gluten protein) was added to a 37uC 0.01N HCl solution containing 0.6 mg/mL pepsin over a course of 15 min, resulting in a final pH of ,4.5 and a final measured gluten protein concentration of ,15 mg/mL. After this addition phase, the simultated gastric digests were incubated for 60 min at 37uC, quenched by boiling 5 min, and stored at 220uC. The concentration of 33-mer or related epitopes remaining in each digest was determined by an indirect competitive ELISA using moAb A1. On day 1 of the procedure, synthetic 33-mer was diluted to 1.6 mg/mL in coating solution (50 mM sodium carbonate/bicarbonate buffer, pH 9.6, 0.02% NaN3) and 100 mL/well was incubated overnight at 4uC in 96-well microtiter plates (Nunc Maxisorp). Synthetic 33-mer standards (0.00625– 51.2 mg/mL) or bread digest dilutions (1:10–1:163,840) were prepared in StartingBlock T20 (TBS) blocking buffer (Pierce). An equal volume of A1 moAb diluted 1:5,000 in blocking buffer was added to each standard or sample dilution, and these mixtures were incubated overnight at 4uC. Final dilutions in mixtures were: 1:10,000 A1 moAb and either 0.003125–25.6 mg/mL 33-mer standards or 1:20–1:327,680 digest dilutions. On day 2, antigen- coated plates were washed thrice with washing buffer prior to blocking and between all subsequent steps. Plates were blocked with 200 mL/well blocking buffer for 1 h at room temperature. MoAb/standard and moAb/sample preincubations were added to the wells in triplicate (100 mL/well) and incubated 3 h at room temperature. Goat anti-mouse IgG-alkaline phosphatase conjugate (Chemicon) was diluted 1:1,000 in blocking buffer and 200 mL/ well was incubated 3 h at room temperature. Freshly prepared substrate solution (5 mg/mL pNPP, 50 mM sodium carbonate/ bicarbonate buffer, pH 9.8, 1mM MgCl2, 0.02% NaN3) was added (200 mL/well) and the absorbance at 405 nm was measured every 6 seconds for 5 minutes. The initial rate (mA405/min) of p- nitrophenolate production in each well was determined from 31 datapoints. For each digest, the initial rates were plotted against the log10 of the corresponding digest dilutions, yielding a sigmoidal curve; the standard curve was similarly processed. The concen- tration of 33-mer in each digest was determined from those digest References 1. Alaedini A, Green PHR (2005) Narrative review: celiac disease: understanding a complex autoimmune disorder. Ann Int Med 142: 289–299. 2. Robins G, Howdle PD (2005) Advances in celiac disease. Curr Opin Gastroenterol 21: 152–161. 3. Logan RF, Tucker G, Rifkind EA, Heading RC, Ferguson A (1983) Changes in clinical features of coeliac disease in adults in Edinburgh and the Lothians 1960- 79. 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Statistical analysis Peptide and prolamin curves were obtained by plotting percentage of maximum absorbance against logarithm of antigen concentration. The software package Sigma Plot 9.0 (Systat Software, Inc., Point Richmond, CA, U.S.A.) was used to calculate the IC50. The limit of detection was defined here as reagent blank- 3x standard deviations of reagent blank. The limit of quantifica- tion was defined here as reagent blank-10x standard deviations of reagent blank. The CR was calculated as follows: (IC50 of the antigen for which the moAb was raised/IC50 of each antigen assayed)X100. The reproducibility (inter-plate variability) was assessed by measuring the standard curve for different gliadin samples on two separate ELISA plates on different days. The reproducibility (intra-plate variability) was calculated by measuring the standard curve eight times for the same gliadin sample on a single ELISA plate. 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