Can Ali AĞCA, Artem TYKHOMYROV, Victor NEDZVETSKY, Sergiy SHEMET

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 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.

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Amero SA, James TC, Elgin SC (1988). Production of antibodies using proteins in gel bands. Methods Mol Biol 3: 355-362.
Bradford MM (1976). Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
Butenas S, Parhami-Seren B, Gissel MT, Gomperts ED, Fass DN, Mann KG (2009). Potency and mass of factor VIII in FVIII products. Haemophilia 15: 63-72.
Chandler WL, Ferrell C, Lee J, Tun T, Kha H (2003). Comparison of three methods for measuring factor VIII levels in plasma. Am J Clin Pathol 120: 34-39.
Chitlur M, Young G (2016). Global assays in hemophilia. Semin Hematol 53: 40-45.
Clifton JG, Huang F, Kovac S, Yang X, Hixson DC, Josic D (2009) Proteomic characterization of plasma-derived clotting factor VIII-von Willebrand factor concentrates. Electrophoresis 30: 3636-3646.
Fang H, Wang L, Wang H (2007). The protein structure and effect of factor VIII. Thromb Res 119: 1-13.
Goyal R, Mathur SK, Gupta S, Goyal R, Kumar S, Batra A, Hasija S, Sen R (2015). Immunohistochemical expression of glial fibrillary acidic protein and CAM5.2 in glial tumors and their role in differentiating glial tumors from metastatic tumors of central nervous system. J Neurosci Rural Pract 6: 499-503.
Gringeri A (2011). Factor VIII safety: plasma-derived versus recombinant products. Blood Transfus 9: 366-370.
Hollestelle MJ, Thinnes T, Crain K, Stiko A, Kruijt JK, van Berkel TJ, Loskutoff DJ, van Mourik JA (2001). Tissue distribution of factor VIII gene expression in vivo - a closer look. Thromb Haemost 86: 855-861.
Jacquemin M, Neyrinck A, Hermanns MI, Lavendhomme R, Rega F, Saint-Remy JM, Peerlinck K, Van Raemdonck D, Kirkpatrick CJ (2006). FVIII production by human lung microvascular endothelial cells. Blood 108: 515-517.
Johnston A, Thorpe R (1982). Immunochemistry in Practice. London, UK: Blackwell Scientific Publications Ltd.
Lenting PJ, van Mourik JA, Mertens K (1998). The life cycle of coagulation factor VIII in view of its structure and function. Blood 92: 3983-3996.
Lindgren A, Wadenvik H, Tengborn L (2002). Characterization of inhibitors to FVIII with an ELISA in congenital and acquired haemophilia A. Haemophilia 8: 644-648.
Lollar P (2003). The factor VIII assay problem: neither rhyme nor reason. J Thromb Haemost 1: 2275-2279.
Mazurkiewicz-Pisarek A, Płucienniczak G, Ciach T, Płucienniczak A (2016). The factor VIII protein and its function. Acta Biochim Pol 63: 11-16.
Montgomery RR, Shi Q (2012). Platelet and endothelial expression of clotting factors for the treatment of hemophilia. Thromb Res 129: 46-48.
Mumaw MM, de la Fuente M, Noble DN, Nieman MT (2014). Targeting the anionic region of human protease-activated receptor 4 inhibits platelet aggregation and thrombosis without interfering with hemostasis. J Thromb Haemost 12: 1331-1341.
Obergfell A, Sturm A, Speer CP, Walter U, Grossmann R (2006). Factor VIII is a positive regulator of platelet function. Platelets 17: 448-453.
Ouafik L, Sauze S, Boudouresque F, Chinot O, Delfino C, Fina F, Vuaroqueaux V, Dussert C, Palmari J, Dufour H et al. (2002). Neutralization of adrenomedullin inhibits the growth of human glioblastoma cell lines in vitro and suppresses tumor xenograft growth in vivo. Am J Pathol 160: 1279-1292.
Recht M, Nemes L, Matysiak M, Manco-Johnson M, Lusher J, Smith M, Mannucci P, Hay C, Abshire T, OBrien A et al. (2009). Clinical evaluation of moroctocog alfa (AF-CC), a new generation of B-domain deleted recombinant factor VIII (BDDrFVIII) for treatment of haemophilia A: demonstration of safety, efficacy, and pharmacokinetic equivalence to full- length recombinant factor VIII. Haemophilia 15: 869-880.
Shi Q, Wilcox DA, Fahs SA, Kroner PA, Montgomery RR (2003). Expression of human factor VIII under control of the platelet- specific alphaIIb promoter in megakaryocytic cell line as well as storage together with VWF. Mol Genet Metab 79: 25-33.
Sohrabi S, Akbarzadeh A, Norouzian D, Farhangi A, Mortazavi M, Mehrabi MR, Chiani M, Saffari Z, Ghassemi S (2011). Production and purification of rabbits polyclonal antibody against Factor VIII. Indian J Clin Biochem 26: 354-359.
Stoscheck CM (1990). Quantitation of protein. Methods Enzymol 182: 50-68.
Turner NA, Moake JL (2015) Factor VIII is synthesized in human endothelial cells, packaged in Weibel-Palade bodies and secreted bound to ULVWF strings. PLoS One 10: e0140740.
Windyga J, Rusen L, Gruppo R, OBrien AC, Kelly P, Roth DA, Arkin S (2010). BDDrFVIII (Moroctocog alfa [AF-CC]) for surgical haemostasis in patients with haemophilia A: results of a pivotal study. Haemophilia 16: 731-739.
Yarovoi HV, Kufrin D, Eslin DE, Thornton MA, Haberichter SL, Shi Q, Zhu H, Camire R, Fakharzadeh SS, Kowalska MA et al. (2003). Factor VIII ectopically expressed in platelets: efficacy in hemophilia A treatment. Blood 102: 4006-4013.