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Enzyme immunoassays are currently the methods of choice for gluten control in foods labelled as gluten free , providing a mechanism for assessing food safety for consumption by coeliac and other allergic patients. However, their limitations, many of them associated to the reactivity of the different antibodies used and their degree of specificity, have prevented the establishment of a standardised method of analysis. We explore new methods for quantitatively determining gluten content in foods based on the use of two recently described aptamers, raised against a 33-mer peptide recognised as the immunodominant fragment from α2-gliadin. The assays use the target peptide immobilised onto streptavidin-coated magnetic beads in combination with a limited amount of biotin-aptamer in a competitive format, followed by streptavidin-peroxidase labelling of the aptamer that remains bound to the magnetic beads. The enzyme activity onto the beads, measured by chronoamperometry in disposable screen-printed electrodes, is inversely related to the target concentration in the test solution. We find that while the assay using the aptamer with the highest affinity towards the target (Gli 4) achieves low detection limits (~0.5 ppm) and excellent analytical performance, when challenged in samples containing the intact protein, gliadin, it fails in detecting the peptide in solution. This problem is circumvented by employing another aptamer (Gli 1), the most abundant one in the SELEX pool, as a receptor. The proposed assays allow the convenient detection of the allergen in different kinds of food samples, including heat-treated and hydrolysed ones. The obtained results correlate with those of commercially available antibody-based assays, providing an alternative for ensuring the safety and quality of nominally gluten-free foods. Graphical Abstract Electrochemical magnetoassay for gluten determination using biotin-aptamers as receptors

Prolamins, alcohol soluble storage proteins of the Triticeae tribe of Gramineae family, are known as gliadin, secalin and hordein in wheat, rye and barley respectively. Prolamins were extracted from fifteen cultivars using DuPont protocol to study their physiochemical, morphological and structural characteristics. SDS-PAGE of prolamins showed well resolved low molecular weight proteins with significant amount of albumin and globulin as cross-contaminant. The β-sheet (32.72–37.41%) and β-turn (30.36–37.91%) were found higher in gliadins, while α-helix (20.32–28.95%) and random coil (9.05–10.28%) in hordeins. The high colloidal stability as depicted by zeta-potential was observed in gliadins (23.5–27.0 mV) followed secalins (11.2–16.6 mV) and hordeins (4.1–7.8 mV). Surface morphology by SEM illustrated the globular particle arrangement in gliadins, sheet like arrangement in secalins and stacked flaky particle arrangement in hordeins fraction. TEM studies showed that secalin and hordein fractions were globular in shape while gliadins in addition to globular structure also possessed rod-shaped particle arrangement. XRD pattern of prolamin fractions showed the ordered crystalline domain at 2θ values of 44.1°, 37.8° and 10.4°. The extracted prolamins fractions showed amorphous as well as crystalline structures as revealed by XRD and TEM analysis. Space saving hexagonal molecular symmetry was also observed in TEM molecular arrangement of prolamins which has profound application in development of plant-based polymers and fibres.

Although the economic value of wheat flour is determined by the complement of gluten proteins, these proteins have been challenging to study because of the complexity of the major protein groups and the tremendous sequence diversity among wheat cultivars. The completion of a high-quality wheat genome sequence from the reference wheat Chinese Spring recently facilitated the assembly and annotation of a complete set of gluten protein genes from a single cultivar, making it possible to link individual proteins in the flour to specific gene sequences. In a proteomic analysis of total wheat flour protein from Chinese Spring using quantitative two-dimensional gel electrophoresis combined with tandem mass spectrometry, gliadins or low-molecular-weight glutenin subunits were identified as the predominant proteins in 72 protein spots. Individual spots were associated with 40 of 56 Chinese Spring gene sequences, including 16 of 26 alpha gliadins, 10 of 11 gamma gliadins, six of seven omega gliadins, one of two delta gliadins, and nine of ten LMW-GS. Most genes that were not associated with protein spots were either expressed at low levels in endosperm or encoded proteins with high similarity to other proteins. A wide range of protein accumulation levels were observed and discrepancies between transcript levels and protein levels were noted. This work together with similar studies using other commercial cultivars should provide new insight into the molecular basis of wheat flour quality and allergenic potential.

Wheat gluten is responsible for the unique baking properties of wheat flour, but it also causes wheat-related disorders in predisposed individuals. Different commercially available gluten materials are commonly used for a variety of assays, but a detailed characterization of their composition is missing in many cases. This is why we aimed to provide an in-depth analysis of three commonly used gliadin and gluten materials from two different batches using gel electrophoretic and chromatographic techniques. The gliadin material did not show the typical qualitative and quantitative protein composition and does not appear to be representative of wheat gliadin. The two gluten materials had the expected protein composition, but both showed large batch-to-batch variability regarding total protein content. Since these variations result in different biochemical, immunological, and functional behaviors, it is important to analyze at least the total protein content of each material and each batch.

Wheat flour cannot be tolerated by those who suffer allergies to gluten. Human pathologies associated with grain proteins have increased worldwide in recent years, and the only effective treatment available is a lifelong gluten-free diet, which is complicated to follow and detrimental to gut health. This manuscript describes the development of wheat bread potentially suitable for celiac patients and other gluten-intolerant individuals. We have made bread using wheat flour with very low content of the specific gluten proteins (near gliadin-free) that are the causal agents for pathologies such as celiac disease. Loaves were compared with normal wheat breads and rice bread. Organoleptic, nutritional, and immunotoxic properties were studied. The reduced-gliadin breads showed baking and sensory properties, and overall acceptance, similar to those of normal flour, but with up to 97% lower gliadin content. Moreover, the low-gliadin flour has improved nutritional properties since its lysine content is significantly higher than that of normal flour. Conservative estimates indicate that celiac patients could safely consume 67 grams of bread per day that is made with low-gliadin flour. However, additional studies, such as feeding trials with gluten-intolerant patients, are still needed in order to determine whether or not the product can be consumed by the general celiac population, as well as the actual tolerated amount that can be safely ingested. The results presented here offer a major opportunity to improve the quality of life for millions of sufferers of gluten intolerance throughout the world.

Gliadin is a fraction of gluten, known to trigger celiac disease in susceptible people. To date, the life-long gluten-free diet is used for the prevention of this disease. Hence, methods for gluten control in foods are of significant importance. Being one of the most-used methods used for this purpose, ELISA should use high-affinity antibodies to gliadin peptides involved into celiac process. This study investigates the characteristics of a novel anti-gliadin antibody X6. We found the QXQPFPXP site to be a recognized epitope that provides specific binding of the antibody to cereal prolamins involved in celiac disease manifestation. A specificity study using immunoblotting shows the recognition of wheat, barley and rye proteins—as well as α-gliadin homologs from non-edible cereals (Dasypyrum villosum). Reactivity to avenin was less pronounced, as this protein does not contain the PFP motif most critical for antibody recognition. The proteins of Zea mays and Setaria italica were not recognized by X6. X6-based ELISA highly correlated with R5 and G12, which are Codex Alimentarius standards in the quantitative assessment of gluten content (Pearson’s R = 0.86 and 0.87, respectively). Qualitative assessment revealed no significant differences between R5 and G12 and X6.

The classical Osborne wheat protein fractions (albumins, globulins, gliadins, and glutenins), as well as several proteins from each of the four subunits of gliadin using SDS-PAGE analyses, were determined in the grain of five bread (T. aestivum L.) and five durum wheat (T. durum Desf.) genotypes. In addition, content of tryptophan and wet gluten were analyzed. Gliadins and glutenins comprise from 58.17% to 65.27% and 56.25% to 64.48% of total proteins and as such account for both quantity and quality of the bread and durum wheat grain proteins, respectively. The ratio of gliadin/total glutenin varied from 0.49 to 1.01 and 0.57 to 1.06 among the bread and durum genotypes, respectively. According to SDS-PAGE analysis, bread wheat genotypes had a higher concentration of α + β + γ-subunits of gliadin (on average 61.54% of extractable proteins) than durum wheat (on average 55.32% of extractable proteins). However, low concentration of ω-subunit was found in both bread (0.50% to 2.53% of extractable proteins) and durum (3.65% to 6.99% of extractable proteins) wheat genotypes. On average, durum wheat contained significantly higher amounts of tryptophan and wet gluten (0.163% dry weight (d.w.) and 26.96% d.w., respectively) than bread wheat (0.147% d.w. and 24.18% d.w., respectively).

The aim of this work was to assess the ability of Near Infrared Spectroscopy (NIRS) to distinguish wheat lines with low gliadin content, obtained by RNA interference (RNAi), from non-transgenic wheat lines. The discriminant analysis was performed using both whole grain and flour. The transgenic sample set included 409 samples for whole grain sorting and 414 samples for flour experiments, while the non-transgenic set consisted of 126 and 156 samples for whole grain and flour, respectively. Samples were scanned using a Foss-NIR Systems 6500 System II instrument. Discrimination models were developed using the entire spectral range (400-2500 nm) and ranges of 400-780 nm, 800-1098 nm and 1100-2500 nm, followed by analysis of means of partial least square (PLS). Two external validations were made, using samples from the years 2013 and 2014 and a minimum of 99% of the flour samples and 96% of the whole grain samples were classified correctly. The results demonstrate the ability of NIRS to successfully discriminate between wheat samples with low-gliadin content and wild types. These findings are important for the development and analysis of foodstuff for celiac disease (CD) patients to achieve better dietary composition and a reduction in disease incidence.

Highly sensitive detection of biomolecules is of paramount interest in many fields including biomedicine, safety and eco-pollution. Conventional analyses use well-established techniques with detection limits ~1 pM. Here we propose a pyro-concentrator able to accumulate biomolecules directly onto a conventional binding surface. The operation principle is relatively simple but very effective. Tiny droplets are drawn pyro-electro-dynamically and released onto a specific site, thus increasing the sensitivity. The reliability of the technique is demonstrated in case of labelled oligonucleotides diluted serially. The results show the possibility to detect very diluted oligonucleotides, down to a few hundreds of attomoles. Excellent results are shown also in case of a sample of clinical interest, the gliadin, where a 60-fold improved detection limit is reached, compared with standard ELISA. This method could open the way to a mass-based technology for sensing molecules at very low concentrations, in environmental as well as in diagnostics applications. Biomolecule detection is important in many fields, but obviously becomes increasingly difficult as concentrations decrease. Here, the authors demonstrate a method to dispense and concentrate biomolecules onto a target slide, greatly improving the detection limits.

Gluten is the main storage protein in grains and consists of gliadin and glutenin occurring in the same ratio. Persons suffering from intolerances, including celiac disease, must avoid foods containing gluten or products containing wheat, barley, and rye. Accordingly, gluten detection is of high interest for the food safety of celiac patients. This study was designed to determine the concentrations of gluten in foods labeled \"gluten free\" available in the United States. Seventy-eight samples labeled gluten free were collected and analyzed using a gliadin competitive enzyme-linked immunosorbent assay. The gluten content was calculated based on the assumption of the same ratio between gliadin and glutenin. Forty-eight (61.5%) of the 78 samples contained less than the limit of quantification of 10 mg/kg for gluten. In addition, 14 (17.9%) of the 78 samples labeled gluten free contained less gluten than the guidelines established by the Codex Alimentarius for gluten-free labeling (20 mg/kg). However, 16 samples (20.5%) did contain gluten levels of ≥20 mg/kg, ranging from 20.3 to 60.3 mg/kg. In particular, five of eight breakfast cereal samples showed gluten contents higher than 20 mg/kg. These results may be of concern, as gluten sensitivity is known to vary among celiac disease patients.