Rapid analysis of wheat gluten composition using a triple ELISA.

BACKGROUND: Gluten composition is an important quality parameter of wheat flour. Reversed-phase high-performance liquid chromatography (RP-HPLC) is a state-of-the-art method for its analysis. As this is a very labour-intensive and time-consuming procedure, alternative faster methods are desirable. Enzyme-linked immunosorbent assay (ELISA) is a high-throughput method often used for the analysis of gluten traces in gluten-free products. In this proof-of-principle study, we introduce an experimental triple ELISA for the relative quantitation of gliadins, high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) of one wheat flour extract. RESULTS: The results of 80 common wheat flour samples obtained from the triple ELISA and RP-HPLC were correlated. The results for gliadins (r = 0.69) and HMW-GS (r = 0.81) showed a medium and high correlation, respectively. Only a very weak correlation of ELISA and RP-HPLC results was observed for LMW-GS (r = 0.49). Results for glutenins (r = 0.69) and gluten (r = 0.72) had a medium correlation. The gliadin/glutenin ratio (r = 0.47) and LMW-GS/HMW-GS ratio (r = 0.40) showed a weak or no correlation. The gliadin, LMW-GS and gluten contents were lower and the HMW-GS content was higher in the ELISA measurement compared to RP-HPLC. CONCLUSION: The quantitation of gliadins and HMW-GS by the experimental triple ELISA showed comparable results to RP-HPLC, whereas no strong correlation between the results from the two methods was found for LMW-GS. Overall, the experimental triple ELISA is suitable for relative gluten quantitation, especially for the analysis of large sample sets. Further work will focus on improving the experimental procedure of the ELISA. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Wheat-dependent exercise-induced anaphylaxis: subtypes, diagnosis, and management.

Wheat-dependent exercise-induced anaphylaxis (WDEIA) is an IgE-mediated food allergy with allergic symptoms ranging from intermittent urticaria to severe anaphylaxis that occurs when wheat ingestion is combined with augmenting cofactors such as exercise, non-steroidal anti-inflammatory drugs, or alcohol. In most cases, patients are identified by sensitization to ω5-gliadins in the gluten fraction of wheat. ω5-gliadin-negative subtypes of WDEIA are often difficult to diagnose and may be caused by Tri a 14 (wheat lipid transfer protein), after percutaneous sensitization with hydrolyzed wheat proteins, or, in rare cases, by cross-reactivity to grass pollen. Diagnosis is established based on the patients' history in combination with serum IgE profile, skin testing, basophil activation tests, and challenge tests with cofactors. Individual dietary counselling remains the central pillar in the management of WDEIA patients. A completely wheat-free diet is a possible option. However, this appears to promote tolerance less than continued regular consumption of gluten-containing cereals in the absence of cofactors. All patients should have an emergency set for self-treatment including an adrenaline autoinjector and receive adequate instruction. More data are needed on sublingual immunotherapy for WDEIA, a potentially promising therapeutic prospect. This article provides an overview of current knowledge on the diagnosis and management of WDEIA including an optimized challenge protocol using wheat gluten and cofactors.

Mass spectrometry of in-gel digests reveals differences in amino acid sequences of high-molecular-weight glutenin subunits in spelt and emmer compared to common wheat.

High-molecular-weight glutenin subunits (HMW-GS) play an important role for the baking quality of wheat. The ancient wheats emmer and spelt differ in their HMW-GS pattern compared to modern common wheat and this might be one reason for their comparatively poor baking quality. The aim of this study was to elucidate similarities and differences in the amino acid sequences of two 1Bx HMW-GS of common wheat, spelt and emmer. First, the sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) system was optimized to separate common wheat, spelt and emmer Bx6 and Bx7 from other HMW-GS (e.g., 1Ax and 1By) in high concentrations. The in-gel digests of the Bx6 and Bx7 bands were analyzed by untargeted LC-MS/MS experiments revealing different UniProtKB accessions in spelt and emmer compared to common wheat. The HMW-GS Bx6 and Bx7, respectively, of emmer and spelt showed differences in the amino acid sequences compared to those of common wheat. The identities of the peptide variations were confirmed by targeted LC-MS/MS. These peptides can be used to differentiate between Bx6 and Bx7 of spelt and emmer and Bx6 and Bx7 of common wheat. The findings should help to increase the reliability and curation status of wheat protein databases and to understand the effects of protein structure on the functional properties. Graphical abstract.

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

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.

Influence of baking conditions on the extractability and immunochemical detection of wheat gluten proteins.

Food processing conditions affect the accurate detection of gluten by ELISA, which is of importance for proper gluten-free labelling. We prepared different wheat flour-based and incurred baked goods (bread, crispbread, pretzel) to investigate the influence of baking conditions and alkali treatment on gluten quantitation by ELISA using different extraction solvents. Protein composition and extractability were determined (SDS-PAGE, RP-HPLC, GP-HPLC). The extraction solvents showed different performances; none of them could compensate the effect of baking on the detection. Dough preparation, baking and additional alkali treatment decreased protein extractability under reducing and non-reducing conditions. High temperature combined with alkali treatment resulted in the lowest protein extractabilities (<77% for bread crust, <61% for pretzel crust) due to the formation of disulfide and non-disulfide gluten crosslinks. There was no clear correlation between the protein composition and the extractability of alcohol- and SDS-soluble proteins of the baked goods. Thus, this research shows that gluten extractability rather than gluten composition is crucial for detection by ELISA in baked goods.

Proteomic Characterization of Wheat Protein Fractions Taken at Different Baking Conditions.

Food processing conditions affect the structure, solubility, and therefore accurate detection of gluten proteins. We investigated the influence of dough, bread, and pretzel making on the composition of different wheat protein fractions obtained by Osborne fractionation. The albumin/globulin, gliadin, and glutenin fractions from flour, dough, crispbread, bread, and pretzel were analyzed using RP-HPLC, SDS-PAGE, and untargeted nanoLC-MS/MS. This approach enabled an in-depth profiling of the fractionated proteomes and related compositional changes to processing conditions (mixing, heat, and alkali treatment). Overall, heat treatment demonstrated the most pronounced effect. Label-free quantitation revealed significant changes in the relative abundances of 82 proteins within the fractions of bread crumb and crust in comparison to flour. Certain gluten proteins showed shifts or reductions in particular fractions, indicating their incorporation into the gluten network through SS and non-SS cross-links. Other gluten proteins were enriched, suggesting their limited involvement in the gluten network formation.

Prediction of wheat gluten composition via near-infrared spectroscopy.

Gluten composition is an important quality parameter for wheat flour, because it is strongly correlated to baking quality. Wheat proteins are commonly extracted stepwise and analysed using RP-HPLC-UV to determine the gluten composition. This procedure is very time-consuming and labour-intensive. Therefore, a new, fast and easy method to quantitate gluten proteins was established using NIR spectroscopy (NIRS). PLS-regression models were calculated containing 207 samples for calibration and 169 for test set validation. Albumin/globulin (ALGL), gluten, gliadin and glutenin content was predicted with a root mean square error of prediction (RMSEP) of 2.01 mg/g, 6.09 mg/g, 4.25 mg/g and 3.50 mg/g, respectively. High-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) were predicted with a RMSEP of 1.12 mg/g and 2.38 mg/g. The relative error was too high for ALGL, LMW-GS and HMW-GS, but that of gluten, gliadins and glutenins was in a range comparable to the reference method. Therefore, the new NIRS method can be used to estimate the gluten composition of wheat flour, including the gliadin/glutenin and the LMW-GS/HMW-GS ratio.

Lipidomic insights into the reaction of baking lipases in cakes.

Lipases are promising improvers of cake batter and baking properties. Their suitability for use in various cake formulations cannot be predicted yet, because the reactions that lead to macroscopic effects need to be unravelled. Therefore, the lipidome of three different cake recipes with and without lipase treatment was assessed by ultra high performance liquid chromatography-mass spectrometry before and after baking. By comparing the reaction patterns of seven different lipases in the recipes with known effects on texture, we show that lipase substrate specificity impacts baking quality. Key reactions for the recipes were identified with the help of principal component analysis. In the eggless basic cake, glyceroglycolipids are causal for baking improvement. In pound cake, lysoglycerophospholipids were linked to textural effects. Lipase substrate specificity was shown to be dependent on the recipe. Further research is needed to understand how recipes can be adjusted to achieve optimal lipase substrate specificity for desirable batter and baking properties.

Microscopic analysis of gluten network development under shear load-combining confocal laser scanning microscopy with rheometry.

A comprehensive in-situ analysis of the developing gluten network during kneading is still a gap in cereal science. With an in-line microscale shear kneading and measuring setup in a conventional rheometer, a first step was taken in previous works toward fully comprehensible gluten network development evaluation. In this work, this setup was extended by an in-situ optical analysis of the evolving gluten network. By connecting a laser scanning microscope with a conventional rheometer, the evaluation of the rheological and optical protein network evolution was possible. An image processing tool for analyzing the protein network was applied for evaluating the gluten network development in a wheat dough during the shear kneading process. This network evaluation was possible without interruption or invasive sample transfer comparing it to former approaches. The shear kneading system was able to produce a fully developed dough matrix within 125% of the reference dough development time in a classical kneader. The calculated network connectivity values from frequency testing ranged over all samples was in good agreement with traditional kneaded wheat dough just over peak consistency.

Breeding from 1891 to 2010 did not increase the content of amylase/trypsin-inhibitors in wheat (Triticum aestivum).

The prevalence of hypersensitivities towards wheat has increased in the last decades. Apart from celiac disease these include allergic and other inflammatory reactions summarized under the term non-celiac wheat sensitivity. One suspected trigger is the family of amylase/trypsin-inhibitors (ATIs), non-gluten proteins that are prominent wheat allergens and that activate the toll-like receptor 4 on intestinal immune cells to promote intestinal and extra-intestinal inflammation. We therefore quantified 13 ATIs in 60 German hexaploid winter wheat cultivars originating from 1891 to 2010 and harvested in three years by targeted liquid chromatography-tandem mass spectrometry combined with stable isotope dilution assay using specific marker peptides as internal standards. The total ATI content and that of the two major ATIs 0.19 and CM3 did not change from old cultivars (first registered from 1891 to 1950) to modern cultivars (1951-2010). There were also no significant changes in ATI distribution.

Improvement of cake baking properties by lipases compared to a traditional emulsifier.

Lipases are commonly used as clean-label improvers for bread. However, their potential use in cakes with different formulations remains unknown. The aim was to analyze the effects of seven baking lipases on three different cake formulations (an eggless cake, a pound cake with eggs and a yeast-based cake) in comparison to a traditional emulsifier. Product density, water loss during baking and product texture were assessed. If and to what extent the product quality was improved depended on both the lipase and the cake formulation. Lipase-induced effects mostly exceeded those of the emulsifier and were most pronounced in formulations without intrinsic emulsifiers like eggs. The lipases differed in their extent of improvement, hinting at the importance of their specific reactivity patterns and the resulting range of interactions with macromolecules. Further research is needed to unravel the mechanistic background of baking quality improvement in cakes.

Basophil Activation to Gluten and Non-Gluten Proteins in Wheat-Dependent Exercise-Induced Anaphylaxis.

Wheat-dependent exercise-induced anaphylaxis (WDEIA) is a cofactor-induced wheat allergy. Gluten proteins, especially ω5-gliadins, are known as major allergens, but partially hydrolyzed wheat proteins (HWPs) also play a role. Our study investigated the link between the molecular composition of gluten or HWP and allergenicity. Saline extracts of gluten (G), gluten with reduced content of ω5-gliadins (G-ω5), slightly treated HWPs (sHWPs), and extensively treated HWPs (eHWPs) were prepared as allergen test solutions and their allergenicity assessed using the skin prick test and basophil activation test (BAT) on twelve patients with WDEIA and ten controls. Complementary sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), high-performance liquid chromatography (HPLC), and mass spectrometry (MS) analyses revealed that non-gluten proteins, mainly α-amylase/trypsin inhibitors (ATIs), were predominant in the allergen test solutions of G, G-ω5, and sHWPs. Only eHWPs contained gliadins and glutenins as major fraction. All allergen test solutions induced significantly higher %CD63+ basophils/anti-FcεRI ratios in patients compared with controls. BAT using sHWPs yielded 100% sensitivity and 83% specificity at optimal cut-off and may be useful as another tool in WDEIA diagnosis. Our findings indicate that non-gluten proteins carrying yet unidentified allergenic epitopes appear to be relevant in WDEIA. Further research is needed to clarify the role of nutritional ATIs in WDEIA and identify specific mechanisms of immune activation.

Water Absorption Capacity Determines the Functionality of Vital Gluten Related to Specific Bread Volume.

Vital gluten is often used in baking to supplement weak wheat flours and improve their baking quality. Even with the same recipe, variable final bread volumes are common, because the functionality differs between vital gluten samples also from the same manufacturer. To understand why, the protein composition of ten vital gluten samples was investigated as well as their performance in a microbaking test depending on the water content in the dough. The gluten content and composition as well the content of free thiols and disulfide bonds of the samples were similar and not related to the specific bread volumes obtained using two dough systems, one based on a baking mixture and one based on a weak wheat flour. Variations of water addition showed that an optimal specific volume of 1.74-2.38 mL/g (baking mixture) and 4.25-5.49 mL/g (weak wheat flour) was reached for each vital gluten sample depending on its specific water absorption capacity.

A Portable Gluten Sensor for Celiac Disease Patients May Not Always Be Reliable Depending on the Food and the User.

A strict lifelong gluten-free (GF) diet is currently the only known effective treatment for celiac disease (CD), an inflammatory disorder of the small intestine with a worldwide prevalence of about 1%. CD patients need to avoid wheat, rye, and barley and consume GF foods containing <20 mg/kg of gluten. However, strict adherence to a GF diet tends to reduce the quality of life of CD patients compared to the general population and may lead to fear of inadvertent gluten consumption, especially when eating out. To help alleviate risk of gluten exposure, a portable gluten sensor was developed by Nima Labs that allows CD patients to test foods on site prior to consumption. With very limited independent information on the analytical performance of the Nima sensor available so far, our aim was to evaluate the reliability of the sensor using a variety of different foods with defined gluten content. All samples were tested with the sensor and analyzed by enzyme-linked immunosorbent assay as reference method. Of the 119 samples with gluten content ranging from 2 to 101,888 mg/kg tested in total, the sensor showed 80 positive (67.2%), 37 negative (31.1%) and 2 invalid results at the first of three consecutive measurements. The detection rate for samples containing ≥20 mg/kg of gluten was 90%. Samples containing 2 mg/kg of gluten or below consistently tested negative, but samples with a gluten content between 2 to 20 mg/kg of gluten may either test positive or negative. Overall, the performance of the sensor was acceptable in our study, but we observed systematic variation between different users that also appeared to depend on the sample being tested. This highlights the need to improve user education especially regarding the effect of sampling, testing limitations in case of partially hydrolyzed, fractionated or fermented gluten and training users on how to perform the test in a way that gluten will be reliably detected.

Old and modern wheat (Triticum aestivum L.) cultivars and their potential to elicit celiac disease.

One potential explanation for the increasing prevalence of celiac disease (CD) over the past decades is that breeding may have inadvertently changed the immunoreactive potential of wheat. To test this hypothesis, we quantitated four CD-active peptides, namely the 33-mer and peptides containing the DQ2.5-glia-α1a/DQ2.5-glia-α2 (P1), DQ2.5-glia-α3 (P2) and DQ2.5-glia-γ1 (P3) epitopes, in a set of 60 German hexaploid winter wheat cultivars from 1891 to 2010 and grown in three consecutive years. The contents of CD-active peptides were affected more by the harvest year than by the cultivar. The 33-mer and P1 peptides showed no tendency regarding their absolute contents in the flour, but they tended to increase slightly over time when calculated relative to the α-gliadins. No trends in relative or absolute values were observed for the P2 and P3 peptides derived from α- and γ-gliadins. Therefore, the immunoreactive potential of old and modern wheat cultivars appears to be similar.

The basophil activation test differentiates between patients with wheat-dependent exercise-induced anaphylaxis and control subjects using gluten and isolated gluten protein types.

BACKGROUND: Oral food challenge using gluten and cofactors is the gold standard to diagnose wheat-dependent exercise-induced anaphylaxis (WDEIA), but this procedure puts patients at risk of an anaphylactic reaction. Specific IgE to ω5-gliadins as major allergens and skin prick tests to wheat may yield negative results. Thus, we designed a proof-of-principle study to investigate the utility of the basophil activation test (BAT) for WDEIA diagnosis. METHODS: Different gluten protein types (GPT; α-, γ-, ω1,2- and ω5-gliadins, high-molecular-weight glutenin subunits [HMW-GS] and low-molecular-weight glutenin subunits [LMW-GS]) and gluten were used in different concentrations to measure basophil activation in 12 challenge-confirmed WDEIA patients and 10 control subjects. The results were compared to routine allergy diagnostics. Parameters analyzed include the percentage of CD63+ basophils, the ratio of %CD63+ basophils induced by GPT/gluten to %CD63+ basophils induced by anti-FcεRI antibody, area under the dose-response curve and test sensitivity and specificity. RESULTS: GPT and gluten induced strong basophil activation for %CD63+ basophils and for %CD63+/anti-FcɛRI ratio in a dose-dependent manner in patients, but not in controls (p < 0.001, respectively). BAT performance differed from acceptable (0.73 for LMW-GS) to excellent (0.91 for ω5-gliadins) depending on the specific GPT as evaluated by the area under the receiver operating characteristic curve. Patients showed individual sensitization profiles. After determination of the best cut-off points, ω5-gliadins and HMW-GS showed the best discrimination between patients and controls with a sensitivity/specificity of 100/70 and 75/100, respectively. CONCLUSION: This study shows the alternative role of BAT in better defining WDEIA and the causative wheat allergens. The best BAT parameters to distinguish WDEIA patients from controls were %CD63+ basophil values for ω5-gliadins and HMW-GS.

Predicting vital wheat gluten quality using the gluten aggregation test and the microscale extension test.

Vital gluten is a by-product of wheat starch production and commonly used in bread making, but its quality is difficult to predict. The most accurate method to determine vital gluten quality is the baking experiment, but this approach is time- and labor-intensive. Therefore, the aim was to identify faster and easier ways to predict vital gluten quality. Three different approaches, the gliadin/glutenin ratio, the gluten aggregation test and the microscale extension test, were assessed for their predictive value regarding the baking performance of 46 vital gluten samples using two recipes. Hierarchical clustering classified the vital gluten samples into 23 samples with good, 15 with medium and eight with poor quality. Protein-related parameters, such as the gliadin/glutenin ratio, were not reliable to predict gluten quality, because the correlations to the bread volumes were weak. The gluten aggregation test and the microscale extension test were reliable methods to predict vital gluten quality for use in baking based on a scoring system. Both methods need less material, time and labor compared to baking experiments. Especially, maximum torque, peak maximum time, the ratio between peak30 and peak180 as well as the corresponding distance at maximum resistance to extension seem to be suitable alternatives to predict vital gluten quality.

Preparation of Validation Materials for Estimating Gluten Recovery by ELISA According to SMPR 2017.021.

BACKGROUND: In September 2017, the AOAC INTERNATIONAL Stakeholder Panel for Alternative Methods adopted Standard Method Performance Requirement (SMPR®) 2017.021, "Quantitation of Wheat, Rye, and Barley Gluten in Oats," as guidance for the validation of methods for measuring gluten in oat products. The SMPR requires prospective methods to demonstrate adequate recovery (50-200%) based on the analysis of a set of reference samples. OBJECTIVE: This document provides specific methods and data on the preparation of such validation materials and their analysis by an R5 ELISA kit to demonstrate the SMPR recovery estimation procedure. METHODS: Seven reference samples were made by spiking wheat, rye, and barley into gluten-free oat flour at two levels, 10 and 20 mg/kg. The levels of gluten were determined by a wet chemical method based on the Codex Alimentarius definition of gluten. RESULTS: The recoveries for wheat, rye, and barley were 122, 425, and 349%, respectively, for the R5 ELISA kit. The wet chemical method for estimating gluten in a sample of pure grain demonstrated repeatability relative SDs ranging from 1.40 to 2.75%. CONCLUSIONS: The reference materials are suitable to estimate ELISA kit responses to wheat, rye, and barley and calculate recoveries. HIGHLIGHTS: A series of oat flours spiked with wheat, rye, and barley flours were developed to be used as reference materials. A wet chemical method was established to estimate gluten contents based on the Codex definition. The reference materials are available for purchase to support further method development and validation.

Comparative Characterization of Gluten and Hydrolyzed Wheat Proteins.

Hydrolyzed wheat proteins (HWPs) are widely used as functional ingredients in foods and cosmetics, because of their emulsifying and foaming properties. However, in individuals suffering from celiac disease or wheat allergy, HWPs may have a modified immunoreactivity compared to native gluten due to changes in molecular structures. Although a variety of HWPs are commercially available, there are no in-depth comparative studies that characterize the relative molecular mass (Mr) distribution, solubility, and hydrophilicity/hydrophobicity of HWPs compared to native gluten. Therefore, we aimed to fill this gap by studying the above characteristics of different commercial HWP and gluten samples. Up to 100% of the peptides/proteins in the HWP were soluble in aqueous solution, compared to about 3% in native gluten. Analysis of the Mr distribution indicated that HWPs contained high percentages of low-molecular-weight peptides/proteins and also deamidated glutamine residues. We also found considerable differences between the seven HWPs studied, so that each HWP needs to be studied in detail to help explain its potential immunoreactivity.