Further Steps Toward the Development of Gluten Reference Materials – Wheat Flours or Protein Isolates?

Abstract

Celiac disease is a gluten-induced hypersensitivity reaction that requires a lifelong gluten-free diet. Gluten-free foods must not contain more than 20 mg/kg gluten as laid down by Codex Alimentarius. Measuring the presence of gluten with routine immunoanalytical methods in food is a serious challenge as many factors affect accurate determination. Comparability of the results obtained with different methods and method validation are hindered by the lack of a widely accepted reference material (RM). The core questions of RM development from wheat are the number of cultivars to be included and the format of gluten (i.e., flour, gluten, or gliadin isolates) to be applied. Therefore, the aim of our work was to produce an appropriate gluten RM from wheat. For this, five previously selected wheat cultivars and their blend were used to produce flours, gluten and gliadin isolates under laboratory conditions. Protein content, protein composition and responses to different ELISA methods were compared and widely evaluated in our study. The protein contents of the flours were 12.1–18.7%, those of the gluten isolates 93.8–97.4% and those of the gliadin isolates 72.7–101.9%. The gluten and gliadin isolates had similar protein profiles as the source flours. By comparing the different wheat cultivars and their protein isolates, we found that the isolation had a smaller effect on protein composition than genetic variability. The choice of a blend would be more suitable for the production of a RM in case of flours and also isolates. The immunoanalytical results showed that the isolation had an effect on the analytical results, but its extent depended on the ELISA method. The use of flour would be more applicable in this regard, but handling of the material and long-term stability should also be considered in the final decision of gluten RM production.

Investigation of the effects of sample preparation on gluten quantitation in rye and barley flours

“…Currently the available standard materials, e.g. PWG gliadin (van Eckert et al, 2006(van Eckert et al, , 2010, and a reference candidate wheat flour mixture (Hajas et al, 2018;Schall et al, 2020aSchall et al, , 2020b are based on wheat proteins, which sets further difficulties for gluten determination in rye and barley as the ELISA antibodies bind their proteins with different affinities. Therefore, rye and barley should also be included in the reference material development (Lester, 2008;Tanner et al, 2013;Lexhaller et al, 2017;Huang et al, 2020;Xhaferaj et al, 2020;Makharia et al, 2022).…”

Investigation of different extraction conditions on the efficiency of gliadin extraction and determination by ELISA method

“…These methods are based on immunochemical reactions, in which antibodies recognize one or more short peptide sequences (epitopes) of gluten proteins. These reactions result in very specific and sensitive determinations [13][14][15].…”

“…Most commercial ELISA test kits are based on monoclonal antibodies. ELISA is very expensive, and the reproducibility of the results varies depending on the ELISA test kit type [15,16].…”

Barley C-Hordein as the Calibrant for Wheat Gluten Quantification

“…Based on these strategies and led by the PWG, five cultivars were selected after characterisation as the basis for the development of a new reference material [ 15 , 16 ]. The gluten composition of these five cultivar flours varied between harvest years, but a blend of the five cultivars overcame this variability and showed advantages over use of a single cultivar in ELISA responses [ 17 , 18 ]. A gluten isolate or a gliadin isolate from the blend of the five cultivars showed the same protein composition as the native flour [ 18 ].…”

“…The gluten composition of these five cultivar flours varied between harvest years, but a blend of the five cultivars overcame this variability and showed advantages over use of a single cultivar in ELISA responses [ 17 , 18 ]. A gluten isolate or a gliadin isolate from the blend of the five cultivars showed the same protein composition as the native flour [ 18 ]. The varying gluten composition by genotype and environmental factors certainly increased the difficulty of satisfying the criteria of reference material.…”