Analysis of epitope structure of PSP94 (prostate secretory protein of 94 amino acids): (II). Epitope mapping by monoclonal antibodies.
PSP94 has shown potential to be a serum biomarker for evaluating prostate cancer. Studies of the epitope structure is crucial for this endeavour. In this article, we have used 15 different monoclonal antibodies (MAb) to analyse the epitope structure of PSP94 and to compare with the results obtained from our previous work using polyclonal antibody and recombinant PSP94. Firstly, we determined the relative activities of the 15 MAb population by direct and competitive ELISA. The two predominant MAbs (MAb PSP-6 and -19) in 15 MAbs were selected for further studies of the epitope structure. By comparing the binding activities of recombinant GST-PSP94 and natural PSP94 with MAbs, and by comparing their affinity with MAbs in an in vitro denaturing experiment, PSP94 was shown to have a similar, prevalently linear epitope structure as we demonstrated by polyclonal antibody. Using recombinant GST fusion protein with PSP94 and with each half of the N- and C-terminal 47 amino acids (GST-PSP-N47/C47) in E. coli cells, the different epitopes recognized by 15 monoclonal antibodies were delineated and the polar distribution of the epitope structure of PSP94 was characterized. Results of direct ELISA of recombinant N47 and C47 and their competitive binding against natural PSP94 (competitive ELISA) showed that the N- and C-termini represent the immuno-dominant and immuno-recessive area separately. A majority of the monoclonal antibodies (12/15) showed preferential binding of the N-terminal sequence of the PSP94 protein. Using GST-PSP-N47 as a standard protein, an epitope map of the 15 monoclonal antibodies was obtained. The results of this study will help to define the clinical utility of PSP94.
Xuan
J W
JW
Department of Surgery, University of Western Ontario, London, Canada.
Wu
D
D
Guo
Y
Y
Huang
C L
CL
Wright
G L
GL
Chin
J L
JL
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000737:chemistry*; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D008297:Male; D051379:Mice; D011471:Prostatic Neoplasms / Q000276:immunology; D029584:Prostatic Secretory Proteins; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins; D029589:Seminal Plasma Proteins
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D029584:Prostatic Secretory Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029589:Seminal Plasma Proteins; C043076:beta-microseminoprotein; D005982:Glutathione Transferase