synthetic 33-mer peptide used to calibrate the competitive ELISA on bread digests was synthesized using Boc/HBTU chemistry on solid-phase as previously described [38]. Pepsin was purchased from American Laboratories (Omaha, NE). EP-B2 and SC PEP were prepared as previously described [15,17,39]. Enzyme stock concentrations and specific activities are the same as previously reported [20]. Anti-33-mer moAbs The A1 moAb, G12 moAb and their derived Horseradish Peroxidase (HRP)-conjugated moAb (A1-HRP and G12-HRP) were used in this study [23]. A1, G12 and A1-HRP moAb concentrations are 3.6 mg/mL, 1 mg/mL and 0.6 mg/mL, respectively. G12-HRP moAb concentration is the same as previously reported [23]. Determination of the moAbs A1 and G12 recognition sequence within the 33-mer peptide In order to determine the A1 and G12 recognition sequence, several fragments of DNA codifying for peptides representing the whole 33-mer sequence were constructed by synthesis of the following overlapping oligonucleotides: Leu57-Pro63, 59- GATCTGCAGTTACAACCGTTTCCGTA-39, and 59-AGCT- TACGGAAACGGTTGTAACTGCA-39; Pro63-Tyr69, 59- GATCCGCAGCCACAACTGCCGTATTA-39, and 59-AGCT- TAATACGGCAGTTGTGGCTGCG-39; Gln64-Pro70, 59- GATCAGCCACAACTGCCGTATCCGTA-39, and 59- AGCT- TACGGATACGGCAGTTGTGGCT-39; Pro65-Gln71, 59- GATCCACAACTGCCGTATCCGCAATA-39, and 59- AGCT- TATTGCGGATACGGCAGTTGTG-39; Gln66-Pro72, 59- GATCAACTGCCGTATCCGCAACCATA-39, and 59-AGCT- TATGGTTGCGGATACGGCAGTT-39; Leu67-Gln73, 59- GATCTGCCGTATCCGCAACCACAGTA-39, and 59-AGCT- TATTATGGTTGCGGATACGGCA-39; Pro68-Leu74, 59- GATCCGTATCCGCAACCACAGTTGTA-39, and 59-AGCT- TACAATTATGGTTGCGGATACG-39; Pro82-Phe89, 59- GATCCGTATCCTCAACCGCAACCGTTTTA-39, and 59- TAAAACGGTTGCGGTTGAGGATACG-39. These synthetic DNAs were designed according to the typical codon usage of E. coli to maximize the expression. The resultant synthetic DNA fragments were assembled by hybridization, and cloned into the BamHI-HindIII digested pALEXb plasmid (Biomedal S.L., Sevilla, Spain). This plasmid contained the Pm promoter of the Cascade expression system [40,41] and the C-LYTAG for affinity purification of fusion peptides to a choline binding domain (Biomedal S.L., Sevilla, Spain). The resultant pALEXb-derivative plasmids were checked by sequencing, and transformed into the Cascade expression host E. coli REG1 (Biomedal S.L., Sevilla, Spain). A plasmid for the expression of a C-LYTAG-33mer fusion was constructed in a previous work [23] and used in this study as positive control. Cultures were then induced by addition of 2 mM salicylate and incubated 5 h at 30uC. The soluble protein extracts were used for the determination of the A1 and G12 recognition sequence by indirect ELISA. Preparation of gliadin solution Gliadin (Sigma, St. Louis, Missouri, U.S.A.) was prepared in 60% (v/v) aqueous ethanol at 1 mg/mL. Preparation of prolamins from wheat, barley, rye, oat, corn, and rice flours Prolamins from wheat, barley, rye, oat, corn, and rice flours were extracted by mixing 0.5 g of flour with 5 mL 60% (v/v) MoAbs to Toxic Gluten Peptides ethanol in a rotary shaker for 1 h at room temperature. The suspension was then centrifuged at 2,500 g for 10 min; the supernatant was separated, and protein concentration was measured by the Bradford method. The required concentration of these samples was prepared in 0.02 M PB containing BSA (5 mg/mL) before use in immunoassays. Indirect enzyme-linked immunosorbent assay A Maxisorp microtiter plate (Nunc, Roskilde, Denmark) was coated overnight at 4uC with 100 mL/well of the appropriate antigen (C-LYTAG-33-mer protein, gliadin, hordein, secalin, avenin, oryzein, zein or soluble protein extracts containing peptide fusions) serially diluted with 0.1 M of Na2CO3-NaHCO3 (pH 9.6). After washing with PBS containing 0.05% Tween-20 (washing buffer), 300 mL of blocking buffer (washing buffer supplemented with 5% non-fat dry milk) was added to each well, and incubated for 1 h at room temperature. Plates were incubated for 1 h at room temperature with the G12 moAb (1:1,000 in the blocking solution), A1 moAb (1:500 in the blocking solution) or 6B5L1 moAb (1:1,000 in the blocking solution). After washing, goat anti-mouse IgG-peroxidase antibody (Biomedal S.L., Sevilla, Spain) was added (1:2,000 in the blocking solution). Plates were washed again, and the substrate solution (TMB liquid substrate system, Sigma, St. Louis, Missouri, U.S.A.) was added. After 30 min dark incubation, the reaction was stopped with 1 M sulfuric acid. Absorbance at 450 nm was measured in the microplate reader UVM340 (Asys Hitech GmbH, Eugendorf, Austria). Competitive enzyme-linked immunosorbent assay G12 competitive ELISA was carried out according to Moro´n et al. [23]. For the A1 competitive ELISA, Maxisorp microtiter plates (Nunc, Roskilde, Denmark) were coated with 100 mL/well of Sigma gliadin solution (5 mg/mL) in 0.1 M of Na2CO3- NaHCO3 (pH 9.6), and incubated overnight at 4uC. Plates were washed with washing buffer and blocked with blocking solution for 1 h at room temperature. Gliadin and samples serially diluted in PBS containing 3% BSA (100 mL) and 100 mL solution of A1 moAb conjugated to HRP (1:10,000 in PBS containing 3% BSA) were preincubated 3 h at room temperature with gentle mixing, and then added to the wells. After 40 min incubation at room temperature, the plates were washed, and 100 mL per well of substrate solution (TMB liquid substrate system, Sigma, St. Louis, Missouri, U.S.A.) was added. After dark incubation for 30 min at room temperature, color development was stopped with 1 M sulfuric acid (100 mL per well), and the absorbance was measured at 450 nm (microplate reader UVM340, Asys Hitech GmbH, Eugendorf, Austria). Western blot reactivity of the A1 moAb For the Western blot of cereal extracts, after one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE), proteins were electrotransferred on to polyvinylidene difluoride (PVDF) membranes, incubated directly with A1-HRP moAb, and developed by ECL Western Blotting Analysis System immunodetection (Amersham Biosciences, Little Chalfont, Buck- inghamshire, U.K.). Whole-wheat bread digests and indirect competitive ELISA for 33-mer in digests Whole-wheat bread digests were carried out as previously described [20]. Briefly, whole-wheat bread (1 g) presoaked in specified amounts of EP-B2 and SC PEP (units of activity/mg PLoS ONE | www.plosone.org 11 May 2008 | Volume 3 | Issue 5 | e2294