by a competitive assay (Figure 5A). The peptides assayed for A1 were more numerous than for G12 due to the longer heptapeptide recognition sequence recognition contained in the 33-mer and due to the suspected broader specificity of A1 for other prolamin sequences based on indirect ELISA assays (Figure 2). Figure 5 shows the affinity of the A1 moAb for the different peptides assayed; the IC50 was used to compare the affinity of A1 for each peptide. Notably, two peptides present in secalin and hordein (QQPFPQP and QLPFPQP, Figures 5C and 5D, respectively) showed higher affinity for the A1 moAb than did the 33-mer- derived recognition sequence peptide (QLPYPQP). This suggests that the fourth residue in the recognition sequence is substantially important to A1 recognition, whereas the second position is not. Consistent with this, gliadin peptides QLPYPQP and QQPYPQP showed comparable affinity for the moAb (Figure 5B). The affinity of the anti-33-mer A1 moAb for epitopes present in celiac-toxic ce- reals decreased as follows: QLPFPQP.QQPFPQP.QLPYPQP. QQPYPQP&QQPYPQE. The affinity for the sequence included in the wheat gliadin 33- mer was not as high as for QQPFPQP, which is one of the most abundant sequences in secalin and hordein, similar to the 33-mer epitope in gliadin. This may explain why, despite its lower affinity for 33-mer peptide relative to the G12 moAb, the A1 moAb had higher sensitivity for the whole range of toxic cereals tested in this study. The A1 moAb may therefore be useful as a sensitive detection tool for identifying celiac-toxic peptides in complex foodstuffs. Preliminary attempts to find an avenin epitope gave no positive results (Figure 5E). The prolamins in oats represent much less of the total seed proteins than in the other cereals [24]. Furthermore, the amount of proline residues contained in avenins (10%) is about two-thirds that in the prolamins of wheat (gliadins and glutenins), barley (hordeins), and rye (secalins). In any case, we tested certain previously proposed potential avenin epitopes located in the avenin regions with the highest content of proline residues, regions also rich in glutamine, but could not obtain any reactivity to the A1 moAb. To study the relative importance of glutamine and proline residues in epitope selection by the A1 moAb, single substitutions or deletions were made to these amino acids in the recognition sequence (QLPYPQP; Figure 6A). We performed the analysis with the A1 moAb rather than with the G12 moAb because A1 has higher sensitivity for prolamins from toxic cereals. When the first glutamine of the A1 recognition sequence was eliminated (LPYPQP), the affinity for A1 decreased significantly, consistent with the results from epitope scanning with the C-LYTAG fusions (Figure 3). Substitutions of each proline residue in the recognition sequence with a serine residue decreased A1 affinity markedly (Figures 6A and 6B). This effect was most marked when the substitution was made in the second proline position (QLPYSQP), resulting in a CR that was practically zero. These results indicate that the initial glutamine residue and all three prolines of the epitope QLPYPQP were important for its recognition by A1, suggesting that this moAb could serve as a tool for monitoring enzymatic degradation of toxic peptides by potentially therapeutic glutamine and proline specific proteases. Use of moAb A1 to monitor gluten detoxification by candidate glutenases Oral administration of glutamine and proline specific proteases (i.e. glutenases) represents a potential therapeutic alternative (or adjunct) to a gluten-free diet [27,28]. However, validation of the efficacy of these enzymes at detoxifying gluten in vitro must precede clinical testing, and such validation currently relies on low- MoAbs to Toxic Gluten Peptides throughput, technically challenging cell culture-based assays [19,20,27] or on polyclonal anti-gliadin antibody-based ELISA assays that are only grossly quantitative [20]. A competitive ELISA using a anti-33-mer moAb would enable high-throughput, highly quantitative testing of gluten detoxification by candidate thera- peutic glutenases. 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. Analysis of the recognition of anti-33-mer moAbs for deamidated and innate gluten peptides CD is closely associated with genes that code for human leukocyte antigens DQ2 and DQ8. These have been shown to bind with high affinity to gliadin-derived peptides in which specific glutamine residues in key positions have been converted to glutamic acid by transglutaminase 2-mediated deamidation [1,16]. A moAb capable of discriminating between native and deamidated gluten peptides would be a valuable research tool for monitoring the fate of digested prolamin peptides. To test the relative sensitivity of each moAb for the deamidated 33-mer peptide, a peptide (QPQLPYPQP) was designed that represented a region of recognition common to the two moAbs, together with the same peptide deamidated (QPELPYPQP) (Figure 8A). The affinities of the A1 and G12 moAbs for these peptides were determined by a competitive assay in which immobilized gliadin was challenged with peptides as competitors. The affinity of the G12 moAb for the deamidated peptide was about forty times higher than that of the A1 moAb (Figure 8A). However, both moAbs recognized the non- deamidated peptide with .100-fold greater affinity than they did the deamidated peptide (Figure 8A). In combination with a previously characterized, commercially available moAb that has 20-fold greater affinity for the deamidated form of an overlapping gluten peptide than for its non-deamidated counterpart [29], moAbs G12 and A1 may be useful for future studies on transglutaminase 2-mediated gluten peptide deamidation. The innate immune response to gluten plays a key role in the development of CD [30,31]. This response is mediated by interleukin 15 (a typical cytokine of the innate immune system) and elicited by the toxic peptide, p31-49 (19-mer), derived from alpha-gliadin [31]. To test whether the anti-33-mer A1 and G12 moAbs recognized peptide p31-49, competitive ELISAs with each moAb were performed. The A1 moAb was able to detect p31-49 (IC50 3.18 mg/mL) (Figure 8B). The G12 moAb showed no PLoS ONE | www.plosone.org 6 May 2008 | Volume 3 | Issue 5 | e2294