Production and characterization of polyclonal antibodies to human recombinant domain B-free antihemophilic factor VIII

Abstract

Moroctocog alpha is a human B-domain deleted recombinant factor VIII (BDDrFVIII), which represents a new generationof pure antihemophilic products. We describe here an optimized procedure for polyclonal anti-FVIII-antibody production withthe use of BDDrFVIII as an immunogen. The main immunochemical characteristics of the produced antibodies and their potentialbiomedical applications are also reported. Rabbits were immunized with BDDrFVIII as an emulsion with Freund s adjuvant or withantigen immobilized in polyacrylamide gel (PAAG). Antibody titers in immune sera were assayed by enzyme-linked immunosorbentassay (ELISA). IgG purification was performed by affine chromatography on protein A-sepharose. Immune sera and IgG were testedby immunoblotting with the use of human plasma of healthy donors and people with hemophilia A, platelet lysates, and commercialplasma-derived concentrates as sources of FVIII-related antigens. FVIII-producing human umbilical vein cells were processed forimmunocytochemical staining with the use of purified anti-FVIII-antibodies. Immunization of rabbits with PAAG-trapped antigeninduced more potent immune response compared to the standard immunization procedure with Freund s adjuvant. The lowestworking amount of immune IgG, measured by ELISA, was ~50 ng. Immunoblotting demonstrated that anti-BDDrFVIII antibodieseffectively recognize the whole FVIII molecule (320 kDa), as well as different truncated polypeptides thereof, and are suitable forimmunocytochemical analysis of FVIII-producing cells. An optimized procedure for the production of polyclonal antibodies againstFVIII with the use of PAAG-immobilized BDDrFVIII (moroctocog alpha) was proposed and successfully validated. The producedantibodies are suitable for detecting and measuring FVIII-related antigens and may have various biomedical applications.