An antibody that binds to primary specific pocket-associated structure in the active site of bovine thrombin.
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 IgG(1) and kappa 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.
Kinjoh
Kiyohiko
K
1st Department of Physiology, School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan. kkinjoh@med.u-ryukyu.ac.jp
Nakamura
Mariko
M
Zeng
Gang
G
Kosugi
Tadayoshi
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D001120:Arginine / Q000031:analogs & derivatives; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005340:Fibrinogen / Q000378:metabolism; D006825:Hybridomas / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D010875:Pipecolic Acids / Q000494:pharmacology; D011485:Protein Binding; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D013154:Spleen / Q000166:cytology; D013449:Sulfonamides; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D010875:Pipecolic Acids; D013449:Sulfonamides; D005340:Fibrinogen; D001120:Arginine; D013917:Thrombin; C027962:diisopropylphosphoryl-thrombin; C031942:argatroban