Toward the Assessment of Food Toxicity for Celiac Patients: Characterization of Monoclonal Antibodies to a Main Immunogenic Gluten Peptide (original) (raw)
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Fine specificity of monoclonal antibodies against celiac disease-inducing peptides in the gluteome
The American journal of clinical nutrition, 2008
In celiac disease patients, peptides derived from dietary gluten are recognized by HLA-DQ2-restricted CD4(+) T cells, which results in inflammation. Such immune-stimulatory peptides are found in both gliadins and glutenins. Monoclonal antibodies (mAbs) against these peptides can be used to screen food for the presence of such peptides. We aimed to determine the specificity of 5 mAbs raised against T cell stimulatory peptides found in alpha- and gamma-gliadins and in low- and high-molecular-weight glutenins and to compare it with the specificity of patient-derived T cells. The reactivity of the mAbs with gluten peptides, enzymatic gluten digests, and intact gluten proteins was determined and compared with that of gluten-specific T cells by using a combination of immunologic and biochemical techniques. Furthermore, the reactivity of the mAbs with gluten homologues in barley, rye, and oat was determined. The specificity of the mAbs largely overlaps with that of gluten-specific T cells....
Background: Celiac disease is an immune-mediated enteropathy caused by the ingestion of gluten, a protein fraction found in certain cereals. Immunotoxic gluten peptides that are recalcitrant to degradation of digestive enzymes appear to trigger celiac syndromes. A 33-mer peptide from ␣-2 gliadin has been identified as a principal contributor to gluten immunotoxicity. A gluten-free diet is the usual first therapy for celiac disease patients; therefore, the characterization and quantification of the toxic portion of the gluten in foodstuffs is crucial to avoid celiac damage. Objective: We aimed to develop immunologic assays as a novel food analysis tool for measuring cereal fractions that are immunotoxic to celiac disease patients. Design: The design focused on the production of monoclonal antibodies against the gliadin 33-mer peptide and the development of enzyme-linked immunosorbent assays (ELISAs) and Western blot analysis with the use of novel antibodies. Results: A sandwich ELISA method showed a detection limit for wheat, barley, and rye of 1 ppm prolamine. However, the method required a sample that was ͧ1 order of magnitude greater for the detection of low-toxic oats, and there was no signal with the safe cereals maize and rice. A competitive ELISA method was also developed for detection of the toxic peptide in hydrolyzed food, which had a detection limit of 0.5 ppm gliadin. Conclusions: Both ELISAs designed for use with the toxic gliadin 33-mer peptide suggested a high correlation between the presence of the peptide and the amount of cereal that was toxic to celiac disease patients. The sensitivity was significantly higher than that of equivalent methods recognizing other gluten epitopes.
American Journal of …, 2008
Background: Celiac disease is an immune-mediated enteropathy caused by the ingestion of gluten, a protein fraction found in certain cereals. Immunotoxic gluten peptides that are recalcitrant to degradation of digestive enzymes appear to trigger celiac syndromes. A 33-mer peptide from ␣-2 gliadin has been identified as a principal contributor to gluten immunotoxicity. A gluten-free diet is the usual first therapy for celiac disease patients; therefore, the characterization and quantification of the toxic portion of the gluten in foodstuffs is crucial to avoid celiac damage. Objective: We aimed to develop immunologic assays as a novel food analysis tool for measuring cereal fractions that are immunotoxic to celiac disease patients. Design: The design focused on the production of monoclonal antibodies against the gliadin 33-mer peptide and the development of enzyme-linked immunosorbent assays (ELISAs) and Western blot analysis with the use of novel antibodies. Results: A sandwich ELISA method showed a detection limit for wheat, barley, and rye of 1 ppm prolamine. However, the method required a sample that was ͧ1 order of magnitude greater for the detection of low-toxic oats, and there was no signal with the safe cereals maize and rice. A competitive ELISA method was also developed for detection of the toxic peptide in hydrolyzed food, which had a detection limit of 0.5 ppm gliadin. Conclusions: Both ELISAs designed for use with the toxic gliadin 33-mer peptide suggested a high correlation between the presence of the peptide and the amount of cereal that was toxic to celiac disease patients. The sensitivity was significantly higher than that of equivalent methods recognizing other gluten epitopes.
2000
Background: In celiac disease patients, peptides derived from di- etary gluten are recognized by HLA-DQ2-restricted CD4 T cells, which results in inflammation. Such immune-stimulatory peptides are found in both gliadins and glutenins. Monoclonal antibodies (mAbs) against these peptides can be used to screen food for the presence of such peptides. Objective: We aimed to determine the specificity of 5 mAbs
Toxic, Immunostimulatory and Antagonist Gluten Peptides in Celiac Disease
Current Medicinal Chemistry, 2009
Celiac disease (CD) is an increasingly diagnosed, permanent autoimmune enteropathy, triggered, in susceptible individuals, by the ingestion of gluten, the alcohol -soluble protein fraction of some cereals, such as wheat, rye and barley. The main protein of wheat gluten is called gliadin, the similar proteins of rye and barley are secalin and hordein, respectively. Approximately 96% of CD patients express the HLA molecule DQ2, while the remainder mostly express the less common haplotype DQ8, reflecting the pivotal role of these molecules in the pathogenesis of CD. Because of their aminoacid sequence and tri-dimensional structure, gluten peptides selectively bind to these HLA alleles present on the surface of antigen presenting cells and then they are presented to the T lymphocytes in intestinal mucosa, thus starting the inflammatory immune response. CD is defined by the characteristic histological changes of small bowel mucosa: villous atrophy, crypts hyperplasia and T cells infiltration of the lamina propria, along with the increase of the number of intraepithelial lymphocytes. The withdrawal of the gluten-containing food from the diet determines a complete recovery of the intestinal mucosa, whereas the reintroduction causes a relapse of the disease.
Measurement of gluten using a monoclonal antibody to a coeliac toxic peptide of A gliadin
Gut, 1998
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.
Analytical and functional approaches to assess the immunogenicity of gluten proteins
Frontiers in Nutrition
Gluten proteins are the causative agents of celiac disease (CD), a lifelong and worldwide spread food intolerance, characterized by an autoimmune enteropathy. Gluten is a complex mixture of high homologous water-insoluble proteins, characterized by a high content of glutamine and proline amino acids that confers a marked resistance to degradation by gastrointestinal proteases. As a consequence of that, large peptides are released in the gut lumen with the potential to activate inflammatory T cells, in CD predisposed individuals. To date, several strategies aimed to detoxify gluten proteins or to develop immunomodulatory drugs to recover immune tolerance to gluten are under investigation. This review overviews the state of art of both analytical and functional methods currently used to assess the immunogenicity potential of gluten proteins from different cereal sources, including native raw seed flours and complex food products, as well as drug-treated samples. The analytical design ...
The Journal of Immunology, 2009
The identification of gluten peptides eliciting intestinal T cell responses is crucial for the design of a peptide-based immunotherapy in celiac disease (CD). To date, several gluten peptides have been identified to be active in CD. In the present study, we investigated the recognition profile of gluten immunogenic peptides in adult HLA-DQ2 ؉ celiac patients. Polyclonal, gliadin-reactive T cell lines were generated from jejunal mucosa and assayed for both proliferation and IFN-␥ production in response to 21 peptides from wheat glutenins and ␣-, ␥-, and-gliadins. A magnitude analysis of the IFN-␥ responses was performed to assess the hierarchy of peptide potency. Remarkably, 12 of the 14 patients recognized a different array of peptides. All ␣-gliadin stimulatory peptides mapped the 57-89 N-terminal region, thus confirming the relevance of the known polyepitope 33-mer, although it was recognized by only 50% of the patients. By contrast, ␥-gliadin peptides were collectively recognized by the great majority (11 of 14, 78%) of CD volunteers. A 17-mer variant of 33-mer, QLQPFPQPQLPYPQPQP, containing only one copy of DQ2-␣-I and DQ2-␣-II epitopes, was as potent as 33-mer in stimulating intestinal T cell responses. A peptide from-gliadin, QPQQPFPQPQQPFPWQP, although structurally related to the ␣-gliadin 17-mer, is a distinct epitope and was active in 5 out of 14 patients. In conclusion, these results showed that there is a substantial heterogeneity in intestinal T cell responses to gluten and highlighted the relevance of ␥and-gliadin peptides for CD pathogenesis. Our findings indicated that ␣-gliadin (57-73), ␥-gliadin (139-153), and-gliadin (102-118) are the most active gluten peptides in DQ2 ؉ celiac patients.