Evaluation of the discriminatory potential of antibodies created from synthetic peptides derived from wheat, barley, rye and oat gluten

Abstract

Celiac disease (CD) is triggered by ingestion of gluten-containing cereals such as wheat, barley, rye and in some cases oat. The only way for affected individuals to avoid symptoms of this condition is to adopt a gluten-free diet. Thus, gluten-free foodstuffs need to be monitored in order to ensure their innocuity. For this purpose, commercial immunoassays based on recognition of defined linear gluten sequences are currently used. These immunoassays are designed to detect or quantify total gluten regardless of the cereal, and often result in over or underestimation of the exact gluten content. In addition, Canadian regulations require a declaration of the source of gluten on the label of prepackaged foods, which cannot be done due to the limitations of existing methods. In this study, the development of new antibodies targeting discrimination of gluten sources was conducted using synthetic peptides as immunization strategy. Fourteen synthetic peptides selected from unique linear amino acid sequences of gluten were bioconjugated to Concholepas concholepas hemocyanin (CCH) as protein carrier, to elicit antibodies in rabbit. The resulting polyclonal antibodies (pAbs) successfully discriminated wheat, barley and oat prolamins during indirect ELISA assessments. pAbs raised against rye synthetic peptides cross-reacted evenly with wheat and rye prolamins but could still be useful to successfully discriminate gluten sources in combination with the other pAbs. Discrimination of gluten sources can be further refined and enhanced by raising monoclonal antibodies using a similar immunization strategy. A methodology capable of discriminating gluten sources, such as the one proposed in this study, could facilitate compliance with Canadian regulations on this matter. This type of discrimination could also complement current immunoassays by settling the issue of over and underestimation of gluten content, thus improving the safety of food intended to CD and wheat-allergic patients.

Evaluation of Gluten Protein Profiles in Hydrolyzed Food Products by a Multiplex-Competitive Enzyme-Linked Immunosorbent Assay

“…The cluster pattern reflects the importance of grain source of gluten (wheat, rye, or barley) in affecting the overall gluten protein/peptide response profiles of the hydrolyzed samples. Previous studies have highlighted the importance of the gluten grain source in affecting the results of gluten ELISA methods, and one recent study proposed the use of antibodies in immunoassays that can distinguish between different grain sources of gluten to overcome the issues of over- and underestimation. − The results obtained in this study indicate that accurate quantitation of gluten in hydrolyzed food products may also need the use of gluten grain specific reference materials and/or antibodies in immunoassays. Processing conditions may have impact on the overall protein/peptide response profiles of hydrolyzed samples, as observed in the case of HWP that are used for food and those used for preparing media.…”