Immunopeptidometric assay for enzymatically active prostate-specific antigen

Background: Determinations of certain forms of prostate-specific antigen (PSA) have been shown to increase the specificity for prostate cancer (PCa). One such variant, proteolytically active PSA, is a potentially useful tumor marker, but it is not specifically recognized by antibodies. Using phage display libraries, we previously identified a family of peptides that bind specifically to active PSA. We used these to develop an immunopeptidometric assay (IPMA) that specifically detects this form of PSA. Methods: Microtitration plates coated with a PSA antibody were used to capture PSA, and a PSA-binding glutathione S-transferase (GST) fusion peptide was used as a tracer. Bound tracer was detected with an antibody to GST labeled with a europium chelate. PSA isoenzymes with high and low enzymatic activity were used to study binding specificity. Results: The IPMA detected enzymatically active PSA but not internally cleaved PSA and pro-PSA, which are enzymatically inactive. The assay detected 1-10% of free PSA in serum from PCa patients. Conclusions: Peptides identified by phage display can be used to develop assays with unique specificities for enzymatically active PSA. IPMA represents a new assay principle with wide potential utility. (C) 2004 American Association for Clinical Chemistry.