ANALYSIS OF EPITOPE STRUCTURE OF PSP94 (PROSTATE SECRETORY PROTEIN OF94 AMINO-ACIDS) .2. EPITOPE MAPPING BY MONOCLONAL-ANTIBODIES

PSP94 has shown potential to be a serum biomarker for evaluating prost ate cancer. Studies of the epitope structure is crucial for this endea vour. In this article, we have used 15 different monoclonal antibodies (MAb) to analyse the epitope structure of PSP94 and to compare with t he results obtained from our previous work using polyclonal antibody a nd recombinant PSP94. Firstly, we determined the relative activities o f the 15 MAb population by direct and competitive ELISA. The two predo minant MAbs (MAb PSP-6 and -19) in 15 MAbs were selected for further s tudies of the epitope structure. By comparing the binding activities o f recombinant CST-PSP94 and natural PSP94 with MAbs, and by comparing their affinity with MAbs in an in vitro denaturing experiment, PSP94 w as shown to have a similar, prevalently linear epitope structure as we demonstrated by polyclonal antibody. Using recombinant CST fusion pro tein with PSP94 and with each half of the N- and C-terminal 47 amino a cids (GST-PSP-N47/C47) in E. coli cells, the different epitopes recogn ized by 15 monoclonal antibodies were delineated and the polar distrib ution 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-ter mini represent the immuno-dominant and immune-recessive area separatel y. A majority of the monoclonal antibodies (12/15) showed preferential binding of the N-terminal sequence of the PSP94 protein. Using GST-PS P-N47 as a standard protein, an epitope map of the 15 monoclonal antib odies was obtained. The results of this study will help to define the clinical utility of PSP94. (C) 1997 Wiiey-Liss, Inc.