting of IgG₁ and κ light chain, was purified using protein A affinity chromatography. Purified MAb_CC2 prolonged the fibrin forming time of bovine thrombin and inhibited the release of fibrinopeptide A from rabbit fibrinogen. In addition, it was found that argatroban partially, but competitively, interfere the binding between MAb_CC2 and bovine thrombin. It was then considered that MAb_CC2 would bind to the molecular structure associating primary specific pocket in the active site of bovine thrombin.