Download PDF
Thromb Haemost 1988; 60(03): 415-418
DOI: 10.1055/s-0038-1646982
Original Article
Schattauer GmbH Stuttgart

A Monoclonal Antibody-Based Qunatitative Enzyme Immunoassay for the Determination of Plasma Fibrinogen Concentartions

E Hoegee-de Nobel
The Gaubius Institute TNO, Leiden, The Netherlands
,
M Voskuilen
The Gaubius Institute TNO, Leiden, The Netherlands
,
E Briët
*   The Academic Hospital, Leiden, The Netherlands
,
E J P Brommer
The Gaubius Institute TNO, Leiden, The Netherlands
,
W Nieuwenhuizen
The Gaubius Institute TNO, Leiden, The Netherlands
› Author Affiliations
Further Information

Publication History

Received 25 May 1988

Accepted after revision 18 July 1988

Publication Date:
30 June 2018 (online)

Also available at
  PDF Download Permissions and Reprints

Summary

The most commonly used fibrinogen assays in the clinic are clotting rate assays, e.g. the Clauss method. Such functional assays may be disturbed by e.g. heparin, anticoagulant fibrinogen degradation products (FgDP) and in the case of a dysfi-brinogenemia. Immunological methods would not suffer from these interferences. However, immunological assays for fibrinogen, which do not measure FgDPs, do not exist.

To set up such an enzyme immunoassay (EIA) we developed two monoclonal antibodies. The first monoclonal antibody (G8) has its epitope in the carboxyl-terminal 150 amino acid stretches of the fibrinogen Aa-chains. G8 is used to coat the wells of microtitration plates, and is the capture antibody in this EIA. The second antibody (Y18) has been described by us previously (Blood 1985; 66: 503). It is directed against fibrinopeptide A, covalently bound to the ±-chains i.e. against the amino-terminal stretches of the A±-chains. Y18 is conjugated with horse-radish peroxidase, and used as tagging antibody.

The EIA does not react with, and is not interfered by FgDP such as purified fragments X and Y, up to a concentration of 800 μg/ml. An FgDP mixture such as generated by Streptokinase treatment of plasma does not respond. Fibrin degradation products (whole blood lysate) up to 800 μg/ml do not interfere nor do heparin, EDTA or oxalate. The time-to-result of the EIA is only 45 minutes. Some patient plasmas yielded dose-response curves which are not parallel with the calibration curve of the EIA. An explanation for this phenomenon could not be given. Our fibrinogen EIA may be especially suitable to monitor patients with conditions which favour proteolytic damage to fibrinogen such as thrombolytic therapies.

keywords

Enzyme immunoassay - Fibrinogen - Monoclonal antibodies
 

  • References

  • 1 Lowe GD O, Drummond MM, Lorimer AR. et al Relation between extent of coronary artery disease and blood viscosity. Br Med J 1980; 281: 673-674
    PubMedSearch in Google Scholar
  • 2 Hamsten A, Blomback M, Wiman B. et al Haemostatic function in myocardial infarction. Br Heart J 1986; 55: 58-66
    CrossrefPubMedSearch in Google Scholar
  • 3 Meade TW, North WR S, Chakrabarti R. et al Haemostatic function and cardiovascular death: early results of a prospective study. Lancet 1980; i: 1050-1054
    PubMedSearch in Google Scholar
  • 4 Wilhelmsen L, Svadsudd K, Korsan-Bengtsen K, Larsson B, Welin L, Tibblin G. Fibrinogen as a risk factor for stroke and myocardial infarction. N Engl J Med 1984; 311: 501-505
    CrossrefPubMedSearch in Google Scholar
  • 5 Meade TW, Mellows S, Bruzovic M. et al Haemostatic function and ischaemic heart disease: principal result of the Northwick Park heart study. Lancet 1986; ii: 533-537
    PubMedSearch in Google Scholar
  • 6 Kannel WB. et al Fibrinogen and risk of cardiovascular disease. J Am Med Assoc 1987; 258: 1183-1186
    CrossrefPubMedSearch in Google Scholar
  • 7 Hamsten A, Iselius L, de Faire U, Blomback M. Genetic and cultural inheritance of plasma fibrinogen concentration. Lancet 1987; 1031; 988-991
    PubMedSearch in Google Scholar
  • 8 Doolittle RF. Fibrinogen and fibrin. Sci Am 1981; 245: 92-101
    PubMedSearch in Google Scholar
  • 9 Lipinska I, Lipinski B, Gurewich W. Fibrinogen heterogeneity in human plasma. Electrophoretic demonstration and characterization of two major fibrinogen components. J Lab Clin Med 1974 84. 509-516
    PubMedSearch in Google Scholar
  • 10 Holm B, Godal HC. Quantitation of the three normally occurring plasma fibrinogens in health and during socalled “acute phase” by SDS-electrophoresis of fibrin obtained from EDTA-plasma. Thromb Res 1984; 35: 279-290
    CrossrefPubMedSearch in Google Scholar
  • 11 Mosesson MW, Galanakis DK, Finlayson JS. Comparison of human plasma fibrinogen subfractions and early plasmic fibrinogen derivatives. J Biol Chem 1974; 249: 4656-4664
    PubMedSearch in Google Scholar
  • 12 Phillips HM. Isolation of size homogeneous preparations of high molecular weight and low molecular weight fibrinogens. Can J Biochem 1981; 59: 332-342
    CrossrefPubMedSearch in Google Scholar
  • 13 Holm B, Nilsen DW T, Kierulf P, Godal HC. Purification and characterization of 3 fibrinogens with different molecular weights obtained from normal human plasma. Thromb Res 1985; 37: 165-176
    CrossrefPubMedSearch in Google Scholar
  • 14 Ellis BC, Stransky A. A quick and accurate method for the determination of fibrinogen in plasma. J Lab Clin Med 1961; 58: 477-488
    PubMedSearch in Google Scholar
  • 15 Bang HO. Quantitative determination of fibrinogen in plasma. Scand J Clin Lab Invest 1975; 9: 205-207
    PubMedSearch in Google Scholar
  • 16 Ratnoff OD, Menzie C. A new method for the determination of fibrinogen in small samples of plasma. J Lab Clin Med 1951; 37: 316-320
    PubMedSearch in Google Scholar
  • 17 Blomback B. On the properties of fibrinogen and fibrin. Arkiv Kemi 1958; 12: 99-108
    PubMedSearch in Google Scholar
  • 18 Bonsnes RW, Sweeney WJ. A rapid simple semiquantitative test for fibrinogen employing thrombin. Am J Obst Gynecol 1956; 70: 334-340
    PubMedSearch in Google Scholar
  • 19 Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol 1957; 17: 237-242
    CrossrefPubMedSearch in Google Scholar
  • 20 Nieuwenhuizen W, Gravesen M. Anticoagulant and calcium-binding properties of high molecular weight derivatives of human fibrinogen, produced by plasmin (fragments X). Biochim Biophys Acta 1981; 668: 81-88
    CrossrefPubMedSearch in Google Scholar
  • 21 Nieuwenhuizen W, Voskuilen M, Hermans J. Anticoagulant and calcium-binding properties of high molecular weight derivatives of human fibrinogen (plasmin fragments Y). Biochim Biophys Acta 1982; 708: 313-316
    CrossrefPubMedSearch in Google Scholar
  • 22 Millar HR, Simpson JC, Stalker AL. An evaluation of the heat precipitation method for plasma fibrinogen estimation. J Clin Pathol 1971; 24: 827-830
    CrossrefPubMedSearch in Google Scholar
  • 23 Kazal LA, Grannis GK, Tocantins LM. Preparation of fibrinogen by glycine precipitation. In Blood Coagulation. Tocantins LM, Kazal LA. eds Grune and Stratton; New York: 1964: 232-238
  • 24 Parfentjev JA, Johnson ML, Clifton EE. The determination of plasma fibrinogen by turbidity with ammonium sulfate. Arch Kinehe m Biophys 1953; 46: 470-480
    PubMedSearch in Google Scholar
  • 25 Fowell AH. Turbidimetric method of fibrinogen assay. Am J Clin Pathol 1955; 25: 340-342
    CrossrefPubMedSearch in Google Scholar
  • 26 Brittin GM, Raffinia H, Raval D, Werner M, Brown B. Evaluation of a single radial immunodiffusion for quantitation of plasma fibrinogen. Am J Clin Pathol 1972; 57: 89-94
    CrossrefPubMedSearch in Google Scholar
  • 27 Catt KJ, Hirsh J, Castelan DJ, Niall ND, Tregear GW. Radioimmunoassay of fibrinogen and its proteolysis products. Thromb Diath Haemorrh 1968; 20: 1-6
    PubMedSearch in Google Scholar
  • 28 Exner T, Burridge J, Power P, Richard KA. An evaluation of currently available methods for plasma fibrinogen. Am J Clin Pathol 1979; 51: 521-527
    PubMedSearch in Google Scholar
  • 29 van Ruijven-Vermeer IA M, Nieuwenhuizen W. Purification of rat fibrinogen and its constituent chains. Biochem J 1978; 169: 653-658
    CrossrefPubMedSearch in Google Scholar
  • 30 Doolittle RF, Cassman KG, Cottrell BA, Friezner SJ, Hucko JT, Takagi T. Amino acid sequence studies on the a-chain of human fibrinogen. Characterization of 11 cyanogen bromide fragments Biochemistry 1977 16. 1703-1709
    PubMedSearch in Google Scholar
  • 31 Koopman J, Haverkate F, Koppert PW, Nieuwenhuizen W, Brom-mer EJ P, van der Werf WG C. New enzyme immunoassay of fibrin-fibrinogen degradation products in plasma using a monoclonal antibody. J Lab Clin Med 1987; 109: 75-84
    PubMedSearch in Google Scholar
  • 32 Koppert PW, Huijsmans CM G, 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
    PubMedSearch in Google Scholar
  • 33 Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256: 495-497
    CrossrefPubMedSearch in Google Scholar
  • 34 McKearn TJ. Cloning of hybridoma cells by limiting dilution in fluid phase. In: Monoclonal Antibodies Hybridomas; A New Dimension in Biological Analyses Kennett RH, McKearn TJ, Bechtol KB. eds Plenum Press; New York: 1980: 374
  • 35 Radi J. Immunoglobulin levels and abnormalities in aging humans and mice. In: Immunological Techniques Applied to Aging Research Adler WH, Nordin AA. eds CRC Press Inc; Boca Raton, CA: 1981: 122-139
  • 36 Ey PL, Prowse SJ, Jenkin CR. Isolation of pure IgGl5 IgG2a, and IgG2b immunoglobulins from mouse serum using protein A Sepha-rose. Immunochem 1978; 15: 429-436
    CrossrefPubMedSearch in Google Scholar
  • 37 Koppert PW, Kuipers W, Hoegee-de Nobel B, Brommer EJ P, Koopman J, Nieuwenhuizen W. A quantitative enzyme immunoassay for primary fibrinogenolysis products in plasma. Thromb Haemostas 1987; 57: 25-28
    PubMedSearch in Google Scholar
  • 38 Astrup T, Brakman P, Nissen U. The estimation of fibrinogen A revision. Scand J Clin Lab Invest 1965; 17: 57-65
    CrossrefPubMedSearch in Google Scholar