A novel and sensitive method for the detection of T cell stimulatory epitopes of a/b- and g-gliadin (1)
CD is an immune disease caused by T cells recognising gluten derived peptides presented by HLA-DQ2 or -DQ8 molecules. T cell stimulatory peptides have been identified by us and others, and these originate from proline and glutamine rich regions in a-gliadin, g-gliadin, and low (LMW) and high (HMW) molecular weight glutenins. Tissue transglutaminase (tTG) converts glutamine residues in gluten peptides into glutamic acid which facilitates gluten peptide binding to HLA-DQ2 or -DQ8.
BALB/c mice were immunised with TTd coupled peptides. For both T cell stimulatory peptides, several specific mAbs were obtained.
The mAbs were used to develop a competition assay. For the a-gliadin T cell epitope, a mAb was selected that was obtained after immunisation with peptides encoding amino acids 59–69 of a-gliadin. This mAb is referred to as anti-glia-a2/9 hereafter. For g-gliadin, a mAb was selected that was obtained after immunisation with amino acids 147–159 of g-gliadin. This mAb is referred to as anti-glia-c1 hereafter.
The detection limit was determined using the European gliadin reference (IRMM-480)26 as standard. In this way, sensitive assays were developed in which the gliadin reference was detected in the range 100 ug/ml to 12 ng/ml for both the glia-a2/9 T cell epitope and the glia-c1 T cell epitope. Extraction of 2 g food/20 ml of 40% aqueous ethanol, and a minimal sample dilution of 1:20, a gliadin content as low as 2.4 ppm (or 4.8 ppm gluten) can be detected.
The minimal amino acid sequence recognised by the anti-glia-a2/9 mAb was LQPFPQPQ which covers most of the glia-a9 T-cell epitope. Similarly, we found that the minimal sequence recognised by the anti-glia-c1 mAb was QQRPFI which overlaps with the C terminal amino acids of the glia-g1 T-cell epitope.
A higher concentration of peptides is required to reach the same level of competition as found for intact proteins. This indicates that the affinity of the anti- glia-a2/9 mAb is lower for peptides than for intact proteins. For the glia-c1 competition assay, no difference was found between detection of the peptide in intact proteins and the 25-mer synthetic peptide.
Both methods detected the glia-a2/9 epitope in protein preparations of barley, wheat, rye, and triticale, but not in oats. Both methods however detected the glia-c1 T cell epitope not only in the cereals barley, wheat, rye, and triticale but also in oats.
High levels of both a- and g-gliadins were detected in products 11, 12, and 16 whereas low levels were detected in food products 1, 2, 4, 13, 14, and 18. In a product in which maize starch (product No 15) was used, levels of gliadin were very low (g-gliadin) or not detectable (a-gliadin).
As peptides with only 11 amino acids are large enough to stimulate T cells, it is important to ensure the absence of such remaining peptides in food products and in gluten free food products in particular.