MoAbs to Toxic Gluten Peptides 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 mean6standard deviation of n = 4 assays. IC50 values of the moAbs to both antigens are indicated. doi:10.1371/journal.pone.0002294.g001 CD4+ T cells in CD patient-derived small intestine biopsies [9– 12]. To elicit a T-cell response, most gliadin epitopes must undergo a transglutaminase 2-mediated deamidation of certain glutamine residues to glutamate residues [10]. Among the main dietary proteins, gluten is unique in that it contains approximately 15% proline and 35% glutamine residues [13]. This high proline and glutamine content prevents complete proteolysis by gastric and pancreatic enzymes, such that long oligopeptides that are toxic to CD patients build up in the small intestine. One peptide in particular, the 33-mer from a-2 gliadin (residues 57–89), contains 6 T-cell epitopes, is highly proteolyti- cally resistant, and is a principal contributor to gluten immuno- toxicity [14]. It has been proposed that oral administration of a therapeutic dose of a suitably formulated prolyl endopeptidase (SC PEP from Sphingomonas capsulata) and glutamine specific endopro- tease (EP-B2, a cysteine endoprotease from germinating barley seeds) might counter the toxic effects of certain quantities of ingested gluten [15–20]. At present, the prescription of a gluten-free diet is the only therapy for CD patients. However, it is not easy to maintain a diet with zero gluten content because gluten contamination in food is commonplace [21]. Gluten is a common ingredient in the human diet; after sugar, it is perhaps the second most widespread food substance in Western civilization. Since about 10% of gluten seems to be made up of potentially toxic gliadin peptides [22], it is desirable to quantify the amount of these peptides ingested by a CD patient, so that the factual toxicity of the gluten present in foods can be established more precisely. In a previous work, we obtained monoclonal antibodies (moAb) (G12 and A1) against the gliadin 33-mer peptide [23]. The aims of the current study were two-fold. First, we sought to characterize the sequence specificity of these anti-33-mer moAbs using a panel of overlapping fusion peptides based on the 33-mer sequence. Second, we evaluated the practical efficacy of these moAbs as analytical tools for quantifying food toxicity for CD patients. Toward this end, we showed that the reactivity of each moAb with a variety of cereal storage proteins correlated with the immunotoxicity of those dietary grains from which the proteins were extracted. Additionally, we showed with one of these moAb (A1) that reductions in the 33-mer content of commercial whole- wheat bread caused by treatment with candidate therapeutic glutenases can be quantified using a simple, high-throughput competitive ELISA. Our results establish A1 and G12 anti-33- mer moAbs as specific and reliable tools for detecting foods potentially harmful for CD patients. Results Detection of gliadin immunogenic peptide by anti-33- mer moAbs The 33-mer peptide from a-2 gliadin is a principal contributor to gluten immunotoxicity [14]. Thus the production of moAbs against this toxic gluten peptide could be of great importance in both research and diagnosis. In a previous work, we obtained moAbs against the 33-mer peptide (A1 and G12 moAbs) [23]. To test the relative sensitivity of each moAb for the 33-mer peptide, we immobilized different concentrations of the C-LYTAG-33-mer polypeptide, and detected with A1 and G12 moAb in an indirect ELISA. The affinity of each moAb for the antigen was quantified by calculation of the concentration of the antigen giving a 50% reduction of the peak signal in the ELISA (IC50). The sensitivity of the G12 moAb for the toxic 33-mer peptide was about eight times higher than that of A1 (Figure 1A). To test for moAb specificity, we studied the cross-reactivity values (CR) of these moAb against commercial gliadin, also by indirect ELISA. The G12 moAb presented an IC50 of almost double that obtained with the A1 moAb, suggesting that A1 had broader reactivity with gliadin epitopes than G12, which is more specific for the 33-mer (Figure 1B). Characterization of A1 and G12 moAbs sensitivity for celiac-toxic cereals In a previous work, we investigated whether the G12 moAb was able to detect the presence of gliadin 33-mer related epitopes in prolamins from various cereals [23]. The results indicated that the moAb showed CR against prolamins of wheat, barley, rye and oats PLoS ONE | www.plosone.org 2 May 2008 | Volume 3 | Issue 5 | e2294