Identification of epitopes of trichosanthin by phage peptide library

The phage displayed random peptide library has recently emerged as a powerf ul technique for analyzing Ab-Ag interactions. In this study, the method wa s employed to identify epitopes of trichosanthin. Two monoclonal Abs (4B5, 2E9) which recognized different epitopes of trichosanthin (TCS) were select ed and a phage-peptide library with nine amino acids (9 aa) was used to scr een the positive phage clones that have high affinity to the mAbs. Two grou ps of phage clones that carried peptide-specific binding to mAbs were ident ified by the screen. The identified phage clones carried peptide-specific b inding to 4B5 and 2E9 mAbs were immunized in mice. To evaluate mimotope of selected phages, the specific binding activity to TCS was measured in the s erum from phage-immunized mice. They all showed positive results. The conse rved interaction motifs were deduced from the peptide sequences of each gro up of selected phage clones. When compared the motif sequence with the sequ ence of TCS, it was predicted that 4B5-corresponding epitope was located at 27-37 aa of TCS protein and 2E9-corresponding epitope was located at 41-48 aa of TCS. The predicted sequence of 4B5-corresponding epitope was further con firmed by site-directed mutation of TCS protein. The data showed that the expressed TCS protein mutated in 4B5-corresponding epitope was unable t o bind 4B5 mAb. The results suggested that the phage display peptide librar y is useful to identify Ag epitopes and to raise Ab in disease diagnosis an d treatment. (C) 2001 Academic Press.