Enzyme-linked immunosorbent assay of urokinase-type plasminogen activator (uPA) in cytosolic extracts of human breast cancer tissue

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Abstract
Summary The enzyme urokinase-type plasminogen activator (uPA) plays a role in cancer invasion, and high levels of uPA in detergent extracts of mammary cancer tissue have been reported to be associated with a poor prognosis. We have explored the possibility of using mammary cancer cytosol extracts routinely prepared for steroid receptor analysis for retrospective prognostic studies of uPA. A sandwich enzyme-linked immunosorbent assay (ELISA) for uPA was developed, using polyclonal catching antibodies and a mixture of three biotinylated monoclonal detecting antibodies, that were selected to recognize free uPA, inhibitor-bound uPA, and uPA bound to its cell surface receptor. The assay detects active uPA and its inactive proenzyme form, pro-uPA, equally well. The limit of detection is approximately 1 pg of pro-uPA in a volume of 100 µl, and there is a linear dose-response up to 100 pg pro-uPA. The efficiency in extracting uPA of a neutral non-detergent buffer used to prepare cytosol extracts was compared with that of 4 other buffers. There was a pronounced difference in the efficiency, the most efficient being a pH 4.2 buffer containing the non-ionic detergent Triton X-100, while the least efficient was the buffer used to prepare cytosols. Nevertheless, uPA immunoreactivity was readily measurable in the cytosols, and there was a close correlation between the amounts of uPA extracted under optimal conditions and those routinely used for steroid hormone receptor analysis. While the amount of uPA extracted under the latter conditions constituted only about 12% of that optimally extractable, we conclude that the ELISA described herein can be used to retrospectively analyze the potential prognostic value of uPA-concentrations in cytosols prepared for analysis of steroid hormone receptors.