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ELISA for complexes between urokinase‐type plasminogen activator and its receptor in lung cancer tissue extracts
Author(s) -
de Witte Hans,
Pappot Helle,
Brünner Nils,
GrøndahlHansen Jan,
HøyerHansen Gunilla,
Behrendt Niels,
GuldhammerSkov Birgit,
Sweep Fred,
Benraad Theo,
Danø Keld
Publication year - 1997
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/(sici)1097-0215(19970729)72:3<416::aid-ijc8>3.0.co;2-s
Subject(s) - urokinase receptor , microbiology and biotechnology , chemistry , plasminogen activator , receptor , monoclonal antibody , polyclonal antibodies , antibody , urokinase , supar , cancer research , biology , biochemistry , immunology , endocrinology , genetics
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. Int. J. Cancer 72:416–423, 1997. © 1997 Wiley‐Liss, Inc.