COMPLEXES BETWEEN UROKINASE-TYPE PLASMINOGEN-ACTIVATOR AND ITS RECEPTOR IN BLOOD AS DETERMINED BY ENZYME-LINKED-IMMUNOSORBENT-ASSAY

Complexes between urokinase-type plasminogen activator (uPA) and its r eceptor (uPAR) were assessed in plasma and serum from 39 breast cancer patients and from 20 healthy individuals, applying a recently develop ed enzyme-linked immunosorbent assay (ELISA) for the analysis of these complexes in tumor tissue extracts. The assay is based on a combinati on of rabbit polyclonal anti-uPA antibodies for catching and a mouse a nti-uPAR monoclonal antibody (MAb) for detection. The specificity of t he assessment of uPA:uPAR complexes was verified by simultaneous analy sis of the individual blood samples in corresponding non-sense ELISA f ormats, in which either the anti-uPA catching antibody or the anti-uPA R detecting antibody was substituted with an irrelevant antibody. Asse ssment of native uPA:uPAR complexes was ascertained by demonstrating t he absence of any de novo formation of uPA:uPAR complexes in plasma an d serum during the sample incubation step in the ELISA, as verified by the use of a peptide antagonist for uPAR. Plasma and serum samples co ntained almost identical levels of uPA:uPAR complexes. The levels of u PA:uPAR complexes were found to be significantly lower in serum from b reast cancer patients compared to the serum of healthy donors, while t he levels of(total) uPAR in plasma from breast cancer patients were si gnificantly higher than in plasma from the healthy controls. In additi on, the free, uncomplexed uPAR levels, estimated by subtraction of uPA :uPAR complex levels from (total) uPAR levels, were significantly elev ated in plasma as well as in serum from breast cancer patients compare d to healthy individuals. The uPA:uPAR complex levels were highly comp arable to the uPA levels analyzed in the same plasma and serum samples , indicating that most if not all of the uPA present in these samples is complexed with uPAR. (C) 1998 Wiley-Liss, Inc.