Immunological characterization of factor VIII inhibitors by a sensitive micro-ELISA method

A micro-ELISA method for the immunological characterization of factor VIII (F. VIII) inhibitors is described. Microplates sensitized with purified monospecific anti-von Willebrand factor (vWF) IgG were firstly incubated with a commercial F. VIII concentrate and then with the samples containing the F. VIII inhibitors to be characterized. Peroxidase conjugated monospecific antisera were used to determine the Ig class and the light chain type. Alkaline phosphatase conjugated monoclonal antibodies were employed to investigate the IgG subclasses. The F. VIII inhibitors from 8 hemophiliacs and 1 patient with systemic lupus erythematosus (SLE) were characterized. All of them belonged to the IgG class but one case simultaneously exhibited another inhibitor of the IgA class. Eight inhibitors analyzed for light chains resulted to be polyclonal. In 7 cases the inhibitors belonged solely to the IgG4 subclass while in the other 2 cases contained all the 4 IgG subclasses. The method appears to be simple, accurate and highly sensitive (detecting as low as 0.156 Bethesda Units/ml) for the immunological characterization of F. VIII inhibitors.