IgG subclass identification of inhibitors to factor IX in haemophilia B patients

Inhibitors to factor IX in haemophilia B are IgG antibodies. It is of interest to know the subclass type of these antibodies since the biological activity of the four IgG subclasses are different. Subclass identification has been difficult due to the lack of specific antibodies to IgG subclasses. Monoclonal hybridoma antibodies to these subclasses are now available. Here we present the results on the subclass identification of inhibitors from six patients with severe haemophilia B (titres 0.8-800 mu/ml). Plasma from one patient was obtained before inhibitor could be detected by coagulation assays. He had circulating complexes between factor IX and inhibitor. The technique used was crossed immunoelectrophoresis combined with an enzyme-linked immunoassay. This technique allows the subclass determination of both low-titre and high-titre inhibitors. All inhibitors gave a very strong reaction with antibody to IgG4, and four inhibitors also reacted with antibody to IgG1. Three of these inhibitors gave a faint reaction with antibody to IgG2 also, whereas no inhibitor contained any detectable IgG3. The finding of IgG4 as the main subclass is important since IgG4 antibodies do not bind complement. The presence of more than one subclass in the inhibitors demonstrates the heterogeneous nature of the inhibitors. This was demonstrated also at a stage of inhibitor development when the inhibitor could not be detected by coagulation assay.