From: Food Quality & Safety magazine, August/September 2012

Gluten-Free: How Can You Prove It?

by Elisabeth Hammer

Over the past decade, the demand for gluten-free food has soared, and more and more of these products can now be found in stores. The number of consumers who have difficulties with digesting gluten has grown to around 10%.

These individuals show varying degrees of sensitivity toward gluten, but their situation generally improves when they follow a gluten-free diet. Furthermore, increasing numbers of consumers believe that a gluten-free diet is more healthful. But what is gluten? How can it be toxic? And how can gluten be detected in food?

What Is Gluten?

The name “gluten” is derived from the Latin word for glue, and it refers to the composite of the proteins called prolamins and glutelins, found in wheat, barley, rye, oats, and their crossbred varieties. Prolamins are defined as the fraction that can be extracted using 40-70% of ethanol, and this fraction is called gliadin, hordein, secalin, or avenin, respectively, depending on the grain variety. In general, prolamins and glutelins are estimated to occur in the same ratio in gluten.

Worldwide, gluten is an important source of nutritional protein, both in foods prepared directly from gluten-containing sources and as an additive to foods otherwise low in protein. Gluten contributes texture and form to food products, due to its physicochemical properties. Mixed with water and kneaded, gluten forms a viscoelastic dough with a special protein network, which is responsible for the shape of bakery products.

Celiac Disease

Since the transition from the hunter-gatherer lifestyle to the beginning of agriculture 10,000 years ago, cereals have been a main pillar of the human diet, which raises the question of why gluten is causing increasing levels of health problems now. Approximately 1% of the world’s population is affected by celiac disease, an immune-mediated enteropathy caused by the ingestion of gluten.

Interestingly, the condition occurs more frequently in women than in men. Symptoms are diverse and not confined to the gastrointestinal tract. Examples include not only diarrhea, abdominal pain, flatulence, indigestion, and weight loss, but also irritability, depression, and anxiety. However, all these symptoms are considered to be unreliable as an indicator for the disease. Celiac disease can be diagnosed by screening for certain antibodies in the serum.

Another recommended diagnosis involves a biopsy of the mucosa and the small intestine to affirm damage, because the disease leads to the destruction of villi, microscopic, finger-like projections in the small intestine. Because intestinal villi are responsible for the absorption of nutrients, malnourishment may occur and, if it continues on a long-term basis, may lead to developmental delays, osteoporosis, or nutrient deficiencies, along with other problems.

Celiac disease, in which the immune system responds inappropriately to dietary gluten, is an autoimmune disorder to which people can be genetically predisposed. The enzyme called tissue transglutaminase modifies gluten peptides by deamidation so that T-cell epitopes are formed. This stimulates the immune system and cross reacts with the small intestine tissue, causing an inflammatory reaction that leads to the truncation of the villi. The majority of proteins responsible for such an immune reaction are prolamins. The strongest response is directed toward a 2-gliadin fragment that is 33 amino acids long, and is a principal contributor to gluten immunotoxicity. This so-called 33-mer is highly resistant to breakdown by digestive enzymes and is, therefore, a suitable molecule for use as an analytical marker. Homologues that have been found in food grains that are toxic for celiac patients are absent in nontoxic grains.

The only effective treatment for celiac disease up to now has been a lifelong gluten-free diet. Unfortunately, gluten is found in most foods, making this diet challenging to maintain. “Hidden” gluten used as a protein filler can be found in unexpected products such as pharmaceuticals, sausages, sauces, and desserts. In addition, gluten-free products may contain gluten due to cross contamination during milling, storage, and production. Gluten-free food is usually based on rice, maize, or buckwheat, as well as purified starch that contains low levels of gluten. It is very difficult to set limits, because sensitivity varies from individual to individual. According to scientific studies, the ingestion of gluten should be maintained at a level below 50 mg per day.

Legislation and Standards

The Codex Alimentarius Commission started to discuss recommendations for limits in the late 1970s, culminating in the 2008 CODEX Standard for Foods for Special Dietary Use for Persons Intolerant to Gluten (CODEX STAN 118 – 1979). This recommendation was taken into European legislation through Commission Regulation (EC) No 41/2009 of 20 January 2009, concerning the composition and labeling of foodstuffs suitable for people intolerant to gluten. In contrast to other food allergens, thresholds for glutens have been defined. Food labeled as gluten-free must not exceed 20 ppm, whereas food containing low levels of gluten must be lower than 100 ppm. A proposed rule for gluten-free labeling of foods is in preparation in the U.S.

The only effective treatment for celiac disease up to now has been a lifelong gluten-free diet. Unfortunately, gluten is found in most foods, making this diet challenging to maintain. “Hidden” gluten used as a protein filler can be found in pharmaceuticals, sausages, sauces, and desserts.

Gluten Analysis

With regulations in place, appropriate detection methods for gluten in food are needed. Several technologies that give qualitative and quantitative results, such as specific antibody-based tests (e.g., enzyme-linked immunosorbent assays or lateral flow assays), polymerase chain reaction methods, and newer concepts like mass spectrometry are available—all with varying degrees of commercialization. An analytical test system should preferably be able to detect epitopes involved in celiac disease.

The fact that gluten is a complex mixture of proteins and occurs in a wide range of unprocessed as well as processed matrices creates a huge challenge in terms of correct quantification, making it difficult to find a suitable reference material. In 1985, the Working Group on Prolamin Analysis and Toxicity was founded in Europe. Its first task was to establish a recognized gluten standard for gliadin.

By extracting gliadins from a selection of the most common wheat varieties, the group managed to get a reference material. The IRMM [Institute for Reference Material and Measurements] initially accepted the group’s gliadin standard as a certified reference material, but later withdrew its acceptance because of discussions on the selection and the number of wheat species used to generate it, as well as purity issues. However, because it is still the only reference that has some acceptance, it has been widely used for calibration of test systems.

Enzyme-linked immunosorbent assays are the recommended method for the detection of gluten in food, and a large number of test kits are available commercially. Immunological methods apply antibodies that have been raised against different prolamin fractions or specific sequences that are harmful. Different test kits do not necessarily give similar results, for several reasons. These include different specificities of the polyclonal and monoclonal antibodies used, different extraction methods, and different materials used to calibrate the assays.

Numerous monoclonal and polyclonal antibodies have been developed for gluten testing, but only some are accepted on a broader basis. In the late 1980s, the Skerritt antibody was developed. This monoclonal antibody was raised against wheat gliadin from an Australian wheat variety and recognizes high molecular weight glutenin subunits and the heat stable subfraction called ω-gliadins, which makes the Skerritt antibody suitable for gluten analysis in processed foods. Even so, because the quantitation is based on the amount of ω-gliadins, which differ among cereal species, results can differ considerably. Moreover, the Skerritt antibody only has a weak response to hordein.

Another monoclonal antibody used for the detection of gluten is the R5 antibody, developed by Professor Enrique Mendez in Spain. The R5 antibody was raised against rye secalin, but showed strong cross reactivity to wheat gliadin. It also detects proteins from soy and lupin that are not harmful prolamins, however. The change in direction from detecting prolamins to detecting immunotoxic peptides that play a role in the pathogenesis of celiac disease led to the development of a next generation of antibodies. The G12 antibody employed in the AgraQuant Gluten G12 ELISA and AgraStrip Gluten G12 Lateral Flow Test belongs to this next generation.

Gluten is a complex mixture of proteins and occurs in a wide range of unprocessed and processed matrices, creating a huge challenge in terms of correct quantification.

Next-Generation Analysis

The G12 antibody specifically recognizes the 33-mer of the gliadin protein present in gluten. This toxic fragment was identified by the University of Stanford and published in 2002 in a paper in Science. The G12 antibody, which was raised against this 33-mer peptide using knowledge gained from this publication, recognizes the hexapeptide sequence QPQLPY and similar peptides found in barley, rye, and oats.

In contrast, the R5 antibody was raised against a secalin extract; later, the epitope it reacts with was identified as the QQPFP pentapeptide. The distinction between the two antibodies relates to the fact that the G12 antibody specifically targets the toxic fragment that triggers the autoimmune reaction in celiac patients rather than a peptide sequence unrelated to clinical outcomes. It was confirmed during validation studies that G12 does not give false positive signals with soy and is suitable for measuring gluten in products containing soy. There is also no cross reactivity to maize or rice.

There is an ongoing debate about whether or not oats are safe. Several publications have concluded that certain oat varieties may cause an autoimmune response in celiac patients. During the validation of the AgraQuant Gluten G12 ELISA test and the AgraStrip Gluten G12 Lateral Flow Test, positive and negative responses to oat varieties were observed. The positive results appear to be a specific reaction of the antibody with the toxic fragment rather than a nonspecific response. Therefore, the G12 antibody may shed new light on this debate by recognizing oat varieties that trigger a response in celiac patients. The Spanish Celiac Association recently awarded the 6th National Prize for Research on Celiac Disease to a scientific team that used the G12 antibody to identify oat varieties containing low levels of gluten.

Celiac patients depend on the correct labeling of gluten-free food to maintain their health. Ensuring the safety of food is a demanding task, fueling new developments in detection methods for gluten. Results obtained from new immunochemical test systems based on the G12 antibody should be considered closer to the ideal of a food safety test, because they establish the important link between celiac disease and the detection of the immunotoxic peptides.

Elisabeth Hammer is a product manager with Romer Labs.



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