Monoclonal Antibodies As Probes Of Factor VIII/Von Willebrand Factor
Thrombosis and Haemostasis
Monoclonal antibodies are essential for the isolation and specific assay of the various components of Factor VIII/von Willebrand Factor (F.VIII/vWF) and for the immunochemical analysis of the surface epitopes of this protein. In this study, we have produced and characterized a panel of thirty-eight antibody synthesizing hybridomas. Mice were hyperimmunized with human F.VIII/vWF and three days after a 10-20 jig booster dose, spleen cells were harvested and fused with P3X63Ag8 or SP2/0 myeloma
... or SP2/0 myeloma cells. All antibodies reacted by solid phase ELISA with purified F.VIII/ vWF and normal cryoprecipitate and did not bind to cryoprecipitate from severe vWD, fibrinogen, or fibronectin. Antibody titers (20 μl) varied from lO2 to 106. Seven antibody producing hybridomas derived from P3X63Ag8 myeloma cells were cloned, recloned, and were stable in ascites growth. Two of these partially blocked VIIIR:RCo while the mixture of the seven exhibited significant inhibition, suggesting a cooperative effect in steric hindrance or induction of allosteric effects on the VIIIR:RCo function of the molecule. Three out of thirty-one hybridomas derived from SP2/0 myeloma cells blocked Factor VIII activity. A spatial map of the epitopes at the surface of F.VIII/vWF was derived from competitive displacement studies of the monoclonal antibodies on immobilized F.VIII/vWF. Three types of results were observed : no displacement, indicating independence of two epitopes; partial displacement, indicating steric proximity; and complete displacement, indicating epitope identity or extremely close proximity.