Barley hordein isolates as candidate reference materials for gluten quantitation – a proof-of-concept study

Barley hordeins trigger celiac disease (CeD), but current ELISA methods frequently overestimate gluten from barley because they rely on wheat-based reference materials (RM). To address this, we produced four hordein isolates, namely prolamins, acetonitrile/water-extractable proteins (AWEP), gluten and glutelins, from a blend of seven European and one Canadian barley cultivars. Using SDS-PAGE, HPLC and shotgun proteomics, we confirmed that blending eight cultivars successfully mitigated genetic and environmental variability in protein content and composition. Complementary analyses revealed that the isolates were comparable in protein composition to the respective barley flour fractions, except some divergence in glutelins. These well-characterized hordein isolates are proposed as suitable candidates for a standardized barley RM. Establishing such an RM is essential for accurate gluten quantitation in gluten-free compliance monitoring to reduce the variation of results within and between test kits and thus enhance food safety for consumers with CeD.