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

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.

Conflict of interest statement

Competing Interests: Ángel Cebolla is a founder and stockholder in Biomedal S.L., which is developing the moAbs described in this article to detect gluten in food. Marina Ferragud is a staff scientist in Biomedal S.L. The rest of the authors don't have a personal or financial conflict of interest.

Figures

Figure 1. Standard curve of the detection of C-LYTAG-33-mer polypeptide (A) and Sigma gliadin (B) by indirect ELISA with use of moAbs G12 (black) and A1 (white).

Each point of the curve represents the mean±standard deviation of n = 4 assays. IC50 values of the moAbs to both antigens are indicated.

Figure 2. Comparative reactivity of prolamins from wheat, barley, rye, oats, corn and rice from indirect ELISAs using moAbs G12 (black) and A1 (white).

Each point of the curve shows the mean of n = 3 assays. IC50 and CR values of the moAbs to prolamins are indicated. N.A.: Not applicable.

Figure 3. Analysis of anti-33-mer moAbs recognition regions in recombinant 33-mer peptide fragments expressed in E. coli.

A. Nucleotide sequences and the deduced amino acid sequences of the encoded peptide fusions to C-LYTAG. B. Detection of C-LYTAG-peptide fusions by an indirect ELISA with the use of moAbs G12, A1 and 6B5L1.

Figure 4. Relative affinity of moAb G12 for different peptide variants derived from its recognition region (QPQLPY).

A. Amino acid sequences of the peptides. The G12 recognition sequence in the 33-mer peptide is in bold face. IC50 and CR values of the moAb G12 to peptides are indicated. B. Competition assay measuring the affinity of the moAb G12 for the peptides. Two separate assays were performed with the antibody, each with three repetitions. C. Localization of the peptides in the α-gliadin (accession number: JQ1047), γ-secalin (accession number: ABO32294.1) and C-hordein (accession number: AAA92333.1) sequences. The same color code for labelling the peptides has been used in A, B and C.

Figure 5. Relative affinity of moAb A1 for different peptide variants derived from its recognition region (QLPYPQP).

A. Amino acid sequences of the peptides. The A1 recognition sequence in the 33-mer peptide is in red. B, C, D and E. Competition assay for detection of the affinity of the moAb A1 for the peptides and their localization in α-gliadin (B; accession number: JQ1047), γ-secalin (C; accession number: ABO32294.1), C-hordein (D; accession number: AAA92333.1) and avenin (E; accession number: AAA32716.1). Two separate assays were performed with the moAb, each with three repetitions. IC50 values of the moAb A1 to peptides are indicated. N.A.: Not applicable. The color code for labelling the peptides is the same as that used in A.

Figure 6. Relative affinity of moAb A1 for peptide variants of the 33-mer recognition region (QLPYPQP) featuring single amino acid sustitutions.

A. Amino acid sequences of the peptides. The A1 recognition sequence in the 33-mer peptide is in bold face. IC50 and CR values of the moAb A1 to peptides are indicated. B. Competition assay for the detection of the affinity of the moAb A1 for the peptides. Two separate assays were performed with the antibody, each with three repetitions. The color code for labelling the peptides is the same as that used in A.

Figure 7. 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.

Figure 8. Relative affinity of the anti-33-mer moAbs for different peptides involved in the immune responses to gluten in CD patients.

A. Competition assay for detection of the affinity of G12 and A1 moAbs for a peptide containing recognition common regions (QPQLPYPQP) to the two moAbs and its deamidated counterpart (QPELPYPQP). IC50 and CR values of the anti-33-mer moAbs to peptides are indicated. B. Competition assay for detection of the affinity of the moAbs G12 and A1 for the innate peptide, p31-49. The 33-mer and the p31-49 peptide sequences are underlined and in bold face, respectively. Wheat alpha-gliadin (α-gliadin, accession number: JQ1047). Two separate assays were performed with the antibodies, each with three repetitions (A and B).