1. Study of IgE cross-reactivity of Blomia tropicalis (Bt) and Dermatophagoides farinae (Df) Grp 2, 3 and 5 recombinant allergen Supervisor: Dr Chew Fook Tim Prepared by: Chua Gek Huey

2. Outline Background Hypothesis Objectives Methods Trouble shooting Results/ Discussion Future Work Acknowledgement

3. Background 1 in 3 people in the UK 40 to 50 million people in the United States 1 in 5 school children in Singapore asthmatic

4. Symptoms Asthma Rhinitis (nose) Dermatitis (skin) Conjunctivitis (eyes) Anaphylaxis

5. Overview of allergic immune response Source: The HOPGENE Program for Genomic Applications (copyright of John Hopkins University)

6. What is an allergen? Protein Criteria: Stimulate IgE production bind to at least one IgE release mediators such as histamine and leukotriene

7. Sources of allergens Food Pollen Mould Insects Dust mites

8. Treatment Pharmacotherapy Antihistamine Steroids Immunotherapy (Allergy shots) Injection of tiny amts of allergen in increasing doses Increase in IgG Decrease in IgE

9. Allergen characterization Complete characterization of allergen extracts difficult: Extract diversity Variability in patient responses Cross-reactivity A D C B D C E

10. Hypothesis Allergens of same group (homologous) may have unique epitopes, common epitopes different affinitiy for IgE binding in different serum

11. Objective Blomia tropicalis vs Dermatophagoides farinae To evaluate the degree of cross-reactivity between Blo t 2 vs Der f 2 Blo t 3 vs Der f 3 Blo t 5 vs Der f 5

12. Methods Competitive Inhibition Fluorescent allergosorbent (FAST) Radioallergosorbent (RAST) Enzyme-linked immunosorbent assay (ELISA) Western blot Immuno-dot blot (least serum needed)

13. Materials Human sera: 42 Italian, 55 Singaporean Recombinant allergen (Bt & Df Grp 2, 3 & 5) pET32 E. coli (BL21) Expressed as a fusion protein with his-tag, s-tag & trxA

14. Immuno-dot blot IgE Bt x Df x Bt Df pET Positive ctrl Negative ctrl System negative control : serum from non-allergenic individual alkaline phosphatase anti-human IgE, alkaline phosphatase substrate

15. Competitive inhibition Inhibitor replaced by pET vector for zero-inhibition SeruminhibitorDiluant Test allergen

16. Competitive inhibition concentration of inhibitor: 2  g/  l, 4  g/  l, 6  g/  l IgE Bt x Df x Bt Df pET No inhibition Inhibit with Bt x Increasing conc. Inhibit with Df x Increasing conc. Detect allergens

17. Problem 1 Low reactivity of positive control IgE conc. = 250IU Cause: 2 o ab

18. Problem 2 0 inhibition has lower reactivity than those with inhibition Inhibit with Bt x Increasing conc. rBt 2 rDf 2

19. Results/Discussion rBt 2rDf 2 Serum=Singaporean Sera (2220) Complete inhibition for both allergens Both allergens contain same epitopes which bind to IgE of this sera IgE rBt 2 rDf 2 pET

20. Results/Discussion rBt 2 rDf 2 Serum=pool (I221, I86, 230, 22523, I146) rBT2 unable to inhibit rDF2 rDF2 is able to inhibit rBT2 completely rDF2 contains some unique epitopes not in rBT2 IgE rBt 2 rDf 2 pET

21. Results/Discussion Most Italian serum not reactive to Blo t allergens but strongly reactive to Der f allergens Blo t & Der f contain different IgE binding epitopes

22. Proposal Standardize volume of diluted serum used Separate every dot into different well

23. Bioinformatics Hydropathic plots Dotplots Blastp unique short amino-acid sequences conserved within mites

24. Hydropatic plots (Group 2)

25. Dot plot (Group 2)

26. Bioinformatics (Group 2) Der f 2Der f 2C I I H R G K (-0.0714, 0.035) Blo t 2C I I H K G K (0.0143, 0.0266) Der p 2C I I H R G K (-0.0714, 0.035) Gly d 2 CV I H R G K (-0.1143, 0.0358) Eur m 2 CV I H R G T (0.3429, 0.0440) allergen A precursor CV I H K G K (-0.0286, 0.0274) [Psoroptes ovis] Lep D 1CV I H R G E (-0.0571, 0.0314) Tyr p 2CV I H K S K (-0.0857, 0.0386) Homo sapiensC I I N R G K (-0.1143, 0.0320) similar to bA494O16.1 (KIAA0637) EIIP: electron-ion interaction potential Allergen (hydropathic value, EIIP)

27. Bioinformatics Der f 2Der f 2LVGDNGVLAC Blo t 2LVGDHGVVAC Lep d 2LVGDHGVMAC Lep D 1L IGDHGVMAC Gly d 2 L IGEHGVLAC Streptococcus LVGDHGLVAN pneumoniae

28. Conclusion Homologous Bt and Df allergens contain unique and common IgE binding epitopes Different sera have different cross- reactivity profile

29. Future work Cross-reactivity Clustering of sera base on Cross-reactivity profile Bioinformatics tools to predict binding epitopes

30. Acknowledgement Dr Chew Fook Tim Dr Shang Huishen Ms Ong Seow Theng All Labmates (BII) Dr Aylwin Ng Dr Li Kuobin Ms Wong Siew Chin Ms Lim Yunping

32. References [1] American Academy of Allergy, Asthma and Immunology (AAAAI). The Allergy Report: Science Based Findings on the Diagnosis & Treatment of Allergic Disorders, 1996-2001. [2] Lancet. Worldwide variation in prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and atopic eczema: ISAAC. The International Study of Asthma and Allergies in Childhood (ISAAC) Steering Committee, 1998 Apr 25;351(9111):1225-32. [3] Goh DYT, Chew FT, Quek SC, Lee BW. Prevalence and severity of asthma, rhinitis, and eczema in Singapore school-children. Arch Dis Child 1996; 74:131-5. [4] Chatkin MN, Menezes AM, Victora CG, Barros FC. High prevalence of asthma in preschool children in Southern Brazil: A population-based study. Pediatr Pulmonol 2003 Apr;35(4):296-301. [5] Leung R, Wong G, Lau J, Ho A, Chan JK, Choy D, Douglass C, Lai CKEur. Prevalence of asthma and allergy in Hong Kong schoolchildren: an ISAAC study. Respir J 1997 Feb;10(2):354-60. [6] Shohat T, Green MS, Davidson Y, Livne I, Tamir R, Garty BZ. Differences in the prevalence of asthma and current wheeze between Jews and Arabs: results from a national survey of schoolchildren in Israel. Ann Allergy Asthma Immunol. 2002 Oct;89(4):386-92. [7] Johannes Ring, Ursula Kramer, Torsten Schafer, Heridrun Behrendt. Why are allergies increasing? Current Opinion in Immunology 2001. 13:701- 708. [8] Janeway, Charles A.; Travers, Paul; Walport, Mark; Shlomchik, Mark. Immunobiology. 5th ed. 2001. [9] Finegold I. Immunotherapy and asthma. J Asthma. 2003;40 Suppl:31-5. [10] Arlian LG, Morgan MS, Neal JS. Dust mite allergens: ecology and distribution. Curr Allergy Asthma Rep. 2002 Sep;2(5):401-11.

33. References [11] Chew FT, Lim SH, Goh DY, Lee BW. Sensitization to local dust-mite fauna in Singapore. Asian Pac J Allergy Immunol. 1999 Sep;17(3):179-88. [12] Hage-Hamsten M. Dermatophagoides siboney and Blomia tropicalis dust mites of subtropical and tropical areas. Clin Exp Allergy 1995;25:905-7. [13] Chew FT, Yi FC, Chua KY, Fernandez-Caldas E, Arruda LK, Chapman MD, et al. Allergenic differences between the domestic mites Blomia tropicalis and Dermatophagoides pternyssinu. Clin Exp Allergy 1999;29:982-8. [14] Morgan MA, Arlian LG, Fernandez-Caldas E. Cross-allergenicity of the house dust mites Euroglyphus maynei and Blomia tropicalis. Ann Allergy Asthma Immunol 1996;77:386-92. [15] Johansson E, Schmidt M, Johansson SG, Machado L, Olsson S, Hage-Hamsten M. Allergenic crossreactivity between Lepidoglyphus destructor and Blomia tropicalis. Clin Exp Allergy 1997;27:691-9. [16] Ferrandiz R, Casas R, Dreborg S. Cross-reactivity between Dermatophagoides siboney and other domestic mites. II. Analysis of individual cross- reacting allergens after SDS-PAGE and Western blotting inhibition. Int Arch Allergy Immunol 1998;116:206-14. [17] Kuo IC, Cheong N, Trakultivakorn M, Lee BW, Chua KY. An Extensive study of human IgE cross-reactivity of Blo t 5 and Der p 5. J Allergy Clin Immunol. 2003 Mar;111(3):603-9. [18] Saul B. Needleman and Christian D. Wunsch. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol., 48:443-453, 1970. [19] Smith T.F. and Waterman M.S. Identification of common molecular subsequences. J. Mol. Biol., 147:195-197, 1981. [20] Kyte J., Doolittle R.F. Amino acid scale: Hydropathicity. J. Mol. Biol. 157:105-132(1982). [21] Helfman, Dotplot Patterns: a Literal Look at Pattern Languages, TAPOS, 2(1):31-1,1995.