Writing in the Talanta journal, lead author Maria C. Mena and her colleagues from the Madrid-based Proteomics Facility, Centro Nacional de Biotecnologia, noted that the only treatment for coeliac disease (intolerance to gluten proteins in wheat, barley and possibly oats) was to follow a strict, life-long gluten-free diet.
The usual method for determining gluten content in ‘gluten-free’ foods (as accepted by CODEX or Codex Alimentarius Commission) was an R5 antibody-based sandwich ELISA, combined with a cocktail-extraction solution, they said.
But this technique required at least two toxic epitopes in the protein, the scientists added, while in hydrolysed foods (beers, baby foods, syrups) proteins were fragmented during food processing and converted into peptides in which only one toxic epitope might appear.
Given that sandwich R5 ELISA could yield incorrect results, Mena et al. wrote,“it was necessary to develop a new competitive immunoassay that, together with a reliable, compatible extraction solution, would provide a complete gluten analysis in any kind of food.”
Commercial foods tested
The team analysed commercial foods both labeled and not labeled ‘gluten free’, including wheat beers and home-made breads spiked with gliadins (gluten proteins), using sandwich R5 ELISA and their new competitive R5 ELISA. Foods were extracted using 60% ethanol/water, the cocktail solution or a new extracting solution called UPEX (universal prolamin and glutelin extractant solution).
Results showed limits of detection and quantification of the competitive assay were 0.36 and 1.22ng/ml of gliadins respectively, which led the scientists to hail a “better technique than the sandwich R5 ELISA for detecting gliadins quantitatively in hydrolysed foods”.
Using the competitive assay with the UPEX extraction buffer, the researchers found gliadin levels ranging from 68-218ppm (parts per million) in six commercially available beers, well above the CODEX level of 20ppm, while baked goods such as pasta, breadcrumbs and cake tested also exceeded this limit.
Mena told BeverageDaily.com:“[R]egarding the assay, for beers it is recommended to use the competitive ELISA system instead of the sandwich type, because most of the gluten proteins are hydrolysed.”
Mena added: “The main advantages of the new extraction method (UPEX) is that is useful to extract gluten in foods that have been heat-treated during their manufacture, and it is suitable for analysis with a competitive ELISA system, and therefore for analysis of hydrolysed foods.”
Can firms trust traditional test?
Asked how big a problem she thought it could be for food or beverage firms relying on the Sandwich R5 ELISA to make gluten-free or low-gluten claims, Mena said: “When Sandwich R5 ELISA is used to quantify gluten in hydrolysed foods, the resulting values may be underestimated, and therefore it is necessary to use the Competitive R5 ELISA to give reliable results.”
Nevertheless, if gluten-free food manufacturers were “completely sure” that all the proteins were not hydrolysed, then the Sandwich R5 ELISA still offered reliable results, she added.
So did some gluten-free manufacturers need to re-evaluate their risk assessment procedures in light of the work undertaken by Mena and her colleagues, and should CODEX changes be contemplated regarding a test standard?
“Although the Sandwich R5 ELISA is now the accepted method, the Codex Alimentarius also stated that for the detection of hydrolysed gluten a modification of the R5 assay (competitive ELISA) has to be applied (Codex Alimentarius Commission, 2006, ALINORM 06/29/23),” she said.
We also asked Mena to what extent it would be a problem for any authority complaining of gluten levels above the 20ppm threshold in products labeled ‘gluten free’, as measured by the current ELISA assay, to enforce this in court, given its potential inaccuracy in respect of hydrolysed foods?
Mena said: “When applying any method of analysis, it is assumed that the results are reliable taking into account the sensitivity and specificity of the method. In addition, it is necessary to use the method more appropriate for the type of sample.”
Title: ‘Comprehensive analysis of gluten in processed foods using a new extraction method and a competitive ELISA based on the R5 antibody’.
Authors: M.C Mena, M.Lombardia, A.Hernando, E.Mendez, J.P Albar
Source: Talanta, 91 (March 15, 2012) 33-40, http://dx.doi.org/10.1016/j.talanta.2011.12.073