ENZYME-LINKED-IMMUNOSORBENT-ASSAY OF UROKINASE-TYPE PLASMINOGEN-ACTIVATOR (UPA) IN CYTOSOLIC EXTRACTS OF HUMAN BREAST-CANCER TISSUE

The enzyme urokinase-type plasminogen activator (uPA) plays a role in cancer invasion, and high levels of uPA in detergent extracts of mamma ry cancer tissue have been reported to be associated with a poor progn osis. We have explored the possibility of using mammary cancer cytosol extracts routinely prepared for steroid receptor analysis for retrosp ective prognostic studies of uPA. A sandwich enzyme-linked immunosorbe nt assay (ELISA) for uPA was developed, using polyclonal catching anti bodies and a mixture of three biotinylated monoclonal detecting antibo dies, that were selected to recognize free uPA, inhibitor-bound uPA, a nd uPA bound to its cell surface receptor. The assay detects active uP A 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 yl, 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 cy tosol 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 the re was a close correlation between the amounts of uPA extracted under optimal conditions and those routinely used for steroid hormone recept or analysis. While the amount of uPA extracted under the latter condit ions constituted only about 12% of that optimally extractable, we conc lude that the ELISA described herein can be used to retrospectively an alyze the potential prognostic value of uPA-concentrations in cytosols prepared for analysis of steroid hormone receptors.