Studies on a monoclonal antibody to human factor viii coagulant activity, with a description of a facile two-site factor VIII coagulant antigen assay.
IgG was purified from the ascites tumor fluid obtained from mice injected with a monoclonal cell line secreting antibody that inhibited VIII:C. With a modified Bethesda assay method (18 hr, 4 degrees C), the titer of the purified IgG was 14,000 U/mg. In a fluid-phase IRMA for VIII:CAg utilizing the Fab fragment prepared from the monoclonal IgG, two high-titer human anti-VIII:C inhibitors (IgG fractions) showed no demonstrable competition for the monoclonal VIII:CAg binding site. Conversely, neither human antibody (125I-Fab fragment) was displaced from its VIII:CAg binding site by the monoclonal IgG molecule. When the monoclonal antibody was used in a fluid-phase IRMA, slightly decreased VIII:CAg levels were found in serum. A facile one-step, two-site IRMA using Sepharose-bound human anti-VIII:C and labeled monoclonal IgG was designed. With this assay, in contrast to the finding with the fluid-phase IRMA, both the rate and apparent level of binding of VIII:CAg "sandwiched" between the two antibodies were increased approximately twofold in serum compared to the native plasma. A similar increase in rate and apparent level of binding was also found after thrombin treatment of VIII:C/vWf relative to the untreated control preparation.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification; D000915:Antibody Affinity; D000941:Antigens / Q000032:analysis / Q000276:immunology*; D002417:Cattle; D005169:Factor VIII / Q000032:analysis / Q000276:immunology*; D006467:Hemophilia A / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000302:isolation & purification; D010949:Plasma / Q000276:immunology; D011863:Radioimmunoassay; D013917:Thrombin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000941:Antigens; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D005169:Factor VIII; D013917:Thrombin