The R5 ELISA is a mechanism used to test for gluten in final food products. It is commercially available in two forms: the R5 sandwich ELISA and the R5 competitive ELISA. Until now, the R5 competitive ELISA has not been scientifically validated, prompting a recent study to determine the validity of the test. Previously, in-house validation data by the manufacturer has been the only method available.
The below responses are from Dr. Peter Koehler, Vice Director of the German Research Center for Food Chemistry in Freising, Germany and chairman of the Prolamin Working Group (PWG). Dr. Clyde Don, owner of Foodphysica in the Netherlands and chair of the Protein and Enzymes Technical Committee of AACC International also contributed to these responses. Both Drs. Koehler and Don played a role in this study.
Q. Can you explain what the R5 sandwich ELISA and the R5 competitive ELISA are? What’s the difference? Can you provide examples of when it is best to test using the R5 sandwich ELISA versus the R5 competitive ELISA?
A. ELISA stands for ‘Enzyme Linked Immuno Sorbent Assay’ and is an immunochemical method to detect and quantify a protein. For an ELISA an antibody raised against the protein to be analyzed is required. For gluten detection several antibodies are currently available and in this case the R5 antibody was used.
It is essential to know that gluten is not one protein but a complex mixture of several hundred individual proteins. Additionally, these proteins come from three different species, wheat, rye and barley. ELISAs usually detect only one type of gluten protein, and based on the percentage of this protein type in total gluten, the content is then converted to the total gluten content by multiplying by a factor. Most antibodies detect the prolamin fraction of gluten (equal to a fraction soluble in aqueous alcohol) and the Codex Alimentarius states that the prolamin content is usually 50% of total gluten. Thus, a conversion factor of two is currently used. In general, the regulations of the Codex Alimentarius are globally used as a reference and guideline for national legislators.
The R5 antibody has been raised towards omega-secalin from rye and also detects related protein groups of wheat (omega-gliadin) and barley (C-hordein). By suitable calibration it can be used to quantify the prolamin fraction of gluten. The gluten content is then calculated as: prolamin x 2 = gluten.
ELISAs have been commercialized in two different versions, sandwich and competitive. The sandwich version is suitable for the quantitation of intact proteins because it requires at least two binding sites (epitopes) for the antibody. The competitive version only requires one epitope and is, thus, suitable for detecting smaller-sized protein fragments, which are present in partially degraded gluten.
Q. The study talks about using the R5 competitive ELISA to test for partially hydrolyzed gluten (gluten peptides). Can you define partially hydrolyzed gluten? In which types of food and drink is this most commonly found?
A. Partially hydrolyzed gluten is gluten that has undergone processing leading to a partial degradation of the intact proteins. In the food sector, this processing step is usually a fermentation (e.g. in beer, the germination and subsequent enzyme action during mashing), in which enzymes cleave the initial protein to yield fragments (gluten peptides). Depending on the process these fragments can be large or small. This is relevant in foods such as beer, (alcohol-free) malt drinks, hypoallergenic food based on gluten, soy sauce, sourdough and starch/glucose syrup.
Q. This study looked at the sensitivity of the R5 competitive ELISA for measuring gluten in fermented foods and beverages. How did the investigators determine which specific fermented products to study?
A. We looked at the commercially most relevant cereal-based foods that had undergone fermentation during processing. Thus, it was very obvious to study beer, starch syrup and sourdough.
Q. There has been much controversy concerning beer that is brewed with barley, but claims to remove the gluten in the brewing process. Has this study proved the R5 competitive ELISA to be reliable in detecting gluten in these “gluten-removed” beverages? If such beverages test negative for gluten using the R5 competitive ELISA, can these beers now be confidently deemed as “gluten-free”?
A. This controversy is still there and the topic is lively discussed. What we can say is that the assay was able to reproducibly quantify gluten in the beer tested. In Europe, this test is accepted by legislators who allow a gluten-free claim on the product. There are a number of beers with a gluten-free claim on the market and no adverse observations and complaints by the consumers have evolved so far.
In North America and Australia there is a different view on this topic, in particular because mass-spectrometry based methods are evolving and these methods allow gluten protein fragments to be detected in those beers. There is controversy on the medical significance of these fragments as no intervention studies are available confirming or disproving potential toxicity of these fragments. In Australia the detection of gluten, no matter of the concentration, inhibits a gluten-free claim. In the U.S. no final threshold for a gluten-free claim has been set by the FDA so far. In addition, the U.S. Alcohol and Tobacco Tax and Trade Bureau (TTB) currently does not accept the R5 competitive method as valid for beer because it doesn’t use a proper calibrator. Currently, a peptic-tryptic digest of a mixture of wheat, rye and barley prolamins (equal to partially hydrolyzed gluten) is used as a reference. The TTB will only accept a fermented gluten reference material, which is not available to date. Therefore, a gluten-free claim for (wheat or barley-based) “deglutenized” beer found to be gluten-free by the R5 competitive ELISA is not allowed.
Q. What type of impact do you believe this study will have on gluten-free manufacturers?
A. In the U.S. a considerable impact will only be given after a gluten-free legislation has been put into action and the FDA has approved the method as being valid.
Q. How does this information affect consumers and their decisions to purchase various food and drink labeled as gluten-free?
A. The same restrictions as addressed in the answer directly above apply here as well. Of course, organizations at national levels like the FDA in the U.S. or other national agencies in Australia and Canada may take more time to consider the advised threshold and methods. But this will also bring a certain period of time of uncertainty for people with celiac disease from country to country.