New ELISA for quantitation of human urokinase receptor (CD87) in cancer.

  • Authors:
    • M Kotzsch
    • T Luther
    • N Harbeck
    • D Ockert
    • V Lutz
    • F Noack
    • D Grossmann
    • S Albrecht
    • M D Kramer
    • A Lossnitzer
    • M Grosser
    • M Schmitt
    • V Magdolen
  • Corresponding author:
  • View Affiliations

  • Published online on: Sunday, October 1, 2000
  • Pages: 827-861 DOI: 10.3892/ijo.17.4.827

Abstract

The serine protease urokinase-type plasminogen activator (uPA), its inhibitor (PAI-1), and its receptor (uPAR; CD87) facilitate cancer cell invasion and metastasis. Whereas uPA and PAI-1 antigen levels determined in tumor tissue extracts of breast cancer patients correlate with disease recurrence and overall survival, the prognostic relevance of uPAR is still a matter of debate. We established two new sandwich-type enzyme-linked immunosorbent assay (ELISA) formats (HU/IIIF10-ELISA and HU/HD13-ELISA) using the epitope-defined monoclonal antibody (mAb) IIIF10 or the conformation-dependent mAb HD13.1, a polyclonal chicken antibody (HU277), and recombinant soluble uPAR (CHO-suPAR) as the standard. The lower detection limit of the assays was at 0.16 ng/ml, with a linear dose-response up to 5 ng/ml of uPAR antigen. Both ELISA formats showed good reproducibility and recovery. The intra-assay and the inter-assay variation coefficients were respectively 4.3% and 11.7% (HU/IIIF10-ELISA) and 4.0% and 10.7% (HU/HD13-ELISA). The recovery rate of uPAR in cell lysates spiked with CHO-suPAR was above 82% and 88%, respectively. With these new ELISA formats, uPAR antigen content in breast cancer tissue extracts and tumor cell lysates was determined and compared to a commercially available ELISA (ADI-ELISA). By all of the three uPAR ELISA formats CHO-suPAR and uPAR present in lysates of non-malignant epithelial cells and stimulated monocytes were quantified with similar sensitivity. Interestingly, in breast cancer cell lines of epitheloid origin a higher uPAR antigen content was determined by the HU/IIIF10-ELISA than the HU/HD13- or ADI-ELISA formats. In lysates of fibroblastic breast cancer cell lines similar uPAR values were obtained with the HU/IIIF10- and ADI-ELISA formats, whereas with the HU/HD13-ELISA significantly lower uPAR concentrations were determined. The prognostic relevance of tumor uPAR antigen was evaluated in 199 primary breast cancer patients with a median follow-up of 24 months. uPAR antigen values above the cut-off of 3.33 ng/mg protein as determined by the HU/IIIF10-ELISA were significantly correlated with short disease-free survival (p=0.025). Results obtained by the other two ELISA formats (HU/HD13-ELISA and ADI-ELISA) were not associated with prognosis. Our findings stress the need of well-characterized antibodies, which detect both uPAR of non-malignant and tumor cells, in setting up a uPAR-ELISA useful for assessing breast cancer patient prognosis.
Journal Cover

October 2000
Volume 17 Issue 4

Print ISSN: 1019-6439
Online ISSN:1791-2423

2013 Impact Factor: 2.773
2014 I.F. (Expected) ≥ 3.258 Ranked #30/202 Oncology
(total number of cites)

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
APA
Kotzsch, M., Luther, T., Harbeck, N., Ockert, D., Lutz, V., Noack, F. ... Magdolen, V. (2000). New ELISA for quantitation of human urokinase receptor (CD87) in cancer.. International Journal of Oncology, 17, 827-861. http://dx.doi.org/10.3892/ijo.17.4.827
MLA
Kotzsch, M., Luther, T., Harbeck, N., Ockert, D., Lutz, V., Noack, F., Grossmann, D., Albrecht, S., Kramer, M., Lossnitzer, A., Grosser, M., Schmitt, M., Magdolen, V."New ELISA for quantitation of human urokinase receptor (CD87) in cancer.". International Journal of Oncology 17.4 (2000): 827-861.
Chicago
Kotzsch, M., Luther, T., Harbeck, N., Ockert, D., Lutz, V., Noack, F., Grossmann, D., Albrecht, S., Kramer, M., Lossnitzer, A., Grosser, M., Schmitt, M., Magdolen, V."New ELISA for quantitation of human urokinase receptor (CD87) in cancer.". International Journal of Oncology 17, no. 4 (2000): 827-861. http://dx.doi.org/10.3892/ijo.17.4.827