Gluten Testing: Current Detection Methods—And Their Limits

An estimated 5% of the world’s population is currently affected by gluten-related disorders, and the prevalence of these conditions continues to grow. Along with celiac disease (CD), other gluten-related disorders that make gluten-free products attractive and necessary for many consumers include dermatitis herpetiformis, gluten ataxia, wheat allergy, and non-celiac gluten sensitivity. When looking at CD alone, evidence shows a significantly increased incidence over the past few decades in industrialized counties, particularly in females and children.

Specifically for people with CD, ensuring a gluten-free diet is the only safeguard to ward off disease progression. As such, food processors and manufacturers are under strict regulations regarding which products can be labeled gluten free to ensure their safety for consumers.

One challenge for food manufacturers is how labeling requirements vary globally. Food producers in the U.S. must comply with FDA regulations that, following an international regulation set by the Codex Alimentarius Commission, set a limit of 20 mg/kg gluten as the amount a product can contain and still be labeled “gluten free.” The Codex Alimentarius Commission also allows food products to be labeled “very low gluten” if they contain 20 mg/kg to100 mg/kg of gluten. Ensuring products meet this standard for appropriate labeling is critical and is accomplished by rigorous adherence to and understanding of current testing methods.

This is not a new issue for food producers, but it’s one that will continue to drive the need for improved methods of detection given the high stakes for a growing consumer population.

Current Gluten Testing Methods

Current testing methods loosely fall into three categories, all of which have advantages and disadvantages (see “Table 1,”), according to Sachin Rustgi, PhD, assistant professor of molecular breeding in the department of plant and environmental sciences at the College of Agriculture, Forestry and Life Sciences at Clemson University Pee Dee Research and Education Center in Florence, S.C.

Table 1

Two of these methods, both immunological, are currently approved by the Prolamin Working Group of the Codex Alimentarius Commission and supported by the FDA: the R5 antibody raised against omega-secalin from rye (rye gluten compliment) and the G12 antibody developed against the wheat alpha 2-gliadin 33 amino acid peptide (highly immunogenic peptide). Dr. Rustgi described these methods along with the others listed above in a 2019 review article of gluten detection methods published in Nutrients.

“Immunoassays are currently the primary methods used by the food industry,” says Steve Taylor, PhD, professor emeritus in the department of food science and technology, and retired founding director of the Food Allergy Research and Resource Program at the University of Nebraska in Lincoln. Available in both quantitative and qualitative (lateral flow devices) formats, the most popular quantitative gluten methods are excellent, according to Dr. Taylor. “Based on either R5 or G12 monoclonal antibodies, [these tests] are so very specific, and [there’s] not much of a chance of a false positive,” he says, adding that qualitative methods based on the same antibodies are quite good.

He cautions that methods based on older Skerritt antibodies are not as highly recommended because they can miss barley. Along with R5 and G12, Skerritt is a monoclonal antibody used for gluten testing. He also says that issues sometimes found with the food matrix are less problematic with gluten methods, especially when Mendez cocktail is used.

Dr. Taylor doesn’t see many gaps in these immunoassay methods but cites three large issues that remain important in improving detection: how to manage particulate contamination in a product, questions around testing used frying oil, and detection of gluten residues in fermented and hydrolyzed foods.

Calling it the proverbial needle in the haystack problem, Dr. Taylor says that the problem with particulate contaminant is that if a particle containing gluten is found in a food sample, the test will be positive, but if you miss the particle, then the test may not be positive. “This issue is magnified with gluten testing because the sample size for the immunoassay is 0.25 mg, which is rather small [and, by contrast] most allergen immunoassays have a sample size of 5 g, or 20 times larger,” he says. “With the small sample size, you could easily miss the presence of particles, and much depends on the homogeneity of distribution of gluten particles in the overall sample.” For powders, the particulate issue is not as serious as it could be with larger particles such as crumbs, he adds.