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
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  • 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

2012 Impact Factor: 2.657
Ranked #31/196 Oncology
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APA
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. (2000). New ELISA for quantitation of human urokinase receptor (CD87) in cancer.. International Journal of Oncology, 17(4), 827-861.
MLA
Kotzsch, Luther, Harbeck, Ockert, Lutz, Noack, Grossmann, Albrecht, Kramer, Lossnitzer, Grosser, Schmitt, and V Magdolen. "New ELISA for quantitation of human urokinase receptor (CD87) in cancer.." International Journal of Oncology International Journal of Oncology 17.4 (2000): 827-861.
Chicago
Kotzsch, Luther, Harbeck, Ockert, Lutz, Noack, Grossmann, Albrecht, Kramer, Lossnitzer, Grosser, Schmitt, and V Magdolen. "New ELISA for quantitation of human urokinase receptor (CD87) in cancer.." International Journal of Oncology International Journal of Oncology 17 no. 4 (2000): 827-861.