Comparative analysis of prolamin and glutelin fractions from wheat, rye, and barley with five sandwich ELISA test kits

Abstract

The safety of gluten-free foods is essential for celiac disease (CD) patients to prevent serious complications. Enzyme-linked immunosorbent assays (ELISAs) are recommended for gluten analysis to monitor the compliance of gluten-free products to the Codex threshold of 20 mg gluten/kg. However, due to the specific features of each gluten ELISA test kit, the results often deviate systematically and largely depend on the characteristics of the antibody. This comprehensive study assessed the specificities and sensitivities of three monoclonal (R5, G12, and Skerritt) and two polyclonal antibodies to the alcohol-soluble prolamin and alcohol-insoluble glutelin fractions of gluten from wheat, rye, and barley, all of which harbor CD-active epitopes. Reversed-phase high-performance liquid chromatography served as independent reference method to quantify gluten protein concentrations and allow comparisons of different gluten fractions within one kit and between kits. Wheat prolamins were detected quite accurately by all antibodies, but high variability between antibody specificities and sensitivities was observed for rye and barley prolamins and rye glutelins, and the largest discrepancies were found for wheat and barley glutelins. The gluten content (sum of prolamins and glutelins) was either overestimated up to six times (rye) or underestimated up to seven times (barley). Overestimation of gluten contents may unnecessarily limit the availability of gluten-free products, but underestimation represents a serious health risk for CD patients. It is important to consider these differences between antibodies used in kits and consider what each kit is capable of measuring, especially with samples where the source of gluten is unknown.

Preparation of a Defined Gluten Hydrolysate for Diagnosis and Clinical Investigations of Wheat Hypersensitivities

“…Due to the stretched conformation of reduced gluten proteins, their SDS-PAGE mobility is known to be restricted compared to the globular proteins used in the marker, so that, e.g., HMW-GS (M r : 67–88 × 10 3 based on known amino acid sequences) appeared between M r : 85–120 × 10 3 , as has been reported before [ 5 , 30 ]. The contents, elution profiles of GLIA and GLUT and their ratio (1.88) were very similar to those reported for common wheat flour [ 39 ]. In contrast, ALGL, usually present in wheat flours with contents of 150–200 mg/g, were almost absent due to the washing steps during gluten-starch separation.…”

Characterization and Relative Quantitation of Wheat, Rye, and Barley Gluten Protein Types by Liquid Chromatography–Tandem Mass Spectrometry

“…Table S2 lists the CP contents of the GPTs isolated from wheat, rye and barley flours and the proportions of each GPT within total gluten. The Osborne fraction values are based on flour weight; the proportions of GPTs are based on total gluten content (Lexhaller et al, 2016;Lexhaller et al, 2017). The results corresponded well to those reported previously (Gellrich et al, 2003;Kerpes et al, 2016;Schalk et al, 2017) identification of Protein Groups in the Gluten Protein Types…”

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

“…The results obtained also suggest, in accordance with previous research (Allred and Ritter 2010;van Eckert et al 2010;Sharma 2012), that assay antibodies could interact with glutenin proteins, mainly HMW-GS. That certain antibodies also react with the glutelin fraction has already been shown by Rallabhandi et al (2015) and Lexhaller et al (2016). Therefore it cannot be simply assumed that the gliadin fraction is the sole target for gluten quantification.…”

“…Recently, another ELISA, based on the G12 monoclonal antibody (mAb), has been accepted by AOAC as an official first action method (Halbmayr-Jech et al 2015). However, there is inconsistency in the measurement results obtained by different commercially available ELISA kits, resulting in a lack of comparability between measurement results (Mena et al 2012;Sharma 2012;Bugyi et al 2013;Lexhaller et al 2016). The intended measurand in this case is gluten.…”