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

TY - JOUR

T1 - ELISA for complexes between urokinase-type plasminogen activator and its receptor in lung cancer tissue extracts

AU - de Witte, H

AU - Pappot, H

AU - Brünner, N

AU - Grøndahl-Hansen, J

AU - Hoyer-Hansen, G

AU - Behrendt, N

AU - Guldhammer-Skov, B

AU - Sweep, Fred

AU - Benraad, T

AU - Danø, K

PY - 1997/7/29

Y1 - 1997/7/29

N2 - 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.

AB - 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.

KW - Animals

KW - Antibodies, Monoclonal

KW - Carcinoma, Squamous Cell

KW - Cross-Linking Reagents

KW - Enzyme-Linked Immunosorbent Assay

KW - Humans

KW - Immunosorbent Techniques

KW - Lung Neoplasms

KW - Mice

KW - Rabbits

KW - Receptors, Cell Surface

KW - Receptors, Urokinase Plasminogen Activator

KW - Sensitivity and Specificity

KW - Urokinase-Type Plasminogen Activator

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

M3 - Journal article

C2 - 9247284

SN - 0898-6924

VL - 72

SP - 416

EP - 423

JO - International Journal of Cancer. Supplement

JF - International Journal of Cancer. Supplement

IS - 3

ER -