Determination of the complex between urokinase and its type-I inhibitor in plasma from healthy donors and breast cancer patients

Abstract

Background: The complex between urokinase (uPA) and its type-1 inhibitor (PAI-1) is formed exclusively from the active forms of these components; thus, the complex concentration in a biological sample may reflect the ongoing degree of plasminogen activation. Our aim was to establish an ELISA for specific quantification of the uPA:PAI-1 complex in plasma of healthy donors and breast cancer patients. Methods: A kinetic sandwich format immunoassay was developed, validated, and applied to plasma from 19 advanced- stage breast cancer patients, 39 age-matched healthy women, and 31 men. Results: The assay detection limit was <2 ng/L, and the detection of complex in plasma was validated using immunoabsorption, competition, and recovery tests. Eighteen cancer patients had a measurable complex concentration (median, 68 ng/L; range, <16 to 8700 ng/L), whereas for healthy females and males the median signal values were below the detection limit (median, <16 ng/L; range, <16 to 200 ng/L; P <0.0001). For patient plasma, a comparison with total uPA and PAI-1 showed that the complex represented a variable, minor fraction of the uPA and PAI-1 concentrations of each sample. Conclusion: The reported ELISA enables detection of the uPA:PAI-1 complex in blood and, therefore, the evaluation of the complex as a prognostic marker in cancer.