Innovative approach to low-level gluten determination in foods using a novel sandwich enzyme-linked immunosorbent assay protocol

OBJECTIVES: There is currently much call for a reliable enzyme-linked immunosorbent assay (ELISA) protocol for determining gluten in foods to serve as a basis for further Codex Alimentarius regulations. Given its ability to recognize the potential coeliac-toxic epitope QQPFP, which occurs repeatedly in alpha-, gamma- and omega-gliadins, hordeins and secalins, the monoclonal antibody R5 raised against a secalin extract may prove to be an essential tool for gluten analysis. This study was designed to develop a highly sensitive and specific sandwich ELISA to quantify low levels of wheat, barley and rye prolamins in foods for coeliacs. METHODS: Simple sandwich ELISA based on the use of a single monoclonal antibody (R5) as both the coating and detection was developed. A quantitative cocktail gluten-extraction procedure for heat-processed foods was also tested. RESULTS: R5-ELISA was able to identify gliadins, hordeins and secalins with assay sensitivities of 0.78, 0.39 and 0.39 ng/ml, respectively. The assay's detection limit was 1.5 ng gliadins/ml (1.56 ppm gliadins, 3.2 ppm gluten). The system proved insensitive to the non-coeliac-toxic cereals maize, rice and oats, and was non-cultivar-dependent. It was also able to detect gliadins and hordeins in unprocessed and heat-processed wheat- and barley-based products, and to estimate the gluten content of hydrolysed foods. CONCLUSION: We present a new generation of a robust sandwich R5-ELISA with good reproducibility (8.7%) and repeatability (7.7%). Its gluten-detection limit of 3.2 ppm is lower than the existing threshold of 20-200 ppm. The ELISA, which is equally sensitive to barley, wheat and rye prolamins, is compatible with the quantitative cocktail extraction procedure for heat-processed foods. Along with the cocktail procedure, the Working Group on Prolamin Analysis and Toxicity is currently evaluating an R5-ELISA system as proposed by the Codex Alimentarius Commission.     

Measurement of wheat gluten and barley hordeins in contaminated oats from Europe, the United States and Canada by Sandwich R5 ELISA

OBJECTIVES: We have investigated the extent of contamination with wheat, barley, rye or a mixture of these cereals in a large number of grains and commercial oats. We have also attempted to identify the type of cereal contaminant. METHODS: Sandwich R5 ELISA (using either gliadins or hordeins as standards), western blot, matrix-assisted laser desorption/ionization time-of-flight mass spectrometric and quantitative real-time PCR (Q-PCR) techniques have been used to analyze a total of 134 oats, comprising grains and commercial oat products collected from Europe, the United States and Canada. RESULTS: Twenty-five of the 134 pure, uncontaminated oat varieties were found to have undetectable levels of gluten, whereas most of the 109 grains and commercial oat products were mainly contaminated with mixtures of wheat, barley and rye, barley being the predominant contaminant. The percentages of these cereals in the oat samples have been calculated by specific wheat, barley and rye Q-PCR systems. The oat samples were grouped according to the avenin spectra determined by the mass spectrometric technique. The data confirmed that contaminated oat foods, based on the same variety, could have different levels of wheat, barley and rye contamination. CONCLUSION: This study has verified that contamination with wheat gliadins or barley hordeins in oat samples can be measured by the Sandwich R5 ELISA, using either gliadins or hordeins as standards, and also the importance of using confirmatory techniques (such as western blot, Q-PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) to confirm that most oats are contaminated with mixtures of wheat, barley and rye.     

A novel and sensitive method for the detection of T cell stimulatory epitopes of alpha/beta- and gamma-gliadin

BACKGROUND: It is now generally accepted that coeliac disease (CD) is caused by inflammatory T cell responses to gluten peptides bound to HLA-DQ2 or -DQ8 molecules. There is overwhelming evidence that CD patients can mount T cell responses to peptides found in both alpha-gliadin and gamma-gliadin molecules. Assays that would detect the presence or absence of such peptides in food would thus be accurate indicators of safety for consumption by CD patients. AIMS: The development of a sensitive method to detect T cell stimulatory epitopes of alpha-gliadin and gamma-gliadin molecules in food products. METHODS: Monoclonal antibodies (mAb) were raised against peptides encoding the T cell stimulatory epitopes of alpha-gliadin (amino acids (aa) 59-71) and aa gamma-gliadin (aa 142-153 and aa 147-159). These mAb competition assays were developed that quantitatively detect T cell stimulatory epitopes present on both intact proteins and peptides of sizes recognisable by CD4(+) T cells. RESULTS: With the mAb based competition assays, T cell epitopes were detected in pepsin/trypsin digests of wheat proteins and ethanol extracts of various food products, with detection levels lower than those reached with gluten specific T cells. Moreover, the presence of T cell stimulatory epitopes was also detected in preparations of barley, rye, and triticale, other cereals known to be toxic for CD patients. CONCLUSIONS: A new antibody based method has been developed, detecting the presence of T cell stimulatory gluten peptides. This can be used to further ensure the safety of food consumed by CD patients.     

Is Candida albicans a trigger in the onset of coeliac disease?

Coeliac disease is a T-cell-mediated autoimmune disease of the small intestine that is induced by ingestion of gluten proteins from wheat, barley, or rye. We postulate that Candida albicans is a trigger in the onset of coeliac disease. The virulence factor of C albicans-hyphal wall protein 1 (HWP1)-contains aminoacid sequences that are identical or highly homologous to known coeliac disease-related alpha-gliadin and gamma-gliadin T-cell epitopes. HWP1 is a transglutaminase substrate, and is used by C albicans to adhere to the intestinal epithelium. Furthermore, tissue transglutaminase and endomysium components could become covalently linked to the yeast. Subsequently, C albicans might function as an adjuvant that stimulates antibody formation against HWP1 and gluten, and formation of autoreactive antibodies against tissue transglutaminase and endomysium.     

Development of a general procedure for complete extraction of gliadins for heat processed and unheated foods

OBJECTIVES: In the past, one of the major problems in gluten analysis has been the unavailability of an efficient, universal, extraction procedure of gliadins - the alcohol-soluble proteins of gluten - from both heat processed and unprocessed products. This study was designed to develop a universal, extraction procedure capable of extracting the totality of gliadins from both unprocessed and heat processed foods for coeliac patients. METHODS: A simple quantitative extraction solution containing 250 mM 2-mercaptoethanol and 2 M guanidine hydrochloride ('cocktail'), was developed to extract gliadins from heated foods. RESULTS: The diluted reducing and disaggregating agents reaching the micro plate at low concentration do not affect the ELISA system based on the R5 monoclonal antibody. The recovery of gliadins extracted by the cocktail from spiked samples was nearly complete, with an average mean value of 95.5%, which is clearly superior to 44.4% obtained with conventional 60% aqueous ethanol. The cocktail always yielded either slightly similar or higher values than 60% aqueous ethanol depending on the type of foods: 1.1-fold in unheated foods, 1.4-fold in wheat starches and 3.0-fold in heated foods. False positives or negatives were never observed using the cocktail solution. CONCLUSION: We present a general complete gliadin extraction procedure based on reducing and disaggregating agents for both heated and unheated foods as a crucial tool for gliadin analysis. The new extraction solution is used for corresponding proteins from rye (secalins) and barley (hordeins). The cocktail was employed as the extraction method in the international ring trial evaluation of sandwich R5-ELISA as proposed by the Codex Alimentarius and organized by the Working Group on Prolamin Analysis and Toxicity.     

Mapping of gluten T-cell epitopes in the bread wheat ancestors: implications for celiac disease

BACKGROUND AND AIMS: Celiac disease is a prevalent disorder characterized by a chronic intestinal inflammation driven by HLA-DQ2 or -DQ8-restricted T cells specific for ingested wheat gluten peptides. The dominant T-cell responses are to epitopes that cluster within a stable 33mer fragment formed by physiologic digestion of distinct alpha-gliadins. Celiac disease is treated by excluding all gluten proteins from the diet. Conceivably, a diet based on baking-quality gluten from a wheat species that expresses no or few T-cell stimulatory gluten peptides should be equally well tolerated by the celiac patients and, importantly, also be beneficial for disease prevention. METHODS: To identify baking quality, harmless wheat, we followed the evolution of the wheat back to the species that most likely have contributed the AA, BB, and DD genomes to the bread wheat. Gluten were extracted from a large collection of these ancient wheat species and screened for T-cell stimulatory gluten peptides. RESULTS: Distinct differences in the intestinal T-cell responses to the diploid species were identified. Interestingly, we found that the fragments identical or equivalent to the immunodominant 33mer fragment are encoded by alpha-gliadin genes on the wheat chromosome 6D and thus absent from gluten of diploid einkorn (AA) and even certain cultivars of the tetraploid (AABB) pasta wheat. CONCLUSIONS: These findings have implications for celiac disease because they raise the prospect of identifying or producing by breeding wheat species with low or absent levels of harmful gluten proteins.     

Combination enzyme therapy for gastric digestion of dietary gluten in patients with celiac sprue

BACKGROUND AND AIMS: Celiac sprue is a multifactorial disease characterized by an inflammatory response to ingested gluten in the small intestine. Proteolytically resistant, proline- and glutamine-rich gluten peptides from wheat, rye, and barley persist in the intestinal lumen and elicit an immune response in genetically susceptible persons. We investigated a new combination enzyme product, consisting of a glutamine-specific endoprotease (EP-B2 from barley) and a prolyl endopeptidase (SC PEP from Sphingomonas capsulata), for its ability to digest gluten under gastric conditions. METHODS: The ability of this combination enzyme to digest and detoxify whole-wheat bread gluten was investigated. In vitro and in vivo (rat) experimental systems were developed to simulate human gastric digestion, and the resulting material was analyzed by high-performance liquid chromatography, enzyme-linked immunoabsorbent assay, and patient-derived T-cell proliferation assays. RESULTS: The analysis revealed that EP-B2 extensively proteolyzes complex gluten proteins in bread, whereas SC PEP rapidly detoxifies the residual oligopeptide products of EP-B2 digestion. In vitro dose variation data suggests that an approximate 1:1 weight ratio of the 2 enzymes should maximize their synergistic potential. The efficacy of this 2-enzyme glutenase was verified in a rat model of gastric gluten digestion. CONCLUSIONS: By combining 2 enzymes with gastric activity and complementary substrate specificity, it should be possible to increase the safe threshold of ingested gluten, thereby ameliorating the burden of a highly restricted diet for patients with celiac sprue.     

A monoclonal antibody that recognizes a potential coeliac-toxic repetitive pentapeptide epitope in gliadins.

OBJECTIVES: Antibodies that detect coeliac-toxic prolamins from wheat, barley and rye are important tools for controlling the diet of coeliac disease patients. Recently, a monoclonal antibody R5 that recognizes wheat gliadin, barley hordein and rye secalin equally was described. In this study, the epitope recognized by R5 was investigated. METHODS: Both a phage-displayed heptapeptide library and overlapping peptides spanning the sequence of alpha- and gamma-type gliadins (pepscan) were screened for binding of R5. RESULTS: Both techniques yielded comparable pentapeptide consensus sequences (phage display QXPW/FP; pepscan QQPFP). According to recent observations, this peptide stretch may be of key importance in the pathogenicity of coeliac disease. This sequence occurs repetitively in prolamins (in gamma- and omega-type prolamins more frequently than in alpha-type prolamins) together with several homologous peptide stretches, which are recognized less strongly. CONCLUSIONS: R5 seems to be a good candidate for the specific detection of putative coeliac disease-active sequences in prolamins and thus represents a valuable tool for the quality control of gluten-free food.     

Intestinal T-cell responses to high-molecular-weight glutenins in celiac disease

BACKGROUND & AIMS: The chronic, small intestinal inflammation that defines celiac disease is initiated by a HLA-DQ2 restricted T-cell response to ingested gluten peptides after their in vivo deamidation by tissue transglutaminase (TG2). To date, celiac disease can only be treated by a lifelong abstinence from foods that contain wheat, rye, or barley; better therapeutic options are hence needed. An attractive target would be to identify nontoxic wheat cultivars or components thereof with intact baking qualities. Because these qualities are mainly determined by the high molecular weight (HMW) glutenin proteins of gluten, it is critical to know if these proteins are toxic or, more specifically, if they will trigger the activation of T cells in the celiac lesion. METHODS: Different, highly purified HMW glutenins were isolated from wheat cultivars or expressed as recombinant proteins. The proteins were first tested for recognition by a large panel of gluten-specific T-cell lines established from celiac lesions and then applied during ex vivo challenges of celiac biopsies to allow for a direct identification of HMW specific T cells. RESULTS: Intestinal T-cell responses to TG2-deamidated HMW glutenins but not the corresponding native proteins were detectable in 9 of the 22 adult and childhood celiac disease patients tested. CONCLUSIONS: T cells within celiac lesions frequently recognize deamidated HMW glutenin proteins. This finding questions the possibility of implementing these proteins in novel food items destined to be nontoxic for celiac disease patients.     

Celiac sprue

Celiac sprue, celiac disease, or gluten-sensitive enteropathy, is a malabsorption disorder of the small intestine that occurs after ingestion of wheat gluten in genetically susceptible individuals. This disease is characterized by intestinal malabsorption associated with villous atrophy of the small intestinal mucosa, clinical and histological improvement after adherence to strict gluten free diet, and relapse when gluten is reintroduced. Celiac sprue has a high prevalence in Western Europe and North America where it is estimated to affect 1:120 to 1:300 individuals. The pathogenesis of celiac sprue is related to inappropriate intestinal T-cell activation in HLA-DQ2 positive individuals triggered by antigenic peptides from wheat gluten or prolamins from barley and rye. Although previously thought to be mainly a disease of childhood onset, the diagnosis is increasingly being made in adults. There are a wide variety of presentations, which range from asymptomatic forms to severe diarrhea, weight loss and nutritional deficiencies. Extraintestinal manifestations including anemia, osteopenia or neurological disorders and associated conditions such as diabetes or hypothyroidism are commonly present. The availability of highly sensitive and specific serologic markers has dramatically facilitated the diagnosis of celiac sprue. However, the demonstration of characteristic histological abnormalities in a biopsy specimen of the small intestine remains the mainstay of diagnosis. Treatment consists of life-long avoidance of dietary gluten to control symptoms and to prevent both immediate and long-term complications.     

A conserved repetitive DNA element located in the centromeres of cereal chromosomes

Repetitive DNA sequences have been demonstrated to play an important role for centromere function of eukaryotic chromosomes, including those from fission yeast, Drosophila melanogaster, and humans. Here we report on the isolation of a repetitive DNA element located in the centromeric regions of cereal chromosomes. A 745-bp repetitive DNA clone, pSau3A9, was isolated from sorghum (Sorghum bicolor). This DNA element is located in the centromeric regions of all sorghum chromosomes, as demonstrated by fluorescence in situ hybridization. Repetitive DNA sequences homologous to pSau3A9 also are present in the centromeric regions of chromosomes from other cereal species, including rice, maize, wheat, barley, rye, and oats. Probe pSau3A9 also hybridized to the centromeric region of B chromosomes from rye and maize. The repetitive nature and its conservation in distantly related plant species indicate that the pSau3A9 family may be associated with centromere function of cereal chromosomes. The absence of DNA sequences homologous to pSau3A9 in dicot species suggests a faster divergence of centromere-related sequences compared with the telomere-related sequences in plants.     

Future therapeutic options for celiac disease

Celiac disease is a disorder of the small intestine caused by an inappropriate immune response to wheat gluten and similar proteins of barley and rye. At present, the only available treatment is a strict gluten-exclusion diet; hence the need for alternative treatments. Recent advances have improved our understanding of the molecular basis for this disorder and there are several attractive targets for new treatments. Oral enzyme supplementation is designed to accelerate gastrointestinal degradation of proline-rich gluten, especially its proteolytically stable antigenic peptides. Complementary strategies aiming to interfere with activation of gluten-reactive T cells include the inhibition of intestinal tissue transglutaminase activity to prevent selective deamidation of gluten peptides, and blocking the binding of gluten peptides to the HLA-DQ2 or HLA-DQ8 molecules. Other possible treatments include cytokine therapy, and selective adhesion molecule inhibitors that interfere with inflammatory reactions, some of which are already showing promise in the clinic for other gastrointestinal diseases.     

Natural variation in toxicity of wheat: potential for selection of nontoxic varieties for celiac disease patients

BACKGROUND & AIMS: Celiac disease (CD) is an intestinal disorder caused by T-cell responses to peptides derived from the gluten proteins present in wheat. Such peptides have been found both in the gliadin and glutenin proteins in gluten. The only cure for CD is a lifelong gluten-free diet. It is unknown, however, if all wheat varieties are equally harmful for patients. We investigated whether wheat varieties exist with a natural low number of T-cell-stimulatory epitopes. METHODS: Gluten proteins present in public databases were analyzed for the presence of T-cell-stimulatory sequences. In addition, wheat accessions from diploid (AA, SS/BB, and DD genomes), tetraploid (AABB), and hexaploid (AABBDD) Triticum species were tested for the presence of T-cell-stimulatory epitopes in gliadins and glutenins by both T-cell and monoclonal antibody-based assays. RESULTS: The database analysis readily identified gluten proteins that lack 1 or more of the known T-cell-stimulatory sequences. Moreover, both the T-cell- and antibody-based assays showed that a large variation exists in the amount of T-cell-stimulatory peptides present in the wheat accessions. CONCLUSIONS: Sufficient genetic variation is present to endeavor the selection of wheat accessions that contain low amounts of T-cell-stimulatory sequences. Such materials may be used to select and breed wheat varieties suitable for consumption by CD patients, contributing to a well-balanced diet and an increase in their quality of life. Such varieties also may be useful for disease prevention in individuals at risk.     

Genetic and functional analysis of pyroglutamyl-peptidase I in coeliac disease

Coeliac disease (CD) is an enteropathy caused by an immune reaction towards wheat gluten and similar proteins from barley and rye. It was shown that some gluten peptides spontaneously form N-terminal L-pyroglutamate. This modification could potentially make gluten more resistant to proteolytic degradation within the intestine. Pyroglutamyl-peptidase I (PGPEPI) is an enzyme that hydrolytically removes the L-pyroglutamyl residues that render the modified proteins and peptides more sensitive to degradation by other proteases. Interestingly, we found that the PGPEP1 gene is located in a CD susceptibility locus. As an impaired enzyme function caused by genetic alterations might increase the amount of immunogenic gluten peptides, we conducted a comprehensive functional genomics analysis of PGPEP1, including DNA sequencing, genetic association testing, and quantifying RNA expression. We also determined the enzymatic activity of PGPEPI in duodenal biopsies. Our results uniformly indicate that PGPEP1 is not involved in the aetiology and pathology of CD.     

Gluten-free diet--what is toxic?

The cornerstone of treatment of coeliac disease is a gluten-free diet devoid of proteins from wheat, rye, barley and related cereals. Oats are tolerated by most patients with coeliac disease but are not totally innocent. There are considerable differences between individual patients with respect to clinical and mucosal responses to gluten challenge. In vitro and in vivo testing has identified synthetic peptides that are toxic to the coeliac small intestinal mucosa. This toxicity overlaps at least partly to the known epitopes that are recognised by small intestinal T-cells. However, the clinical significance of several of these epitopes is unclear, as is the maximum level of gluten intake that can be recommended to be safe for patients with coeliac disease. Future efforts may lead to better understanding of the disease processes as well as possible new therapeutic options.     

Effect of the timing of gluten introduction on the development of celiac disease

Celiac disease(CD) is a permanent auto-immune enteropathy,triggered in genetically predisposed individuals by the ingestion of dietary gluten.Gluten is the alcohol-soluble protein component of the cereals wheat,rye and barley.CD is a multifactorial condition,originating from the interplay of genetic and environmental factors.The necessary environmental trigger is gluten,while the genetic predisposition has been identified in the major histocompatibility complex region on chromosome 6p21,with over 90% of CD ...     

Maladie cœliaque

Celiac disease is an enteropathy due to gluten intake in genetically predisposed individuals (HLA DQ2/DQ8). Celiac disease occurs in adults and children at rates approaching 1% of population in Europe and USA. Clinical features observed in celiac disease are extremely various and anaemia, oral aphthous stomatis, amenorrhea or articular symptoms may be the only presenting manifestations. Diagnosis relies on the evidence of histological villous atrophy in proximal small bowel and the presence of specific serum antibodies. Treatment relies on eviction of gluten (wheat, barley, rye) from diet. Gluten free diet allows prevention of malignant complications such as small bowel adenocarcinoma and lymphoma, and osteopenia. The main cause of resistance to gluten free diet is its poor observance. If not the case, serious complications of celiac disease, such as clonal refractory celiac sprue and intestinal T-cell lymphoma should be suspected. Current therapeutic challenges concern alternative to gluten free diet and new efficient treatments of lymphomatous complications.     

Pathomechanisms in celiac disease

Celiac disease is an inflammatory disorder of the small intestine caused by an immune response to ingested wheat gluten and similar proteins of rye and barley. It affects at least 1 in 200 individuals, corresponding to roughly three million patients in Western Europe and Northern America alone. Data accumulated since the discovery of gluten specific T cells in the intestine of celiac disease patients the early 1990s have allowed the deciphering of the interplay between the triggering environmental factor, gluten, the main genetic risk factor, the HLA-DQ2/8 haplotypes and the autoantigen; the enzyme tissue transglutaminase (tTG). This established a key role of adaptive immunity orchestrated by lamina propria T cells responding to a set of gluten derived peptides. More recent work points to an important contribution of innate immunity triggered by a distinct gluten peptide and driven by the proinflammatory cytokine Interleukine-5 (IL-15). Together, these observations provide a unique explanation for the disease inducing capacity of gluten.     

Measurement of gluten using a monoclonal antibody to a coeliac toxic peptide of A-gliadin

BACKGROUND: Future European Community regulations will require a sensitive and specific assay for measurement of coeliac toxic gluten proteins in foods marketed as gluten-free. To avoid spurious cross reactions with non-toxic proteins, specific antibodies and target antigens are required. A synthetic 19 amino acid peptide of A gliadin has been shown to cause deterioration in the morphology of small intestinal biopsy specimens of coeliac patients in remission. AIMS: To develop an assay for detection of gluten in foods, based on measurement of a known toxic peptide. METHODS: A monoclonal antibody raised against the toxic A gliadin peptide, with a polyclonal anti-unfractionated gliadin capture antibody, was used to develop a double sandwich enzyme linked immunosorbent assay (ELISA) for the measurement of gluten in foods. RESULTS: Standard curves for gliadin and for rye, barley, and oat prolamins were produced. The sensitivity of the assay was 4 ng/ml of gliadin, 500 ng/ml for rye prolamins, and 1000 ng/ml for oat and barley prolamins. The assay could detect gluten in cooked foods, although at reduced sensitivity. Prolamins from coeliac non-toxic rice, maize, millet, and sorghum did not cross react in the assay. A variety of commercially available gluten-free foods were analysed; small quantities of gluten were detected in some products. CONCLUSION: The assay may form the basis of a sensitive method for measurement of gluten in foods for consumption by patients with coeliac disease.     

Effective shutdown in the expression of celiac disease-related wheat gliadin T-cell epitopes by RNA interference

Celiac disease (CD) is an enteropathy triggered by the ingestion of gluten proteins from wheat and similar proteins from barley and rye. The inflammatory reaction is controlled by T cells that recognize gluten peptides in the context of human leukocyte antigen (HLA) DQ2 or HLA-DQ8 molecules. The only available treatment for the disease is a lifelong gluten-exclusion diet. We have used RNAi to down-regulate the expression of gliadins in bread wheat. A set of hairpin constructs were designed and expressed in the endosperm of bread wheat. The expression of gliadins was strongly down-regulated in the transgenic lines. Total gluten protein was extracted from transgenic lines and tested for ability to stimulate four different T-cell clones derived from the intestinal lesion of CD patients and specific for the DQ2-α-II, DQ2-γ-VII, DQ8-α-I, and DQ8-γ-I epitopes. For five of the transgenic lines, there was a 1.5–2 log reduction in the amount of the DQ2-α-II and DQ2-γ-VII epitopes and at least 1 log reduction in the amount of the DQ8-α-I and DQ8-γ-I epitopes. Furthermore, transgenic lines were also tested with two T-cell lines that are reactive with ω-gliadin epitopes. The total gluten extracts were unable to elicit T-cell responses for three of the transgenic wheat lines, and there were reduced responses for six of the transgenic lines. This work shows that the down-regulation of gliadins by RNAi can be used to obtain wheat lines with very low levels of toxicity for CD patients.