Alloantibodies to Factor IX in Haemophilia B Characterized by Crossed Immunoelectrophoresis and Enzyme‐conjugated Antisera to Human Immunoglobulins

S ummary. Incubation of factor IX with non‐precipitating alloantibodies to factor IX gives rise to soluble complexes between factor IX and the alloantibodies. These complexes appear as a factor IX molecule with a reduced mobility in crossed immunoelectrophoresis against a rabbit antiserum to factor IX. This factor IX immunoprecipitate was used for the study of alloantibodies to factor IX from five patients with severe haemophilia B (antibody titres 0·1–800 units/ml plasma). Incorporation of antiserum to κ light chains or λ light chains of human immunoglobulin G in an intermediate gel in crossed immunoelectrophoresis gave a reduction of the factor IX precipitin arc, indicating the presence of immunoglobulin G alloantibodies containing both κ and λ light chains in complex with factor IX. Incubation of the factor IX immunoprecipitate with peroxidase‐conjugated antisera to the same immunoglobulins, and staining for peroxidase activity, confirmed the presence of immunoglobulin G containing both types of light chains in the factor immunoprecipitate. It is concluded that all five alloantibodies were polyclonal immunoglobulin G antibodies. The technique had the advantage that the light chain types of low‐titre antibodies could be determined, and it may be suitable for further characterization of alloantibodies to factor IX if antibodies to immunoglobulin subclasses are available.