Rye secalin isolates to develop reference materials for gluten detection

Gluten-free products must not contain more than 20 mg/kg of gluten to be safe for consumption by celiac disease patients. Almost all analytical methods are calibrated to wheat, wheat gluten or gliadin, and there is no rye-specific reference material available. The aim of this study was to assess the effect of the harvest year on rye gluten composition and to generate distinct rye isolates to serve as calibration standards. Four different extraction procedures of a specific rye cultivar mixture were tested yielding prolamins (PROL), glutelins (GLUT), gluten (G) and acetonitrile/water-extractable proteins (AWEP). The isolates were characterized using different methods such as RP-HPLC, GP-HPLC, SDS-PAGE and LC-MS/MS. The isolates were evaluated in the R5 ELISA which resulted in the following response order: PROL$_{iso}$ > AWEP$_{iso}$ > G$_{iso}$ > GLUT$_{iso}$. This paper represents a significant step towards improving gluten analysis, particularly in the context of rye-contaminated gluten-free products.