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 immuno-dominant N-terminus and an immuno-recessive C-terminus.
Xuan
J W
JW
Department of Surgery, University of Western Ontario, London, Canada.
Wu
D
D
Guo
Y
Y
Garde
S
S
Baijal-Gupta
M
M
Chin
J L
JL
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000276:immunology; D001483:Base Sequence; D001667:Binding, Competitive; D014408:Biomarkers, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011471:Prostatic Neoplasms / Q000276:immunology; D029584:Prostatic Secretory Proteins; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins; D029589:Seminal Plasma Proteins
D000906:Antibodies; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D029584:Prostatic Secretory Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029589:Seminal Plasma Proteins; C043076:beta-microseminoprotein; D005982:Glutathione Transferase