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Urokinase receptor in breast cancer tissue extracts. Enzyme-linked immunosorbent assay with a combination of mono- and polyclonal antibodies

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Summary

Urokinase plasminogen activator (uPA) is a proteolytic enzyme involved in degradation of the extracellular matrix during cancer invasion. The levels of uPA and its inhibitor PAI-1 in tumor extracts have previously been demonstrated to be of prognostic value in breast cancer as well as other types of cancer. We have previously characterized a specific cell surface receptor for uPA (uPAR) which strongly enhances the catalytic activity of uPA and is expressed during mammary cancer invasion. In order to quantitate uPAR in breast cancer tissue, we have now developed a sensitive enzyme-linked immunosorbent assay (ELISA), with polyclonal catching antibodies and three monoclonal detecting antibodies. The detection limit of the assay is approximately 0.16 fmol of uPAR in a volume of 100 µl (1.6 pM). There is a linear relationship between signal and uPAR concentration up to at least 6.6 fmol per 100 µl (66 pM). Both free uPAR and uPAR in complex with uPA is detected. The recovery of an internal uPAR standard in breast cancer tissue extracts is above 87%. The intra-assay and inter-assay variation coefficients are 7% and 13%. In order to find a suitable buffer for extraction of various components of the uPA-system from breast cancer tissue, we tested buffers which previously have been used for optimal extraction of estrogen receptor (A), uPA (B), and uPAR (C). Buffer A and B extracted approximately 30% and 50%, respectively, of the amount of uPAR extracted with buffer C. Extracts of samples of breast cancer tissue from 94 patients all contained uPAR in amounts above the detection limit of the present assay, which appears suitable for studies of the potential prognostic value of uPAR in this disease. Significant correlations were found between uPAR, uPA and PAI-1 tumor levels.

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Authors and Affiliations

  1. Finsen Laboratory, Rigshospitalet, Strandboulevarden 49, DK-2100, Copenhagen Ø., Denmark

    E. Rønne, G. Høyer-Hansen, N. Brünner, H. Pedersen, K. Danø & J. Grøndahl-Hansen

  2. Department of Pathology, Bispebjerg Hospital, Copenhagen, Denmark

    F. Rank

  3. Department of Medicine, Division of Oncology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA

    C. K. Osborne & G. M. Clark

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  1. E. Rønne
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  2. G. Høyer-Hansen
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  3. N. Brünner
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  4. H. Pedersen
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  8. K. Danø
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  9. J. Grøndahl-Hansen
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Rønne, E., Høyer-Hansen, G., Brünner, N. et al. Urokinase receptor in breast cancer tissue extracts. Enzyme-linked immunosorbent assay with a combination of mono- and polyclonal antibodies. Breast Cancer Res Tr 33, 199–207 (1995). https://doi.org/10.1007/BF00665944

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