Antigenicity of Wheat Prolamins: Detailed Epitope Analysis using a Panel of Monoclonal Antibodies

Abstract

Putative continuous epitopes, recognised by five panels of monoclonal antibodies (MAb) with differing specificities for gliadins and glutenin subunits, were identified using overlapping nonapeptides. These peptides corresponded to the entire sequence of an α/β-gliadin, a γ-gliadin, an ω-prolamin (homologous to ω-gliadin), a low molecular weight glutenin subunit (L MrGS) and several high molecular weight glutenin subunits (HMr GS). Antibodies that bound to γ- or ω-gliadins, L MrGS or HMr GS bound to the peptides at similar concentrations used normally in direct ELISA, but little binding to the peptides was seen for several antibodies that bound specifically to small groups of α/β-gliadins. Epitopes for these antibodies in α/β-gliadin may be discontinuous (i.e. derived from amino acid residues that are brought together by folding of the polypeptide chain or by juxtaposition of two polypeptide chains), since binding of these antibodies to gliadins was greatly decreased following the reduction of intra-molecular disulphide bonds. While some regions in particular subunits were immunodominant, such as the cysteine–cysteine containing peptide found in the central domain of many prolamins, a diversity of reaction patterns was found. Cross-reaction of antibody with peptides from other prolamin families was often due to binding to a peptide having significant sequence homology, but in some cases no homology was obvious. Some major trends were as follows. Antibodies which bound to most or all H MrGS recognised the central repeat region, while those that were selective for one or two subunits bound to epitopes in the unique N- and/or C-terminal domains. A high proportion of the epitopes recognised by MAb to α-, β-, ω-gliadins and L MrGS contained cysteine; these MAb may be useful in detecting covalent binding sites within or between subunits. Although a number of MAb bound a wide range of gliadins and GS, several of these recognised single (and differing) epitopes in the target proteins. However, comparatively few MAb recognised epitopes from either the N- or C-terminal regions of the target proteins. Several explanations are possible; either these regions are buried in the immunogen and not accessible for antibody production or alternatively the repeat sequences are immunodominant.