Celiac disease-specific prolamin peptide content of wheat relatives and wild species determined by ELISA assays and bioinformatics analyses

Abstract

Enzyme-linked immunosorbent assays (ELISAs) are widely used to determine gluten contamination in gluten-free and low gluten food samples. ELISA assays developed using monoclonal antibodies against known toxic peptides have an advantage in the identification of toxic prolamin content in protein extracts of different food samples, as well as raw materials. R5 and G12 monoclonal antibodies specific for two known toxic peptides used in commercially available gluten ELISA assays were applied to test toxic peptide contents in wheat relatives and wild wheat species with different genome composition and complexity. Although the R5 peptide content showed some correlation with ploidy levels in Triticum species, there was a high variance among Aegilops species. Some of the analysed diploid Aegilops species showed extremely high R5 peptide contents. Based on the bioinformatics analyses, the R5 peptide was present in most of the sulphur rich prolamins in all the analysed species, whereas the G12 epitope was exclusively present in alpha gliadins. High variation was detected in the position and frequency of epitopes in sequences originating from the same species, thus highlighting the importance of genotypic variation within species. Identification of new prolamin alleles of wheat relatives and wild wheat species is of great importance in order to find germplasm for special end-use quality purposes as well as development of food with reduced toxicity.

Label‐Free Proteomic Analysis of Wheat Gluten Proteins and Their Immunoreactivity to ELISA Antibodies

“…The G12 mAb was observed to react to both gliadin and glutenin protein fractions (Sharma 2012). Studies have determined that the epitope recognized by the G12 mAb is QPQLPY (Morón et al 2008), which is present mainly in a-gliadin (Gell et al 2015) (F12-F20, Figures 2C and 3C). The a20 mAb recognizes the minimal amino acid sequence RPQQPYP present in gliadins and has shown crossreactivity with other gliadin epitopes found in aand g-gliadin but not toward the HMW-GS (Mitea et al 2008).…”

Determination of Gluten Peptides Associated with Celiac Disease by Mass Spectrometry

“…Triticum species exhibits an important genetic variability, resulting in different toxicities, what can be a promising alternative for obtaining suitable varieties for consumption by celiac patients [19,41,42]. Higher levels of immunogenic peptides related to CD were attributed to a modern Canadian wheat when compared to old varieties of common wheat and tetraploid wheat [43].…”

Gluten weight in ancient and modern wheat and the reactivity of epitopes towards R5 and G12 monoclonal antibodies

“…Based on G12 data, Gell et al . () also reported only 1–2% gluten in two bread wheat samples of Chinese and Hungarian origin although 6–8% gluten was found according to R5.…”

“…According to this report, durum wheat had only 1.6% gluten, which is much lower than dry gluten weight normally found in durum wheat (Table 2). Based on G12 data, Gell et al (2015) also reported only 1-2% gluten in two bread wheat samples of Chinese and Hungarian origin although 6-8% gluten was found according to R5.…”

“…Calculation of the gluten content by multiplying gliadin content by factor 2 also contributed to its overestimation because this factor varies according to wheat type (Wieser & Koehler, 2009). In gluten-free food analyses, data obtained with different ELISA commercial kits for 'gluten' detection would be difficult to compare because they are based on the selective reactivity of different wheat gluten epitopes, not all toxic peptides and including noncoeliac-active epitopes (Diaz-Amigo & Popping, 2013;Bruins Slot et al, 2015;Gell et al, 2015). Antibodies for ELISA R5 and G12 both show variable specificity towards different gluten-rich cereals (Rallabhandi et al, 2015).…”