A modified extraction protocol enables detection and quantification of celiac disease-related gluten proteins from wheat (1)
Exposure to gluten proteins leads to damage of the small intestine, which causes a range of symptoms including altered bowel habits, malnutrition and weight loss. The only way to avoid symptoms is to maintain a life-long strict gluten-free diet. In wheat, gluten proteins are composed of monomeric gliadins and polymeric glutenins, which are present in approximately equal amounts. A drawback of these immunoassays is their extraction proto- col. It uses only 60% aqueous ethanol, because the presence of reducing agents would interfere with the immunoassay. However, the use of reducing agents (2-mercaptoethanol, DTT) has been shown to improve the extraction efficiency of glutenins. Further studies have shown that the use of reducing agents (2- mercaptoethanol and guanidiniumchloride) in a 1:100 dilution does not affect immunoreactivity and is used in the R5 capture ELISA.
Three ‘gliadin’ extracts were obtained by sequential extraction with 50% isopropanol. Finally, a ‘glutenin’ extract was obtained by extraction of the residue with 50% isopropanol, 50mM Tris–HCl (pH 7.5) containing 1% DTT. The average protein content in the ‘1st gliadin extract’ of the four hexaploid wheat varieties was 1.21 mg/ml. In the ‘2nd gliadin extract’ the average protein con- tent was 0.71mg/ml, while in the ‘3rd gliadin extract’ it was only 0.02 mg/ml, demonstrating that all ‘gliadins’ were extracted from the flour before the final glutenin extraction.
Extractions were performed on the flour of the four wheat varieties and the average protein content of the 1st 60% ethanol extracts was 0.33mg/ml.
Extraction of the residue with 25 mM Tris–HCl (pH 8.0) containing 2% SDS (1st residual extract) and with 25 mM Tris–HCl (pH 8.0) containing 2% SDS and 1% DTT (2nd residual extract) resulted in an average protein concentration of 0.84 mg/ml and 0.08 mg/ml, respectively.
RIDASCREENGliadin competitive ELISA showed no major differences between the two different extracts of each variety, only differences among the varieties.
A protein database search (http://www.ncbi.nlm.nih.gov/) of gluten protein sequences from T. aestivum was searched for the presence of the sequences recognized by the mAbs R5 (QQPFP, QQQFP, LQPFP and QLPFP), Glia-alpha 9 (QPFPQPQ) and LMW-2 (QSPF). The higher signals in immunoblotting in both the 1st gluten extracts (50% iso-propanol) and 2nd gluten extracts (50% iso-propanol, 50 mM Tris–HCl (pH 7.5) containing 1% DTT) compared to 60% ethanol extracts, when using the LMW glutenin mAb. No HMW-GS sequences were found that contained QQPFP, QQQFP, LQPFP, or QLPFP sequences.
Note: Low-molecular weight glutenin subunits (LMW-GS) and high-molecular weight glutenin subunits (HMW-GS).
The R5 mAb recognizes different small peptide sequences (QQPFP, QQQFP, LQPFP, and QLPFP) which are present mainly in gliadin proteins. The sequence LQPFP, which is strongly recognized by the R5 mAb, is present in the T-cell stimulatory proteolysis-resistant 33-mer. However, this sequence is only present in alpha-gliadins. The RIDASCREEN Gliadin competitive ELISA is used to detect ‘gluten’ in general in food products.