Received on June 4, 2004
Accepted on July 30, 2004

Proteomics and Protein Markers

Specific Immunoassays for Detection of Intact and Cleaved Forms of the Urokinase Receptor

¹ Finsen Laboratory, Copenhagen, Denmark, and Current address: Schering Oy, Turku, Finland
² Finsen Laboratory, Copenhagen, Denmark

^* To whom correspondence should be addressed. E-mail: gunilla{at}finsenlab.dk.

Background:The cell surface receptor (uPAR) for urokinase plasminogen activator (uPA), is a strong prognostic marker in several types of cancer. uPA cleaves the three-domain protein uPAR(I-III) into two fragments: uPAR(I), which contains domain I; and uPAR(II-III), which contains domains II and III. Established immunoassays measure a combination of uPAR forms. Our aim was to design immunoassays for specific quantification of the individual forms of uPAR.

Methods: Using appropriate combinations of epitope-mapped monoclonal antibodies (Mabs) for capture and europium-labeled detection Mabs we designed two-site sandwich time-resolved fluorescence immunoassays (TR-FIAs): TR-FIA 1 to measure uPAR(I-III) alone; TR-FIA 2 to measure both uPAR(I-III) and uPAR(II-III); and TR-FIA 3 to measure uPAR(I). To avoid detection of uPAR(I-III) in TR-FIA 3, we used a combination of the peptide uPAR antagonist AE120 and a domain I antibody, R3. AE120 blocks the binding of R3 to uPAR(I-III). In contrast, AE120 does not interact with liberated domain I and therefore does not interfere with the binding of R3 to uPAR(I).

Results: The limits of quantification (CV <20%) determined by adding the proteins to uPAR-depleted plasma were <3 pmol/L in all three assays. The interassay CVs were <11%, and recoveries were between 93% and 105%. Cross-reactivities of purified proteins in the three TR-FIAs were no more than 4%. Studies on chymotrypsin cleavage of uPAR and size-exclusion chromatography of plasma with and without added protein further supported the specificity of the assays.

Conclusions: The three novel TR-FIAs accurately quantify uPAR(I-III) alone, uPAR(I-III) together with uPAR(II-III), and uPAR(I), respectively, in biological samples, including blood plasma, and thus are well suited for studies of the diagnostic and prognostic value of individual uPAR forms in cancer patients.