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. doi:10.1371/journal.pone.0002294.g006 affinity for the 19-mer peptide (Figure 8B). These results were consistent with our previous identification of the QQPYPQP peptide, included in p31-49, as a permissive epitope for the A1 moAb (Figure 5B). Therefore, this moAb shows an interesting range of peptide recognition that includes gliadin peptides involved in the both the adaptive and innate immunological responses in CD. Discussion In this work, we characterized two moAbs raised against the immunotoxic gliadin 33-mer peptide, each of which showed suitable epitope recognition for fundamental and practical applications in CD research. The most straightforward of these is that these moAbs may be used to detect immunotoxic gluten epitopes in foodstuffs via immunological assays. In a previous work we obtained moAbs (G12 and A1) against the 33-mer, a peptide from a-2 gliadin that is a principal contributor to gluten immunotoxicity [14]. Here, we characterized the precise site of peptide recognition and the practical efficacy of these moAbs as MoAbs to Toxic Gluten Peptides analytical tools for quantifying food toxicity for CD patients. ELISAs based on moAbs G12 and A1 exhibited broad specificity toward prolamins toxic to CD patients, along with a high degree of sensitivity, accuracy, and reproducibility. The A1 moAb was particularly sensitive to immunotoxic prolamins, with highest affinity for barley and rye, followed by wheat. The G12 moAb was more specific for the 33-mer than was A1, but it also recognized all immunotoxic prolamins, with highest affinity for barley and wheat, followed by rye. Both moAbs detected oat prolamins, albeit with substantially reduced sensitivity. To provide a basis for these observed sensitivities, we identified the hexapeptide (QPQLPY) and heptapeptide (QLPYPQP) epitopes recognized by G12 and A1 moAbs, respectively, and studied the relative affinity of these moAbs for peptide variants of their recognition sequence. This analysis suggested that A1 showed higher affinity for prolamins of wheat, barley, rye and oats than did G12 as a consequence of the prevalence of the A1 recognition sequence and related sequences in these cereals. The ability of G12 and A1 to detect oats is of potential practical importance. Oats cause damage to the mucosa in a subset of CD patients [2,6]. In contrast to previously described anti-prolamin moAbs (e.g. R5 and anti-glia-a2/9) [32,33], G12 and A1 both detected oats although with lower sensitivity than the prolamins from wheat, barley and rye. Peptides recognized by these moAbs were confirmed by western blot (data not shown), exhibiting a pattern not indicative of contamination by other cereals. The reduction in G12 and A1 sensitivity toward oat prolamins relative to immunotoxic prolamins from wheat, rye, and barley may be due to the lower prolamin content in total oat seed protein compared to the other cereals [24]. Moreover, the potential sequences for moAb recognition could be less prevalent in avenins than in gliadins, hordeins, and secalins. Regardless, ELISA assays using G12 and A1 exhibited high sensitivity toward toxic wheat, rye, and barley prolamins; reduced but significant sensitivity toward oat prolamins, which are moderately toxic in a subset of CD patients; and no sensitivity toward non-toxic prolamins from corn and rice. Together, these observations reveal a direct correlation between assay signal and potential toxicity of food samples for CD patients. This correlation suggested that an ELISA using a moAb specific for immunotoxic gluten epitopes could be used to quantitatively monitor the enzymatic detoxification of gluten. The use of glutamine and proline specific proteases (EP-B2 and SC PEP, respectively) is currently being explored as an oral therapy for CD [15–20]. Because our results suggested that the initial glutamine residue and all three prolines in the recognition sequence for A1 were substantially important for A1 recognition, we used this moAb to determine the concentration of immunotoxic epitopes remaining in wheat bread after its treatment with varying amounts of EP-B2 and SC PEP. Decreases in A1 epitopes due to enzymatic digestion by glutenases were consistent with previously reported decreases in T cell activation caused by identical enzymatic treatment of wheat bread [20]. Therefore, the A1 moAb immunoassay may be used to monitor gluten detoxification in enzymatic therapy studies by detecting that part of the digested food that remains toxic for a CD patient. The 33-mer gliadin peptide and other main prolamin epitopes are deamidated at specific glutamine residues by transglutaminase 2, considerably enhancing their affinity for HLA DQ2 or DQ8 molecules [10,12]. This transformation is suggested to be critical toward rendering gluten immunotoxic. We determined the affinities of G12 and A1 moAbs for a synthetically deamidated gluten peptide, and found that both moAbs recognized the deamidated peptide with .100-fold less affinity than they did the PLoS ONE | www.plosone.org 8 May 2008 | Volume 3 | Issue 5 | e2294