title: Fast and efficient characterization of an anti-gliadin monoclonal antibody epitope related to celiac disease using resin-bound peptides.
abstract: Celiac disease is a permanent intolerance to gluten. A strong indication for celiac disease is the presence of antibodies specific for gliadin, a main component of gluten. Using a deamidated immunogenic gliadin fragment, corresponding to amino acid residues 58-73 (LQPFPQPQLPYPQPQ) of gliadin a monoclonal anti-gliadin antibody has previously been generated. In this study we present an alternative approach for fast and efficient epitope mapping of the monoclonal anti-gliadin antibody, using gliadin peptide (Lys57)-Glu65-[gliadin 58-73] as template. This approach involves generation of a number of N-terminally truncated peptide analogues in one synthesis, and the use of these resin-bound peptides for rapid screening, in order to identify the minimal peptide length required for antibody recognition. The peptides were assembled on a TentaGel resin equipped with an acid-stable HMBA linker, which allows cleavage of the side-chain protecting groups but leaves the peptide attached to the resin. If required, the peptides may be cleaved from the support under mild basic conditions. Using this approach and competitive inhibition assays we identified the immunodominant epitope as the PELPYPQPQ sequence, which was located in the C-terminal end of the gliadin peptide. Our results show that the N-terminal proline residue and the C-terminal glutamine residues are essential for antibody recognition in addition to the deamidated glutamine residue.
authors.affiliation: Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark.
authors.forename: Nicole Hartwig
authors.initials: NH
authors.lastname: Petersen
authors.forename: Paul Robert
authors.initials: PR
authors.lastname: Hansen
authors.forename: Gunnar
authors.initials: G
authors.lastname: Houen
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001324:Autoantigens / Q000235:genetics / Q000276:immunology; D001667:Binding, Competitive; D002446:Celiac Disease / Q000235:genetics / Q000276:immunology*; D018604:Epitope Mapping / Q000379:methods*; D005903:Gliadin / Q000235:genetics / Q000276:immunology*; D006801:Humans; D016056:Immunodominant Epitopes / Q000235:genetics / Q000276:immunology; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D011137:Polystyrenes; D012117:Resins, Synthetic
chemical_list: D000911:Antibodies, Monoclonal; D001324:Autoantigens; D016056:Immunodominant Epitopes; D010446:Peptide Fragments; D011137:Polystyrenes; D012117:Resins, Synthetic; C101297:Tentagel resin; D005903:Gliadin