f 416 r Tri a 19 Omega5 Gliadin

f 416 r. Tri a 19; Omega-5 Gliadin - a risk marker for severe immediate or exerciseinduced reactions in patients with suspected wheat allergy 2007 -03 -29 1

Classification of wheat proteins Albumin Water/salt soluble Globulin Wheat proteins Water/salt insoluble (Gluten 85%) Gliadins (α-, β-, γ-, ω-1, 2, 3, 4, ω-5) Glutenins (HMW*-, LMW-) 2007 -03 -29 2

n What is on Immuno. CAP® f 416 r. Tri a 19; Omega-5 Gliadin? Tri a 19; Omega-5 Gliadin from wheat (Triticum aestivum). n Omega-5 gliadin is a storage protein and a major allergen among water/salt–insoluble proteins, with limited presence on f 4 Wheat Immuno. CAP. n Omega-5 gliadin can be found to some extent on f 79 Gluten and to a higher extent on f 98 Gliadin Immuno. CAP. n The immunodominant peptide epitopes of omega-5 gliadin have been identified. n A c. DNA encoding a hydrophilic portion of Tri a 19, containing major epitopes for Ig. E antibodies, has been cloned and expressed in E. 3 2007 -03 -29 coli and the purified recombinant product

Possible clinical utility of Immuno. CAP® f 416 r. Tri a 19; Omega-5 Gliadin n Identify patients at risk of developing severe immediate reactions to wheat. n Identify patient at risk of developing anaphylaxis to wheat in relation to exercise (WDEIA). 2007 -03 -29 4

Severe immediate reactions to wheat n Are important to identify, especially in children. n ~ 80% of patients with severe immediate reactions have Ig. E antibodies to both omega-5 gliadin and wheat. n The remaining 20% of patients have Ig. E antibodies to other components in wheat and will thus be positive with f 4 Wheat Immuno. CAP. 2007 -03 -29 5

Doctors diagnosis of wheat allergy and levels of Ig. E ab to omega-5 gliadin in 88 children sensitized to wheat 37/44 (84%) > 0. 35 k. UA/l 100 12/44 (27%) > 0. 35 k. UA/l k. UA /L 10 1 0. 1 2007 -03 -29 No wheat allergy n = 44 Wheat allergy n = 44 6 Reference: Ito K et al. Poster presented at AAAAI in San Diego 2007

Challenge outcome and levels of Ig. E ab to omega-5 gliadin in children sensitized to wheat p<0. 001 100 k. UA /L 10 1 0. 1 Negative Challenge/History n = 44 2007 -03 -29 Mild n=5 Moderate n=8 Positive Challenge 7 Reference: Ito K et al. Poster presented at AAAAI in San Severe n=8

Wheat-dependent, exercise-induced anaphylaxis (WDEIA) n Wheat allergy induced by physical exercise. n Symptoms are typically generalized urticaria and severe allergic reactions such as anaphylactic shock or hypotension. n Skin tests and in vitro Ig. E ab tests for wheat are not always satisfactory. n Challenge test + intense physical exercise is the gold std for diagnosis, but is inconvenient and dangerous. 2007 -03 -29 8

Major allergens in WDEIA n ~80% of patients with WDEIA have Ig. E ab to omega-5 gliadin. n ~20% of patients with WDEIA have Ig. E ab to high molecular weight glutenin (HMWglutinin). n 30 -50% of patient with WDEIA are, however, negative with f 4 Wheat Immuno. CAP, but a majority are positive to omega-5 gliadin. 2007 -03 -29 9

Ig. E ab to different wheat proteins in patients with WDEIA Peptide A = Omega-5 gliadin Peptide B = HMW-glutinin 2007 -03 -29 10

Investigation of risk for severe immediate or exercise-induced reactions in patients with suspected wheat™allergy Test with Immuno. CAP Allergen: f 4 Wheat + f 416 r. Tri a 19; Omega-5 Gliadin Test results f 4 neg / f 416 neg Test results f 4 neg / f 416 pos Test results f 4 pos / f 416 neg Is there a history of exercise-induced symptoms? Yes Conclusion No sensitization to High risk for exercisewheat; symptoms are induced reactions probably caused by due to wheat another food or due to a different mechanism 2007 -03 -29 No Conclusion High risk for severe immediate reactions due to wheat 11 Conclusion High risk for severe immediate or exercise-induced reactions due to wheat Conclusion Low risk for severe immediate or exercise-induced reactions due to wheat

Recommended test procedure in patients with suspected wheat allergy f 4 Wheat + f 415 r. Tri a 19; Omega-5 gliadin n The clinical relevance of a positive f 4 Wheat test are sometimes difficult to interpret. n Omega-5 gliadin is found to be a risk marker for severe immediate reactions including WDEIA (80% pos). n 30 -50% of patients with WDEIA have no Ig. E abs to wheat (f 4). n f 4 neg & f 416 neg tests: no sensitization to wheat is found; another food or mechanism is probably the cause. n f 4 neg & f 416 pos tests: high risk for severe immediate reactions including WDEIA due to wheat. n f 4 pos & f 416 pos tests: high risk for severe immediate reactions including WDEIA due to wheat. f 4 pos & f 416 neg tests: low risk for severe immediate reactions including WDEIA due to wheat. However, many of these patients will 12 2007 -03 -29 have immediate type reactions such as skin and gastrointestinal n

A case report n Age: 57 n Gender: Male n Race group: White n Country of birth: Zambia n Living in: South Africa n Occupation: Engineer, Project Manager (often very stressful) n Activities: Camping, Cycling, Scuba diving, Hiking 2007 -03 -29 13

Anamnesis n Onset three years ago, June 2003. n 15 adverse reactions between June -03 and Feb n n n 06. Reaction one hour after eating with extreme itching from hairline to armpits, buttocks and groin where hives break out. Steadily increasing hart rate (180 beats vs. 80 normally). Tight chest with labored breathing. Loss of conscious ones with cardiac arrest. Mild reactions on a few occasions that were stopped with 3 antihistamine tablets. 14 2007 -03 -29

What is the offending allergen (s)? Immuno. CAP® tested in South Africa 13 Nov 2003 n Total Ig. E 1601 k. U/l n Phadiatop positive n Cat 1. 09 k. UA/l n Mixed Cockroach 0. 56 k. UA/l n Negative test results*: Dog, mites, trees, weeds, timothy grass, mould, egg, milk, soy, peanut, wheat, fish and shrimp 2007 -03 -29 31 March 2004 n Total Ig. E 1818 k. U/l n Phadiatop positive n Milk 0. 58 k. UA/l n Negative test results*: soy, peanut, wheat, fish, shrimp, cheese * Negative < 0. 35 k. UA/l 15

What is the offending allergen (s)? - Immuno. CAP® tested in South Africa 11 March 2006 n Total Ig. E 2315 k. U/l n Mixed cockroach 0. 81 k. UA/l (tropomyosin) n Avocado & pear negative* (Chitinase) n Pineapple negative* (Profilin) n Sunflower seed negative* (2 S albumin) n Peach negative* (LTP) n Strawberry negative* (Bet v 1) n Salmon negative* (Calcium binding protein/Polcalcin) n Yeast negative* (Enolase) n Bell pepper/paprika negative* (Thaumatin protein) * Negative < 0. 35 k. UA/l 2007 -03 -29 16

What is the offending allergen (s)? - Immuno. CAP® tested at Phadia in Sweden Fall 2006 n Phadiatop 0. 47 PAU/l n Wheat 3. 3 k. UA/l n Gluten 4. 4 k. UA/l n Pork 0. 81 k. UA/l n Gliadin 30. 5 k. UA/l n Mushroom 0. 1 k. UA/l n Omega-5 Gliadin 30. 5 k. UA/l n Sesame 0. 19 k. UA/l n Black pepper 0. 13 k. UA/l n Negative test results*: n Olive 0. 15 k. UA/l tomato, Gly m 4, Ara h 8, n Casein 0. 19 k. UA/l onion, grape, mustard, alfan Bovine lactoferrin 0. 64 lactalbumin, betak. UA/l lactoglobuline, CCD; MUXF 3 n Beef 1 k. UA/l n Anisakis 0. 15 k. UA/l * Negative < 0. 1 k. UA/l n Echinococcus 0. 91 17 2007 -03 -29 k. UA/l

Summary case report n Omega-5 gliadin from wheat was finally found to be the offending allergen. n Since wheat was tested negative already in South Africa it was not at once retested at Phadia. n To test for gluten (f 79) was a last try to find the offending allergen. n The positive gluten result (4. 4 k. UA/L) led into testing with gliadin (AGf 98) but also omega-5 gliadin (f 416) that was under development. n When omega-5 gliadin was tested positive (30. 5 k. UA/L) the wheat test was repeated and now found positive (3. 3 k. UA/L). n Similar levels of Ig. E ab to gliadin (AGf 98) and omega-5 gliadin (f 416), indicate that the amount of omega-5 gliadin is fairly 18 good represented on 2007 -03 -29 gliadin Immuno. CAP AGf 98.

Information material available New Horizon 1/2007; article & clinical leaflet, product leaflet and test chart See more info in Marketing Info No. 6 & 7/2007 -03 -29 19

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