Food Safety Magazine

SIGNATURE SERIES | August 2012

Gluten-free: How Can You Prove It?

By Elisabeth Hammer, M.Sc., Romer Labs


Over the past decade, the demand for gluten-free food has soared and, therefore, more and more of these products can be found in the stores. The spectrum of consumers with difficulties in digesting gluten has grown to around 10 percent. These individuals show varying degrees of sensitivity towards gluten, but their situation generally improves when following a gluten-free diet. Furthermore, there is a growing perception amongst increasing numbers of consumers that a gluten-free diet is better for you. But what is gluten? Why 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 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 percent of ethanol, and this fraction is called gliadin, hordein, secalin or avenin, respectively, depending on the grain variety. In general, it can be estimated that prolamins and glutelins occur in the same ratio in gluten.

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

Celiac Disease
Following 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 as to why gluten is causing increasing levels of health problems nowadays. Approximately 1 percent of the world’s population is affected by celiac disease—an immune-mediated enteropathy caused by the ingestion of gluten. Interestingly, it is more frequent in women than in men. Symptoms are diverse and not confined to the gastrointestinal tract. Examples are not only diarrhoea, abdominal pain, flatulence, indigestion or weight loss, but also irritability, depression and anxiety. However, all these symptoms are considered unreliable as an indicator for the disease. Celiac disease can be diagnosed by a screening for certain antibodies in the serum. Another recommended diagnosis is a biopsy of the mucosa and the small intestine to affirm damage, as the disease leads to the destruction of microscopic, finger-like projections in the small intestine that are called villi. As intestinal villi are responsible for the absorption of nutrients, malnourishment is a problem that—on a long-term basis—may lead to development delays, osteoporosis or nutrient deficiencies, amongst other problems. Celiac disease is a genetically predisposed autoimmune disorder, in which the immune system responds inappropriately to dietary gluten. The enzyme called tissue transglutaminase modifies gluten peptides by deamidation in a way 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 towards an α2-gliadin fragment that is 33 amino acids long and 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 have been found in food grains that are toxic for celiac patients, but are absent in nontoxic grains.

The only effective treatment for coeliac disease up to now has been a lifelong gluten-free diet. This is challenging to maintain, as gluten is very common in food. “Hidden” gluten that is 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 still 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 below 50 mg per day.

Legislation and Standards
The Codex Alimentarius Commission started to discuss recommendations for limits in the late 1970s, cumulating 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 labelling of foodstuffs suitable for people intolerant to gluten. In contrast to other food allergens, thresholds have been defined. Food labelled as gluten-free must not exceed 20 ppm, whereas food containing low levels of gluten has to be lower than 100 ppm. A proposed rule for gluten-free labelling of foods is in preparation in the U.S.

Gluten Analysis
As there are regulations in place, there is a need for appropriate detection methods for gluten in food. Several technologies such as specific antibody based tests e.g. enzyme linked immunosorbent assays (ELISA) or lateral flow assays, polymerase chain reaction methods and newer concepts like mass spectrometry are available—all with varying degrees of commercialization—giving both qualitative and quantitative results. An analytical test system should preferably be able to detect epitopes that are involved in coeliac disease.
The fact that gluten is a complex mixture of proteins and that it occurs in a wide range of unprocessed as well as processed matrices creates a huge challenge in terms of correct quantification and makes it difficult to find a suitable reference material. In 1985, the Prolamin Working Group (PWG) was founded in Europe. Its first task was to establish a recognized gluten—respectively gliadin—standard. By extracting gliadins from a selection of the most common wheat varieties, they managed to get a reference material. The Institute for Reference Material and Measurements initially accepted the PWG gliadin 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, it is still the only reference that has some acceptance and has been widely used for calibration of test systems.

ELISA is the recommended method for the detection of gluten in food and a large number of test kits are available commercially. In immunological methods, antibodies are applied 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 for the calibration of the assays.

Numerous monoclonal and polyclonal antibodies have been developed for gluten testing, but only some of them 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, as the quantitation is based on the amount of ω-gliadins, which differ among cereals species, this can cause considerable differences in results. Moreover, the Skerritt antibody only has a weak response to hordein.

Another monoclonal antibody for the detection of gluten is the R5 antibody, which was developed by Professor Mendez in Spain. The R5 antibody was raised against rye secalin, but showed strong cross reactivity to wheat gliadin. However, it also detects proteins from soy and lupin that are not harmful prolamins. The change in direction to detecting immunotoxic peptides that play a role in the pathogenesis of celiac disease from the detection of prolamins, 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.

The Next Generation of Gluten 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 was raised against this 33-mer peptide using knowledge gained from this publication, and 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 and 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 any false positive signals with soy and is, therefore, suitable for measuring gluten in products containing soy. There is also no cross reactivity to maize or rice.

There is an on-going debate whether oats are safe. Several publications conclude that certain oat varieties may cause an autoimmune response in celiac patients. During the validation of AgraQuant® Gluten G12 ELISA test and 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 non-specific response. Therefore, the G12 antibody may shed new light on this debate by recognizing oat varieties that trigger a response in coeliac patients. The Spanish Coeliac Association has 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, in this regard.

Conclusions
Celiac patients depend on the correct labelling of gluten-free food in order to maintain their health. Ensuring the safety of food is a demanding task and, therefore, new developments in the field of detection methods for gluten are ongoing. The results obtained from new immunochemical test systems based on the G12 antibody should be considered to be closer to the ideal of a food safety test as they establish the important link between celiac disease and detection of the immunotoxic peptides.

Elisabeth Hammer, M.Sc., is product manager of Romer Labs. For more information, visit www.romerlabs.com.

Resources
Shan L., Ø. Molbergv, I. Parrot, F. Hausch, F. Filiz, G.M. Gray, L.M. Sollid and C. Khosla. 2002. Structural basis for gluten intolerance in celiac sprue. Science 297(5590):2275–2279.

Morón B., M.T. Bethune, I. Comino, H. Manyani, M. Ferragud, M.C. López, A. Cebolla, C. Khosla and C. Sousa. 2008. Toward the assessment of food toxicity for celiac patients: Characterization of monoclonal antibodies to a main immunogenic gluten peptide. PLoS ONE. 3(5):e2294.

Morón B., A. Cebolla, H. Manyani, M. Alvarez-Maqueda, M. Megías, M. Del Carmen Thomas, M.C. López and C. Sousa. 2008. Sensitive detection of cereal fractions that are toxic to celiac disease patients by using monoclonal antibodies to a main immunogenic wheat peptide. Am J Clin Nutr 87(2):405–414.

Arentz-Hansen H., B. Fleckenstein, Ø. Molberg, H. Scott, F. Koning, G. Jung, P. Roepstorff, K.E.A. Lundin and L.M. Sollid. 2004. The molecular basis for oat intolerance in patients with celiac disease. PLoS Medicine 1(1):e1.