NEW. AgraQuant Gluten G12. Gluten. Celiac Disease. Celiac disease is an immunemediated

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$from Celiac disease is an immunemediated enteropathy caused by the ingestion of gluten, a protein fraction found in certain cereals.$

1 from Celiac disease is an immunemediated enteropathy caused by the ingestion of gluten, a protein fraction found in certain cereals. Celiac disease occurs in genetically predisposed persons and leads to the destruction of the microscopic finger-like projections of the small intestine, called villi. The disease is triggered by the ingestion of peptides from wheat, barley, rye, their crossbred varieties and in some cases oats. It currently affects roughly 1% of the world s population. Immunotoxic gluten peptides, namely a fragment called 33- mer, which are resistant to degradation of digestive enzymes, appear to trigger celiac syndrome. Homologues of this peptide were found in every food grain that is toxic to celiac disease patients, but were absent in all nontoxic food grains. A monoclonal antibody, called G12, which detects the 33-mer from α2-gliadin and has been identified as the principal contributor to gluten immunotoxicity), was developed. The sensitivity of the G12 antibody is significantly higher than that of equivalent methods recognising other gluten epitopes. G12 based immunotechniques appear to be a pragmatic approach to determining immunotoxicity. NEW AgraQuant Gluten G12 Epidermis Hypodermis Cross Cells Tube Cells Seed Coat (Testa) Nucellar Tissue Aleurone Cell Endosperm Germ Bran Gluten Gluten is a composite of the proteins gliadin and glutenin. These exist, conjoined with starch, in the endosperms of some grass-related grains, like wheat, rye, barley, oats and maize. Gliadin and glutenin comprise about 80% of the protein contained in wheat seed. Being insoluble in water, they can be purified by washing away the associated starch. 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. Table 1. Nomenclature of Protein Classes in different Cereal Species Fraction Wheat Rye Barley Oats Maize Albumin soluble in water Globulin soluble in NaCl solution Prolamin soluble in 40-70% EtOH Glutenin insoluble Hairs of Brush Leucosin Edestin Celiac Disease Gliadin Secalin Hordein Avenin Zeain Glutelin Secalinin Hordeinin Avenalin Zeanin Celiac disease has also several other names, including: cœliac disease (with œ ligature), c(o)eliac sprue, non-tropical sprue, endemic sprue, gluten enteropathy or gluten-sensitive enteropathy, and gluten intolerance. The term celiac derives from the Greek κοιλιακός (koiliakόs, abdominal ), and was introduced in the 19th century in a translation of what is generally regarded as an ancient Greek description of the disease by Aretaeus of Cappadocia.

2 NEWfrom It is an immune mediated reaction to prolamins (mainly gliadin), whereby, upon exposure to gliadin, the enzyme tissue transglutaminase modifies the protein, and the immune system cross-reacts with the small-bowel tissue, causing an inflammatory reaction. That leads to truncation of the villi, lining the small intestine (called villous atrophy). This then interferes with the absorption of nutrients. The only known effective treatment is a lifelong gluten-free diet. While the disease is caused by a reaction to wheat proteins, it is not the same as wheat allergy. Considering all clinical patterns of celiac disease (active, silent, latent forms) it has a prevalence of 1:100 to 1:200, hence it is a relatively common disease. Prolamins are storage proteins, rich in proline (prol-) and glutamine (-amin), that dissolve in alcohols and are resistant to proteases and peptidases of the gut. One region of α-gliadin stimulates membrane cells of the intestine, the enterocytes, to allow larger molecules pass around the sealant between cells. The disruption of these tight junctions allow peptides larger than three amino acids to enter circulation. The strongest and most common adaptive response to gliadin is directed toward an α2-gliadin fragment of 33 amino acids in length (see sequence below) 1. LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (L = leucin, Q = Glutamine, P = Proline, F = Phenylalanine, Y = Tyrosine) Once the intestinal wall absorbs 33-mer peptides, antigen presenting cells in celiac sprue patients signal other white blood cells to attack. The result is an eventual destruction of absorption cells and villi in the intestinal wall. Severe celiac disease leads to the characteristic symptoms of pale, loose and greasy stool (steatorrhoea), weight loss or failure to gain weight (in young children). Some people can have less severe manifestations of celiac disease and may show symptoms that are much more subtle and occur in other organs, rather than the bowel itself. Finally, it is possible to have celiac disease without any symptoms whatsoever. Many adults with subtle symptoms only have fatigue or anaemia. At present, the only effective treatment known is a life-long gluten-free diet. However, a recent review concluded that consumption of less than 10 mg of gluten per day is unlikely to cause histological abnormalities. Legislation The Codex Alimentarius Committee started to discuss recommendations for limits in the late 1970 s culmulating in the 2008 CODEX Standard for Foods for Special Dietary Use for Persons intolerant to Gluten (CODEX STAN ). This recommendation was taken over into European legislation with COMMISSION REGULA- TION (EC) No 41/2009 of 20 January 2009 concerning the composition and labelling of foodstuffs suitable for people intolerant to gluten. Here it is stated, that foodstuffs for people intolerant to gluten shall not contain a level of gluten exceeding 100 mg/kg and foodstuffs with a level not exceeding 20 mg/kg can be labeled gluten-free. For the US, Federal Register Proposed Rule - 72 FR 2795 January 23, 2007: Food Labeling; Gluten-Free Labeling of Foods, also featuring a limit of 20 ppm gluten in food is in preparation. Health Canada is proposing an Regulations Amending the Food and Drug Regulations (1220 Enhanced Labelling for Food Allergen and Gluten Sources and Added Sulphites), as published in the Canada Gazette, Vol 142, No. 30 (2008). 1 Anderson et al, Nat Med 2000;6: ; Arentz-Hansen et al. J Exp Med 2000; 191: Shan et al. Science 2002; 297: Page 2

3 AgraQuant Gluten G12 - ELISA Gluten Analysis The analysis of gluten has a long and confusing history. First of all, when talking about gluten analysis, in reality, people talk about prolamin analysis, as gluten consists of soluble prolamins and insoluble glutelins. In reality, the prolamin concentration is determined and based on that, the gluten concentration is calculated. The lack of a clearly defined Gluten reference material has also created a challenging environment for Gluten analysis. However, the latest discussions about celiac disease and gluten analysis, led from the concept of gluten detection to detection of potential relative toxicity of gluten for the safety of celiac food consumers. In 1985, the Prolamin Working Group (PWG) was founded in Europe 2. 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, this material was later modified before presented to the Institute for Reference Material and Measurements (IRMM) in Geel (Belgium). The IRMM initially accepted the PWG gliadin to become a certified reference material, but later withdrew because of discussions on the selection and the number of the wheat species used to generate it and also because of purity issues. Over the years, a few different antibodies became standard in gluten testing. Initially, the Skerritt antibody was most commonly used and tests based on it had Type 1 method status. It was replaced by the R5 antibody, an antibody developed in cooperation with the PWG. The R5 antibody was raised against rye secalin, but showed strong cross reactivity to wheat gliadin. It became quickly the consensus antibody, seen by many as the one, giving the correct answers. However, the change of the concept, detecting the immunotoxic peptides, playing a role in the pathogenesis of celiac disease, instead of detection of prolamins, led to the development of a next generation of antibodies. The G12 antibody belongs to this new generation. Table 3. Comparison of 3 gliadin antibodies Antibody G12 R5 Skerritt Specificity QPQLPY (wheat gliadin) QQPFP (rye secalin) HMG - high molecular weight glutenins (wheat) Detects also rye, barley wheat, barley rye, barley Crossreactivity oat (if toxic) soy* * controversial reports The G12 antibody specifically recognizes the toxic fragment of the gliadin protein present in gluten. This fragment (called 33-mer) was identified by the University of Stanford and published in a paper 2002 in Science. Using the knowledge from this publication, the G12 antibody was raised against this 33-mer peptide. In contrast, the R5 antibody was raised against a secalin extract. The epitope it reacts with was later identificed 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 auto-immune reaction in celiac patients, rather than a peptide sequence unrelated to clinical outcomes. 2 The Prolamine Working Group was founded in 1985 with the aim to coordinate research on laboratory gluten analysis in food and on clinical evaluation of celiac patients sensitivity to prolamins. It is a group of physicians, chemists, nutritionists and food scientists from different countries which successfully cooperates with the starch-producing industry, with manufacturers of gluten-free products, with manufacturers of test systems for gluten analysis in food, with national and international celiac societies and with official organizations such as the Codex Alimentarius Committee on Nutrition and Food for Special Dietary Uses. In order to provide a gliadin standard the Prolamine working group has produced PWG gliadin as the basis for further standardization. Page 3

4 NEWfrom Detection of Oats There is an ongoing debate concerning the presence or absence of gluten in oat. In fact, current antibodies were not recommended for the detection of gluten in oat. The G12 antibody has shed light on this debate due to its specificity for potentially immunotoxic sequences. G12 shows also a higher affinity to these sequences, which makes it much more sensitive, compared to other antibodies. It seems, that G12 can recognize oat varieties which trigger response in celiacs, while other oat varieties which are not causing any celiac response are not detected 3. For this, the Spanish Celiac Association from Madrid has recently awarded the 6th National Price for Research on Celiac Disease to a scientific team that used the G12 antibody to select oat varieties that contain very low levels of gluten that are suited for their low pathogenicity for celiac patients. References Anderson RP, Degano P, Godkin AJ, Jewell DP, Hill AV. In vivo antigen challenge in celiac disease identifies a single transglutaminase-modified peptide as the dominant A-gliadin T-cell epitope. Nat Med (2000), 6: Shan L, Molberg Ø, Parrot I, Hausch F, Filiz F, Gray GM, Sollid LM, Khosla C. Structural basis for gluten intolerance in celiac sprue. Science (2002), 297(5590): Arentz-Hansen H, Fleckenstein B, Molberg Ø, Scott H, Koning F, Jung G, Roepstorff P, Lundin KEA, Sollid LM. The Molecular Basis for Oat Intolerance in Patients with Celiac Disease. PLoS Medicine (2004), medicine.plosjournals.org. Garcìa E, Llorente M, Hernando A, Kieffer R, Wieser H, Méndez E. Development of a general procedure for complete extraction of gliadins for heat processed and unheated foods. Eur. J. Gastroenterol. Hepatol. (2005), 17: van Eckert R, Berghofer E, Ciclitira PJ, Chirdo F, Denery-Papini S, Ellis HJ, Ferranti P, Goodwin P, Immer U, Mamone G, Méndez E, Mothes T, Novalin S, Osman A, Rumbo M, Stern M, Thorell L, Whim A, Wieser H. Towards a new gliadin reference material isolation and characterisation. Jurnal of Cereal Science (2006) 43, Proceedings of the 21st Meeting Working Group on Prolamin Analysis and Toxicity, September , Trieste, Italy; Verlag Wissenschaftliche Scripten, Zwickau (2007) Morón B, Bethune MT, Comino I, Manyani H, Ferragud M, López MC, Cebolla A, Khosla C, Sousa C. Toward the assessment of food toxicity for celiac patients: characterization of monoclonal antibodies to a main immunogenic gluten peptide. PLoS ONE (2008), 3(5):e2294. Morón B, Cebolla A, Manyani H, Alvarez-Maqueda M, Megías M, Thomas Mdel C, López MC, Sousa C. 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. (2008), 87(2): Comino I, Real A, de Lorenzo L, Cornell H, López-Casado MÁ, Barro F, Loritte P, Torres MI, Cebolla À, Sousa C. Diversity in oat potential immunogenicity: basis for the selection of oat varieties with no toxicity in coeliac disease. Gut (2011), doi /gut Product Description The AgraQuant Gluten G12 is a 96 well Sandwich ELISA test kit which includes following items: Package Insert Certificate of Performance 5 standards (0, 4, 20, 80, 200 ppm), calibrated to the Prolamin Working Group (PWG) Gliadin. Gluten G12 antibody coated microwells Ready to use Extraction Solution 5x concentrated Diluent Buffer 10x concentrated Wash Buffer Ready to use Conjugate, Substrate and Stop Solutions 1 sachet of Fish Gelatin Limit of Detection: 2 ppm gluten Measurement Range: ppm gluten 3 Comino I, Real A, de Lorenzo L, Cornell H, López-Casado MÁ, Barro F, Loritte P, Torres MI, Cebolla À, Sousa C. Diversity in oat potential immunogenicity: basis for the selection of oat varieties with no toxicity in coeliac disease. Gut (2011), doi /gut Page 4

5 AgraQuant Gluten G12 - ELISA Validation Data Crossreactivity Food Sample Results in ppm Gluten Nuts Pecan < 4 Walnut < 4 Almond < 4 Cashew < 4 Macadamia < 4 Peanut < 4 Hazelnut < 4 Pine Nuts < 4 Pistachio < 4 Oils Hazelnut Oil < 4 Walnut Oil < 4 Vegetable Oil < 4 Sunflower Oil < 4 Seeds Golden Linseed < 4 Brown Linseed < 4 Poppy Seeds < 4 Sesame < 4 Naturally Gluten Free Foods Mustard < 4 Soya Mince < 4 Soya Beans < 4 Millet < 4 Buckwheat < 4 Rice Flour < 4 Quinoa < 4 Food Sample Results in ppm Gluten Starches Tapioca Starch < 4 Potato Starch < 4 Misc. Fennel < 4 Chickpea < 4 Soy Sauce < 4 Coriander < 4 Black Eyed Peas < 4 Puy Lentils < 4 Cardamon < 4 Food labbeled as Gluten Free Beers Wheat Flour > 200 Cookie Mix < 4 Chocolate Bar < 4 Cake Mix < 4 Shortbread 5.7 Breadcrumbs < 4 Pancake Mix < 4 Cake Mix < 4 Soy Sauce < 4 Hoegaarden > 200 (Wheat Beer) Pedigree (Ale) < 4 Budvaar (Lager) < 4 Pedigree (Ale) < 4 (not extracted) As expected the AgraQuant Gluten G12 kit clearly detects gluten in wheat flour and wheat beer. Moreover, no crossreactivity with the tested nuts, oils, seeds, starches etc. was observed. Processed and spiked samples Comparative data of AgraQuant Gluten G12 ELISA and an R5 ELISA with processed and spiked food samples show comparable performance. Some examples are displayed in the table. Sample AgraQuant Gluten G12 ELISA Gluten [ppm] R5 ELISA Gluten [ppm] Fruity Muesli (Baby Food) Maize flour (naturally contaminated) Honey and Oat Cookie (gluten free) Pita Bread (gluten free) 4.3 <5 English Muffins (gluten free) 4.8 <5 Spaghetti (gluten free) 6.9 <5 Shortbread (gluten free) <4 <5 Chocolate Brownie (gluten free) <4 <5 Cherry Bakewell (gluten free) <4 <5 Bread >80 Corn Flakes >200 >80 Crisps (5 ppm spike) 4.2 <5 Chocolate (5 ppm spike) 5.0 <5 Page 5

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