Epitope discovery using bacteriophage display: The minimum epitope of an anti-IRBP antibody

The purpose of this study was to determine, using random peptide library (R PL) technologies. the minimal epitope requirements of the mouse monoclonal anti-interphotoreceptor-retinoid-binding protein antibody, H3B5, This previ ously characterized antibody is used as an example to examine whether RPL's offer a relatively easy and rapid route to obtaining detailed epitope mapp ing data. A pentadecamer random peptide library (RPL) displayed on the major coat pro tein (gene 8) of filamentous bacteriophage (F88-4-15) was used as a target for selection by the anti-IRBP monoclonal antibody, H3B5. Three rounds of l ibrary selection were performed, and 90 of the resultant RPL clones were ex amined for affinity to H3B5 by enzyme-linked immunosorbent assay (ELISA). D NA sequencing of ELISA positive clones provided sequence of the region enco ding the random peptide. After three rounds of selection of the RPL, 76.7% of clones examined intera cted with H3B5, 17.7% did not show significant binding and 6.6% bound to co ntrol antibody also. The essential elements of the peptide epitope were det ermined by sequence comparison of 24 clones to be the four amino-acid seque nce (Aspartic or glutamic acid)-Proline-Arginine-(leucine, Isoleucine or Va line). This motif [(D/E) PR (L/I/V)] is in agreement, but at greater resolu tion, than previous synthetic peptide studies where the motif AASEDPRL was identified. Other motifs were found which bound to H3B5 but did not share p rimary structure similarities (peptidomimetics). Selection from a RPL has rapidly defined the minimal requirements for the H 3B5 epitope in fine detail, Such a process offers great potential for inves tigating antibody-antigen interactions and core sequences of an epitope, an d enables the identification of motifs in other proteins which may be recog nized by the antibody, providing information on possible cross-reactivity, (C) 1999 Academic Press.