Characterization of four monoclonal antibodies to factor VIII coagulant protein and their use in immunopurification of factor VIII.

Four monoclonal anti-VIII:C antibodies were obtained from the fusion of the splenocytes of one Balb/C mouse with a specific activity ranging from 2.3 to 45,000 U/mg when purified from ascitic fluid. Only one antibody was able to inhibit completely Factor VIII:C in normal plasma. The four antibodies could bind Factor VIII:CAg in plasma and commercial concentrate both in liquid and solid phase, and were suitable for immunopurification of Factor VIII:C. Three antibodies competed with polyclonal anti-VIII:CAg Fab' in a liquid phase IRMA, and all of them were able to displace their own binding to Factor VIII:CAg. Competition studies between monoclonal antibodies for the binding to Factor VIII:CAg were performed and showed the recognition of different epitopes and various functional impact. These studies indicate that at least one antibody, with the lowest anti-VIII:C titer clearly recognizes a different epitope of VIII:C than those recognized by the others. Affinity constants ranged from 10(9) to 10(10) l/mole.