title: Epitope discovery using bacteriophage display: the minimum epitope of an anti-IRBP antibody.
abstract: The purpose of this study was to determine, using random peptide library (RPL) technologies, the minimal epitope requirements of the mouse monoclonal anti-interphotoreceptor-retinoid-binding protein antibody, H3B5. This previously characterized antibody is used as an example to examine whether RPL's offer a relatively easy and rapid route to obtaining detailed epitope mapping data.A pentadecamer random peptide library (RPL) displayed on the major coat protein (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 library selection were performed, and 90 of the resultant RPL clones were examined for affinity to H3B5 by enzyme-linked immunosorbent assay (ELISA). DNA sequencing of ELISA positive clones provided sequence of the region encoding the random peptide. After three rounds of selection of the RPL, 76.7% of clones examined interacted with H3B5, 17.7% did not show significant binding and 6.6% bound to control antibody also. The essential elements of the peptide epitope were determined by sequence comparison of 24 clones to be the four amino-acid sequence (Aspartic or glutamic acid)-Proline-Arginine-(Leucine, Isoleucine or Valine). This motif [(D/E) PR (L/I/V)] is in agreement, but at greater resolution, than previous synthetic peptide studies where the motif AASEDPRL was identified. Other motifs were found which bound to H3B5 but did not share primary structure similarities (peptidomimetics). Selection from a RPL has rapidly defined the minimal requirements for the H3B5 epitope in fine detail. Such a process offers great potential for investigating antibody-antigen interactions and core sequences of an epitope, and enables the identification of motifs in other proteins which may be recognized by the antibody, providing information on possible cross-reactivity.
authors.affiliation: Division of Ophthalmology, University Hospital, Queens Medical Centre, Nottingham, NG7 2UH, UK.
authors.forename: P J
authors.initials: PJ
authors.lastname: Tighe
authors.forename: A
authors.initials: A
authors.lastname: Powell-Richards
authors.forename: H F
authors.initials: HF
authors.lastname: Sewell
authors.forename: D
authors.initials: D
authors.lastname: Fischer
authors.forename: L
authors.initials: L
authors.lastname: Donoso
authors.forename: H S
authors.initials: HS
authors.lastname: Dua
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001324:Autoantigens / Q000276:immunology*; D001435:Bacteriophages / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D005136:Eye Proteins / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism; D012177:Retinol-Binding Proteins / Q000276:immunology*; D000099214:Retinol-Binding Proteins, Interstitial
chemical_list: D000911:Antibodies, Monoclonal; D001324:Autoantigens; D000939:Epitopes; D005136:Eye Proteins; D019151:Peptide Library; D010455:Peptides; D012177:Retinol-Binding Proteins; D000099214:Retinol-Binding Proteins, Interstitial