1. WAC 2011 Debates 4, 6 December 2011: 10:45 - 11:45 Skin test or in-vitro Test for Food Allergy? IgE “In vitro” Motohiro Ebisawa, MD, PhD

2. Disclosures of Motohiro Ebisawa, MD, PhD 1)Employee of Sagamihara National Hospital 2)Academic activities WAO: Board member at large, AAAAI: International Assembly vice-chair Japanese Society of Allergology: Board member Japanese Society of Pediatric Allergy: Board member 3)Grant support from Japanese government, Ministry of Labor, Health, and Welfare for 12 years as PI 4)No Conflicts of Interests

3. 3 RAST: First Generation RAST 1 st on the market in 1974, considerable variability & questionable quantification-no longer in use and term is no longer appropriate Allergen bound to paper disc All antibody isotypes bind: Ig of A,M,G,E class Bound IgE detected with polyclonal I 125 Anti-IgE Results reported as log-related classes or arbitrary units by interpolation of heterologous IgE anti-birch pollen curve Hamilton R, Adkinson F. J Allergy Clin Immunol 2004;114:213-25.

4. Historical Manual Chemistries RAST = disc allergosorbent 1 o (transitioned) 1968 Hycor Hy-Tec (paper disc based) FAST = Allergenics/Biowhittaker, fluorescent allergosorbent test MAST = Hitachi: thread pipette EAST = Sanofi Dignostics Pasteur Magic Lite = ALK/Corning/Bayer Matrix = Abbott Historical Semi-automated Chemistries Alastat, Diagnostic Products Corp. (biotinylated-allergen) AutoCAP, Pharmacia (Allergen insolubilized on sponge) “The Pearls and Pitfalls of Diagnostic Allergy Testing” developed by the ACAAI/AAAAI Specific IgE Test Task Force (SETTaF)

5. ImmunoCAP (250, 1000): Phadia (changed from Pharmacia, Jan 06) HyTec-288: Hycor Biomedical- Agilent (June 07) Immulite 2000/2500: Siemens Medical Solutions Diagnostics (Jan 07)

6. The antibody binds to the allergen on the solid phase Enzyme anti-IgE detects bound IgE All assays report in similar units (kUa/L) with comparable analytical sensitivities of 0.1 kUa/L All assays primarily use allergens from extracts

7. ImmunoCAP perceived as gold standard for in vitro IgE testing ”The Pharmacia CAP system is in world wide use and is a de facto standard to which other methods are compared” Dolen WK. Allergy 2003; 58: 717-723 ”The predicitive values associated with clinical evidence for ImmunoCAP cannot be applied to Turbo-MP and Immulite”NIH/NIAID Food allergy guideline. J Allergy Clin Immunol 2010; 126:S1-S58

8. In-vitro In-vivo IgE Antibody SPT High sensitivity*YesYes High specificity*YesYes High reproducibilityYesYes Quantitative results in kIU/L^YesNo WHO Standard calibratedYesNo Quality assurance test programYesNo Can be used independentlyYesNo of pharmaceutical treatment Can be used independentlyYesNo of patient skin status Time factor1-7 days15-30 minutes Cost factormore expensiveinexpensive Usefulness in motivating patientsobscuredramatic *Results may vary between specific bioassays ^Although all are expressed with same units, cannot compare results between different bioassays “The Pearls and Pitfalls of Diagnostic Allergy Testing” developed by the ACAAI/AAAAI Specific IgE Test Task Force (SETTaF)

9. IgE testing in vitro Standardization of reagents! No common standardization exists for SPT extracts Huge variability between extracts from different producers, and also from the same producer Blood tests are standardized to WHO ref World wide proficiency testing assure low CV% with ImmunoCAP IgE Van Ree R. JACI 2007; 119: 270-277

10. Probability of Reacting to a Food at a Given IgE Value Retrospective study Prospective study Logit model using log(kU A /L) Reference: Calculated from Sampson and Ho, J Allergy Clin Immunol 1997; 100: 444-51

11. Probability of failed challenge in relation to the specific IgE antibody levels for egg and milk respectively divided into three age groups Egg (n=764)Milk (n=861) T. Komata, L. Soderstorm, M.P. Borres, H. Tachimoto,and M.Ebisawa J Allergy Clin Immunol, 119(5); 1272-1274

12. Predicted threshold values for 90% and 95% probabilities for failing oral 359 challenge, for children below 1 years of age, 1 year of age and 2 years or older T. Komata, L. Soderstorm, M.P. Borres, H. Tachimoto,and M.Ebisawa J Allergy Clin Immunol, 119(5); 1272-1274 (U A /ml ） n PPV 90%95% < 1 year Egg2156.413.0 Milk2233.65.8 1 year Egg18710.923.0 Milk17720.838.6 2 years or older Egg36217.030.0 Milk27533.857.3

13. Positive and negative decision points using specific IgE antibody measurements obtained from challenge with raw or heated egg white H. Ando, A Urisu et al J Allergy Clin Immunol, 122 ;583-588 (U A /ml ） Raw egg whiteHeated egg white Specific IgE Egg whiteOvomucoidEgg whiteOvomucoid Positive decision point 7.385.2130.710.8

14. Probability curves for wheat and soybean 0.10.3131030100 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Specific IgE antibody concentration (kU A /L) Probability for failed challenge Wheat (n= 277) 0.10.3131030100 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Specific IgE antibody concentration (kU A /L) Probability for failed challenge Soybean (n= 272) T. Komata, L. Soderstorm, M.P. Borres, H. Tachimoto,and M.Ebisawa Allergol Int. 2009

15. Probability curves of Wheat and its age effect 0.10.3131030100 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Specific IgE antibody concentration (kU A /L) Probability for failed challenge Wheat (n=277) T. Komata, L. Soderstorm, M.P. Borres, H. Tachimoto,and M.Ebisawa Allergol Int. 2009 <1 y >=1 y

16. Plant Food Allergens Pollen cross- reactive components* LTPPollen non-cross-reactive components** PeanutAra h 8Ara h 9Ara h 1; Ara h 2; Ara h 3 Arah 4; Ara h 6; Ara h 7 Ara h 5 HazelnutCor a 1Cor a 8Cor a 9 Cor a 11 Cor a 2 SoybeanGly m 4Gly m 1Gly m 5 Gly m 6 Gly m 3 WheatTri a 12Tri a 14Tri a 19 (ω-5 gliadin) Tri a 21 - alfa gliadin Tri a 26 - HMW glutenin Tri a 28 - AAI dimer 0.19 Ana risk PRP-10 Profilin 16

17. Allergen components in wheat gluten albumins and globulins gliadinsglutenins Tri a 15 - AAI monomer Tri a 28 - AAI dimer Tri a 29, 30 - AAI tetramer Tri a 12 - profilin Tri a 14 - LTP Tri a 18 - hevein-like Tri a 25 - thioredoxin Tri a 33 - serpin Homologs to components in timothy Tri a 19 - omega-5 gliadin Tri a 21 - alfa/beta gliadin Tri a gamma gliadin Tri a omega-2 gliadin Tri a 26 - HMW glutenin Tri a 36 - LMW glutenin 17

18. Subjects and Methods 343 patients with suspected wheat allergy from 3 different hospitals in Japan. Age range: 6 months - 20.4 years. Median age: 2.3 years old. Oral wheat challenge were performed for 339 children. Four children were included with a recent convincing case history of anaphylaxis in relation to wheat. Ebisawa M, Söderström L, Ito K, Shibata R, Sato S, Tanaka A, Borres M and Morita E, EAACI ’09

19. Skin113 (82%) Cough50 (36%) Wheeze28 (20%) Gastrointestinal9 (7%) Anaphylaxis3 (2%) Nausea9 (7%) OAS10 (7%) Summary of positive reactions by challenge 138 children had positive reactions Results

20. Ebisawa M et al,Int Arch Allergy Immunol, 2011(in press) IgE to wheat and ω-5 gliadin in wheat allergics and non-wheat allergics WA= wheat allergics 137 challenge positives 36 convincing history NoWA= no wheat allergics 78 challenge negative 60 convincing history

21. Probability curves for the outcome of wheat allergy at a given IgE value for ω-5 gliadin for all children and for children ≤ 1 year and >1 years of age Ebisawa M et al,Int Arch Allergy Immunol, 2011(in press)

22. Plant Food Allergens Pollen cross- reactive components* LTPPollen non-cross-reactive components** PeanutAra h 8Ara h 9Ara h 1; Ara h 2; Ara h 3 Arah 4; Ara h 6; Ara h 7 Ara h 5 HazelnutCor a 1Cor a 8Cor a 9 Cor a 11 Cor a 2 SoybeanGly m 4Gly m 1Gly m 5 Gly m 6 Gly m 3 WheatTri a 12Tri a 14Tri a 19 (ω-5 gliadin) Tri a 21 - alfa gliadin Tri a 26 - HMW glutenin Tri a 28 - AAI dimer 0.19 Ana risk PRP-10 Profilin 22

23. Allergen components in soy bean Allergen Cupin superfamilyProlamin superfamily PR-10Profilin 7S globulin /Vicillin 11S globulin /Legumin 2S albuminLTP Soy Gly m 5Gly m 6 Gly m 2S Albumin Gly m 1Gly m 4Gly m 3 Corresponding peanut allergen Ara h 1Ara h 3Ara h 2Ara h 9Ara h 8Ara h 5 23

24. IgE to Gly m 5 and Gly m 6 (soy) predict severe reactions Holzhauser et al, JACI 2009 IgE to Gly m 5 and/or Gly m 6 were found in 86% of the cases with anaphylaxis against soy 24

25. Ito, Ebisawa et al, JACI 2011 Gly m 5 & Gly m 6 Are Associated with Systemic Reactions in Soybean-allergic Japanese Children 25

26. Diagnostic value of measuring IgE to soybean 2S albumin in clinical assessment of soybean allergic Japanese children Quantitative measurement of IgE to 2S albumin from soybean in sera from 19 Japanese children allergic to soybean and 36 non- symptomatic controls Comparison of IgE level between symptomatic and non-symptomatic groups, the median levels are indicated. Sigrid Sjölander et al, EAACI 2009

27. Plant Food Allergens Pollen cross- reactive components* LTPPollen non-cross-reactive components** PeanutAra h 8Ara h 9Ara h 1; Ara h 2; Ara h 3 Arah 4; Ara h 6; Ara h 7 Ara h 5 HazelnutCor a 1Cor a 8Cor a 9 Cor a 11 Cor a 2 SoybeanGly m 4Gly m 1Gly m 5 Gly m 6 Gly m 3 WheatTri a 12Tri a 14Tri a 19 (ω-5 gliadin) Tri a 21 - alfa gliadin Tri a 26 - HMW glutenin Tri a 28 - AAI dimer 0.19 Ana risk PRP-10 Profilin 27 *Birch tree pollen, Timothy grass pollen for wheat ** Storage seed proteins, albumins and globulins

28. Peanut Ara h 2 Conglutin Ara h 1 Vicilin Ara h 3 Glycin Ara h 4 Glycin Ara h 5 Profilin Ara h 6 Conglutin Ara h 7 Conglutin Ara h 8 PR-10 Ara h 9 nsLTP Ara h 10 Oleosin Ara h 11 Oleosin Annica Önell Nov 2010 Peanut components

29. 29 Nicolaou et al. JACI. March 2011.. With components you correctly identify 97.5% of the peanut allergics

30. Allergy testing in the 21st century For primary care by eye or instrument For specialists

31. Quantitative sIgE reduces the risk and need for oral food challenge Sampson et al reduced the need with 40% Österballe et al reduced the need with 60% Sampson and Ho. J Allergy Clin Immunol 1997; 100: 444-51 Österballe M. J Allergy Clin Immunol 2003;112:196-201 “Shifted to Immuno CAP from SPT, more and more” by Dr. Hugh Sampson APAPARI 2011, in Fukuoka (Japan)