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

✍ Scribed by Hans de Witte; Helle Pappot; Nils Brünner; Jan Grøndahl-Hansen; Gunilla Høyer-Hansen; Niels Behrendt; Birgit Guldhammer-Skov; Fred Sweep; Theo Benraad; Keld Danø

• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: French
• Weight: 131 KB
• Volume: 72
• Category: Article
• ISSN: 0020-7136
• DOI: 10.1002/(sici)1097-0215(19970729)72:3<416::aid-ijc8>3.0.co;2-s

✦ Synopsis

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. Int.