Specificity of Monoclonal Antibodies to Factor VIII: C

Summary

Three monoclonal antibodies (42 IgG, 47 IgG, 56 IgG) towards factor-VIII: C (VIII :C) have been produced. In ELISA for VIII:C-antigen (VIII:CAg), 47 IgG showed higher affinity for VIII: CAg than 42 IgG and 56 IgG. In solid phase immunoiso-lation of iodinated VIII :C diluted in EDTA buffer, the three monoclonals, like human VIII: C inhibitors, bound the 77/80 kD-light chain of VIII: C. In the absence of EDTA, 56 IgG bound the heavy chain-light chain complex of VIII: C, while 47 IgG was only able to bind the light chain. When coupled on Sepharose, 56 IgG adsorbed coagulation active VIII :C, while 47 IgG was only able to adsorb coagulation inactive VIII: CAg. In coagulation assay 56 IgG inhibited with 20 BU/mg while 42 IgG and 47 IgG inhibited with 4 BU/mg. A mixture of 42 IgG and 56 IgG showed a synergistic effect and inhibited with 50 BU/mg total IgG. In radioimmunoassay a human VIII: C inhibitor was able to inhibit the VIII: C binding of 42 IgG and 56 IgG but not of 47 IgG. The monoclonals did not inhibit each other. On the contrary, 56 IgG increased the binding of 42 IgG to VIII: C.

Keywords

Factor VIII :C - Monoclonal antibodies - Factor VIII antibodies - Factor VIII: CAg

• References

• 1 Hoyer LW. The factor VIII complex: Structure and function. Blood 1981; 58: 1-13
• 2 Chavin SI. Factor VIII: Structure and function in blood clotting. Am J Hematol 1984; 16: 297-306
• 3 Gitschier J, Wood WI, Goralka TM, Wion KL, Chen EY, Eaton DH, Vehar GA, Capon DJ, Lawn RM. Characterization of the human factor VIII gene. Nature 1984; 312: 326-330
• 4 Wood WI, Capon DJ, Simonsen CS, Eaton DL, Gitschier J, Keyt B, Seeburg PH, Smith DH, Hollingshead P, Wion KL, Delwart E, Tuddenham EG D, Vehar GA, Lawn RM. Expression of active human factor VIII from recombinant DNA clones. Nature 1984; 312: 330-337
• 5 Toole JJ, Knopf JL, Wozney JM, Saltzman LA, Buecker JL, Pittman DD, Kaufman RJ, Brown E, Shoemaker C, Orr EC, Amphlett GW, Foster WB, Coe ML, Knutson GJ, Fass DN, Hewick RM. Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature 1984; 312: 342-347
• 6 Truett MA, Blacher R, Caput D, Chu C, Craine B, Dina D, Halle well RA, Hartog K, Kuo CH, Laybourn P, Masiarz FR, Merry weather JP, Najarian RC, Potter SJ, Puma J, Quiroga M, Rail LB, Randolph A, Urdea MS, Valenzuela P, Dahl HH, Favaloro J, Hansen JJ, Nordfang O, Ezban M. Polypeptide composition of human coagulation factor VIII. C and molecular cloning of its genomic and cDNA sequences, DNA in press
• 7 Vehar GA, Keyt B, Eaton D, Rodriguez H, O’Brien DP, Rotblat F, Opperman H, Keck R, Wood WI, Harkins RN, Tuddenham EG D, Lawn RM, Capon DJ. Structure of human factor VIII. Nature 1984; 312: 337-342
• 8 Weinstein M, Chite L, Deykin D. Analysis of factor VIII coagulant antigen in normal, thrombin-treated and hemophilic plasma. Proc Natl Acad Sci USA 1981; 8: 5137-5141
• 9 Gawryl MS, Hoyer LW. Immunologic studies of antihemophilic factor (AHF, VIII :C). Clin Immunol Immunopathol 1982; 23: 517-526
• 10 Tran TH, Duckert F. Dissociation of factor VIII procoagulant antigen VIII: CAg and factor VIII related antigen VIIIR: Ag-Influence of divalent cation binding on the binding of VIII :CAg and VIIIR :Ag. ThrombHaemostas 1983; 50: 547-551
• 11 Nordfang O, Ezban M, Dinesen B. Reactivity of Factor VIII inhibitors in a micro ELISA for factor VIII: CAg and in solid phase immunoiso-lation of VIII: CAg. Thromb Haemostas 1985; 53: 346-350
• 12 Cooper HA, Lee D, Lamb MA, Wagner RH. Structure-function of the factor VIII complex studied with an immobilized heteroantiserum to VIII :C. Br J Haematol 1980; 44: 149-160
• 13 Tran TH, Marbet GA, Duckert F. Rabbit antibodies against the procoagulant activity (VIII :C) of human factor VIII. Thromb Haemostas 1981; 46: 699-705
• 14 Muller HP, van Tilburg NH, Derks J, Klein-Breteler E, Bertina RM. A monoclonal antibody to VIII: C produced by a mouse hybridoma. Blood 1981; 58: 1000-1006
• 15 Brown JE, Thuy LP, Carton CL, Hougie C. Studies on a monoclonal antibody to human factor VIII coagulant activity, with a description of a facile two-site factor VIII coagulant antigen assay. J Lab Clin Med 1983; 101: 793-805
• 16 Stel HV, Veerman EC I, Huisman JG, Janssen MC, van Mourik JA. A rapid one-step immunoradiometric assay for factor VIII-procoagulant antigen utilizing monoclonal antibodies. Thromb Haemostas 1983; 50: 860-863
• 17 Fass DN, Knutson GJ, Katzmann JA. Monoclonal antibodies to porcine factor VIII coagulant and their use in the isolation of active coagulant protein. Blood 1982; 59: 594-600
• 18 Tuddenham EG D, Trabold NC, Collins JA, Hoyer LW. The properties of factor VIII coagulant activity prepared by immunosorbent chromatography. J Lab Clin Med 1979; 93: 40-53
• 19 Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256: 495-497
• 20 Dinesen B, Feddersen C. An enzyme immunoassay (ELISA) for the quantitation of human factor VIII coagulant antigen (VIII: CAg). Thromb Res 1983; 31: 707-718
• 21 O’Brien DP, Goodall AH, Rotblat F, Tuddenham EG D. Monoclonal antibodies to VIII :C. Preparation and application. Thromb Haemostas 1983; 50: 306 (Abstr).
• 22 Fulcher CA, Roberts JR, Zimmerman TS. Thrombin proteolysis of purified factor VIII procoagulant protein. Blood 1983; 61: 807-811
• 23 Underwood PA, Kelly JF, Harman DF, MacMillan HM. Use of protein A to remove immunoglobulin from serum in hybridoma culture media. J Immunol Meth 1983; 60: 33-45
• 24 Kuo G, Craine B, Masiarz F, Rail L, Truett M, Valenzuela P, Nordfang O, Ezban M. Studies on the molecular structure of human factor VIII :C. Thromb Haemostas 1983; 50: 262 (Abstr).
• 25 Tuddenham EG D. Factor VIII from non-human sources. Abstracts of the 18th Congress of the International Society of Blood Transfusion. S. Karger, Basel 1984: 17 (Abstr)
• 26 Ehrlich PH, Moyle WR, Moustafa ZA, Canfield RE. Mixing two monoclonal antibodies yields enhanced affinity for antigen. J Immunol 1982; 128: 2709-2713