FVIII inhibitor IgG subclass and FVIII polypeptide specificity determined by immunoblotting

Abstract

We used immunoblotting of purified factor VIII coagulant protein (FVIII) to localize FVIII inhibitor epitopes in 76 inhibitor plasmas to either the 92-kd FVIII polypeptide (and its 54-kd and/or 44-kd thrombin fragments), the 80-kd polypeptide (and its 72-kd thrombin fragment), or both of these polypeptides. We also used immunoblotting to examine the immunoglobulin class and subclass content of 12 inhibitors with monoclonal antibodies specific for human IgG subclasses and IgM. Seven hemophilic (alloantibody) and five spontaneous (autoantibody) inhibitors contained IgG-1 and IgG-4 antibody; one of the spontaneous inhibitors also contained IgG-3. In one hemophilic inhibitor, the IgG-4 component reacted strongly with the 92-kd and 80-kd polypeptides, whereas the IgG-1 component reacted only minimally with the 92-kd polypeptide although its reactivity with the 80-kd polypeptide was strong. Another hemophilic inhibitor was affinity purified and subjected to quantitative radial immunodiffusion, and the presence of IgG-1 and IgG-4 antibody was confirmed. We conclude that the inhibitors examined are not monoclonal but are probably of restricted polyclonal origin and that different IgG subclasses in an inhibitor plasma can have different degrees of FVIII polypeptide reactivity.