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Toward the assessment of food toxicity for celiac patients: characterization of monoclonal antibodies to a main immunogenic gluten peptide.

Morón B, Bethune MT, Comino I, Manyani H, Ferragud M, López MC, Cebolla A, Khosla C, Sousa C - PLoS ONE (2008)

Bottom Line: Although G12 affinity for the 33-mer was superior to A1, the sensitivity for gluten detection was higher for A1.This observation correlated to the higher number of A1 epitopes found in prolamins than G12 epitopes.Activation of T cell from gluten digested by glutenases decreased equivalently to the detection of intact peptides by A1 antibody.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain.

ABSTRACT

Background and aims: Celiac disease is a permanent intolerance to gluten prolamins from wheat, barley, rye and, in some patients, oats. Partially digested gluten peptides produced in the digestive tract cause inflammation of the small intestine. High throughput, immune-based assays using monoclonal antibodies specific for these immunotoxic peptides would facilitate their detection in food and enable monitoring of their enzymatic detoxification. Two monoclonal antibodies, G12 and A1, were developed against a highly immunotoxic 33-mer peptide. The potential of each antibody for quantifying food toxicity for celiac patients was studied.

Methods: Epitope preferences of G12 and A1 antibodies were determined by ELISA with gluten-derived peptide variants of recombinant, synthetic or enzymatic origin.

Results: The recognition sequences of G12 and A1 antibodies were hexameric and heptameric epitopes, respectively. Although G12 affinity for the 33-mer was superior to A1, the sensitivity for gluten detection was higher for A1. This observation correlated to the higher number of A1 epitopes found in prolamins than G12 epitopes. Activation of T cell from gluten digested by glutenases decreased equivalently to the detection of intact peptides by A1 antibody. Peptide recognition of A1 included gliadin peptides involved in the both the adaptive and innate immunological response in celiac disease.

Conclusions: The sensitivity and epitope preferences of the A1 antibody resulted to be useful to detect gluten relevant peptides to infer the potential toxicity of food for celiac patients as well as to monitor peptide modifications by transglutaminase 2 or glutenases.

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Related in: MedlinePlus

Indirect competitive ELISA using moAb A1 to test whole-wheat bread digests for 33-mer content.A. Concentration of 33-mer (µg/mL) in whole-wheat bread digests containing 0.6 mg/mL pepsin supplemented with specified concentrations of recombinant proEP-B2 (U/mg gluten). B. Concentration of 33-mer (µg/mL) in whole-wheat bread digests containing 0.6 mg/mL pepsin and 32 U/mg EP-B2 supplemented with specified concentrations of recombinant SC PEP (U/mg gluten). The concentration of 33-mer in each digest was determined by comparison to a synthetic 33-mer standard curve. Two separate assays were performed with the antibody, each with three repetitions.
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pone-0002294-g007: Indirect competitive ELISA using moAb A1 to test whole-wheat bread digests for 33-mer content.A. Concentration of 33-mer (µg/mL) in whole-wheat bread digests containing 0.6 mg/mL pepsin supplemented with specified concentrations of recombinant proEP-B2 (U/mg gluten). B. Concentration of 33-mer (µg/mL) in whole-wheat bread digests containing 0.6 mg/mL pepsin and 32 U/mg EP-B2 supplemented with specified concentrations of recombinant SC PEP (U/mg gluten). The concentration of 33-mer in each digest was determined by comparison to a synthetic 33-mer standard curve. Two separate assays were performed with the antibody, each with three repetitions.

Mentions: We digested commercial whole-wheat bread under mock gastric conditions for 60 min with pepsin supplemented either with EP-B2 at varied concentrations (Figure 7A), or with a fixed EP-B2 concentration plus varied concentrations of SC PEP (Figure 7B). Dilution series of the quenched digests were prepared in parallel with a calibration dilution series of chemically synthesized 33-mer peptide, and these were tested against fixed 33-mer in an indirect competitive ELISA using moAb A1. Treatment of whole-wheat bread with EP-B2 reduces the concentration of the 33-mer and close analogs by up to 10-fold in a dose-dependent manner (Figure 7A). This is consistent with the observation that EP-B2 cleaves the 33-mer after Gln66, Gln73, and Gln80 [17], cleavages expected to extirpate the affinity of A1 for the resultant fragments (Figures 3 and 6). The combination of EP-B2+SC PEP further reduced antigen concentrations by at least an additional 10-fold to levels undetectable by our methods (Figure 7B). This is again consistent with previously published results, in which EP-B2 substantially detoxified similar bread digests, but the synergistic combination of EP-B2 with SC PEP was required to dramatically reduce the intestinal T cell reactivity of these digests [20]. The intensity of the signal obtained with the A1 moAb in our assay was therefore proportional to the potential damage caused to a CD patient by a commercial gluten source.


Toward the assessment of food toxicity for celiac patients: characterization of monoclonal antibodies to a main immunogenic gluten peptide.

Morón B, Bethune MT, Comino I, Manyani H, Ferragud M, López MC, Cebolla A, Khosla C, Sousa C - PLoS ONE (2008)

Indirect competitive ELISA using moAb A1 to test whole-wheat bread digests for 33-mer content.A. Concentration of 33-mer (µg/mL) in whole-wheat bread digests containing 0.6 mg/mL pepsin supplemented with specified concentrations of recombinant proEP-B2 (U/mg gluten). B. Concentration of 33-mer (µg/mL) in whole-wheat bread digests containing 0.6 mg/mL pepsin and 32 U/mg EP-B2 supplemented with specified concentrations of recombinant SC PEP (U/mg gluten). The concentration of 33-mer in each digest was determined by comparison to a synthetic 33-mer standard curve. Two separate assays were performed with the antibody, each with three repetitions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2386552&req=5

pone-0002294-g007: Indirect competitive ELISA using moAb A1 to test whole-wheat bread digests for 33-mer content.A. Concentration of 33-mer (µg/mL) in whole-wheat bread digests containing 0.6 mg/mL pepsin supplemented with specified concentrations of recombinant proEP-B2 (U/mg gluten). B. Concentration of 33-mer (µg/mL) in whole-wheat bread digests containing 0.6 mg/mL pepsin and 32 U/mg EP-B2 supplemented with specified concentrations of recombinant SC PEP (U/mg gluten). The concentration of 33-mer in each digest was determined by comparison to a synthetic 33-mer standard curve. Two separate assays were performed with the antibody, each with three repetitions.
Mentions: We digested commercial whole-wheat bread under mock gastric conditions for 60 min with pepsin supplemented either with EP-B2 at varied concentrations (Figure 7A), or with a fixed EP-B2 concentration plus varied concentrations of SC PEP (Figure 7B). Dilution series of the quenched digests were prepared in parallel with a calibration dilution series of chemically synthesized 33-mer peptide, and these were tested against fixed 33-mer in an indirect competitive ELISA using moAb A1. Treatment of whole-wheat bread with EP-B2 reduces the concentration of the 33-mer and close analogs by up to 10-fold in a dose-dependent manner (Figure 7A). This is consistent with the observation that EP-B2 cleaves the 33-mer after Gln66, Gln73, and Gln80 [17], cleavages expected to extirpate the affinity of A1 for the resultant fragments (Figures 3 and 6). The combination of EP-B2+SC PEP further reduced antigen concentrations by at least an additional 10-fold to levels undetectable by our methods (Figure 7B). This is again consistent with previously published results, in which EP-B2 substantially detoxified similar bread digests, but the synergistic combination of EP-B2 with SC PEP was required to dramatically reduce the intestinal T cell reactivity of these digests [20]. The intensity of the signal obtained with the A1 moAb in our assay was therefore proportional to the potential damage caused to a CD patient by a commercial gluten source.

Bottom Line: Although G12 affinity for the 33-mer was superior to A1, the sensitivity for gluten detection was higher for A1.This observation correlated to the higher number of A1 epitopes found in prolamins than G12 epitopes.Activation of T cell from gluten digested by glutenases decreased equivalently to the detection of intact peptides by A1 antibody.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain.

ABSTRACT

Background and aims: Celiac disease is a permanent intolerance to gluten prolamins from wheat, barley, rye and, in some patients, oats. Partially digested gluten peptides produced in the digestive tract cause inflammation of the small intestine. High throughput, immune-based assays using monoclonal antibodies specific for these immunotoxic peptides would facilitate their detection in food and enable monitoring of their enzymatic detoxification. Two monoclonal antibodies, G12 and A1, were developed against a highly immunotoxic 33-mer peptide. The potential of each antibody for quantifying food toxicity for celiac patients was studied.

Methods: Epitope preferences of G12 and A1 antibodies were determined by ELISA with gluten-derived peptide variants of recombinant, synthetic or enzymatic origin.

Results: The recognition sequences of G12 and A1 antibodies were hexameric and heptameric epitopes, respectively. Although G12 affinity for the 33-mer was superior to A1, the sensitivity for gluten detection was higher for A1. This observation correlated to the higher number of A1 epitopes found in prolamins than G12 epitopes. Activation of T cell from gluten digested by glutenases decreased equivalently to the detection of intact peptides by A1 antibody. Peptide recognition of A1 included gliadin peptides involved in the both the adaptive and innate immunological response in celiac disease.

Conclusions: The sensitivity and epitope preferences of the A1 antibody resulted to be useful to detect gluten relevant peptides to infer the potential toxicity of food for celiac patients as well as to monitor peptide modifications by transglutaminase 2 or glutenases.

Show MeSH
Related in: MedlinePlus