Analysis Of Gliadins Research Articles

How Important Are Genetic Diversity and Cultivar Uniformity in Wheat? The Case of Gliadins.

Improvements in self-pollinated crops rely on crosses between different genotypes. It has been suggested that the repeated use of "the best" genotypes may lead to the restriction of the genetic diversity of the crop. In wheat, the analysis of gliadin (storage protein) polymorphism has provided evidence that genetic diversity was high and stable throughout the 20th century. Moreover, a worldwide analysis of gliadin polymorphism shows that genetic diversity is structured spatially across countries and their regions. Therefore, the analysis of gliadin genotypes in a given grain sample can provide reliable information about the origin of grains in this sample. An unexpected finding is that many registered common wheat cultivars are genetically non-uniform and composed of authentic biotypes (genotypically related lines originated from the initial cross) in spite of current crop-registration rules that include a strict demand for each new cultivar to be genetically uniform (DUS rules). In summary, the results suggest that each cultivar is the fruit of joint effects of a breeder and of a region's environmental factors. We believe this finding will not be restricted to wheat and suggest there may be a need to re-evaluate relevant rules of cultivar registration for crop species in general.

The Gliadin Analysis by Reversed-Phase High-Performance Liquid Chromatography of Traditional Fermented “Hamoum” Wheat in West Algeria

Our ancestors had always used food fermentation without having a scientific explanation. Their natural phenomena with therapeutic potential became interesting only in the early twentieth century. A comparative study between the electrophoretic profiles of wheat proteins of fermented wheat type Hamoum (FWH) and regular unfermented wheat (NUW) was performed to identify the effect of fermentation realized by the traditional technique using underground storage (locally named Matmora) for 12 months. SDS-PAGE electrophoresis was performed after the sequential extraction of different proteins (albumins/globulins, gliadins and glutenins). The results showed a total degradation of the regular wheat proteins after fermentation. Fermented wheat type Hamoum (FWH) could be a good alternative for people with disorders related to gluten ingestion as compared to regular unfermented wheat (NUW).

Intraspecific divergence of diploid grass Aegilopscomosa is associated with structural chromosome changes.

Aegilopscomosa Smith in Sibthorp et Smith, 1806 is diploid grass with MM genome constitution occurring mainly in Greece. Two morphologically distinct subspecies - Ae.c.comosa Chennaveeraiah, 1960 and Ae.c.heldreichii (Holzmann ex Boissier) Eig, 1929 are discriminated within Ae.comosa, however, genetic and karyotypic bases of their divergence are not fully understood. We used Fluorescence in situ hybridization (FISH) with repetitive DNA probes and electrophoretic analysis of gliadins to characterize the genome and karyotype of Ae.comosa to assess the level of their genetic diversity and uncover mechanisms leading to radiation of subspecies. We show that two subspecies differ in size and morphology of chromosomes 3M and 6M, which can be due to reciprocal translocation. Subspecies also differ in the amount and distribution of microsatellite and satellite DNA sequences, the number and position of minor NORs, especially on 3M and 6M, and gliadin spectra mainly in the a-zone. Frequent occurrence of hybrids can be caused by open pollination, which, along with genetic heterogeneity of accessions and, probably, the lack of geographic or genetic barrier between the subspecies, may contribute to extremely broad intraspecific variation of GAAn and gliadin patterns in Ae.comosa, which are usually not observed in endemic plant species.

Determination of Gliadin as a Measure of Gluten in Food by R5 Sandwich ELISA RIDASCREEN® Gliadin Matrix Extension: Collaborative Study 2012.01.

BackgroundAccording to Codex Alimentarius, food products containing less than 20 mg/kg gluten can be labeled as “gluten-free.” Since 2002, the R5 antibody method allowed determination of gluten levels and led to a huge improvement of products available to celiac disease (CD) patients.MethodThe R5-containing test kit RIDASCREEN® Gliadin in combination with the cocktail solution was endorsed as Codex Type 1 Method in 2006 based on a collaborative study with corn-based bread, rice-based dough, wheat starches, rice, and corn flour. In 2012, the method was approved as First Action Official MethodSM2012.01 with an “in foods” claim. For Final Action in 2016, the matrix claim was reduced to rice- and corn-based matrixes.ObjectiveTherefore, R-Biopharm decided to start a collaborative study to demonstrate the wide applicability of Official Method 2012.01 for the quantitative analysis of gliadin in soy, starches, pseudo cereals, legumes, spices, juice, nut nougat crème, cream cheese, pesto, meat, vegetarian meat alternative, cookies, dessert, cake, fish, bread, candies, and potatoes. Materials for incurring were the MoniQA wheat flour and the PWG gliadin preparation. ResultsGliadin levels ranged from 3.4 up to 27.4 mg gliadin per kg. The results of the collaborative study with 14 participating laboratories showed recoveries ranging from 80 to 130%. Relative reproducibility standard deviations for contaminated samples were between 9.8 and 27.7%.ConclusionsThe collaborative study results confirmed that the method is accurate and suitable to measure gliadin in important gluten-free food matrixes.HighlightsThe title and applicability statement of Official Method 2012.01 were changed as proposed.

Electrophoretic patterns of gliadin as markers of genotypes in the analysis of the durum wheat landrace Kubanka

Background. The collection of durum wheat landraces (Triticum durum Desf.) at VIR contains unique material of the “Russian northern branch”, which is not found in any other collections worldwide. Studying the genetic diversity of such wheat accessions according to their gliadin bands as markers of genotypes is important for identification and conservation of their gene pool authenticity.Materials and methods. For the first time, molecular markers were used to differentiate among 38 accessions of the local durum wheat variety known under the name of “Kubanka”, collected and placed into the VIR collection in the 1910–1940s, and five accessions from the seed genebanks of the USA and Canada. Electrophoretic patterns of gliadin were used as markers of genotypes within the polymorphic cultivar. Recording bands in the form of “protein formulas” allows the researcher to evaluate the polymorphism of each accession and the diversity within the collection. Gliadin analysis was performed on single grains according to the standard method adopted at VIR and approved by ISTA.Results and conclusions. Fourteen major biotypes marked with gliadin bands were identified. Depending on the component composition of the α-zone encoded by alleles of the GLI-2A locus, biotypes were combined into 4 groups. Within the groups, biotypes are determined by alleles of the GLI-1A, GLI-1B, GLI2B loci. Genetically close monotypic accessions and polytypic ones incorporating 2 to 6 biotypes were identified. Group I is typical for the European part of Russia as well as for Kazakhstan and Kyrgyzstan. Accessions of this group can be attributed to the Volga steppe ecotype. Group II biotypes are widespread in Altai Territory, Orenburg and Astrakhan Provinces of Russia; Group III in Stavropol Territory, Russia, and Kyrgyzstan; Group IV only in Altai Territory. The greatest genetic diversity was exhibited by the ‘Kubanka’ accessions from Altai and Krasnodar Territories, and Kyrgyzstan.

Identification of characteristic proteins of wheat varieties used to commercially produce dried noodles by electrophoresis and proteomics analysis

Characteristic proteins of wheat varieties used for commercial production of dried noodles were identified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), acetic acid urea-polyacrylamide gel electrophoresis (AU-PAGE), two dimensional electrophoresis coupled with MALDI-TOF/TOF mass spectrometry analysis (2D-MS). Noodle textural properties prepared from the wheat varieties were also evaluated. The results of SDS-PAGE and texture evaluation of the noodles showed that some high molecular weight glutenin subunits (HMW-GSs), including subunits 5 + 10, 1, 7 + 8, 7 + 9, 7, 13 + 16, 20, 2 + 12, and 4 + 12 were possible desirable subunits of noodle wheat variety. The AU-PAGE analysis of gliadin extracted from the noodle wheat varieties demonstrated that five wheat varieties (SJ, HMT, YJ, DZ, and ZM) consisted of 1BL/1RS translocations. But in view of the noodle textural properties prepared from the five wheat varieties, we can conclude that desirable HMW-GSs existed in the wheat varieties can make up for the defects brought by 1BL/1RS translocations. Further identification of characteristic proteins of the wheat varieties by 2D-MS demonstrated that β-amylase, serpin, and α-amylase inhibitor, etc. were also potential characteristic proteins that affected the application of wheat variety. The study provided useful information for the selection and genetic breeding of wheat variety used to commercially produce dried noodles.

A MALDI-TOF analysis of gliadins and glutenins in “Astana” wheat cultivar

Targeted, quantitative metabolomics can, in principle, provide precise information on intracellular metabolite levels, which can be applied to accurate modeling of intracellular processes required in systems biology and metabolic engineering. However, quantitative metabolite profiling is often hampered by biased mass spectrometry-based analyses caused by matrix effects, the degradation of metabolites and metabolite leakage during sample preparation, and unexpected variation in instrument responses. Isotope Dilution Mass Spectrometry (IDMS) has been proven as the most accurate method for high-throughput detection of intracellular metabolite concentrations, and the key has been the acquisition of the corresponding fully uniformly (U) -13C-labeled metabolites to be measured. Here, we have prepared U-13C-labeled cell extracts by cultivating P. chrysogenum in a fed-batch fermentation with fully U-13C-labeled substrates. Towards this goal, a dynamic fed-batch model describing P. chrysogenum growth and penicillin production was used to simulate the fermentation process and design the fed-batch fermentation media. Further, a case study with extensive intracellular metabolomics data from glucose-limited cultivation of Penicillium chrysogenum under both single and repetitive glucose pulses was illustrated by using the IDMS methods with the prepared U-13C-labeled cell extracts as internal standards. In conclusion, the IDMS method can be incorporated into well-established fast sampling and quenching protocols to obtain dynamic quantitative in vivo metabolome data at the timescales of (tens of) seconds and elucidate the underlying regulatory architecture. The case study revealed gross differences between single and repeated pulses, which suggests that single pulse studies have limited value for understanding of metabolic responses in large-scale bioreactors. Instead, intermittent feeding should be favored.

Effect of high-nitrogen fertilizer on gliadin and glutenin subproteomes during kernel development in wheat (Triticum aestivum L.)

Nitrogen (N), a macronutrient essential for plant growth and development, is needed for biosynthesis of protein and starch, which affect grain yield and quality. Application of high-N fertilizer increases plant growth, grain yield, and flour quality. In this study, we performed the first comparative analysis of gliadin and glutenin subproteomes during kernel development in the elite Chinese wheat cultivar Zhongmai 175 under high-N conditions by reversed-phase ultra-performance liquid chromatography and two-dimensional difference gel electrophoresis (2D-DIGE). Application of high-N fertilizer led to significant increases in gluten macropolymer content, total gliadin and glutenin content, and the accumulation of individual storage protein components. Of 126 differentially accumulated proteins (DAPs) induced by high-N conditions, 24 gliadins, 12 high-molecular-weight glutenins, and 27 low-molecular-weight glutenins were significantly upregulated. DAPs during five kernel developmental stages displayed multiple patterns of accumulation. In particular, gliadins and glutenins showed respectively five and six accumulation patterns. The accumulation of storage proteins under high-N conditions may lead to improved dough properties and bread quality.

Association between gluten protein composition and breadmaking quality characteristics in historical and modern spring wheat

Background and objectivesThirty hard red spring wheat cultivars released between 1910 and 2013 were studied to determine the changes in quality characteristics that occurred over time, and to determine their association with protein composition.FindingsSignificant positive correlations (p ≤ .01) were found between release year and dough quality characteristics, such as farinograph peak time and stability. In size‐exclusion HPLC analysis of proteins, significant positive correlations (p ≤ .01) were observed between release year, glutenin polymeric proteins, and the ω‐gliadin fraction, which also showed significant positive correlations (p ≤ .01) with dough properties. In reverse‐phase HPLC analysis of gliadins, five ω‐gliadin peaks showed significant correlations (p ≤ .05) with year and farinograph characteristics. Mass spectrometry was performed to identify the proteins in these peaks.ConclusionsOverall, the results indicated that breeding efforts improved dough properties, which could be associated with quantitative variations in glutenin polymeric proteins, and certain subfractions of ω‐gliadins.Significance and noveltyGlutenin polymeric proteins, together with specific ω‐gliadin proteins, may play an important role in baking quality of spring wheat.

Cluster Analysis of Historical and Modern Hard Red Spring Wheat Cultivars Based on Parentage and HPLC Analysis of Gluten‐Forming Proteins

Thirty hard red spring (HRS) wheat cultivars released between 1910 and 2013 were analyzed to determine how they cluster in terms of parentage and HPLC analysis of gliadins and unreduced proteins. Markers for reduced height genes were assessed to determine associations between semidwarfism and protein composition, as well as quality characteristics. In terms of parentage, the clusters formed were grouped according to release year and semidwarfism. Farinograph development time and stability showed significant (P ≤ 0.05) differences between clusters, indicating improvement of mixing characteristics over time. Flour ash content was significantly different (P ≤ 0.05) between clusters, indicating improved milling quality. The clusters based on the gliadin reverse‐phase HPLC peak binary data did not group cultivars according to release year and the semidwarf characteristic. However, cultivars were grouped according to year in the absorbance area based dendrogram. The clusters based on absorbance area of size‐exclusion HPLC showed significant (P ≤ 0.05) differences for release year, the semidwarf characteristic, and farinograph stability. Overall, the results indicate that the introduction of reduced height genes accompanied the improvements of dough mixing and breadmaking quality characteristics, without altering the composition of gliadin proteins over the last 100 years of HRS wheat breeding.

Comparative analysis of Agropyron intermedium (Host) Beauv 6Agi and 6Agi2 chromosomes in bread wheat cultivars and lines with wheat–wheatgrass substitutions

A comparative study of wheat–wheatgrass substituted cultivars and lines resistant to leaf rust developed by the Agricultural Research Institute for Southeast Regions (Multi 6R, Belyanka, Favorit, Voevoda, Lebedushka) and Samara Agricultural Research Institute (Tulaikovskaya 5, Tulaikovskaya 10, Tulaikovskaya 100, Tulaikovskaya Zolotistaya) breeding was conducted. A complex analysis using molecular cytogenetic (C-differential banding, fluorescent (FISH) and genomic (GISH) in situ hybridization), molecular (PLUG markers), and biochemical (electrophoretic analysis of gliadins) markers demonstrated that they have a substitition of wheat chromosome 6D by the chromosomes 6Agi and 6Agi2 belonging to the J(=E) Agropyron intermedium (Host) Beauv (=Thinopyrum intermedium (Host) Barkworth & D.R. Dewey) subgenome. In spite of the fact that the chromosomes 6Agi and 6Agi2 differ in the C-banding pattern and demonstrated minor differences in the blocks of gliadin components, they had the identical pattern of pSc119.2 and pAs1 probe distribution and conjugated between themselves with insignificant disturbance. Thus, it was demonstrated that 6Agi and 6Agi2 are homologous chromosomes; however, the question about allelism of their leaf rust resistance genes between themselves requires special studies. Nevertheless, using STS and SCAR markers and taking into account the type of reaction to Puccinia triticina, their non-allelism to the Lr9, Lr19, Lr24, Lr29, Lr38, and Lr47 genes was established. It was revealed that the 6Agi and 6Agi2 chromosomes have a different level of transmission in hybrid F2 populations depending on the hybrid combination gene background.

Development and molecular cytogenetic identification of a new wheat–Leymus mollis Lm#6Ns disomic addition line

AbstractWe developed a new disomic addition line M11028‐1‐1‐5 (2n = 44 = 21” + 1”) from a cross between wheat cv. ‘7182’ and octoploid Tritileymus M47 (2n = 8x = 56, AABBDDNsNs). Cytological observations demonstrated that M11028‐1‐1‐5 contained 44 chromosomes and formed 22 bivalents during meiotic metaphase I. The genomic in situ hybridization (GISH) investigations showed this line contained 42 wheat chromosomes and a pair of L. mollis chromosomes. SSR, EST and PCR‐based landmark unique gene (PLUG) markers were screened to determine the homoeologous relationships of the introduced L. mollis chromosomes in wheat background. Nine markers, i.e. Xwmc256, Xgpw312, Swes123, CD452568, BF483643, BQ169205, TNAC1748, TNAC1751 and TNAC1752, all of which were located on the homoeologous group 6 chromosomes of common wheat, amplified bands unique to L. mollis in M11028‐1‐1‐5. Gliadin analysis also confirmed that the added chromosomes in M11028‐1‐1‐5 were correlated with the sixth group chromosome. This indicated that M11028‐1‐1‐5 contained a pair of introduced L. mollis chromosome belonging to homoeologous group 6, which we designated it as Lm#6Ns disomic addition line. This is the first report of a common wheat–L. mollis disomic addition line.

Genetic marking of glume color in Triticum spelta L. var. caeruleum using gliadins

Using gliadins as genetic markers, Triticum spelta L. var. caeruleum accessions were analyzed to identify genetic control of the dark color of glumes. The research material was F2 and BC1 plants from crosses between spelt accessions and white-glumed common wheat varieties. The segregation for glume color fitted the monogenic control of the trait. The electrophoretic analysis of gliadins in grains from the hybrid plants has shown that the Gli-Alj* allele in the T. spelta var. caeruleum accessions is linked to the allele for the dark (black) color of glumes at the Rg-A1 locus.

Wheat allergy in children evaluated with challenge and IgE antibodies to wheat components.

Wheat sensitization is common but IgE antibodies (IgE-abs) to wheat are not predictive of clinical symptoms in children with suspected wheat allergy. Wheat allergen components other than ω-5gliadin have not been well studied. Our aim was to characterize the clinical profile and investigate the value of adding measurements of IgE-abs to wheat components in a group of children with a doctor's diagnosed wheat allergy. Sixty-three children with a doctor's diagnosis of wheat allergy confirmed sensitization to wheat and, on a wheat elimination diet, went through oral wheat challenges or had a convincing recent history of wheat allergy. IgE-ab to ω-5 gliadin, low molecular weight glutenin (LMW-glutenin), high molecular weight glutenin (HMW-glutenin) and a native gliadin preparation containing α-, β-, γ-, and ω-gliadin (gliadin) were analyzed. Twenty-six children were positive in challenge, while six children were regarded as wheat allergic due to recent anaphylactic reactions. The IgE-ab levels to all four wheat components were significantly higher in the group with wheat allergy compared to the group with no wheat allergy (p<0.0001). Also, the severity of symptoms at challenge correlated with the IgE-ab levels to all four components (p<0.05). IgE-ab levels to ω-5 gliadin correlated best with challenge outcome, and by additional analysis of gliadin, HMW- and LMW-glutenin IgE-abs all challenge positive children could be identified. Many children diagnosed as wheat allergic have outgrown their allergy and are unnecessarily on a wheat-free diet. The levels of IgE-ab to wheat gluten-derived components correlated well with wheat challenge outcome and severity.

Genetic variation of wheat landraces from China as revealed by gliadin and microsatellite markers

A reborn interest has occurred during the last decade toward wheat landraces for broadening genetic basis of modern wheat cultivars. The investigation of molecular traits within and between existing landraces of wheat can help scientists to develop appropriate strategies for their efficient maintenance and exploitation. The present study dealt with the gliadin characterization of forty-seven wheat landraces collected from wheat mainly planted areas of China, each of which was represented by a sample of at least 43 individuals. Twelve accessions selected on the basis of gliadin analysis were investigated further using 21 SSR markers. The results proved that landraces of wheat are a mixture of variable individuals genetically distinguishable from each other. Twelve of the analyzed 47 accessions were observed to be homogeneous, while 35 (74.5%) of them were heterogeneous in their gliadin composition. In total, 122 gliadin pattern were observed. On average, 10.1% (Gst) of the total variation arose from differentiation among regions, and 89.9% was attributed to within-region variation. Furthermore, nineteen of the 21 SSR markers were polymorphic across all the populations. The total number of the amplified DNA products was 110, with a mean of 6.11 alleles per locus. The values of genetic diversity within each landrace population varied from 0.006 to 0.351. Implications for the management of this valuable genetic resource are discussed.

Genetic diversity and relationships among accessions of five crested wheatgrass species (Poaceae: Agropyron) based on gliadin analysis

Agropyron Gaertn. is the most important genus in Triticeae (Poaceae), which includes many forage grasses with high economic value. The genetic diversity and relationships of 36 accessions from five crested wheatgrass species were analyzed by gliadin markers. A total of 54 product bands were detected after acid polyacrylamide gel electrophoresis (A-PAGE), of which 100% were polymorphic. The genetic similarity coefficient based on Nei-Li's method ranged from 0.065 to 0.755 with an average of 0.451. The Shannon diversity information index showed that there was a high level of genetic diversity among the accessions. An unweighted pair group method with arithmetic average (UPGMA) dendrogram was constructed based on the Nei-Li's genetic similarity coefficients, which showed the phylogenetic relationships among accessions of different species. Analysis of molecular variance (AMOVA) showed that the proportion of variance explained by inter- and intraspecific variance was 9.34 and 90.66%, respectively, which revealed that the genetic variations within species were higher than the variations among species. Based on pairwise genetic distances (ΦST) among species, the cluster analysis indicated that A. mongolicum had a low-affinity relationship with other species, while A. fragile showed a close relationship with A. cristatum ssp pectinatum. Finally, the implications of the results for the taxonomy of Agropyron were discussed.

The MSE-proteomic analysis of gliadins and glutenins in wheat grain identifies and quantifies proteins associated with celiac disease and baker's asthma

Precise content of gliadin (Glia) and glutenin (Glu) proteins in wheat grain are largely unknown despite their association with celiac disease, various allergies, and physical processing properties of wheat. Developing methods to quantitatively measure clinically relevant proteins could support advancement in understanding exposure thresholds and clinical study design. The aim of this study was to use a data-independent mass spectrometry (MS(E)) approach for quantifying gliadin and glutenin proteins in wheat grain. The biologically replicated analysis yielded concentrations for 34 gliadin and 22 glutenin proteins. The primary focus of this survey was on measuring celiac disease proteins and baker's asthma associated proteins along with the proteins associated with viscoelastic properties of wheat flour and grain texture. The technical coefficients of variation ranged from 0.12 to 1.39 and indicate that MS(E) proteomics is a reproducible quantitative method for the determination of gliadin and glutenin content in the highly complex matrix of protein extracts from wheat grain. This article is part of a Special Issue entitled: Translational Plant Proteomics.

Gliadin analysis of Elymus nutans Griseb. from the Qinghai-Tibetan Plateau and Xinjiang, China

The diversity and relationships among Elymus nutans Griseb. accessions from the Qinghai–Tibetan Plateau and Xinjiang, China, were analyzed using acid polyacrylamide gel electrophoresis (A-PAGE) in order to estimate the gliadin genetic variability and compare the level of diversity. The cluster analysis separated the accessions into six groups with geographical origins. The results of principal coordinates analysis (PCoA) were almost congruent with the cluster of accessions. The molecular variance analysis (amova) showed that the proportion of variance within and among the Qinghai–Tibetan Plateau and Xinjiang regions was 68.66 and 31.34%, respectively, or 75.99 and 24.01% within and among six political provinces. The distinct geographical divergence of E. nutans was revealed between the Qinghai–Tibetan Plateau and Xinjiang. The ecogeographical conditions such as climate and mountain ranges, genetic drift and mating system could be driving factors for genetic diversity. These results will facilitate the breeding program and germplasm collection and conservation.

Mass spectrometry analysis of gliadins in celiac disease

In recent years, scientific research on wheat gluten proteins has followed three main directions aimed at (1) finding relationships between individual genetic alleles coding for gliadins, high or low molecular weight glutenin subunits, and the viscoelastic dough properties of flour-derived products such as pasta and bread; (2) identifying prolamins and derived peptides involved in celiac disease, a pathological condition in which the small intestine of genetically predisposed individuals is reversibly damaged; and (3) developing and validating sensitive and specific methods for detecting trace amounts of gluten proteins in gluten-free foods for celiac disease patients. In this review, the main aspects of current and perspective applications of mass spectrometry and proteomic technologies to the structural characterization of gliadins are presented, with focus on issues related to detection, identification, and quantification of intact gliadins, as well as gliadin-derived peptides relevant to the biochemical, immunological, and toxicological aspects of celiac disease.

Interference of denaturing and reducing agents on the antigen/antibody interaction. Impact on the performance of quantitative immunoassays in gliadin analysis

Immunoassays are the most commonly used quantitative techniques to determine the gliadin content of food aimed at coeliac patients. Though the minimal amount of gliadins inducing the typical histopathological changes at the intestinal mucosa in coeliacs is still a matter of debate, current research is focussed on the development of methods having higher sensitivities. One of the main drawbacks in gliadin analysis is the low efficiency of the conventional extraction procedure using 60% ethanol. The use of reducing (2-mercaptoethanol) and denaturing (guanidinium chloride) agents has been recommended to improve the extraction efficiency. Owing to the well-known effects of these agents on native conformation of proteins, and their widely reported interference on the antigen/antibody interaction in other systems, we assessed whether gliadin detection by immunoassays is affected by the presence of those agents. Using two ELISA formats with a panel of polyclonal and monoclonal antibodies, we found that recognition by specific antibodies of partially or totally denatured gliadins is severely impaired. The magnitude of the interference depends on the antibodies used and the ELISA format. The impact of such interference was analysed for each step of the immunoassays. 2-mercaptoethanol had a stronger effect than guanidinium chloride, and the antigen became almost undetectable for some assays when both reagents were used in combination. Remarkably, since quantitative results are obtained by comparison with a calibration curve using a native antigen, there is no equivalence between the antigen/antibody interaction occurring in the sample and that in the standard gliadin, leading to underestimation of the actual gliadin content. Therefore, we suggest that not only the effects of reducing and denaturing agents on the antigen during the extraction procedure, but also the effects of residual amounts of these agents on the antigen/antibody interaction should be considered when a quantitative immunoassay is performed.