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 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 microliters, 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Rosenquist
C
C
Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark.
Thorpe
S M
SM
Danø
K
K
Grøndahl-Hansen
J
J
D000906:Antibodies; D000911:Antibodies, Monoclonal; D001943:Breast Neoplasms / Q000201:enzymology*; D002021:Buffers; D002623:Chemistry Techniques, Analytical; D003600:Cytosol / Q000201:enzymology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis* / Q000378:metabolism
D000906:Antibodies; D000911:Antibodies, Monoclonal; D002021:Buffers; D017395:Plasminogen Activator Inhibitor 1; D014568:Urokinase-Type Plasminogen Activator