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.

The interference of antibodies to factor VIII coagulant protein (VIII:C) of 9 nonhemophilic patients with the binding to factor VIII coagulant antigen (VIII:CAg) of a reference hemophilic 125I-Fab' reagent, used in a liquid phase VIII:CAg assay, was studied. The binding competition was estimated from immunoradiometric assay (IRMA) dose-response slope of VIII:CAg present in patient plasma, interference of antibodies with the 125I-Fab' binding to VIII:CAg in normal plasma, and the displacement of antibody from the complexes with VIII:CAg by the 125I Fab'. Antibody populations from three patients were studied in detail; in the VIII:CAg assay, two of them interfered with the 125I-Fab' binding, and one did not (patient 1). The formation of stable complexes between antibodies of each patient and VIII:CAg was demonstrated by protein-A-Sepharose adsorption. The 125I-Fab' binding to VIII:CAg-anti-VIII:CAg IgG complexes indicated that patient 1 antibodies and the 125I-Fab' recognized different antigenic determinants, whereas the other two patient antibodies and 125I-Fab' recognized closely related or identical VIII:CAg determinants. These results demonstrate an apparently selective recognition of at least two distinct VIII:CAg determinants by naturally occurring antibodies, suggesting a possibility of a wider use of these antibodies in studies of the structure and function of factor VIII.

Two non-haemophilic elderly patients who had developed autoantibodies to factor VIII were studied over a period of 9 months to 5 years. Sequential measurements of antibody to factor VIII (anti-VII:C), factor VIII coagulant activity (VIII:C), factor VIII coagulant antigen (VIII:CAg), factor VIII-related antigen (VIIIR:Ag), and factor VIII ristocetin cofactor (VIII:WF) were performed. Before treatment, low VIII:C, normal or increased VIII:CAg and high VIIIR:Ag levels were found and were indicative of the presence of circulating immune complexes. Immunosuppressive therapy induced progressive correction of VIII:C and VIIIR:Ag values. High levels of VIII:CAg subsided in the patient who relapsed. It is suggested that antibodies to factor VIII bind and remove VIII:C from the circulation thereby inducing an increased synthesis of VIII:CAg which may be associated with an augmented release or production of VIIIR:Ag.

An antibody to Factor VIII in non-hemophiliacs is a rare occurrence. The presence of low but detectable Factor VIII: C (VIII: C) and high Factor VIII antigen (VIIIR: Ag), together with high antibody levels, suggests a complex influence of anti- VIII: C on Factor VIII metabolism. We have studied Factor VIII complex in 16 non-hemophilic patients with anti-VIII: C. VIII: C, VIIIR: Ag and VIII: Rcof were measured by standard techniques and VIII: C antigen (VIII: CAg) by a liquid phase immunoradiometric assay using radio labelled anti-VIII:C Fab’ fragments purified from a hemophiliac. The plasma from all patients had low VIII: C (0.01 - 0.24 u/ml) and high VIIIR:Ag (1.4 - 8.8 u/ml) content. VIII:Rcof was approximately 50% of the VIIIR:Ag level. An increase in electrophoretically fast-moving VIIIR: Ag was observed in some cases VIII: CAg was tested in 13 patients. Two patterns of the dose response were observed. In most samples obtained from patients with low affinity anti-VIII:C whose VIII: CAg ranged from 0.19 - 2.45 u/ml, the dose response paralleled that of the control. In patients with high affinity antibodies the dose response curve was less steep than control, suggesting a competition between the patient’s antibody and the hemophilic anti-VIII: C Fab’ fragments for the VIII: C antigenic sites The Factor VTII complex was studied sequentially in 3 patients during immunosuppression. The results showed that : 1) when VIII: C normalizes, VIIIR: Ag levels and electrophoretic mobility progressively return to normal ; 2) levels of VIIIR: Ag are inversely correlated to the VIII: C irrespective of VIII: CAg levels ; 3) high VIII: CAg levels correspond to VIII:C-anti-VIII: C circulating immune complexes. In non-hemophilic patients low VIII: C, high VIII:CAg and high VIIIR: Ag indicate the presence of an anti-VIII: C inhibitor. Measurements of various components of Factor VIII in these patients are not only a means for monitoring the effect of therapy but also provide a rare opportunity to study the metabolism of Factor VIII in man.