and 6B5L1) (Figure 3B). Saturating signals were obtained in the indirect ELISA analysis using the anti-C-LYTAG 6B5L1 moAb for all the analyzed fusion proteins, indicating that all fusion proteins were over-expressed (Figure 3B). With regard to the determination of the sequence of recognition of the anti-33-mer moAbs (G12 and A1), a positive signal was detected only in the bacterial extracts containing the fusion peptides Pro63-Tyr69 (PQPQLPY) and Gln64-Pro70 (QPQ- LPYP) for the G12 moAb and in the bacterial extracts containing the fusion peptide Gln66-Pro72 (QLPYPQP) for the A1 moAb (Figure 3B). These results thus indicate that the region of recognition within the 33-mer peptide for the G12 moAb is QPQLPY (common to the fusion proteins Pro63-Tyr69 and Gln64-Pro70) and that for the A1 moAb is QLPYPQP. Study of the relative affinity of the G12 moAb for different peptide variants derived from the regions of recognition The recognition sequence of the G12 moAb (QPQLPY) is repeated three times within the gliadin 33-mer peptide. To determine the relative affinity of G12 for this epitope, and for similar sequences present elsewhere in toxic prolamins, we MoAbs to Toxic Gluten Peptides constructed hexapeptide variants of the G12 epitope, two of which were designed based on their presence in the prolamins of barley and rye (Figure 4A and 4C). The affinity of the G12 moAb for different hexapeptide variants was determined in a competitive assay in which immobilized gliadin was challenged with QPQLPY-derivative peptides as soluble competitors (Figure 4A). The G12 moAb had high affinity for the peptide QPQLPF, reduced only four-fold relative to the previously identified epitope recognized by this moAb in the 33-mer, QPQLPY (Figure 4B). While the conservative replacement of tyrosine (QPQLPY) with phenylalanine (QPQLPF) did not drastically reduce the affinity of the G12 moAb, substitution with leucine (QPQLPL) reduced the affinity a thousand-fold, indicating the importance of this last position in determining affinity. A dramatic reduction in affinity was also observed for the peptide QPQQPY, such that the affinity of the anti-33-mer G12 moAb decreased as follows: QPQLPY.QPQLPF&QPQLPL.QPQQPY. Determination of the peptide sequence preferences for moAb A1 binding We also studied the relative affinity of the A1 moAb for its recognition sequence (QLPYPQP) and for related peptide variants 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 a-gliadin (accession number: JQ1047), c-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. doi:10.1371/journal.pone.0002294.g004 PLoS ONE | www.plosone.org 5 May 2008 | Volume 3 | Issue 5 | e2294