EPITOPE MAPPING OF ANTI-HIV AND ANTI-HCV MONOCLONAL-ANTIBODIES AND CHARACTERIZATION OF EPITOPE MIMICS USING A FILAMENTOUS PHAGE PEPTIDE LIBRARY

A large filamentous phage library (1 x 10(9) clones) displaying random 30-amino-acid (aa) sequences on the N terminus of the pIII coat prote in was constructed and characterized. Clones in the library were affin ity selected for binding to monoclonal antibodies (mAb) against two vi ral antigens, the HIV gp120 protein and the HCV core protein. The obta ined aa sequences precisely identified the epitopes recognized by the mAb. Binding of peptide-carrying phages to the Ab was demonstrated by ELISA, Western blot and the surface plasmon resonance (SPR) method. Th e mAb-specific peptides were transferred via genetic techniques onto t he N terminus of Escherichia coli alkaline phosphatase (AP), When fuse d to the enzyme, the peptides maintained their ability to bind their r espective mAb, indicating that the peptides contained the necessary co ntact residues for binding, The affinity of the peptides was estimated to be 100 nM by SPR. A comparison is presented of the relative affini ties of phage-derived peptides to the native viral epitopes also displ ayed on the AP scaffold. The approach of transferring epitopes from ph age to AP for further evaluation should be applicable to many other mA b or receptors.