A new R5-ELISA system for gluten analysis in food has been described, with promising results on extraction, sensitivity, specificity and reproducibility.
A quantity as low as 1.5 parts per million of gluten can be detected reliably using a new European reference preparation.
Clinical data on the relation between the quantity of gluten ingested and 'toxic' effects in coeliac disease are not sufficient to back up the current threshold value for gluten-free foods suggested by Codex Alimentarius.
A gluten-free diet is the cornerstone treatment of coeliac disease. Until now, it is not known conclusively whether trace amounts of gluten might be allowed in the diet, as suggested by Codex Alimentarius. Gluten-free foods intended for dietary use can now be analysed reliably for residual gluten by the new R5 enzyme-linked immunosorbent assay (ELISA) system. Some major problems of gluten analysis (sensitivity, specificity, reproducibility) can be solved by the new method. Therefore, the information given by the new test system is relevant and superior to earlier methods. Further clinical studies using small dose challenges and dietary records including gluten analysis are necessary until a more meaningful discussion on standards for gluten-free foods can be started.
Coeliac disease (synonymous gluten-sensitive enteropathy) is an autoimmune disease triggered by the cereal protein gluten. Gluten is defined as the rubbery dough-forming protein that remains when wheat flour is washed to remove starch [1,2]. Gluten is characterized by a uniquely high content of glutamine and proline. Its major fractions, gliadin and glutenin, are storage proteins. The alcohol-soluble gliadin contains mainly monomeric proteins, whereas the insoluble glutenin comprises aggregated proteins. Both fractions consist of numerous partially closely related protein components. Gliadins from wheat, secalins from rye, and hordeins from barley contain repetitive peptide units, some of which have been shown to elicit coeliac-specific 'toxic' effects by small-intestinal organ culture and by T-cell stimulation experiments [3-6]. However, coeliac-disease-eliciting properties and immunogenicity of gluten/gliadin peptides are not totally identical.
A gluten-free diet excluding wheat, rye, barley and traditionally also oats is the conventional cornerstone treatment of coeliac disease. Nevertheless, in coeliac patients the relationship between the quantity of gluten ingested and the severity of clinical symptoms and histological abnormalities is still undefined [7-9]. Individual variation and clinical heterogeneity of coeliac patients pose difficult problems for an attempt to find an acceptable threshold value for trace amounts of gluten to be allowed in gluten-free foods [1]. Challenge studies and dietary survey studies [7,9-13] have shown that a daily intake of 100 mg or more of gliadin was sufficient to elicit typical coeliac changes, whereas a daily intake of gliadin of 4-14 mg did not cause small-intestinal mucosal damage in coeliac patients [10]. Unlike food allergy, in coeliac disease there appears to be a low-level threshold of gluten that might be acceptable for the majority of coeliac patients. Wheat-starch-based bread-flour mixtures containing traces of gluten are more palatable than zero-gluten preparations, and they are preferred by patients in some countries (e.g. the UK and Finland) [11,13]. However, clinical data at present are not sufficient to back up the 200 parts per million (ppm) (200 mg/kg) threshold value suggested by the present Codex Alimentarius standard for foods rendered gluten-free (draft proposal 1998; compare with Stern and colleagues [1]).
Considerable progress has been made in the gluten analysis of food. In the past ten years, several methods for gluten analysis have been developed, based mainly on antibodies recognizing gluten or gliadin. The first of these was an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody directed against ω-gliadin [14]. This antibody recognizes a particularly heat-stable fraction of gliadin and was used in several commercial test kits until recently. However, ω-gliadin content is highly variable in different wheat cultivars and depends on fertilization. The method has weaknesses in poor detection of hordeins and in poor reproducibility, which was due in part to different gliadin preparations used as a standard. A new European reference gliadin was elaborated and characterized in 2001 by the international Working Group on Prolamin Analysis and Toxicity [1,15]. This reference gliadin preparation is now available for comparative analysis and calibration. It is presently under certification by the EC Institute on Reference Methods and Measurements (IRMM). Several other gliadin antibody preparations have been used by other groups [16]. However, problems of sensitivity, specificity and reproducibility have prevailed.
In this issue of the journal, Valdés and colleagues [17] report on a new R5-ELISA system for gluten analysis. The monoclonal antibody R5 was obtained after immunization against ω-secalins and showed a comparable reactivity with gliadins, secalins, and hordeins. It recognizes the pentapeptide QQPFP (one-letter code for amino acids) and homologous sequences that occur repetitively in the 'toxic' prolamins [18]. This is why the antibody can be used as a capture as well as a detection antibody. The development of the powerful R5-ELISA for gluten analysis based on the new European reference gliadin is highly promising in terms of high recovery after extraction, high sensitivity, and low detection limit (1.5 ppm). R5-ELISA is a robust test system with good repeatability and reproducibility.
Recently, knowledge about the molecular basis of gluten intolerance was widened considerably. A peptide sequence in gliadin was identified representing a dominant T-cell epitope. This peptide was stable against breakdown by gastric, pancreatic and intestinal brush-border membrane proteases and was converted by tissue transglutaminase with high selectivity into a potent inducer of gut-derived T-cell lines from coeliac patients [3,5,6,19,20]. The epitope recognized by the R5 antibody is highly homologous to the common core QLPYP of this peptide and R5 was shown to be cross-reactive with it [18]. Thus, R5 obviously recognizes one of the prolamin sequences associated with 'coeliac toxicity'.
An important demand on gluten analysis is detection of prolamin not only in native form but also after food-processing. Prolamins contain several intramolecular disulphide bridges, which are opened during the process of heating (baking) and which form intermolecular cross-links after subsequent cooling. The resulting macromolecular aggregates resist ethanolic extraction and therefore escape detection. Valdés and colleagues [17] describe the use of a solvent cocktail enabling complete extraction of prolamins after baking. More analytical problems arise from partial proteolysis, deamidation and cross-linking of gluten during food-processing. The authors also tested successfully malted barley extracts containing partial proteolysis products. The suitability of the R5-ELISA for smaller peptides, however, remains to be investigated. In this context, competitive assay systems may lead to better results [21]. However, low sensitivity is a disadvantage of these assays.
While some problems of gluten analysis in food (proteolysis in hydrolysed gluten products, matrix effect) cannot be solved by a simple test system, the R5-ELISA for gluten in food may solve some of the common analytical problems. A multicentre collaborative study was completed recently. This work was introduced into discussions within the Codex Alimentarius Committee on Nutrition and Foods for Special Dietary Uses (CCNFSDU) in 2002. Gluten analysis has now reached a new stage and will certainly be of practical help to coeliac patients. Levels as low as 1.5 ppm of gluten can now be detected reliably. However, clinical data are still insufficient to back up a general threshold value of gluten. Further studies using small dose challenges and dietary records based on reliable gluten analysis are necessary. Important new data are to be expected in the next two years forming a basis for a renewed Codex Alimentarius discussion on standards for gluten-free foods with a strong impact on patients, doctors and food industry.