Analysis of epitope structure of PSP94 (prostate secretory protein of 94 amino acids): (I) immuno‐dominant and immuno‐recessive area

PSP94 is a potential biomarker for evaluating patients with prostate carcinoma. We have systematically studied the epitope structure of PSP94 by using a polyclonal antibody against human PSP94. Results of peptide mapping and ELISA tests of dose response to rabbit antiserum against human PSP94 protein showed that only the N‐terminal peptides (N30 and M23) are immunoreactive while all the synthetic peptides (C28, C10) located closer to the C‐terminus are completely devoid of antigenic activity with the polyclonal antibody. These results were confirmed by analysis of reciprocal competitive binding of PSP94 polyclonal antibody by the N‐terminal peptides (N30 and M23) v. either recombinant GST‐PSP94 fusion protein, purified recombinant PSP94, or natural PSP94 protein. To further delineate the antigenic activity of the N‐ and C‐termini, we have also expressed N‐ and C‐terminal half of the whole PSP94 (each 47 peptides) using the E. coli GST expression system. The recombinant N47/C47 peptides were released by thrombin cleavage from the GST fusion protein and characterized by Western blotting experiments. Dose response of the recombinant GST‐PSP‐N47 and ‐C47 peptides to PSP94 polyclonal antibody showed differential binding activities. Competitive binding of these recombinant N47/C47 proteins against the GST‐PSP94 protein demonstrates that the polyclonal antibody has a higher affinity for the N47 peptide than the C47 peptide. Based on the immunological studies of both synthetic peptides and recombinant PSP94‐ N/C terminal proteins, we propose an epitope structure of human PSP94 with an immno‐dominant N‐terminus and an immuno‐recessive C‐terminus. J. Cell. Biochem. 65:172–185. © 1997 Wiley‐Liss, Inc.

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