Evaluation of Monoclonal Antifibrin Antibodies by Their Binding to Human Blood Clots

Summary

The primary goal of this study was to develop a method for evaluating fibrin-specific antibodies as thrombus detecting agents. The apparatus and assay conditions were chosen by testing antibody 64C5, which binds to the amino terminus of the fibrin beta chain, for its ability to bind to human blood clots. Using ¹²⁵I-labeled antibody 64C5, the effects of antibody concentration, clot shape, clot mass, temperature, and flow rate were tested. Increased antibody binding was observed when antibody concentration, clot mass and temperature were increased. Under one set of conditions, six ¹²⁵I-labeled monoclonal antifibrin antibodies (four specific for the beta chain, two specific for the alpha chain) were tested for their binding to retracted clots of human blood. Two radioiodinated antidigoxin antibodies were used as a control. Beta chain-specific antibody 59D8, which provided the highest level of binding to clot, bound 14-fold better than the control antidigoxin antibody. Neither alpha chain-specific antibody bound to clotted blood. To examine the in vivo relevance of the in vitro binding to clots, the uptake of antibody 64C5 was assessed for its binding to human fibrin clotted within the jugular vein of a rabbit. The correlation coefficient between in vitro and in vivo uptake as a function of clot weight was calculated to be 0.91. Thus, in vitro binding of monoclonal antifibrin antibodies to human blood clots was judged to be a realistic method for the comparison and selection of an ideal antifibrin antibody for detailed in vivo testing.

Keywords

Monoclonal antibody - Antifibrin antibody - Fibrin - Thrombus detecting agent

• References

• 1 Reiman AS. Intravenous thrombolysis in acute myocardial infarction. A progress report. N Engl J Med 1985; 312: 915-916
• 2 Kakkar VV, Nicolaides AN, Renney J TG, Friend JR, Clarke MB. ¹²⁵I-labelled fibrinogen test adapted for routine screening for deepvein thrombosis. Lancet 1970; 1: 540-542
• 3 Thakur ML, Welch MJ, Joist JH, Coleman RE. ¹¹¹Indium labeled platelets: studies on preparation and evaluation of in-vitro and in-vivo functions. Thromb Res 1976; 9: 345-357
• 4 Kudryk B, Rohoza A, Ahadi M, Chin J, Wiebe ME. Specificity of a monoclonal antibody for the NH₂-terminal region of fibrin. Molec Immunol 1984; 21: 89-94
• 5 Rosebrough SF, Kudryk B, Grossman ZD, McAfee JG, Subramanian G, Ritter-Hmcirik CA, Witanowski LS, Tillapaugh-Fay G. Radioimmunoimaging of venous thrombi using iodine-131 monoclonal antibody. Radiology 1985; 156: 515-517
• 6 Elms MJ, Bunce IH, Bundesen PG, Rylatt DB, Webber AJ, Masci PP, Whitaker AN. Measurement of crosslinked fibrin degradation products - an immunoassay using monoclonal antibodies. Thromb Haemostas 1983; 50: 591-594
• 7 Rylatt DB, Blake AS, Cottis LE, Massingham WA, Fletcher WA, Masci PP, Whitaker AN, Elms MJ, Bunce IH, Webber AJ, Wyatt D, Bundesen PG. An Immunoassay for human D-Dimer using monoclonal antibodies. Thromb Res 1983; 31: 767-778
• 8 Scheefers-Borchel U, Miiller-Berghaus G, Fuhge P, Eberle R, Heimburger N. Discrimination between fibrin and fibrinogen by a monoclonal antibody against a synthetic peptide. Proc Natl Acad Sci USA 1985; 82: 7091-7095
• 9 Hui KY, Haber E, Matsueda GR. Monoclonal antibodies to a synthetic fibrin-like peptide bind to human fibrin but not fibrinogen. Science 1983; 222: 1129-1132
• 10 Hui KY, Haber E, Matsueda GR. Immunodetection of human fibrin using monoclonal antibody 64C5 in an extracorporeal chicken model. Thromb Haemostas 1985; 54: 524-527
• 11 Collen D, Stassen JM, Verstraete M. Thrombolysis with human extrinsic (tissue-type) plasminogen activator in rabbits with experimental jugular vein thrombosis. J Clin Invest 1983; 71: 368-376
• 12 Matsueda GR, Allen PM. Monoclonal antibodies specific for the amino terminus of fibrin alpha-chains bind to fibrin monomer but not aggregated fibrin (abstract). Fed Proc 1986; 45: 965
• 13 Mudgett-Hunter M, Anderson W, Haber E, Margolies MN. Binding and structural diversity among high-affinity monoclonal anti-digoxin antibodies. Mol Immunol 1985; 22: 477-488
• 14 Parham P. On the fragmentation of monoclonal IgG₁, IgG_2a, and IgG_2b from BALB/c mice. J Immunol 1983; 131: 2895-2902
• 15 Porter RR. The hydrolysis of rabbit y-globulin and antibodies with crystalline papain. Biochem J 1959; 73: 119-127
• 16 Greenwood FC, Hunter WM, Glover JS. The preparation of ¹³¹I-labelled human growth hormone of high specific radioactivity. Biochem J 1963; 89: 114-123
• 17 Frankel ME, Gerhard W. The rapid determination of binding constants for antiviral antibodies by a radioimmunoassay. An analysis of the interaction between hybridoma proteins and influenza virus. Mol Immunol 1979; 16: 101-106
• 18 Okada M, Blombiick B. Factors influencing fibrin gel structure studied by flow measurement. Ann NY Acad Sci (US) 1983; 408: 233-253
• 19 Looney RJ, Abraham GN, Anderson CL. Human monocytes and U937 cells bear two distinct Fc receptors for IgG. J Immunol 1986; 136: 1641-1647