ELISA for complexes between urokinase-type plasminogen activator and its receptor in lung cancer tissue extracts

H de Witte, H Pappot, N Brünner, J Grøndahl-Hansen, G Hoyer-Hansen, N Behrendt, B Guldhammer-Skov, F Sweep, T Benraad, K Danø

    13 Citationer (Scopus)

    Abstract

    A sandwich-type ELISA has been developed for the assessment of complexes between urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in extracts of squamous cell lung carcinomas. The assay is based on a combination of rabbit polyclonal anti-uPA antibodies and a biotinylated mouse anti-uPAR monoclonal antibody (MAb). The detection limit of the assay is approximately 0.5 fmol/ml. A linear dose-response is obtained with up to 40 fmol/ml of uPA:uPAR complexes, while uPA and uPAR separately do not cause any response in the ELISA. A buffer which has been used previously for optimal extraction of uPAR yields the highest amounts of uPA:uPAR complexes. Absorption of tumor extracts with anti-uPA or anti-uPAR MAbs results in a complete disappearance of the ELISA signal, demonstrating the specificity of the ELISA. The recovery of chemically cross-linked uPA:uPAR complexes added to tumor extracts varies between 80% and 105%. The intra- and inter-assay variation coefficients are 5.3% and 9.8%, respectively. Furthermore, a peptide antagonist for uPAR was employed to evaluate de novo uPA:uPAR complex formation during tumor tissue extraction and the immunoassay procedure. Our results strongly indicate that de novo complex formation is a major factor to consider and that complexes analyzed in the presence of this antagonist represent original uPA:uPAR complexes present prior to tumor tissue processing. The present ELISA appears suitable for studying the potential prognostic impact of uPA:uPAR complexes in lung tumor tissue as well as other types of cancer.

    OriginalsprogEngelsk
    TidsskriftInternational Journal of Cancer
    Vol/bind72
    Udgave nummer3
    Sider (fra-til)416-23
    Antal sider8
    ISSN0020-7136
    StatusUdgivet - 29 jul. 1997

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