Download PDF
Thromb Haemost 1996; 76(01): 056-064
DOI: 10.1055/s-0038-1650522
Original Article
Schattauer GmbH Stuttgart

Evaluation of the Fibrin Binding Profile of two Anti-fibrin Monoclonal Antibodies

S Raut
The Division of Haematology, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Herts., UK
,
P J Gaffney
The Division of Haematology, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Herts., UK
› Author Affiliations
Further Information

Publication History

Received: 29 January 1996

Accepted after revision02 April 1996

Publication Date:
10 July 2018 (online)

Also available at
  PDF Download  Permissions and Reprints

Summary

Two anti-fibrin monoclonal antibodies, MAbs 1H10 and 5F3, raised to human freeze-fractured fibrin and thrombin-treated N-terminal disulphide knot (T-NDSK), respectively, were compared for epitope binding to various domains of the fibrinogen/fibrin moiety. Using plasmin-mediated fibrinogen digests, immunoblots showed that both MAbs crossreacted strongly with fragments X and Y, weakly with fragment-E and not at all with fragment D. Purified fragments D and E used in an ELISA confirmed that MAbs 1H10 and 5F3 cross-reacted in a dose-response fashion with the isolated fragment-E, while there was no reaction with fragment-D. The two MAbs were similarly shown to react with fibrin-derived fragment-E. Surface Plasmon Resonance (SPR) technology, employed to further evaluate the epitopes in fibrin, showed that MAb 1 HI0 had a higher affinity for fragment-E (KD = 8.04 × 10-9 M) than MAb 5F3 (KD = 1.13 × 10-8 M). Individual association and dissociation rate constants of 7.97 × 105 M-1S-1 and 3.97 × 10-3S-1, respectively, for MAb 1H10, and 5.16 × 105 M-1s-1 and 3.62 × 10-3s-1, respectively, for MAb 5F3 were also obtained. A SPR inhibition assay confirmed that MAb 1H10 had a greater affinity for fragment-E than MAb 5F3. However individual isolated polypeptide chains of fibrinogen fragment E (E-Aα, E-Bβ, E-γ) showed no reaction with the two antibodies in ELISA, immunoblot or SPR analysis procedures. Furthermore, SPR pair-wise epitope mapping analysis revealed that MAbs 1H10 and 5F3 have in fact distinct epitopes on fragment-E. These distinct epitopes appeared to be a conformational amalgam of linear sequences in two or three of the polypeptide chains of fragment-E, or distinct conformational epitopes on one of the three subunit chains alone.

 

  • References

  • 1 Hui KY, Haber E, Matsueda GR. Immunodetection of human fibrin using monoclonal antibody 64C5 in an extracorporeal chicken model. Thromb Haemost 1985; 54: 524-527
    Article in Thieme ConnectPubMedGoogle Scholar
  • 2 Feitsma RIJ, Blok D, Wasser MNJM, Nieuwenhuizen W, Pauwels EKJ. A new method for 99M Tc-labelling of proteins with an application to clot detection with an anti-fibrin monoclonal antibody. Nucl Med Commun 1987; 8: 771-777
    CrossrefPubMedGoogle Scholar
  • 3 Walker KZ, Khafagi F, Bautovich GJ, Boniface GR, Bundesen PG, Rylatt DB. Antifibrin monoclonal antibodies for radioimmunodetection: preliminary assessment in a rat model system. Thromb Res 1988; 52: 269-278
    CrossrefPubMedGoogle Scholar
  • 4 Tymkewycz PM, Creighton-Kempsford LJ, Gascoine PS, Zanelli GD, Webbon PM, Gaffney PJ. Imaging of human thrombi in the rabbit jugular vein. I: Comparison of two fibrin-specific monoclonal antibodies. Thromb Res 1989; 54: 411-421
    CrossrefPubMedGoogle Scholar
  • 5 Wasser MNJM, Koppert PW, Arndt JW, Emeis JJ, Feitsma RIJ, Pauwels EKJ, Niewenhuizen W. An anti-fibrin monoclonal antibody useful in im-munoscintigraphic detection of thrombi. Blood 1989; 74: 708-714
    PubMedGoogle Scholar
  • 6 Hui KY, Haber E, Matsueda GR. Monoclonal antibodies to a synthetic fibrin-like peptide bind to human fibrin but not to fibrinogen. Science 1983; 222: 1129-1132
    CrossrefPubMedGoogle Scholar
  • 7 Rylatt DB, Blake AS, Cottis LE, Massingham DA, Fletcher WA, Masci PP, Whitaker AN, Elms M, Bunce I, Webber AJ, Wyatt D, Bundesen PG. An immunoassay for human D-dimer using monoclonal antibodies. Thromb Res 1983; 31: 767-778
    CrossrefPubMedGoogle Scholar
  • 8 Kudryk B, Rohoza A, Ahadi M, Chin J, Wiebe ME. Specificity of a monoclonal antibody for the NH2-terminal region of fibrin. Molecular Immunol 1984; 21: 89-94
    CrossrefPubMedGoogle Scholar
  • 9 Gaffney PJ, Creighton-Kempsford LJ, Perry MJ, Callus M, Thorpe R, Spitz M. Monoclonal antibodies to cross-linked fibrin degradation products (XL-FDP). I: Characterisation and preliminary evaluation in plasma. Brit J Haematol 1988; 68: 83-90
    CrossrefPubMedGoogle Scholar
  • 10 Schielen WJG, Voskuilen M, Tesser GI, Nieuwenhuizen W. The sequence Aa-(148-160) in fibrin, but not in fibrinogen, is accessible to monoclonal antibodies. Immunology 1989; 86: 8951-8954
    PubMedGoogle Scholar
  • 11 Schielen WJG, Adams HPHM, van Leuvene K, Voskuilen M, Tesser GI, Nieuwenhuizen W. The sequence y-(312-324) is a fibrin-specific epitope. Blood 1991; 77: 2169-2173
    PubMedGoogle Scholar
  • 12 Tymkewycz PM, Creighton-Kempsford LJ, Gaffney PJ. Generation and partial characterisation of five monoclonal antibodies with high affinities for fibrin. Blood Coagul Fibrinolysis 1993; 4: 211-221
    CrossrefPubMedGoogle Scholar
  • 13 Edgell TA, McEvoy F, Webbon P, Gaffney PJ. Binding profiles of monoclonal antibodies with some animals fibrins. Thromb Res 1992; 65 (Suppl. 01) Suppl 103
    PubMedGoogle Scholar
  • 14 McEvoy F, Webbon PM, Edgell TA, Gaffney PJ. An in vitro circulation model for thrombosis; influences of circulating plasmas on the binding of monoclonal antibodies to human fibrin clots. Fibrinolysis 1994; 9: 309-315
    PubMedGoogle Scholar
  • 15 Lanza GM, Wallace KD, Abondschein D, Scott MJ, Sheehan CK, Cacheris WP, Sharkey AM, Miller JG, Gaffney PJ, Wickline SA. Specific acoustic enhancement of vascular thrombi in vivo with a novel site targeted ultrasonic contrast agent. Circulation 1995; 92 (Suppl. 01) 1-260
    CrossrefPubMedGoogle Scholar
  • 16 Jonsson U, Fagerstam L, Ivarson B, Johnsson B, Karlsson R, Lundh K, Lofas S, Persson B, Roos H, Ronnberg I, Sjolander S, Stenberg E, Stahlberg R, Urbaniczky C, Ostlin H, Malmquist M. Real-time biospecific interaction analysis using surface plasmon resonance and sensor chip technology. Biotechniques 1991; 11: 620-627
    PubMedGoogle Scholar
  • 17 Karlsson R, Michaelsson A, Mattsson L. Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensorbased analytical system. J Immun Methods 1991; 145: 229-240
    CrossrefPubMedGoogle Scholar
  • 18 Koppert PW, Huijsmans CMG, Nieuwenhuizen W. A monoclonal antibody, specific for human fibrinogen fibrinopeptide A-containing fragments and not reacting with free fibrinopeptide A. Blood 1985; 66: 503-507
    PubMedGoogle Scholar
  • 19 Gaffney PJ, Dobos P. A structural aspect of human fibrinogen suggested by its plasmin degradation. FEBS Lett 1971; 15: 13-16
    CrossrefPubMedGoogle Scholar
  • 20 Jamieson GA, Gaffney PJ. Nature of the high molecular weight fraction of fibrinolytic digests of human fibrinogen. Biochim Biophys Acta 1968; 154: 96-109
    CrossrefPubMedGoogle Scholar
  • 21 Nussensweig V, Seligman M, Pelmont J, Grabar P. Les produits de degradation du fibrinogene humaine par la plasmine, I: Separation et proprietes physicochimiques. Annales de l’lnstitut Pasteur 1961; 100: 377-387
    PubMedGoogle Scholar
  • 22 Raut S, Corran PH, Gaffney PJ. Characterisation of the the chains of human fibrinogen isolated by perfusion chromatography using fibrin specific monoclonal antibodies. Thromb Res 1995; 79: 405-413
    CrossrefPubMedGoogle Scholar
  • 23 Raut S, Corran PH, Gaffney PJ. Ultra-rapid preparation of milligram quantities of the purified polypeptide chains of human fibrinogen. J Chromatogr 1994; 660: 390-394
    CrossrefPubMedGoogle Scholar
  • 24 Schagger H, von Jagow G. Tricine-sodium dodecyl sulphate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Analalyt Biochem 1987; 166: 368-379
    PubMedGoogle Scholar
  • 25 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nat Acad Sci USA 1979; 76: 4350-4354
    CrossrefPubMedGoogle Scholar
  • 26 Avrameas S, Ternynck T. Peroxidase labelled antibody and Fab conjugates with enhanced intracelular penetration. Immunochem 1971; 8: 1175-1179
    CrossrefPubMedGoogle Scholar
  • 27 Harlow E, Lane D. Labelling antibodies. In: Antibodies, a laboratory manual Harlow E, Lane D. eds New York, NY: Cold Spring Harbour Laboratory; 1998: 319-358
  • 28 Lofas S, Johnsson B. A novel hydrogel matrix on gold surfaces in surface plasmon resonance sensors for fast and efficient covalent immobilisation of ligands. J Chem Soc Chem Commun 1990; 21: 1526-1528
    CrossrefPubMedGoogle Scholar
  • 29 Merk AB. Ligand immobilisation chemistry. In: BIA Application Handbook. Uppsala, Sweden: Pharmacia Biosensor 1994: 4.1-4.33
    PubMedGoogle Scholar
  • 30 Merk AB. Kinetic and equilibrium theory. In: BIA Technology Handbook. Uppsala, Sweden: Pharmacia Biosensor 1994: A1-A12
    PubMedGoogle Scholar
  • 31 Fagerstam LG, Frostell A, Karlsson R, Kullman M, Larsson A, Malmquist M, Butt H. Detection of antigen-antibody interactions by surface plasmon resonance: Application to epitope mapping. J Mol Recogn 1990; 3: 208-214
    CrossrefPubMedGoogle Scholar