Immunochemical Characterization of a Factor IX Inhibitor Following Anamnestic Response

We previously characterized a human inhibitor for Factor IX in patient P.W.B, with Hemophilia B as an IgG4, λ immunoglobulin of restricted electrophoretic mobility. This restriction to a minor IgG subclass led us to characterize a second Factor IX inhibitor occurring in patient R. J. after an anamnestic response to Factor IX. On preparative zone electrophoresis the inhibitor migrated with a broad zone of mobility in the anodal portion of the γ peak and was restricted to the anodal portion of the IgG containing fractions. Gel filtration on calibrated 1.5 M sepharose columns revealed inhibitor activity in fractions corresponding to a molecular weight of 150,000. The inhibitor was further characterized by the technique of antibody neutralization using monospecific antisera to immunoglobulin classes, subclasses and light chain types in the zone of antibody excess. The inhibitor was completely neutralized by antibody to IgG whereas antisera to IgA, IgM, IgD and IgE had no effect. Neutralization was abolished by absorption of the IgG antiserum with purified IgG. Neutralization with antisera specific for light chains indicated a mixture of light chain types with an estimated ϰ/λ ratio of 6/1. Neutralization with antisera specific for IgG subclasses revealed a mixture of IgG subclasses. The Factor IX inhibitor was thus characterized as a polyclonal IgG immunoglobulin. Sepharose conjugates of R. J. globulin effect complete removal of Factor IX from normal plasma on an immunoabsorbent column and biologically active Factor IX may be eluted with 1600-fold purification.