MoAbs to Toxic Gluten Peptides Analysis of the epitope recognition of G12 and A1 moAb To determine the epitope recognized by the G12 and A1 moAb within the 33-mer peptide, fusions of the C-LYTAG coding sequence of the pALEXb plasmid (Biomedal S.L., Sevilla, Spain) were constructed with coding sequences of hepta- and octapep- tides comprising the complete sequence of the 33-mer peptide (Figure 3A). The resulting plasmids were introduced by transfor- mation into the REG1 strain of Escherichia coli, allowing for over- expression of the encoded fusion proteins upon induction (Biomedal S.L., Sevilla, Spain). The over-expressed bacterial extracts were analyzed by indirect ELISA using the anti-33-mer A1 and G12 moAbs. Similarly, the anti-C-LYTAG 6B5L1 moAb (Biomedal, S.L., Sevilla, Spain) was used to establish that the designed protein was expressed intact in all cases. A reference signal in the bacterial extract containing the C-LYTAG-33-mer fusion protein was observed for all the moAbs assayed (A1, G12 Figure 3. Analysis of anti-33-mer moAbs recognition regions in recombinant 33-mer peptide fragments expressed in E. coli. A. Nucleotide sequences and the deduced amino acid sequences of the encoded peptide fusions to C-LYTAG. B. Detection of C-LYTAG-peptide fusions by an indirect ELISA with the use of moAbs G12, A1 and 6B5L1. doi:10.1371/journal.pone.0002294.g003 PLoS ONE | www.plosone.org 4 May 2008 | Volume 3 | Issue 5 | e2294