Antisera To Human Factor VIII Raised In Balb/C Mice. The Production Of Monoclonal Antibodies To Factor VIII

The aim of this series of experiments is to raise antisera to human Factor VIII in Balb/C mice, study the properties of these antisera, and use the spleen cells from such hyperimmune mice to raise a panel of monoclonal antibodies directed against the varying antigenic determinants of the human factor VIII complex. Antibody activity against factor VIII antigen was determined by a microtitre solid phase radioimmune assay. This involved exposing test antisera to human factor VIII-coated PVC wells, and detection of bound mouse antibody by subsequent exposure to ¹²⁵I labelled rabbit antimouse immunoglobulin. Anticoagulant assays and ristocetin cofactor inhibition assays were based on standard methods adapted to microtitre plates. Balb/C mice were immunized with gel filtered factor VIII complex and boosted immediately prior to fusion. Fusion was performed with the parental line P3-NSI/l-Ag-l in polyethylene glycol (PEG). Compared to rabbits Balb/C mice produced considerably higher anticoagulant activity. Pooled rabbit anti-factor VIII coagulant activity was 1.2 Oxford units/ml whereas the anticoagulant activity of 6 mice ranged between 30-185 Oxford units/ml. Incubation of mouse antisera with human plasma samples and subsequent assay of residual mouse anti-VIII antigen demonstrated that normal and haemophiliac plasma markedly inhibited binding of the antisera whereas plasma samples from patients with Von Willebrand's disease did not. This system has been optimized as a microtitre radioimmune assay for human factor VIII antigen. There is good correlation with the Laurell “rocket” technique (r=0.86; n=14). A number of monoclonal antibodies which bind to the factor VIII complex have been produced. They have no anticoagulant activity nor activity against ristocetin cofactor. They recognize antigens present in plasma fractions after DEAE Sephadex A-50 separation which differ from those fractions recognized by the heterologous Balb/C antisera.