ELISA for complexes between urokinase-type plasminogen activator and its receptor in lung cancer tissue extracts
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ELISA for complexes between urokinase-type plasminogen activator and its receptor in lung cancer tissue extracts. / de Witte, H; Pappot, H; Brünner, N; Grøndahl-Hansen, J; Hoyer-Hansen, G; Behrendt, N; Guldhammer-Skov, B; Sweep, Fred; Benraad, T; Danø, K.
I: International Journal of Cancer. Supplement, Bind 72, Nr. 3, 29.07.1997, s. 416-23.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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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
VL - 72
SP - 416
EP - 423
JO - Acta - Unio Internationalis Contra Cancrum
JF - Acta - Unio Internationalis Contra Cancrum
SN - 0898-6924
IS - 3
ER -
ID: 180823429