THE COMPLEX BETWEEN UROKINASE PLASMINOGEN-ACTIVATOR AND ITS TYPE-1 INHIBITOR IN BREAST-CANCER EXTRACTS QUANTITATED BY ELISA

ELISAs for urokinase plasminogen activator (uPA) and plasminogen activ ator inhibitor type-1 (PAI-1) have shown that tumor levels of these mo lecules are prognostic parameters in breast cancer as well as other ty pes of cancer, These ELISAs measure the total amount of the given comp onent, including preforms, active, inactive, and complex-bound forms, However, the amount of the active forms of a component may more closel y reflect the ongoing level of proteolytic activity and thereby be par ticularly related to prognosis. Since the inactive complex between uPA and PAI-1 can only be formed from the active forms of the individual components, we have developed a sensitive and specific uPA:PAI-1 compl ex ELISA consisting of a sandwich format with two monoclonal antibodie s (MAbs) against PAI-1 as capture antibodies and three biotinylated MA bs against uPA as detector antibodies. The data were collected as kine tic measurements of bound alkaline phosphatase activity. A standard of uPA:PAI-1 complex could be specifically measured in the assay with a detection limit of 8 pg/ml and a linear relationship between signal an d complex concentration up to 4 ng/ml, Neither free uPA nor free PAI-1 were detected by this assay and the addition to the internal standard of free PAI-1 in amounts up to 20 ng/ml or uPA did not reduce the det ection of complex by the assay. This ELISA was applied to extracts fro m 20 individual breast cancers, Each tumor was extracted in two differ ent buffers and the median concentration of uPA:PAI-1 complex in the o ptimal extraction buffer was 0.8 ng/mg protein, range 0.4-2.8 ng/mg pr otein. Extraction of the tumor tissue at a low pH prevented de novo fo rmation of complex from free uPA and PAI-1 in the tissue without desta bilizing preformed uPA:PAI-1 complex. During incubation of the assay p late at neutral pH further uPA:PAI-1 complex formation from free compo nents in the extracts was blocked by p-nitrophenyl guanidinobenzoate ( NPGB). Thus, the present assay selectively quantifies preformed comple x in tumor extracts and will enable us, for the first time, to evaluat e the potential prognostic value of the uPA:PAI-1 complex in cancer.