Immunoprecipitation of recombinant factor VIII fragments with antibodies present in the inhibitor plasma. Recombinant factor VIII fragments corresponding to the factor VIII light chain (80K), the C2 domain (C2), the factor VIII heavy chain (90K), and the A2 domain (A2) were expressed in High Five cells and metabolically labeled as described.26 Binding of antibodies to the metabolically labeled factor VIII fragments was assessed by immunoprecipitation. Bound proteins were eluted and analyzed under reducing conditions on a 10% (wt/vol) SDS-polyacrylamide gel. (A) Reactivity of anti-factor VIII antibodies with factor VIII light chain fragments. Lane 1, CLB-CAg 117; lane 2, control plasma; lane 3, antibodies present in patient's plasma. (B) Reactivity of antibodies with factor VIII heavy chain fragments. Lane 1, CLB-CAg 9; lane 2, control plasma; lane 3, antibodies present in patient's plasma. (C) Subclass determination of anti-factor VIII antibodies. Binding of anti-factor VIII antibodies to metabolically labeled factor VIII light chain was performed using subclass specific antibodies. Lane 1, total IgG; lane 2, IgG1; lane 3, IgG2; lane 4, IgG3; lane 5, IgG4. Molecular weight markers are given at the right of the figure.