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

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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.

Original languageEnglish
JournalInternational Journal of Cancer
Volume72
Issue number3
Pages (from-to)416-23
Number of pages8
ISSN0020-7136
Publication statusPublished - 29 Jul 1997

    Research areas

  • Animals, Antibodies, Monoclonal, Carcinoma, Squamous Cell, Cross-Linking Reagents, Enzyme-Linked Immunosorbent Assay, Humans, Immunosorbent Techniques, Lung Neoplasms, Mice, Rabbits, Receptors, Cell Surface, Receptors, Urokinase Plasminogen Activator, Sensitivity and Specificity, Urokinase-Type Plasminogen Activator

ID: 46435260