The epitopes of four anti-vWF monoclonal antibodies (MoAbs), which inhibit antibiotic ristocetin induced vWF binding to GPⅠb, were investigated and compared with each other. MoAb NMC-4 completely inhibited both the vWF bindings to GPⅠb expressed by ristocetin and snake venom botrocetin at the final concentrations of 10 μg/ml. Another MoAb RFF-VⅢ RAG : 1 also completely inhibited ristocetin-induced vWF binding at the IgG concentration of 10 μg/ml, but showed a partial inhibition (75% at the IgG concentlation of 100 μg/ml) on botrocetin-induced binding. Two other MoAbs, RG46 and 52-K8, inhibited ristocetin-induced vWF binding at the inhibition constant by 50% of 90 μg/ml and 30 μg/ml respectively, but without effect on botrocetin-induced vWF binding. Using the radiolabelled NMC-4 and its binding to vWF immobilized to plastic tubes, the competitive binding assay was performed. In this assay, cold NMC-4 clearly displaced (¹²⁵Ⅰ) NMC-4 binding to solid-phase vWF, and RFF-VⅢRAG : 1 partially blocked the binding (60% at the IgG
concentration of 100 μg/ml), whereas neither RG46 nor 52K-8 blocked this binding. These results indicated that the epitopes of NMC-4 and RFF-VⅢRAG : 1 are in close proximity, but those of RG46 and 52K-8 are different, suggesting the epitope heterogeneity of anti-vWF MoAbs which inhibit ristocetin-induced vWF binding
