STUDIES ON A MONOCLONAL-ANTIBODY TO HUMAN FACTOR-VIII COAGULANT ACTIVITY, WITH A DESCRIPTION OF A FACILE 2-SITE FACTOR-VIII COAGULANT ANTIGEN-ASSAY

  • 1 January 1983
    • journal article
    • research article
    • Vol. 101  (5) , 793-805
Abstract
IgG was purified from the ascites tumor fluid obtained from mice injected with a monoclonal cell line secreting antibody that inhibited VIII:C [procoagulant factor VIII]. With a modified Bethesda assay method (18 h, 4.degree. C), the titer of the purified IgG was 14,000 U/mg. In a fluid-phase IRMA [immunoradiometric assay] for VIII:CAg [factor VIII coagulant antigen] utilizing the Fab fragment prepared from the monoclonal IgG, 2 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 VII:CAg levels were found in serum. A facile 1-step, 2 site IRMA using Sepharose-bound human anti-VIII:C and labeled monoclonal IgG was designed. With this assay, in contrast to finding fluid-phase IRMA, both the rate and apparent level of binding of VIII:CAg sandwiched between the 2 antibodies were increased .apprx. 2-fold 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 [high purity factor VIII concentrates] relative to the untreated control preparation.