Detecting Food Allergens
Jennifer Voyksner was tired of getting sick. She has celiac disease, an autoimmune disease in which the body reacts to gluten, a family of proteins found in wheat and other grains. She would carefully buy foods labeled gluten-free, only to have a reaction that made her sure the products contained gluten.
Voyksner—who is the research director of LCMS Ltd., a private research company in Durham, N.C.—decided to analyze the foods herself. When she did, she indeed found gluten. The levels were below the detection limits of the analyses used in the food industry but high enough to elicit reactions in extremely sensitive individuals like her.
Her experience epitomizes the challenges facing the food industry. Companies and regulators are trying to ensure the safety of a population with a wide range of sensitivities to ingredients that are harmless to most people. To do this, they rely on a handful of analytical methods for finding allergens in food.
Under laws such as the Food Allergen Labeling & Consumer Protection Act of 2004, companies are required to include a “contains” statement that lists in plain language the major allergens in products. The foods considered to be major allergens depend on the population and differ from country to country. In the U.S., that list includes the “big eight”: eggs, milk, wheat, peanuts, soy, tree nuts, fish, and crustacean shellfish. For products that include one or more allergens as ingredients, the statement just reiterates any allergens.
The problems come from foods that don’t have any allergens as intended ingredients. Many of these foods are made in the same facilities—sometimes using the same equipment—as other known allergen-containing products. Companies need to confirm that supposedly allergen-free foods really are allergen-free.
As part of that confirmation strategy, companies often use analytical methods to verify that the equipment has been cleaned adequately, says Lauren S. Jackson, a researcher at the Food & Drug Administration’s Center for Food Safety & Applied Nutrition in Bedford Park, Ill. For example, a single line might be used to process both dairy and fruit drinks, Jackson says. To ensure that no milk gets in the fruit drinks, the company would clean the line and then test the rinse water or equipment surfaces to make sure no residues are present.
Many products bear a voluntary advisory statement that tells consumers they were made in such a facility. A typical warning might say “may contain peanuts” or “this product was produced in a facility that also processes peanuts.”
Such advisory statements aren’t required by the labeling laws. Companies are welcome to use such advisory statements “as long as they’re truthful and not misleading,” says Steven M. Gendel, the food allergen coordinator at another FDA food safety center in College Park, Md. However, FDA expects companies not to use such statements as a substitute for Good Manufacturing Practices, he says.
Another common use for allergen detection methods is to bolster “free from” claims, says Joseph L. Baumert, codirector of the Food Allergy Research & Resource Program (FARRP), a cooperative between the University of Nebraska, Lincoln, and food companies. Companies might use the methods to ensure that their raw materials contain no allergenic residues.
To detect allergens, food companies typically choose from a handful of analytical methods, including immunochemical assays, polymerase chain reaction methods for DNA, and mass spectrometry.
Before 1995, there were no commercial methods for detecting food allergens, says Steve L. Taylor, cofounder and codirector of FARRP. Taylor has focused on developing immunochemical tests known as ELISAs, or enzyme-linked immunosorbent assays. In these methods, antibodies detect specific allergenic proteins or other proteins from the allergenic source.
“ELISA methods continue to be the most popular and perhaps the most robust methods for industry purposes,” Taylor says. “They can be available in formats rugged enough to use within processing facilities.”
But ELISA methods have limitations. Because ELISAs detect proteins, analysts typically assume that they directly measure the allergenic proteins. But that assumption may not always be a good one, says Thomas Holzhauser, a senior scientist in the Division of Allergology at the Paul Ehrlich Institute in Langen, Germany. “In most cases, it’s not really known whether they detect the allergenic proteins,” he says.
In addition to protein-based detection, Holzhauser supports the use of methods that detect DNA. DNA analysis allows specific detection of the plant or animal species. For regulatory purposes, the allergen is peanut, for example, not particular allergy-triggering proteins.
There are two major exceptions, Holzhauser says, where DNA testing is inexact: milk and eggs. In those cases, DNA testing can’t distinguish between cow’s milk and beef or between egg and chicken.
In some cases, DNA tests can succeed where commercial ELISA kits fail. One example is distinguishing between foods subject to mandatory allergen labeling and closely related foods that aren’t considered major allergens. For example, Holzhauser says, commercial ELISA kits can’t distinguish between almonds and apricot kernels, which are used to make persipan, a food similar to marzipan. DNA tests, which can be multiplexed, are particularly suitable as verification methods for tree nuts, he notes, because such tests can differentiate between species of nuts, which must be listed individually in allergen labels.
The third and, at this point, least common method for allergen detection is mass spectrometry. Food scientists tell C&EN that the complexity of the analysis and the need for highly trained operators make MS unlikely to become a frontline test in the food industry, but it has advantages as a backup, confirmatory method.
One such advantage is mass spec’s inherent multiplexing ability. The technique can detect multiple proteins from a single allergenic food, says Bert Popping, director of scientific development for Eurofins Scientific. “And you can target several allergenic plants or organisms in one go,” he says.
For example, last year Popping worked with doctoral student Julia Heick on a project to identify and detect seven allergens baked into bread, including egg, milk, and soy. For each allergen, they chose four peptide markers. “Even if processing destroys one or two of the peptides, you still have two available to confirm the presence of an allergen,” he says.
Popping and Heick compared the MS results with commercial ELISA kits for egg, milk, and soy. In raw materials, MS and ELISAs performed equally well. In the finished product, the ELISA performance was mixed, Popping says, with some kits detecting the allergens and others not. The MS performance with processed materials was more consistent than that of ELISAs.
Another advantage is mass spec’s ability to detect proteins whose structures might have been disrupted during processing such as heating or baking. Such disruptions can prevent antibodies in ELISAs from recognizing particular sequences known as epitopes, says Linda Monaci, a scientist at the Institute of Sciences of Food Production of Italy’s National Research Council. Mass spec methods don’t have the same problem because they can detect either peptides from digests or intact proteins.
Despite the strengths of MS, neither Popping nor Monaci expects the food industry to implement the method in production facilities. But in situations where food companies send samples to a testing lab, MS could become more popular, Popping says.
In fact, Popping believes that the cost of allergen detection in some situations could actually be lower with MS than with ELISAs, not including the cost of the instruments themselves. For products with multiple allergens, each allergen requires a separate ELISA, Popping says. But those allergens could be detected in one MS analysis.
Even with all of the tests available, companies haven’t yet figured out how to use such measurements in voluntary labeling of unintended allergens. The Allergen Bureau, a food industry organization in Australia and New Zealand, is working to give the food industry guidance about how to manage food allergen risk in those situations. In 2005, it introduced a risk-assessment protocol called VITAL (Voluntary Incidental Trace Allergen Labelling). “What set VITAL apart was the fact that it actually recommended levels above which you would declare the levels of an allergen,” says Robin Sherlock, technical manager at Food Allergen Control Training Analysis, in Tennyson, Australia, and a member of the Allergen Bureau’s management committee. Below those levels, allergens wouldn’t be reported. “It was quite controversial at the time.”
Last year, the Allergen Bureau convened a panel of scientific experts to reassess the threshold levels in VITAL. The panel used statistical models and published and unpublished data to determine dose distribution curves for the major allergens. Their recommended threshold levels “will protect 97–99% of all food-allergic individuals,” says FARRP’s Taylor, who chaired the panel. The threshold levels depended on the amount of clinical data available for each allergen.
The panel recommended threshold levels for allergens from around the world, not just Australia and New Zealand. “We wanted it to be appropriate for any country,” Sherlock says. “Because companies are global, they need to be able to adapt to global requirements.” The Allergen Bureau released the recommendations last April.
But Taylor views allergen detection as qualitative rather than quantitative. “Do I care whether an allergen is present at 20 ppm or 10 ppm?” he asks. “If it’s there and it shouldn’t be, it’s a problem if it’s not labeled. I might not care how much. If it’s not supposed to be there, industry doesn’t want to find it at all.”
Nevertheless, Taylor thinks current methods are sensitive enough. “We have pretty sensitive test methods now, down to low parts per million,” he says. “I would argue that we don’t need more sensitive methods to protect the health of allergic consumers.”
Voyksner would disagree, especially for gluten, which, although not an allergen, is treated like one for labeling. “Nobody can seem to agree on what is gluten-free. That’s why the methodology is so important.” Companies can call products gluten-free as long as the products have less than 20 ppm of gluten; the most sensitive ELISAs for gluten have detection limits of 1–5 ppm. “Mass spectrometry’s detection limits can get far lower. By realizing that this is a contamination issue that ELISA would never pick up, we’re going to help people feel safer about what they’re eating.”