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 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.
de Witte
H
H
Department of Experimental and Chemical Endocrinology, University Hospital Nijmegen, The Netherlands. J.dewitte@lev.azn.nl
Pappot
H
H
Brünner
N
N
Grøndahl-Hansen
J
J
Hoyer-Hansen
G
G
Behrendt
N
N
Guldhammer-Skov
B
B
Sweep
F
F
Benraad
T
T
Danø
K
K
D000818:Animals; D000911:Antibodies, Monoclonal; D002294:Carcinoma, Squamous Cell / Q000737:chemistry*; D003432:Cross-Linking Reagents; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007163:Immunosorbent Techniques; D008175:Lung Neoplasms / Q000737:chemistry*; D051379:Mice; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000032:analysis / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D012680:Sensitivity and Specificity; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; C524898:PLAUR protein, human; C524899:Plaur protein, mouse; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator