that are toxic for CD patients [23]. Since the A1 moAb showed even higher sensitivity to wheat gliadin than G12, we evaluated the reactivity of A1 moAb with other cereal grain prolamins. The prolamins from wheat, barley, rye, oats, corn, and rice were extracted, and the samples were analyzed by Western blot, using the A1 moAb. The A1 moAb detected wheat gliadins, barley hordeins, and rye secalins. Oat avenins were also detected by A1, but the sensitivity obtained was lower (data not shown). The A1 moAb did not react to a detectable extent with prolamins extracted from rice (oryzein) and corn (zein), cereals that are non-toxic to CD patients. To obtain quantitative data about the capacity of A1 to detect celiac toxic prolamins, we performed an indirect ELISA with samples of wheat, barley, rye, oats, rice, and corn (Figure 2). The assay proved to be highly specific for wheat, rye and barley, as no signal was observed in samples containing prolamins from rice or corn (Figure 2). The G12 and A1 moAbs detected oats with lower sensitivity, indicating that there are peptides in avenin with sequence similarity to the 33-mer. This is consistent with the identification of proline and glutamine rich epitopes in avenins that are toxic in some CD patients [6]. The lower sensitivity for oat avenins may be due to the lower proportion of oat flour protein content that comprises prolamins relative to the proportion of gliadins, hordeins or secalins in their respective grains [24]. The A1 moAb was clearly more sensitive than the G12 moAb for the detection of the prolamin fractions from wheat, barley, rye, and oats. Although they were targeted at the toxic 33-mer peptide of wheat gliadin, both moAb were more sensitive for barley than for wheat, and the A1 was also more sensitive for the prolamins of rye than for those of wheat. Although both moAb were more sensitive for barley than for wheat (Figure 2), the A1 moAb had almost three-fold higher affinity for barley than did G12. Development of a competitive ELISA assay using the A1 moAb The preparation of many foodstuffs involves heating or enzymatic processes that may partially hydrolyze or deamidate MoAbs to Toxic Gluten Peptides gluten. As a result, the quantity of gluten extracted from foodstuffs processed by heat or fermentation may be underestimated by indirect or sandwich ELISA [25,26]. We therefore developed a competitive ELISA in which the antigen was fixed on the plate and soluble antigens in the sample, were preincubated with a HRP conjugated moAb. Upon addition of this preincubated mixture to the plate, soluble and fixed antigens competed for binding to the HRP conjugated moAb. Thus, a decrease in the signal indicates the presence of antigen in the sample. A competitive ELISA method using G12 moAb was previously developed for detecting toxic peptides in hydrolyzed food down to 0.5 ppm gliadin [23]. Here we developed a similar assay using the A1 moAb. Several assays were carried out to optimize the conditions for the competitive ELISA, including altering the concentration of gliadin fixed on the plate (1–0.01 mg/well), the dilution of A1- HRP moAb used (1:1,000–1:50,000), and the time (15 min to 4 h) and temperature (4uC, room temperature, and 37uC) of preincubation and incubation. A concentration of 0.5 mg/well of gliadin fixed on the plate and a 1:10,000 dilution of the A1-HRP moAb were found to be optimal (data not shown). A preincubation of 3 h and an incubation of 40 min, both at room temperature, were found to be optimal. The resulting highly sensitive competitive assay had a limit of detection of 1.63 ng/mL of gliadin (0.33 ppm of gluten) and a limit of quantification of 6.98 ng/mL of gliadin. IC50 in the standard curve of the competitive assay was 15.76 ng/mL. The standard curve for the detection of toxic gliadin by competitive ELISA under the established conditions showed a good correlation to the data (R2 = 0.99). The repeatability and reproducibility of the method, calculated from various standard curves performed on the same ELISA plate (intra-assay), and on different ELISA plates (inter-assay), were determined. The intra-assay coefficient of variation of the standards situated between 100 and 1.56 ng/mL of gliadin was found to be between 1.37% and 5.21%, while the inter-assay coefficient of variation was between 3.16% and 11.78% for the same standards. Figure 2. Comparative reactivity of prolamins from wheat, barley, rye, oats, corn and rice from indirect ELISAs using moAbs G12 (black) and A1 (white). Each point of the curve shows the mean of n = 3 assays. IC50 and CR values of the moAbs to prolamins are indicated. N.A.: Not applicable. doi:10.1371/journal.pone.0002294.g002 PLoS ONE | www.plosone.org 3 May 2008 | Volume 3 | Issue 5 | e2294