ELISA FOR COMPLEXES BETWEEN UROKINASE-TYPE PLASMINOGEN-ACTIVATOR AND ITS RECEPTOR IN LUNG-CANCER TISSUE-EXTRACTS

A sandwich-type ELISA has been developed for the assessment of complex es between urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in extracts of squamous cell lung carcinomas, The assay is bas ed on a combination of rabbit polyclonal anti-uPA antibodies and a bio tinylated mouse anti-uPAR monoclonal antibody (MAb). The detection lim it of the assay is approximately 0.5 fmol/ml, A linear dose response i s 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 hi ghest 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 recover y of chemically cross-linked uPA:uPAR complexes added to tumor extract s varies between 80% and 105%, The intra- and inter-assay variation co efficients are 5.3% and 9.8%, respectively. Furthermore, a peptide ant agonist for uPAR was employed to evaluate de novo uPA:uPAR complex for mation during tumor tissue extraction and the immunoassay procedure. O ur results strongly indicate that de novo complex formation is a major factor to consider and that complexes analyzed in the presence of thi s antagonist represent original uPA:uPAR complexes present prior to tu mor tissue processing. The present ELISA appears suitable for studying the potential prognostic impact of uPA:uPAR complexes in lung tumor t issue as well as other types of cancer. (C) 1997 Wiley-Liss, Inc.