An immunoradiometric assay for human factor VIII/von Willebrand factor (VIII:vWF) using a monoclonal antibody that defines a functional epitope

Abstract

A murine monoclonal antibody has been produced (RFF-VIII:R/2) that binds specifically to human factor VIII-related antigen (VIII:RAg) in plasma and in vascular endothelial cells but has no reactivity with factor VIII procoagulant antigen (VIII:cAg). This antibody is a potent inhibitor of von Willebrand factor activity (VIII:vWF) in that it can totally neutralize ristocetin-induced aggregation of platelet rich plasma and inhibit platelet adhesion at high flow rates. RFF-VIII:R/2 can be used in a one-stage, fluid phase immunoradiometric assay that can detect VIII:RAg at concentrations of 0.001 u/ml. This method has been used to analyse plasma from patients with von Willebrand's disease (vWD). Results obtained in these patients showed a high degree of correlation between the monoclonally-defined epitope and VIII:vWF levels measured by ristocetin-induced aggregation of washed platelets. This correlation was maintained in those patients with the 'variant' types of vWD who exhibit highly disparate VIII:vWF and VIII:RAg levels when the latter is determined using polyclonal antisera. It appears that this monoclonal antibody recognizes a site on the VIII:RAg molecule which is associated with its interaction with the platelet membrane. Immunoradiometric assays using RFF-VIII:R/2 offer a simplified, reproducible means of detecting functionally-active VIII:RAg as an alternative or supplement to techniques involving platelet interactions.