MoAbs to Toxic Gluten Peptides 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 (a-gliadin, accession number: JQ1047). Two separate assays were performed with the antibodies, each with three repetitions (A and B). doi:10.1371/journal.pone.0002294.g008 non-deamidated peptide. These moAbs may be useful for fundamental studies on the mechanism of transglutaminase 2 mediated deamidation either in vitro or in vivo by discriminating between native and deamidated gluten peptides. Additionally, these moAbs may be used to test the extent to which microbial transglutaminase treatment can abolish gluten toxicity by blocking immunotoxic peptide deamidation sites [34]. Finally, we reported the development of a novel competitive assay using A1, a moAb raised against a 33-mer peptide recognized in vivo to be immunotoxic toward patients with CD. A competitive assay is well-suited for food analysis and monitoring of gluten digestion because it can detect both intact proteins and small protein fragments, the latter of which can be underestimated by a sandwich ELISA [32,35]. The ELISA systems described here showed high reproducibility and repeatability with coefficients of variation below 15%. Taking the minimum working dilution as 1:10, and starting from a sample (obtained by ethanolic (60%) extraction from a foodstuff) at a concentration of 0.1 g/mL of extraction solution, the A1 competitive ELISA developed would enable the detection of the presence of gluten in foodstuffs at values as low as 0.33 ppm of gluten, which is far below the 20 ppm threshold proposed by the Codex Alimentarius Commission [36]. The recommended maxi- mum daily ingestion of gluten is below 50 mg gluten per day. The method reported here could detect several orders of magnitude less concentration than the maximum recommended gluten concentra- tion in the digestive tract (,20 mg/L) for CD patients [37]. The specificity for immunotoxic peptides obtained by developing moAbs G12 and A1 against the immunotoxic 33-mer is an additional advantage of this immunoassay, since anti-gliadin moAbs with no specificity for toxic peptides may produce false negative or positive signals not related to the remaining toxicity of hydrolyzed food. This work suggests that competitive immunoassays using immunotoxic gluten epitope-specific moAbs, such as G12 and A1, are particularly effective at evaluating food safety for CD patients, with likely advantages over assays measuring the amount of any gluten in a given sample, without regard to its immunotoxicity. Materials and Methods Materials Commercially available flours from cultivars of wheat, barley, rye, oats, corn, and rice were employed in this study. The peptides QPQLPYPQP, QPELPYPQP, QPQLPY, QPQLPF, QPQLPL, QPQQPY, QLPYPQP, QQPYPQP, QQPFPQP, QQPYPQE, QLPFPQP, QQPFVQQ, QQMFLQP, QLPYPQS, QLSYPQP, QLPYSQP and LPYPQP were supplied by Biomedal S.L. (Sevilla, Spain). The 19-mer peptide PGQQQPFPPQQPYPQPQPF was provided by Eduardo Arranz (Universidad de Valladolid-C.S.I.C., Valladolid, Spain). Whole-wheat bread (Alvarado St Sprouted Whole-Wheat Bread) was from Alvarado St Bakery (Rohnert Park, CA). The PLoS ONE | www.plosone.org 10 May 2008 | Volume 3 | Issue 5 | e2294