Quantification Of Gluten Research Articles

A UHPLC-MS/MS Method for the Quantification of Wheat Gluten in Commercial Food Products Using Summarized Marker Peptide Contents

A UHPLC-MS/MS Method for the Quantification of Wheat Gluten in Commercial Food Products Using Summarized Marker Peptide Contents

Performance assessment of a new G12/A1 antibody-based rapid ELISA using commercially available and gluten-spiked food samples.

Food products with <20 mg/kg gluten can be labeled 'gluten-free' according to international regulations. Several antibodies-based ELISAs have been develop to track gluten traces in food products. Among them, R5 and G12 antibody-based ELISAs are the frequently used methods. However, these antibodies have certain limitations. We evaluated the accuracy of G12/A1 antibody-based 'Glutentox ELISA Rapid G12' and compared the results with the current reference method i.e., R5 antibody-based 'Ridascreen R5 ELISA'. In the first step, the performance of Glutentox ELISA Rapid G12 kit was inspected by determination of the threshold value i.e., > or <20 mg/kg gluten in different food products. In the second step, quantification accuracy was assessed by quantification of gluten in gluten-free food products spiked with gliadin reference material. In total 47 food products (naturally and labeled gluten-free, and food with traces of gluten) were included. Of them, 29 products were quantified with <20 mg/kg, and 18 with a low level of gluten by both the kits. Six out of 29 gluten-free products were used for the recovery test at different spike levels. Gluten concentration and mean recovery rates of individual kits showed consistency. GlutenTox Rapid G12 ELISA could be an appropriate choice for detecting gluten in food products but needs more in-house validation and collaborative tests.

Validation of the GlutenTox® ELISA Rapid G12 Test Kit for Determination of Gluten in Select Non-Heat Processed Matrixes and Heat Processed Matrixes.

The GlutenTox® ELISA Rapid G12 test kit is a quantitative method designed for the determination of the immunotoxic fraction of gluten in food samples. To obtain AOAC PTM certification for the method for the detection and quantification of gluten from wheat, barley and rye flours in select foods (non-heat processed) and incurred (heat processed) matrixes. The method was evaluated following the "Validation Procedures for Quantitative Gluten ELISA Methods: AOAC Allergen Community Guidance and Best Practices" (1). The validation study was conducted at Hygiena Diagnóstica España using 5 food matrixes (soy flour, corn bread, seasoning mix, rolled oats and evaporated milk) artificially contaminated with gluten from wheat, barley or rye flour at different concentrations: 0, 5, 10, and 20 mg/kg. For each matrix and gluten contamination level, 5 or 6 individually extracted test portions were analyzed. A second bread matrix was prepared by baking a gluten free bread mix spiked at 0, 20 and 30 mg/kg gluten from wheat, barley or rye flour for incurred matrix testing. Ten individually extracted test portions were tested for each incurred bread and contamination level of gluten. The method met the AOAC performance requirements (2) for detection and quantification of wheat gluten in the selected food matrixes, incurred bread sample and spike levels of wheat gluten, showing an acceptable recovery. When tested with barley and rye flours, most of the results showed acceptable recoveries or a slight overestimation, depending on the matrix and gluten concentration. Method developer and independent laboratory results were comparable. The validation study demonstrated the test kit is a reliable, accurate, quick and easy to use method for the detection and quantification of gluten concentration in food and incurred matrixes from wheat, barley and rye flours. Most reagents provided in the kit are at ready-to-use concentrations.

A novel AuNRs self-assembly dimer-based aptasensor for ultrasensitive SERS sensing of wheat gluten

Ultra-sensitive, accurate, and quantitative detection of wheat gluten in food products can provide a basis for correct labeling “gluten-free” on food packages. In this strategy, a gold nanorods (AuNRs) self-assembly dimer-based surface-enhanced Raman scattering (SERS) sensor was constructed to achieve ultra-sensitive and accurate SERS quantification of wheat gluten. The AuNR dimers based-SERS sensor demonstrated satisfactory specificity, accuracy, reproducibility, and storage stability (35 days at 4 °C). Benefitting from the signal amplification function of AuNR dimers and the superiority of SERS, the sensor exhibited a wide detection range from 0.1 to 20 μg/mL with a limit of detection (LOD) of 35.06 ng/mL (S/N = 3) and a limit of quantitation (LOQ) of 116.87 ng/mL (S/N = 10). This strategy successfully achieved accurate quantification of gluten in 17 kinds of food products from five categories of commercially processed foods, providing new opportunities for ultra-sensitive and accurate quantification of gluten in food products throughout the supply chain.

Dual detection of native and deamidated gluten residues using the novel monoclonal antibody, 2D4

Wheat, barley, and rye contain a form of gluten that can trigger symptoms of Celiac Disease (CD) in genetically susceptible individuals. To protect celiac patients from unintentional gluten consumption, numerous countries have adopted regulatory standards that establish gluten limits for foods labeled gluten-free (GF). Enforcement protocols require precise quantification of gluten using antibody-based assays. The dominant assays used throughout the industry rely on the R5 monoclonal antibody (mAb), which recognizes glutamine-containing epitopes selectively present in gluten derived from these three grains. However, various food commodities contain a chemically modified form of gluten in which glutamine residues undergo deamidation, resulting in a more immunotoxic form of gluten. This chemical change essentially ablates the R5 epitopes thus reducing the effectiveness of R5-based detection methods. To address this limitation, we engineered a novel mAb, 2D4, which is uniquely capable of detecting both chemically unmodified and deamidated gluten. In this study, we characterized 2D4 and report that it’s binding profile for wheat, barley, and rye mirrors R5, and additionally, that it displays superior recognition of deamidated gluten compared with R5. Accordingly, the results of these analyses conclude that 2D4 is an improved reagent for use in antibody-based gluten testing assays.

An Effective Label-Free Electrochemical Aptasensor Based on Gold Nanoparticles for Gluten Detection.

Nanomaterials can be used to modify electrodes and improve the conductivity and the performance of electrochemical sensors. Among various nanomaterials, gold-based nanostructures have been used as an anchoring platform for the functionalization of biosensor surfaces. One of the main advantages of using gold for the modification of electrodes is its great affinity for thiol-containing molecules, such as proteins, forming a strong Au-S bond. In this work, we present an impedimetric biosensor based on gold nanoparticles and a truncated aptamer for the quantification of gluten in hydrolyzed matrices such as beer and soy sauce. A good relationship between the Rct values and PWG-Gliadin concentration was found in the range between 0.1–1 mg L−1 of gliadin (corresponding to 0.2–2 mg L−1 of gluten) with a limit of detection of 0.05 mg L−1 of gliadin (corresponding to 0.1 mg L−1 of gluten). The label-free assay was also successfully applied for the determination of real food samples.

Detection and quantification of gluten as allergen in whisky samples by validated immunoassay method

Gluten is an insoluble, elastic thin film like protein typically present in grains such as wheat, barley and oats. The gliadin and glutenin fractions collectively form gluten which is known to causes celiac disease in genetically predisposed individuals. Incidentally, whisky, the World’s most popular alcoholic drink, prepared from fermentation and distillation of aforesaid grains, bears a threat to contain gluten due to improper distillation process and pose a potential threat to individuals having Celiac disease. The present study is aimed to estimate the gluten content in the different brands of whisky samples by means of

Klasszikus témák új megvilágításban: a glutén mint speciális élelmiszerbiztonsági és analitikai kihívás

In the last couple of decades, the nutritional role and perception of gluten became controversial. In one hand, gluten proteins play a central role in determining the baking quality of wheat and other cereals. On the other hand, hypersensitivity reactions triggered by gluten in susceptible individuals have become subjects of growing interest. Of these gluten-related disorders, with an estimated global prevalence of 1%, the most important one is celiac disease (CD), which is an autoimmune disorder accompanied by villous atrophy. CD can manifest in a wide range of symptoms, its only treatment option is a lifelong gluten-free (GF) diet. To support compliance to this diet, current EU legislation maximizes the gluten-content of products sold with a GF label in 20 mg/kg. It necessitates accurate quantification of gluten in this low concentration range. The method-of-choice for this purpose is the immunoanalytical-based ELISA (enzyme-linked immunosorbent assay). However, validation of different ELISA methods and the comparability of their results and, consequently, the reliability of the data they provide is problematic. The major goal of this paper is to introduce the analytical and protein chemistry issues behind this problem and the efforts to improve the conditions of the methodology. We are also including the special role of oats in the GF diet in an attempt to provide the widest possible overview of the food safety and analytical challenges represented by gluten.

Safe use of Wheat Flour by Knowing Gluten Content

There is greater demand for fast food in the form of noodles, pasta, pizza, bread, etc. in younger generation. Wheat is a staple food of many Indians. Wheat flour is the main ingredient of fast food products. The main contents of wheat are proteins and carbohydrates. Gluten is the protein part of wheat flour which gives elasticity and strength to dough. Wheat with high gluten content is preferred by many food industries however gluten is responsible for digestive discomfort and also found to be allergic, under such circumstances it is necessary for us to select wheat with less percentage of gluten for consumption. Hence in present study wheat flour gluten was analysed and the quantification of gluten was done by calculating wet gluten, dry gluten. The gluten index is also studied by using biochemical test (Folin-Lowry method). In all six local varieties of wheat were analyzed for gluten content, and out of them many showed high gluten content. Present study gives an idea to select the variety of wheat to be used for consumption.

Advances in quantification and analysis of the celiac-related immunogenic potential of gluten.

Gluten-free products have emerged in response to the increasing prevalence of gluten-related disorders, namely celiac disease. Therefore, the quantification of gluten in products intended for consumption by individuals who may suffer from these pathologies must be accurate and reproducible, in a way that allows their proper labeling and protects the health of consumers. Immunochemical methods have been the methods of choice for quantifying gluten, and several kits are commercially available. Nevertheless, they still face problems such as the initial extraction of gluten in complex matrices or the use of a standardized reference material to validate the results. Lately, other methodologies relying mostly on mass spectrometry-based techniques have been explored, and that may allow, in addition to quantitative analysis, the characterizationof gluten proteins. On the other hand, although the level of 20mg/kg of gluten detected by these methods is sufficient for a product to be considered gluten-free, its immunogenic potential for celiac patients has not been clinically validated. In this sense, in vitro and in vivo models, such as the organoid technology applied in gut-on-chip devices and the transgenic humanized mouse models, respectively, are being developed for investigating both the gluten-induced pathogenesis and the treatment of celiac disease. Due to the ubiquitous nature of gluten in the food industry, as well as the increased prevalence of gluten-related disorders, here we intend to summarize the available methods for gluten quantification in food matrices and for the evaluation of its immunogenic potential concerning the development of novel therapies for celiac disease to highlight active research and discuss knowledge gaps and current challenges in this field.

Analyzing Gluten Content in Various Food Products Using Different Types of ELISA Test Kits.

Gluten is an insoluble protein produced when glutelins and prolamins, which are found in grains such as wheat, barley, and oats, combine to form an elastic thin film. This dietary gluten can cause severe contraction of the intestinal mucous membrane in some people, preventing nutrient absorption. This condition, called celiac disease (CD), affects approximately 1% of the world’s population. The only current treatment for patients with CD and similar diseases is lifelong avoidance of gluten. To analyze the gluten content in food, various enzyme-linked immunosorbent assay (ELISA) tests are currently used. In this study, the gluten content in various food products was analyzed using different kinds of ELISA test kits. For gluten-free food, three different ELISA test kits mostly yielded values below the limit of detection. However, gluten was detected at 24.0–40.2 g/kg in bread, 6.5–72.6 g/kg in noodles, and 23.0–86.9 g/kg in different powder food samples. A significant difference (p < 0.05) in gluten content was observed for these gluten-containing food products. Reproducibility issues suggest that it is necessary to use several ELISA kits for the accurate detection and quantification of gluten in various food products rather than using one ELISA kit.

Truncated aptamers as selective receptors in a gluten sensor supporting direct measurement in a deep eutectic solvent

Enzyme-linked immunosorbent assays are currently the most popular methods to quantify gluten in foods. Unfortunately, the antibodies used as specific receptors in such methods are not compatible with the usual solvents for the extraction of gluten proteins. In consequence, commercial tests require a high dilution of the sample after the extraction, increasing the limit of quantification and decreasing convenience. In this work, we have rationally truncated an aptamer capable of recognizing gliadin in a deep eutectic solvent (DES). The truncated aptamer is a 19-nucleotides-long DNA that minimizes self-hybridization, allowing the development of an electrochemical sandwich-based sensor for the quantification of gluten in the DES ethaline. The sensor incorporates two identical biotin-labeled truncated aptamers, one of which is immobilized on a carbon screen-printed electrode and the other reports the binding of gliadin after incubation in streptavidin-peroxidase. This sensor can detect gliadin in DES, with a dynamic range between 1 and 100 μg/L and an intra-assay coefficient of variation of 11%. This analytical performance allows the quantification of 20 μg of gluten/kg of food when 1 g of food is extracted with 10 mL of ethaline. We demonstrate the ability of this method to achieve the measurement of gluten in food samples, after the extraction with pure ethaline. The assay is useful for the analysis of residual gluten levels in foods, thus facilitating the evaluation of any potential health risk associated with the consumption of such food by people with celiac disease or other gluten-related disorders.

Gluten Analysis in Processed Foodstuffs by a Multi-Allergens and Grain-Specific UHPLC-MS/MS Method: One Method to Detect Them All

Abstract Background: Celiac disease, a complex, long-term autoimmune disorder and gluten intolerance, is estimated to affect from 1 to 5% of the world’s population. Objective: As a consequence, to protect gluten-sensitive consumers, the development of reliable analytical methods allowing the detection of gluten in various food products is needed. Methods: Currently, ELISA is probably the most widespread used methodology. The method based on the R5 antibody has received type I status in Codex Alimentarius. However, the ELISA method suffers from some limitations, especially concerning quantification of nonwheat gluten. As a consequence, the development of another complementary methodology such as LC–tandem MS (MS/MS) is considered to be essential. Furthermore, this method could also be used for the simultaneous detection of gluten with other allergens, which will constitute a great additional benefit for producers of “free-from” food products and/or having a management policy integrated for several allergies and/or intolerances. Results: A multi-allergen and grain-specific ultra-HPLC coupled to MS/MS method allowing the identification and the discrimination of gluten from seven cereals, simultaneously with the detection and identification of 10 allergens in only one analysis, is thus described here. Conclusions: This method can be used for the analysis of a broad range of foodstuff matrices containing wheat and/or its derivatives, including cereals, flours, heat-treated and foodstuffs, but also more complex samples having undergone fermentation processes (such as beers).

Quantification of Wheat, Rye, and Barley Gluten in Oat and Oat Products by ELISA RIDASCREEN® Total Gluten: Collaborative Study, First Action 2018.15

Abstract Background: Since its introduction to the analytical community, the R5 method to quantify gluten led to a strong improvement of the situation for the food industry and celiac patients. During recent years, some questions arose on the use of the Codex Alimentarius factor of two to convert from prolamins to gluten, an overestimation of rye and barley, inadequate detection of glutelins, and the inhomogeneous distribution of gluten in oats. These limitations of the R5 method, especially when measuring oat samples, led to AOAC Standard Method Performance Requirement (SMPR®) 2017.021, which was approved by stakeholders in 2017. Objective: We present a collaborative study of a method for the quantitative analysis of wheat, rye, and barley gluten in oat and oat products using a sandwich ELISA that is based on four different monoclonal antibodies including the R5 monoclonal anitbody. Methods: The sandwich ELISA detects intact gliadins and related prolamins from rye and barley, high-molecular-weight (HMW) glutenin subunits (GS) from wheat, HMW secalins from rye, and low-molecular-weight (LMW) GS from wheat. It does not detect D-hordeins from barley. Samples are extracted by Cocktail solution, subsequently followed by 80% ethanol, and analyzed within 50 min. Results: The measurement range is between 5 and 80 mg/kg gluten using a calibrator made out of a gluten extract from four different wheat cultivars. The results of the collaborative test with 19 participating laboratories showed recoveries ranging from 99 to 137% for all three grain sources. Relative reproducibility SDs for samples &amp;gt;10 mg/kg gluten ranged from 10 to 53%. Conclusions: The collaborative study results confirmed that the method is accurate and suitable to measure gluten from all three grain sources and has demonstrated performance on oat matrices, which meets the criteria as specified in SMPR 2017.021. Data from in-house validation experiments are available as Annex B to this publication.

Accidental Gluten Contamination in Traditional Lunch Meals from Food Services in Brasilia, Brazil

This study aimed to evaluate the occurrence of gluten contamination in naturally gluten-free meals from food services in the Federal District, Brazil. This is an exploratory cross-sectional quantitative study in which a total of 180 samples of naturally gluten-free dishes were collected from 60 food services in Brazil. The enzyme-linked immunosorbent assay was used for the quantification of gluten. As established by the Codex Alimentarius, the threshold of 20 ppm of gluten was considered as the accepted upper gluten level for gluten-free food. A total of 2.8% (95% CI: 0.3–5.2%) gluten contamination was found in the samples. Among the 60 food services, 6.7% (95% CI: 2.7–10.6%) displayed at least one contaminated food in our sample. The occurrence of gluten contamination in naturally gluten-free preparations was uncommon and low on a quantitative basis.

Quantification of Wheat, Rye, and Barley Gluten in Oat and Oat Products by ELISA RIDASCREEN® Total Gluten: Collaborative Study, First Action 2018.15.

Background: Since its introduction to the analytical community, the R5 method to quantify gluten led to a strong improvement of the situation for the food industry and celiac patients. During recent years, some questions arose on the use of the Codex Alimentarius factor of two to convert from prolamins to gluten, an overestimation of rye and barley, inadequate detection of glutelins, and the inhomogeneous distribution of gluten in oats. These limitations of the R5 method, especially when measuring oat samples, led to AOAC Standard Method Performance Requirement (SMPR®) 2017.021, which was approved by stakeholders in 2017. Objective: We present a collaborative study of a method for the quantitative analysis of wheat, rye, and barley gluten in oat and oat products using a sandwich ELISA that is based on four different monoclonal antibodies including the R5 monoclonal anitbody. Methods: The sandwich ELISA detects intact gliadins and related prolamins from rye and barley, high-molecular-weight (HMW) glutenin subunits (GS) from wheat, HMW secalins from rye, and low-molecular-weight (LMW) GS from wheat. It does not detect D-hordeins from barley. Samples are extracted by Cocktail solution, subsequently followed by 80% ethanol, and analyzed within 50 min. Results: The measurement range is between 5 and 80 mg/kg gluten using a calibrator made out of a gluten extract from four different wheat cultivars. The results of the collaborative test with 19 participating laboratories showed recoveries ranging from 99 to 137% for all three grain sources. Relative reproducibility SDs for samples >10 mg/kg gluten ranged from 10 to 53%. Conclusions: The collaborative study results confirmed that the method is accurate and suitable to measure gluten from all three grain sources and has demonstrated performance on oat matrices, which meets the criteria as specified in SMPR 2017.021. Data from in-house validation experiments are available as Annex B to this publication.

Detection of gluten in a pilot-scale barley-based beer produced with and without a prolyl endopeptidase enzyme

ABSTRACT Immunochemical and mass spectrometric methods were used to examine the gluten composition of a gluten-reduced beer produced by brewing with barley malt in the presence of prolyl endopeptidase (PEP) and a final filtration treatment with diatomaceous earth and perlite. The competitive ELISA is generally considered appropriate for the analysis of hydrolysed gluten, but it is not considered a scientifically valid method for the quantification of gluten in fermented or hydrolysed foods due to the lack of an appropriate reference standard. As no single analytical method can capture the spectrum of gluten-derived products in beer, a comprehensive approach was employed to analyse the intact and hydrolysed fractions of gluten with complementary methods. The combination of PEP addition and diatomaceous earth/perlite filtration was more effective at reducing the concentration of detectable gluten than each of the treatments alone. However, gluten proteins and/or polypeptides were observed in filtered, PEP-treated beers using sandwich ELISA methods, western blot, and bottom-up mass spectrometry. In addition, mass spectrometry results showed that the number of hydrolysed gluten peptides was almost unaffected by the filtration process. Gluten peptides that contained potentially immunopathogenic sequences were identified in the filtered PEP-containing beers by MS. Variability in gluten composition was observed between three replicate pilot-scale productions, suggesting that the gluten profile in beer could differ from batch to batch. As there is uncertainty in the detection and quantification of gluten in hydrolysed and fermented foods, characterisation of hydrolysed gluten by complementary analytical methodologies is recommended.

Gluten Analysis in Processed Foodstuffs by a Multi-Allergens and Grain-Specific UHPLC-MS/MS Method: One Method to Detect Them All.

Background: Celiac disease, a complex, long-term autoimmune disorder and gluten intolerance, is estimated to affect from 1 to 5% of the world's population. Objective: As a consequence, to protect gluten-sensitive consumers, the development of reliable analytical methods allowing the detection of gluten in various food products is needed. Methods: Currently, ELISA is probably the most widespread used methodology. The method based on the R5 antibody has received type I status in Codex Alimentarius. However, the ELISA method suffers from some limitations, especially concerning quantification of nonwheat gluten. As a consequence, the development of another complementary methodology such as LC-tandem MS (MS/MS) is considered to be essential. Furthermore, this method could also be used for the simultaneous detection of gluten with other allergens, which will constitute a great additional benefit for producers of "free-from" food products and/or having a management policy integrated for several allergies and/or intolerances. Results: A multi-allergen and grain-specific ultra-HPLC coupled to MS/MS method allowing the identification and the discrimination of gluten from seven cereals, simultaneously with the detection and identification of 10 allergens in only one analysis, is thus described here. Conclusions: This method can be used for the analysis of a broad range of foodstuff matrices containing wheat and/or its derivatives, including cereals, flours, heat-treated and foodstuffs, but also more complex samples having undergone fermentation processes (such as beers).

Gluten contamination in gluten-free bakery products: a risk for coeliac disease patients.

The present study aimed to assess the safety of gluten-free bakery products for consumption by coeliac patients. Design/setting In the current exploratory cross-sectional quantitative study, a total of 130 samples were collected from twenty-five bakeries in Brasilia (Brazil). For the quantification of gluten, an ELISA was used. The threshold of 20 ppm gluten was considered as the safe upper limit for gluten-free food, as proposed in the Codex Alimentarius. The results revealed a total of 21·5 % of contamination among the bakery products sampled. Sixty-four per cent of the bakeries sold at least one contaminated product in our sample. These findings represent a risk for coeliac patients since the ingestion of gluten traces may be sufficient to adversely impact on their health.

Quantification of gluten in wheat flour by FT-Raman spectroscopy

A procedure for the quantitative determination of gluten in wheat flour based on partial least squares (PLS) treatment of FT-Raman data is described. Results of similar quality were found using a PLS model derived from NIR (near infrared) spectra obtained in DRIFTS (diffuse reflectance infrared Fourier transform spectroscopy) mode and of slightly worse quality from the model constructed based on IR (infrared) spectra registered using a single reflection ATR (attenuated total reflection) diamond accessory. The relative standard errors of prediction (RSEP) were calculated for the calibration, validation and analysed data sets. These errors amounted to 3.2–3.6%, 3.5–3.8% and 4.8–5.7% for the three techniques applied, respectively. The proposed procedures can be used as simple, fast and accurate methods for the quantitative analysis of gluten in flour.