Antigenicity of wheat prolamins: Detailed epitope analysis using a panel of monoclonal antibodies

Putative continuous epitopes, recognised by five panels of monoclonal antib odies (MAb) with differing specificities for gliadins and glutenin subunits , were identified using overlapping nonapeptides. These peptides correspond to the entire sequence of an alpha/beta -gliadin, a gamma -gliadin, an ome ga -prolamin (homologous to omega -gliadin), a low molecular weight gluteni n subunit (LM(r)GS) and several high molecular weight glutenin subunits (HM (r)GS). Antibodies that bound to gamma- or omega -gliadins, LM(r)GS or HM(r )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 normally to small groups of alpha/beta -gliadins. Epitopes for t hese antibodies in alpha/beta -gliadin may be discontinuous (i.e. derived f rom amino acid residues that are brought together by folding of the polypep tide 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 subu nits were immunodominant, such as the cysteine-cysteine containing peptide found in the central domain of many prolamins, a diversity of reaction patt erns was found. Cross-reaction of antibody with peptides from other prolami n families was ofter due to binding to a peptide having significant sequenc e homology, but in some cases no homology was obvious. Some major trends we re as follows. Antibodies which bound to most or all HM(r)GS recognised the central repeat region, while those that were selective for one or two subu nits bound to epitopes in the unique N- and/or G-terminal domains. A high p roportion of the epitopes recognised by MAb to alpha-, beta-, omega -gliadi ns and HM(r)GS contained cysteine; these MAb may be useful in detecting cov alent binding sites within or between subunits. Although a number of MAb bo und a wide range of gliadins and GS, several of these recognised single (an d differing) epitopes in the target proteins. However, comparatively few MA b recognised epitopes from either the N- or C-terminal regions of the targe t proteins. Several explanations are possible; either these regions are bur ied in the immunogen and not accessible for antibody production or alternat ively the repeat sequences are immunodominant.