Prostate-specific antigen: characterization of epitopes by synthetic peptide mapping and inhibition studies Eva Corey,* Sandra K. Wegner, Michael J. Corey, and Robert L. Vessella To improve our understanding of the prostate-specific antigen (PSA) antigenic regions, we studied the associ- ation targets of one anti-PSA polyclonal antibody and 10 anti-PSA monoclonal antibodies (mAbs). We also exam- ined the ability of the mAbs to inhibit PSA enzymatic activity and block the association of PSA with  1 - antichymotrypsin (ACT). Linear epitope mapping with a polyclonal antibody indicated the presence of six major antigenic regions in PSA. Examination of the panel of mAbs established that three of them bind to linear epitopes. Five of the mAbs inhibited >90% of PSA enzymatic activity. However, inhibition of PSA enzymatic activity and hindrance of PSA-ACT associa- tion by mAbs cannot be used to predict whether the mAbs bind to free PSA, the PSA-ACT complex, or both. Some of the mAbs may block PSA-ACT association through peripheral occlusion of the binding site, or through induction of conformational changes in PSA. INDEXING TERMS: linear epitope •  1 -antichymotrypsin • proteolytic activity Despite the widespread use of prostate-specific antigen (PSA) as a clinical marker of prostate cancer [1], little is known about the antigenic determinants of PSA involved in antibody recognition. 1 Information about the locations of the major epitopes on the surface of PSA might yield insight into the antigenic properties of the protein and specific details of the interactions of PSA with particular antibodies, which could in turn lead to methods of developing antibodies with desirable characteristics for use in diagnostic assays, such as higher specificity, equimolar binding to PSA and PSA- 1 -antichymotrypsin (ACT), and specific association only with free (f)-PSA. Antigenic determinants as binding targets of antibod- ies can be divided into two categories: linear (sequential, continuous) and nonlinear (conformational, discontinu- ous). Linear epitopes consist of amino acid residues that are adjacent in the primary sequence; nonlinear epitopes consist of amino acid residues that are separated in the primary structure but are brought into proximity when the protein is in its native form. At present there is no simple way to identify conformational epitopes in the absence of three-dimensional structural information about the monoclonal antibody (mAb)-antigen complex. However, the identity of linear epitopes can be predicted by computer programs that calculate various parameters that have been found to be correlated with antigenic nature in previously studied antigens (e.g., hydrophilic- ity, flexibility, and surface probability [2]). The method postulates that (a) mAbs that bind to linear epitopes react with segments of 5– 8 consecutive amino acid residues, and (b) these epitopes are on the surfaces of molecules that tend to be hydrophilic. However, computational techniques are not yet sophisticated enough to achieve the accuracy of experimental techniques. Other methods of identifying antibody binding sites involve (a) digestion of the antigen, (b) recombinant techniques, (c) phage display, (d) mass spectrometry, and (e) the use of synthetic pep- tides. Fragments from trypsin [3] or papain [4] digestion of antigens have been used to determine antibody binding targets. Numerous attempts with cyanogen bromide cleavage products have been published [4–6]. Van Duijn- hoven et al. reported the use of recombinant DNA tech- niques for epitope mapping [7]. The phage display tech- nique has found use in epitope mapping in determination of the antigenic regions of various proteins [8 –11]. By using modern mass spectrometry techniques, epitopes can be determined as described by Zhao and Chait [12], or Tumor Immunology Laboratory of the Urology Department, School of Medicine of the University of Washington, Seattle, WA 98195. *Address correspondence to this author at: Mailstop 356510, Department of Urology, University of Washington, Seattle, WA 98195. Fax 206-543-1146; e-mail ecorey@u.washington.edu. 1 Nonstandard abbreviations: PSA, prostate-specific antigen; ACT,  1 - antichymotrypsin; f-PSA, free PSA; mAb, monoclonal antibody; EIA, enzyme immunoassay; BSA, bovine serum albumin; RT, room temperature; and HRP, horseradish peroxidase. Received September 11, 1996; revised October 23, 1996; accepted Novem- ber 7, 1996. Clinical Chemistry 43:4 575–584 (1997) Enzymes and Protein Markers 575