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

Moroctocog alpha is a human B-domain deleted recombinant factor VIII (BDDrFVIII), which represents a new generation of pure antihemophilic products. We describe here an optimized procedure for polyclonal anti-FVIII-antibody production with the use of BDDrFVIII as an immunogen. | Turkish Journal of Biology Turk J Biol (2017) 41: 857-867 © TÜBİTAK doi:10.3906/biy-1704-10 http://journals.tubitak.gov.tr/biology/ Research Article Production and characterization of polyclonal antibodies to human recombinant domain B-free antihemophilic factor VIII 1 2,3 4 3, Artem TYKHOMYROV , Victor NEDZVETSKY , Sergiy SHEMET , Can Ali AĞCA * Department of Enzyme Chemistry and Biochemistry, Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, Kyiv, Ukraine 2 Department of Biophysics and Biochemistry, Dnipro National University, Dnipro, Ukraine 3 Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Bingöl University, Bingöl, Turkey 4 Ukrainian Hemophilia Association, Dnipro Regional Chapter, Dnipro, Ukraine 1 Received: 06.04.2017 Accepted/Published Online: 09.09.2017 Final Version: 18.12.2017 Abstract: Moroctocog alpha is a human B-domain deleted recombinant factor VIII (BDDrFVIII), which represents a new generation of pure antihemophilic products. We describe here an optimized procedure for polyclonal anti-FVIII-antibody production with the use of BDDrFVIII as an immunogen. The main immunochemical characteristics of the produced antibodies and their potential biomedical applications are also reported. Rabbits were immunized with BDDrFVIII as an emulsion with Freund’s adjuvant or with antigen immobilized in polyacrylamide gel (PAAG). Antibody titers in immune sera were assayed by enzyme-linked immunosorbent assay (ELISA). IgG purification was performed by affine chromatography on protein A-sepharose. Immune sera and IgG were tested by immunoblotting with the use of human plasma of healthy donors and people with hemophilia A, platelet lysates, and commercial plasma-derived concentrates as sources of FVIII-related antigens. FVIII-producing human umbilical vein cells were processed for immunocytochemical staining with the use of purified anti-FVIII-antibodies. Immunization of rabbits with .