METHOD FOR SELECTING CEREAL SEEDS SUITABLE FOR CONSUMPTION BY COELIAC PATIENTS

Abstract

The present invention relates to a simple method for selecting varieties of seeds from wheat, barley, rye and, in particular, oats, which enables said seeds, whether or not genetically manipulated, to be used in the production of gluten-free foods and beverages suitable for coeliac patients, and also the preparation of excipients for specific drugs. This invention is based on a simple immunological detection system that makes it possible to quantify the most immunogenic peptides of gluten with a view to ascertaining whether the cereal variety, preferably oats, can be used to produce suitable seeds that can form part of the diet of a coeliac patient. Preferred tests would make use of at least one antibody that recognizes the most immunogenic part of prolamines, the reactivity of which antibody vis--vis cereal seeds having been demonstrated to be proportional to the immunogenicity thereof with T-cells from coeliac patients.

Claims

1. A method for cereal seed selection characterized by the use of immunological assays with antibodies that recognize the 33-mer peptide from gliadin so that when they do not exhibit reactivity to gluten extracted from the seeds indicate that are tolerable by celiac patients.

2. The method according to claim 1, wherein the immunological assays are indirect ELISA, competitive ELISA, sandwich ELISA, immunochromatographic strips, fluorescent immunomicroparticles, Western blots, biosensors, using at least one antibody that recognizes the 33-mer peptide of the gliadin.

3. The method according to claim 1 wherein the immunological assays use at least one monoclonal antibody with ability to detect epitopes contained in the 33-mer of the gliadin SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8.

4. The method according to claim 1 in which the immunological assays use at least one of the following monoclonal antibody G12, A1 and R5.

5. The method according to claim 1 in which the immunological assays use the monoclonal antibody G12 conjugated to an enzyme that allows a quantitative assay using chromogenic, fluorogenic or luminescent substrates.

6. The method according to claim 1 characterized in that the immunological assay may use a gliadin pattern, gliadinhydrolyzate, whole 33-mer peptide or part of its sequence of at least 6 amino acids (SEQ ID NO: 2).

7. The method according to claim 1 in which the immunological assays are characterized by the proportionality between the signal obtained with the method according to claim 1 and the potential damage caused by the toxic proteins in celiac individuals measured by cell proliferation and production of IFN- by T cells from celiac patients.

8. The method according to claim 1 in which the seeds are wheat, barley, rye or oats.

9. The method according to claim 1 in which the seeds are oats.

10. The method according to claim 1 in which the seeds are from genetically modified cereals to reduce their immunogenicity degree for celiac patients.

11. (canceled)

12. (canceled)

13. A food which will be labeled as gluten-free comprising cereal varieties selected according to claim 1.

14. A food which will be labeled as gluten-free comprising oat varieties selected according to claim 1.

Description

FIGURES DESCRIPTION

[0023] To complement the description that has being made and in order to help a better understanding of the characteristics of the invention, according to a preferred practical embodiment thereof, it is attached as an integral part of said description, with illustrative and non limited to, the following figures:

[0024] FIG. 1. Evaluation of the presence of amplifiable DNA in oat varieties, as well as in wheat and rice by PCR. Agarose gel of the PCR amplification products of two representative samples of oat, wheat and rice. M: molecular weight marker of DNA (200 bp). Positive control: cereals DNA amplified with primers 18S. The primers used were: a. 18S, b. -avenin (oats), c. -hordein (barley), d. -secalin (rye) and e. -gliadin (wheat).

[0025] FIG. 2. Comparison of avenin fractions extracted from different oat varieties. Avenins spectrum of 9 different varieties obtained by MALDI-TOF MS. Analysis of the protein extract from the different varieties by polyacrylamide gel electrophoresis. M: protein molecular weight marker.

[0026] FIG. 3. Relative affinity of the monoclonal antibody G12 against different varieties of oats. A. Competitive ELISA using G12-HRP antibody to determine the reactivity of this antibody against different varieties of oats. Three tests were performed with three replicates each one. As a positive control gliadin was used. B. IC50 and RC of the different varieties of oats. N. A.: Not applicable. C. Western blot of the toxic fractions of prolamins extracted from oat grains. The membrane was revealed with the antibody G12. The color code used was the same than in A and B. M: protein molecular weight marker.

[0027] FIG. 4. Detection of the concentration of 33-mer peptide in different varieties of oats. The concentration of 33-mer was determined by competitive ELISA using the G12-HRP antibody. Three tests were performed with three replicates each one. Percentage of 33-mer of the variety tested compared to the variety more reactive, OM719.*33-mer concentration below the limit of quantification of the assay (5.4 ng/mL). N. A.: Not applicable.

[0028] FIG. 5. Proliferative response of T cells from celiac patients against deamidated peptides of prolamins from three varieties of oats. PBMCs were exposed for 48 hours to prolamins previously digested and treated with tissue transglutaminase. Values are expressed as units of O.D. at 450 nm. Gliadin and orzenin were used as positive and negative control, respectively. MeanSEM is represented for two trials on two separate days. *Statistically significant differences regarding orzenin (p<0.05).

[0029] FIG. 6. INF- production by T cells from celiac patients exposed to previously digested and deamidated prolamins from three varieties of oats. The production of INF- was assessed by ELISA after 48 hours of incubation. Gliadin and orzenin were used as positive and negative control, respectively. MeanSEM is represented for two trials on two separate days. *Statistically significant differences regarding orzenin (p<0.05).

PREFERRED EMBODIMENT OF THE INVENTION

Example 1. Determination of the Purity of Various Samples of Oats

[0030] The present example shows the importance of checking the purity of oat seeds that are going to be analyzed before determining its immunotoxic potential, since any contamination of other cereals (barley, wheat, etc.) would give false positives. Previous work has suggested that the toxicity of foods elaborated with oats is due to contamination with other cereals toxic for celiac patients (wheat, barley and rye). An example of this is the work published by Hernando et al. (2008, Eur J Gastroenterol Hepatol., 20:545-554) which states that the R5 antibody reactivity against certain foods made with oats is due to contamination of this cereal with wheat, barley or rye (Pulido and al., 2009, Adv Food Nutr res, 57:235-285). For this study, the purity of the samples of different varieties of oats was controlled in two ways. The first one performing a visual examination of individual oat grains to avoid the presence of grains of other cereals. The second form of control was using the polymerase chain reaction (PCR) since it is a highly specific and sensitive method for detecting small amounts of nucleic acid. In food quality studies, PCR has been applied to the detection of genetically modified organisms, pathogenic fungi, microorganisms and viruses as well as for the identification of animal species in meat products or for the detection and discrimination of contamination with cereals in gluten-free food. In general, the presence of cereal proteins is related to the presence of DNA, in this study the PCR technology has been used to detect nucleic acid from various cereals (wheat, rye and barley) in the oat samples.

[0031] To detect contamination of cereals (wheat, rye and barley) in the different varieties of oats, specific target sequences of encoding fragments from gliadin (wheat), secalin (rye), hordein (barley) and avenin (oats) were chosen for the amplification. The nucleotide sequences of these cereal storage proteins genes are well characterized and sequenced, showing a high degree of homology between the sequences of the prolamins genes of wheat, barley and rye. However, it was possible to design pairs of primers that could discriminate between different cereals. The primers used for amplification of -gliadin, -secalin, -hordein and -avenin genes were designed with the software PRIMER3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The specific forward and reverse primers, respectively, were: 5-TCTGCCCTATCAACTTTCGATGGTA-3 and 5-AATTTGCGCGCCTGCTGCCTTCCTT-3 for the 18S ribosomal gene, 5-CAGAAAGCGAGTGGAAAGATGAAAG-3 and 5-GCAAGGAGGACAAAGATGAGGGAA-3 for the gene -gliadin (amplified fragment length of 181 bp), 5-TTTTTCAGAAAGCGAGTTCAATGATG-3 and 5-CGAGGACAAAGATGAGGAAGGTCT-3 for the -secalin gene (amplified fragment length of 181 bp), 5-ATTAATTCCCAAACTGAACGACTA-3 and 5-CATGGCGAACAATGTGAAC-3 for the -hordein gene (amplified fragment length of 164 bp), 5-CGCTCAGTGGCTTCTAAGA-3 and 5-TTTTATTTTATTTGTCACCGCTAC-3 for -avenin gene (amplified fragment length of 104 bp). The oligonucleotides used were provided by Biomedal S.L. (Seville, Spain).

[0032] PCR system was used for the amplification of prolamins genes from wheat, barley, rye and oats. To do this, seeds of oat (Avena sativa) from different varieties: OM719, OE717, OL715, OA729, OH727, OC723, OF720, OR721 and OP722, supplied by different Spanish and Australian commercial sources were used. Wheat (Triticum durum, variety Don Pedro, CSIC, Crdoba, Spain) and rice (Oryza sativa subspecies Japonica, variety J. Sendra, Rice Federation, Seville, Spain) seeds were used as positive and negative control, respectively.

[0033] Grains from different cereals were ground on an electric grinder to obtain homogeneous samples. Nucleic acid extraction was performed using the modified CTAB method. Each sample was frozen with liquid nitrogen and transferred to a 2 mL tube containing 1 mL of extraction buffer [Tris-HCl 100 mM (pH 8.0), NaCl 1.4 M, EDTA 1.4 M (pH 8.0), CTAB 2% (w/v) and 2-mercapto-ethanol (14.3 N)]. Subsequently, samples were incubated at 65 C. for 30 minutes shaking periodically. After incubation, 500 L of chloroform were added and samples were centrifuged at 16,000 g for 15 minutes. The supernatant was transferred to a new 2 mL tube, to which 0.8 volumes of isopropanol were added to precipitate the DNA. The washes were performed with ethanol 70% (v/v). Finally, DNA was eluted in 60 mL of Tris-HCl 10 mmol/L (pH 8.0) containing 20 L/mL of RNase (RNase A Solution, Promega). DNA concentrations were determined by UV absorption at 260 nm and purity of the DNA solution was evaluated by absorption ratio 260/280 nm.

[0034] PCR premix (Biotools B&M Labs, Madrid, Spain) was used with 1 pmol of each primer and 50 ng of DNA template. The amplification reaction was performed under the following conditions: cycle 1, 95 C. for 3 min; cycle 2, 95 C. for 30 s, 52 C. for 30 s and 72 C. for 30 s (repeating the same conditions for 35 cycles); cycle 3, 72 C. for 5 min. PCR products were analyzed by gel electrophoresis at 2% (w/v) agarose MS (Roche Diagnostics, Mannheim, Germany).

[0035] In this way it was determined that the amplified fragment length varied between the different cereals from 104 bp for oats, 164 bp for barley up to 181 bp for wheat and rye. In these experiments, negative results were obtained in oat samples for specific wheat, rye and barley PCR; however, positive results were obtained by amplifying oat samples with specific primers 18S. Wheat amplification showed positive results in the specific PCR for 18S and gliadin. The agarose gel analysis of the DNA products amplified by PCR confirmed that all oat samples were free of contamination with wheat, barley, rye or a mixture of these cereals (FIG. 1).

Example 2. Variability Analysis of the Avenins from Oats Cultivars

[0036] The present example of the application shows that there is a large variability of avenins from different oats cultivars, which in principle is also indicative of a potential diversity in the immunotoxicity. MALDI-TOF MS technology (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) is currently used for the identification of a large number of subunits of glutenin and gliadin in wheat and other cereals (Qian et al., 2008, J Am Mass Spectrom., 19:1542-1550). In this study, we have used this technique together with SDS-PAGE (polyacrylamide gel electrophoresis with sodium dodecyl sulfate) to analyze the content in oat prolamins and to find molecular features that allow the identification and characterization of the different varieties. To do this, flour samples (6 g) of the different varieties of oats were obtained by crushing the seeds, resuspended in 70% ethanol (v/v) (30 mL) and maintained for 24 hours under stirring. After that, the mixture was filtered and prolamins were precipitated by addition of absolute ethanol to obtain a final concentration of 93%. Prolamins were obtained after incubation of the samples at 4 C. for 16 h. After this time it was centrifuged at 8,000 g for 10 minutes and the precipitate containing the proteins under study was collected. The protein concentration was measured by the Bradford method (Bradford, 1976, Anal Biochem., 72:248-254).

[0037] For the analysis of the avenins extractions by MALDI-TOF MS (5 L) 2 L of detergent octyl--D-glucopyrano side was added, and 25 L of saturated sinapinic acid in 30% acetonitrile (v/v) containing 0.1% trifluoroacetic acid (v/v) was used as matrix solution. Sample-matrix mixture was dried for 15 min in a speed-vac centrifuge and then resuspended in 6 L of 60% ethanol with 0.1% trifluoroacetic acid. 2 L of the mixture was placed on a stainless steel probe and dried for 5 min at room temperature. The samples were measured in the PE Biosystems MALDI-TOF Voyager DE-PRO system with a standard configuration. The mass spectrum was recorded in positive linear mode with an acceleration voltage of 25 kV and the final spectrum was obtained by accumulating 200 spectra of single laser shot under irradiation threshold. The equipment was externally calibrated using single and double BSA load signals with molecular masses of 66,430 and 33,215 Da respectively. Identification of avenins by MALDI-TOF MS showed characteristic profiles of protonated masses (Camafeita et al., 1977, J Mass Spectrom., 32:444-449).

[0038] For the analysis by SDS-PAGE, prolamins previously extracted were diluted in running buffer (Tris-HCl 62.5 mM at pH 6.8, 10% glycerol, 2% SDS, 0.001% bromophenol blue and 5% 2-mercapto-ethanol) and denatured by boiling at 100 C. for 5 min This step was repeated a total of three times. Samples were run on 12.5% polyacrylamide gel at a constant voltage of 100 V using Mini Protein system (BioRad Laboratories). The separated proteins in the gel electrophoresis were stained using silver staining.

[0039] The spectrum of avenins fractions from different varieties of oats obtained by MALDI-TOF MS, as well as the electrophoretic distribution of the prolamins observed by SDS-PAGE, disclosed that different varieties showed different protein profiles, which could allow a more comprehensive identification (FIG. 2). Both in the spectrum obtained by MALDI-TOF as by SDS-PAGE, in all cases, the molecular weight distribution was located between 19 and 31 kDa, which agrees with that described to date by others (Chesnut et al., 1989, The Plant Cell, 1:913-924, Hernando et al., 2008, Eur J Gastroenterol Hepatol., 20:545-554). The number and relative intensity of the peaks obtained by MALDI-TOF in the 9 cultivars was highly variable, which indicates that differences exist between the avenins extracted from different cultivars. The variability in size and protein distribution was also evident in SDS gels; the bands obtained differ in electrophoretic mobility and intensity. These results indicate the presence of different subunits of prolamins in the 9 oat varieties which differ in their amino acid composition and size.

Example 3: Differential Reactivity of Anti-Gliadin 33-mer Antibody Against Different Oat Varieties

[0040] This example demonstrates how an antibody that recognizes the peptide 33-mer of gliadin, can have a great variability in reactivity with the different varieties of oats. The example shows how the G12 moAb that was able to detect epitopes related to the 33-mer peptide in several cereal prolamins, reacts differently with different varieties of oats being its reactivity null in some of these varieties. The results indicate that the anti-33-mer moAb shows reactivity against prolamins of wheat, barley and rye, cereals toxic for celiac patients. This antibody was also able to detect avenins present in oats, although the sensitivity obtained in this case was lower, which may be due in part to the smaller proportion of these prolamins with respect to total protein content of this cereal in relation to the proportion of gliadins, hordeins or secalins in their respective grains, and mainly to the lower affinity of this antibody to epitopes of the avenins. G12 antibody did not react with prolamins extracted from rice (orzenin) and corn (zein), cereals that are not toxic for celiac patients. The reduced sensitivity of the G12 antibody against oat prolamins could be due to a lower prevalence of recognition epitopes of the antibody in the avenins with respect to the gliadins, hordeins and secalins.

[0041] To determine whether the moAb G12 showed different reactivity to different varieties of oats, the affinity of the antibody was determined by competitive ELISA. For this assay Maxisorpmicrotiter plates were used (Nunc, Roskilde, Denmark),which were coated with 100 L/well of gliadin solution Sigma (5 g/mL) in 0.1 M carbonate buffer (Na.sub.2CO.sub.3NaHCO.sub.3, pH 9.6), and incubated at 4 C. overnight. The plates were washed with PBS 0.05% Tween 20 and blocked with blocking solution (PBS, 5% skimmed milk) for 1 h at room temperature. Serial dilutions of gliadin and study samples were made in PBS with 3% BSA (100 L) and 100 L of moAb G12 conjugated to HRP (peroxidase) solution was added to each one (1:10,000 in PBS with 3% BSA). Samples were pre-incubated 1 h at room temperature with gentle agitation, and then added to the wells. After 30 minutes of incubation, samples were washed, and 100 L/well of substrate solution (TMB, Sigma, St Louis, Mo., USA) was added. After 30 minutes incubation at room temperature in darkness, the reaction was stopped with 1M sulfuric acid (100 L/well) and the absorbance was measured at 450 nm (UVM340 microplate reader, AsysHitechGmbH Eugendorf, Austria).

[0042] The results obtained showed that different oat varieties presented different reactivity to the G12 antibody (FIG. 3A). Depending on the affinity of the antibody to the different oat varieties, three groups could be clearly distinguished: a group with high affinity to the antibody (OM719, OA729 and OE717 varieties), a group with an intermediate recognition (OH727, OL715 and OC723 varieties) and another that was not recognized by the G12 moAb(OF720, OR721 and OP722 varieties). Results similar to those described were obtained with antibody Al (Morn et al., 2008, PLoS One, 3: e2294).

[0043] In order to quantify the affinity of the antibody G12 for the different varieties of oats, it was determined the antigen concentration with which a reduction of 50% of the maximum signal was obtained (IC50) and cross-reactivity (CR) of each one of them (FIG. 3B). CR was determined as (IC50 of the oat variety that present the highest affinity for the antibody/IC50 of each variety tested)100. OE717 and OA729 varieties showed a CR around 60 and 75% respectively, compared to the most sensitive variety (OM719). Additionally, OH727, OL715 and OC723 with a CR of 25, 24 and 12% respectively, are recognized by the antibody G12 but with less sensitivity. As with negative control (rice), the avenins from varieties OF720, OR721 and OP722 were not recognized by the antibody. The results obtained suggest that OF720, OR721 and OP722 oat varieties could potentially be safe for use in a gluten-free diet in celiac patients.

[0044] With the aim of confirming the results of the competitive ELISA using another immunological technique and identifying reactive protein profile for the anti-33-mer antibody, an assay was performed by immunoblotting using moAb G12. Initially obtained protein extracts were separated by SDS-PAGE gel and then incubated with antibody G12 on PVDF membranes. The extracts were incubated in blocking buffer (TBS with 5% skim milk) overnight and after that G12 antibody was added (dilution 1:5000 in blocking solution). After 3 washes, membranes were incubated with secondary antibody anti-mouse IgG conjugated to phosphatase (Sigma, St. Louis, Mo.) (dilution 1:2000 in blocking solution). The membrane was developed using Sigma-Fast system. The results obtained by Western blot (FIG. 3C) showed that moAb G12 has affinity for OM719, OA729, OE717, OH727, OL715 and OC723 varieties. However, this antibody did not react with OF720, OP722 and OR721. Thus, the variability in the reactivity observed in Western blot analysis confirms the results previously obtained in the competitive ELISA, and it was observed that the protein profiles obtained from reactive varieties differ from each other. OM719 and OE717 varieties presented similar reactive protein fractions; however, OA729, OC723, OL715 and OH727 presented protein profiles which bands differing in molecular weight and intensity. These results suggest the presence of different subunits of prolamins in oat varieties, which differ from each other in terms of amino acid composition and size. Likewise, the differences in reactivity with the monoclonal antibody G12 suggest variations in the protein sequence of the antibody recognition epitopes present in the avenins of different oats varieties.

Example 4: Determination of the Effective Amount of Immunoreactive Peptides in Oats

[0045] This example shows how the equivalent concentration of 33-mer peptide in seeds can be determined using an anti-gliadin 33-mer antibody with a peptide with this sequence as a reference. The 33-mer peptide has been identified as one of the main contributors to the immunotoxicity of gluten (Shan et al., 2002, Science, 297:2275-2279). This peptide from the a-gliadin contains 6 recognition epitopes for T cells and is very resistant to proteolysis. G12 monoclonal antibody is specific for the epitope of 6 amino acids SEQ ID No. 2, which appears 3 times along the peptide sequence of the 33-mer. This monoclonal antibody is able to recognize other immunoreactive peptides present in the gliadin and other toxic prolamins (Morn et al., 2008, PloS One, 3:e2294; Ehren et al., 2009, PLoS One, 4:e6313).

[0046] In order to determine the relative amount of immunotoxic epitopes present in prolamins of different oat varieties, we chose a variety from each group previously identified. For the group with greatest affinity for the antibody we chose OM719, for intermediate reactivity group, we chose the variety OH727, and OF720 from the group not recognized by G12 antibody. The presence of immunoreactive peptides was determined by competitive ELISA G12, using standard peptide 33-mer. The assay was carried out using the same ELISA protocol described above, but in this case 33-mer peptide was used as a standard curve to measure the concentration of toxic epitopes in the samples studied. The concentration of 33-mer in each sample was determined by calculating the equation defined by the standard curve and subsequent extrapolation from the absorbance data obtained for each sample.

[0047] Equivalent presence of 33-mer in the more reactive variety, OM719, was approximately 1.340 ng per mg of avenine (FIG. 4). In the OH727 variety the levels of 33-mer were diminished in the order of 4 times with respect to those of OM719. However, in the case of OF720, the concentration of 33-mer peptide was reduced more than 1300-fold relative to OM719, reaching undetectable levels by our method. These results confirm the data obtained previously by IC50 and cross-reactivity, as well as by Western blot. Likewise, these results indicate the high difference existing between varieties with regarding the presence of immunotoxic sequences for celiac patient and the possibility of finding oat seeds without amounts of detectable 33-mer equivalents.

Example 5. Correlation Between G12 Antibody Reactivity and Immunogenicity of the Different Oats Varieties

[0048] The present example shows how changes in reactivity of the anti-33-mer antibody observed for the different oats varieties are correlated with the degree of immunogenicity of the cereal. Immunogenicity was determined by assays of T-cell proliferation and production of INF-. Avenins ability to induce the immune response was analyzed using peripheral blood lymphocytes of individuals with celiac disease, using wheat gliadin as positive control and rice prolamins as negative control. For this example, three cultivars of oats were selected, one of each group previously identified; thus the variety OM719 represented the group that showed greater affinity for the antibody G12, OH727 the intermediate reactivity group, and OF720 the group that was not recognized by the antibody. It was evaluated whether there was a correlation between immunotoxic potential of the different oats varieties and its reactivity against the antibody G12. To do this the following procedures were carried out:

[0049] 5.1. Digestion with pepsin, trypsin and chymotrypsin. The ethanol soluble protein fraction was extracted from the flour and subjected to sequential digestion with pepsin, trypsin and chymotrypsin (Sigma, St. Louis, Mo., USA). Gastrointestinal digestion was simulated so that the prolamins samples were incubated with 0.6 mg/ml of pepsin in 0.2 M HCl for 1 hour at 37 C. with gentle agitation. After that, pH of the samples was modified with Na.sub.2HPO.sub.4 to pH around 6 to 7, then trypsin and chymotrypsin enzymes were added at a concentration of 0.375 mg/mL. The samples were again incubated for 30 minutes at 37 C. with gentle agitation. For the inactivation of enzymes, the samples were heat treated (100 C. for at least 5 min). Digested prolamins were lyophilized and stored at 20 C. until use.

[0050] 5.2. Deamidation of samples with tissue transglutaminase: specific deamidation of prolamins peptides by tissue transglutaminase is usually necessary for the binding of antigen presenting cells to major histocompatibility complex proteins (DQ2/DQ8) and the subsequent recognition by T cells. Therefore, the avenins peptides from the different oats varieties, as well as gliadin and orzenin controls, were treated for 4 h at 37 C., with 100 g/ml of tissue transglutaminase (Sigma, St. Louis, Mo., USA) in the presence of CaCl.sub.2 2 mM.

[0051] 5.3. Patients: this study was performed after approval by the Ethics Committee of the Hospital Virgen de las Nieves. Small bowel biopsies were obtained by gastrointestinal endoscopy under patients' informed consent for use in research on celiac disease (patients were monitored in the Hospital Virgen de las Nieves in Granada, Spain). Celiac disease patients showed partial or total atrophy of the villi with increase in intraepithelial lymphocytes. Histology positive cases were classified according to Marsh criteria (type I-IV) (Marsh and Crowe, 1995, BaillieresClinGastroenterol., 9:273-293).

[0052] The diagnosis of celiac disease patients was performed by serological test accompanied by intestinal biopsy and confirmation of a clinical response to the exclusion of gluten from the diet. Endomysial antibodies, anti-gliadin antibodies, anti-tissue transglutaminase antibodies and HLA typing specific for celiac disease (HLA-DQ) were determined.

[0053] 5.4. PBMCs and cell culture: peripheral blood mononuclear cells (PBMCs) from patients with active celiac disease who follow a gluten containing diet, were isolated from 6 mL of heparinized blood by gradient centrifugation with HISTOPAQUE (Sigma, St. Louis, Mo., USA) and subsequently grown to obtain a density of 110.sup.6 cells/mL in RPMI-1640 growth medium. After 48 h, the PBMCs were incubated with peptides derived from avenin, gliadin and orzenin (50 g/mL).

[0054] 5.5. Cell proliferation assay: T-cell proliferation was analyzed after 48 hours of incubation using the 5-bromo-2-deoxyuridine ELISA test (BrdU) for cell proliferation (Roche, USA). The results were expressed as optical density (O.D.) at 450 nm. Cell proliferation was determined by BrdU incorporation. This colorimetric test consists in the immunochemical measure of BrdU incorporation, an analogue of thymidine, to a DNA chain being elongated during active synthesis of this nucleic acid.

[0055] 5.6. IFN- production: aliquots of the culture supernatant of T cells were taken after 48 h of incubation for the determination of the production of INF-, and stored at 80 C. A commercial ELISA kit was used for the analysis following the manufacturer's instructions (Thermo Scientific, Spain). Assay sensitivity was less than 2 pg/mL.

[0056] 5.7. Statistical analysis of the cell proliferation assays and production of INF-: each experiment was performed in duplicate on different days. Data are expressed as meanstandard error of the mean (SEM). All statistical analysis were performed using the program STATGRAPHICS for Windows. When the interaction was significant, differences between groups were assessed by ANOVA test. To compare individual means Bonferroni test was used corrected with Student's t. A statistical probability of p<0.05 was considered significant.

[0057] PBMC proliferation exposed to different avenins, and the positive and negative controls, gliadin and rice prolamins respectively, was expressed in O.D. (FIG. 5). Thus, after 48 hours of exposure to the different avenins fragments, higher proliferation of T cells is found in the cultures incubated with the variety OM719 and gliadin (OD.=0.35 and 0.47 respectively), and also with OH727 (OD.=0.24). Thus, it is clear that gliadin and OM719 showed a higher reactivity being those with higher immunogenic potential (FIG. 5). Incubation with the variety OF720 showed increased cell proliferation (OD=0.12) similar to the negative control (orzenin peptides) (OD.=0.11).

[0058] The INF- is the main cytokine involved in the inflammatory response of celiac disease. For that reason the secretion of IFN- was analyzed in the culture medium after exposure of peripheral T lymphocytes to deaminatedavenin peptides (FIG. 6). According to this analysis, gliadin and the variety OM719, were highly immunogenic, with higher values of secretion of INF-y (0.11 IU/mL and 0.09 IU/mL, respectively), while exposure to OH727 induced a lower secretion of INF- (0.07 IU/mL). Finally OF720 and rice were less immunogenic (0.024 IU/mL and 0.015 IU/mL, respectively).

Example 6. Potential Immunotoxic Detection with Cereals with Other Antibodies with Cross-Reactivity Against the 33-mer.

[0059] This example demonstrates how other antibodies which have not been created directly by immunization against 33-mer but which recognize it, although with less specificity, can be used to assess whether the oat seeds may be immunogenic for celiac patients. Although previous work established that R5 antibody reactivity against certain foods made with oats is due to contamination with wheat, barley or rye, and not to a direct recognition of epitopes of this antibody in oat prolamins (Pulido et al., 2009, Adv Food Nutr Res 57:235-285), experiments performed for the present invention demonstrate R5 antibody reactivity with oat seeds with molecularly controlled purity. Following the instructions of the supplier, it was determined the capacity of moAb R5, specific antibody for the sequence QQPFP, to detect oat varieties previously tested. Similarly, the purity of the samples was monitored by PCR in order to avoid false positives.

[0060] The QQPFP peptide values obtained in OM719, OA729, OE717 and OH727 varieties ranged between 25.18 and 97.86 mg peptide/g cereal, which correspond according to supplier instructions to gliadin values between 100 and 400 ppm. In OL715 and OC723 varieties the peptide levels were between 1.5 and 9.2 mg peptide/g cereal respectively (6.3 to 37.2 ppm of gliadin, respectively). Thus the results obtained demonstrate that as the moAb G12 and A1, R5 antibody was able to detect the varieties described. However, in OF720, OR721 Y OP722 varieties gliadin was not detected. These results are equivalent to those already obtained by competitive ELISA G12. The detection of QQPFP like peptides in 6 oat varieties of 9 tested shows that the signal obtained by the R5 antibody in oat samples is due to the recognition of epitopes potentially toxic in prolamins of this cereal, and not to a cross-contamination with other cereals.

Example 7. Detection of Peptides Equivalent to 33-mer in Genetically Engineered Wheat Seed

[0061] This example shows how in cereal seed genetically engineered to suppress the levels of the toxic gliadin, the level of reactivity with anti-33-mer antibody decrease to a proportional extent to the immunogenicity. The transgenic lines published by Gil-Humanes et al. (2010, ProcNatlAcadSci USA, 107:17023-17028) were tested with the monoclonal antibody G12 determining the concentration of toxic peptides present in each of these lines with respect to the controls.

[0062] Transgenic lines 28A, 28B, D783, X387, D770, D793, D894 and X077, transformed with vector pDhp-/; E35, transformed with vector pDhp-/+pghpg8.1; A 1152 and C655 transformed with the pghpg8.1 vector were analyzed with respect to control BW208. The toxic peptide 33-mer concentration was determined by competitive ELISA G12. The immunological assay showed a significant reduction of toxic peptide levels above 90% in the lines 28A, 28B, D783, X387, D770, D793 and X077. In contrast, D894 and E35 lines showed a reduction of 42.42 and 48.57%. Meanwhile, the lines transformed with the vector pghpg8.1 showed no significant reduction in the levels of 33-mer with respect to the line BW208.

[0063] Other transgenic lines were also analyzed as D874, D876, X678 (transformed with vector pDhp-/), C217 and D598 (transformed with vector pghpg8.1) with respect to control BW2003. D874, D876 and X678 present a reduction greater than 95%. However, in lines C217 and D598 the reduction percentage of toxic peptide was 52.21 and 43.96%, respectively.

[0064] This example again shows a correlation between toxic peptide levels obtained with the G12 antibody and T-cell results published by Gil-Humanes et al. (2010, ProcNatlAcadSci USA, 107: 17023-17028) in cancelled gliadins from the transgenic lines used.