Fractionation of human antibody to factor VIII:C: and IRMA for phospholipid binding sites on factor VIII C:Ag

Labelled Fab' fragments, derived from the plasma of a severe haemophiliac with antibody directed against factor VIII clotting antigen (VIII C:Ag), were fractionated by immunoabsorption with, first, a complex of phospholipid (PL) vesicles and factor VIII and, second, with factor VIII alone. Two pools of labelled anti-VIII C:Ag were obtained and were used in immunoradiometric assays (IRMAs) for VIII C:Ag. With one pool (non-PL-site antibody) VIII C:Ag assays were unaffected by pre-incubation of factor VIII with PL vesicles; however, binding of the second pool of antibody to VIII C:Ag was prevented by PL preincubation, indicating that these antibody molecules bind at or near a phospholipid binding site on VIII C:Ag (PL-site antibody). Assays of VIII C:Ag in an intermediate purity factor VIII concentrate with these two antibody pools indicate that more than one third of the VIII C:Ag may be bound to PL.