Download PDF
Thromb Haemost 1985; 54(03): 684-687
DOI: 10.1055/s-0038-1660097
Original Article
Schattauer GmbH Stuttgart

Assay of Human Tissue-Type Plasminogen Activator (t-PA) with an Enzyme-Linked Immunosorbent Assay (ELISA) Based on Three Murine Monoclonal Antibodies to t-PA

Paul Holvoet
The Center for Thrombosis and Vascular Research, University of Leuven, Belgium
,
Hugo Cleemput
The Center for Thrombosis and Vascular Research, University of Leuven, Belgium
,
Désiré Collen
The Center for Thrombosis and Vascular Research, University of Leuven, Belgium
› Author Affiliations
Further Information

Publication History

Received 10 April 1985

Accepted 14 August 1985

Publication Date:
19 July 2018 (online)

  PDF Download  Permissions and Reprints

Summary

An enzyme-linked immunosorbent assay (ELISA) for the measurement of human tissue-type plasminogen activator (t-PA) was developed. Microtiter plates were coated with a mixture of two monoclonal antibodies and bound t-PA was quantitated with a third monoclonal antibody linked to peroxidase. The lower limit of sensitivity of the assay was 0.2 ng of t-PA per ml. The concentration of antigen in citrated plasma of human subjects was found to be 3.4 ± 0.8 ng/ml. The assay had a good reproducibility with values of 3.8, 6.5 and 4.9 percent respectively for the intra-, inter-assay and inter-dilution variation coefficients. The results of the ELISA assay on plasma samples from patients during thrombolytic therapy with t-PA correlated very well, over a wide concentration range, with those obtained with a previously described two-site immuno-radiometric assay (r = 0.96). This ELISA with monoclonal antibodies constitutes a stable and reproducible set of reagents for the measurement of t-PA antigen in biological fluids, avoiding the disadvantages of the use of radioisotopes and of polyclonal antibodies.

Keywords

ELISA - Tissue-type plasminogen activator - Monoclonal antibodies
 

  • References

  • 1 Collen D. On the regulation and control of fibrinolysis. Edward Kowalski Memorial Lecture. Thromb Haemostas 1980; 43: 77-89
    PubMedGoogle Scholar
  • 2 Holmberg L, Kristoffersson AC, Lecander I, Wallén P, Åstedt B. Immunoradiometric quantification of tissue plasminogen activator secreted by fetal organs. Comparison with urokinase. Scand J Clin Lab Invest 1982; 42: 347-354
    CrossrefPubMedGoogle Scholar
  • 3 Holmberg L, Nilsson IM, Åstedt B. Immunoreactive tissue activator and urokinase in plasma after various stimuli. In: Progress in Fibrinolysis, Vol 6. pp 213-8 Davidson JF, Bachmann F, Bouvier CA, Kruithof EKO. (eds) Churchill Livingstone; Edinburgh: 1983
  • 4 Rijken DC, Juhan-Vague I, De Cock F, Collen D. Measurement of human tissue-type plasminogen activator by a two-site immunoradiometric assay. J Lab Clin Med 1983; 101: 274-284
    PubMedGoogle Scholar
  • 5 MacGregor IR, Prowse CV. Tissue plasminogen activator in human plasma measured by radioimmunoassay. Thromb Res 1983; 31: 461-474
    CrossrefPubMedGoogle Scholar
  • 6 Bergsdorf N, Nilsson T, Wallén P. An enzyme linked immunosorbent assay for determination of tissue plasminogen activator applied to patients with thromboembolic disease. Thromb Haemostas 1983; 50: 740-744
    PubMedGoogle Scholar
  • 7 Matsuo O, Kato K, Matsuo C, Matsuo T. Determination of tissue plasminogen activator by an enzyme-immunoassay method. Anal Biochem 1983; 135: 58-63
    CrossrefPubMedGoogle Scholar
  • 8 Rijken DC, Van Hinsbergh VW M, Sens EH C. Quantitation of tissue-type plasminogen activator in human endothelial cell cultures by use of an enzyme immunoassay. Thromb Res 1984; 33: 145-153
    CrossrefPubMedGoogle Scholar
  • 9 Rijken DC, Collen D. Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J Biol Chem 1981; 256: 7035-7041
    PubMedGoogle Scholar
  • 10 Kearney JF, Radbruch A, Liesegang B, Rajewski K. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J Immunol 1979; 123: 1548-1550
    PubMedGoogle Scholar
  • 11 Galfré G, Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol 1981; 73: 3-46
    PubMedGoogle Scholar
  • 12 Ey PL, Prowse SJ, Jenkin CR. Isolation of pure IgG1 IgG2a and IgG2b immunoglobulins from mouse serum using protein A-Sephar-ose. Immunochem 1978; 15: 429-436
    CrossrefPubMedGoogle Scholar
  • 13 Nakane PA, Kawaoi A. Peroxidase labeled antibody. A new method of conjugation. J Histochem Cytochem 1974; 22: 1084-1091
    CrossrefPubMedGoogle Scholar
  • 14 Astrup T, Müllertz S. The fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 1952; 40: 346-351
    CrossrefPubMedGoogle Scholar
  • 15 Axen R, Porath J, Ernback S. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature 1967; 214: 1302-1304
    CrossrefPubMedGoogle Scholar
  • 16 Lijnen HR, Uytterhoeven B, Collen D. Inhibition of trypsin-like serine proteinases by tripeptide arginyl and lysyl chloromethylketo-nes. Thromb Res 1984; 34: 431-437
    CrossrefPubMedGoogle Scholar
  • 17 Juhan-Vague I, Moerman B, De Cock F, Aillaud MF, Collen D. Plasma levels of a specific inhibitor of tissue-type plasminogen activator (and urokinase) in normal and pathological conditions. Thromb Res 1984; 33: 523-530
    CrossrefPubMedGoogle Scholar