Study of Ig E cross-reactivity of Blomia tropicalis

Study of Ig. E 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

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

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

Symptoms Asthma n Rhinitis (nose) n Dermatitis (skin) n Conjunctivitis (eyes) n Anaphylaxis n

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

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

Sources of allergens Food n Pollen n Mould n Insects n Dust mites n

Treatment n Pharmacotherapy n n n Antihistamine Steroids Immunotherapy (Allergy shots) n n n Injection of tiny amts of allergen in increasing doses Increase in Ig. G Decrease in Ig. E

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

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

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

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

Materials Human sera: 42 Italian, 55 Singaporean n Recombinant allergen n (Bt & Df Grp 2, 3 & 5) • p. ET 32 • E. coli (BL 21) • Expressed as a fusion protein with his-tag, s-tag & trx. A

Immuno-dot blot • System negative control : serum from non-allergenic individual • alkaline phosphatase anti-human Ig. E , alkaline phosphatase substrate Ig. E Positive ctrl Bt x Df x Bt Df p. ET Negative ctrl

Competitive inhibition Serum Diluant inhibitor • Inhibitor replaced by p. ET vector for zero-inhibition Test allergen

Competitive inhibition Inhibit with Bt x Detect allergens No inhibition Inhibit with Df x Increasing conc. Ig. E Bt x Df x Bt Df p. ET • concentration of inhibitor: 2 g/ l , 4 g/ l , 6 g/ l

Problem 1 Low reactivity of positive control n Ig. E conc. = 250 IU n Cause: 2 oab n

Problem 2 • 0 inhibition has lower reactivity than those with inhibition Inhibit with Bt x Increasing conc. r. Bt 2 r. Df 2

Results/Discussion r. Bt 2 Ig. E r. Bt 2 r. Df 2 p. ET r. Df 2 • Serum=Singaporean Sera (2220) • Complete inhibition for both allergens • Both allergens contain same epitopes which bind to Ig. E of this sera

Results/Discussion r. Bt 2 Ig. E r. Bt 2 r. Df 2 p. ET r. Df 2 • Serum=pool (I 221, I 86, 230, 22523, I 146) • r. BT 2 unable to inhibit r. DF 2 • r. DF 2 is able to inhibit r. BT 2 completely • r. DF 2 contains some unique epitopes not in r. BT 2

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

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

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

Hydropatic plots (Group 2)

Dot plot (Group 2)

Bioinformatics (Group 2) Allergen (hydropathic value, EIIP) Der f 2 C I I H R G K (-0. 0714, 0. 035) Blo t 2 CI IHKGK (0. 0143, 0. 0266) Der p 2 C 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 1 CV I H R G E (-0. 0571, 0. 0314) Tyr p 2 CV I H K S K (-0. 0857, 0. 0386) Homo sapiens C I I N R G K (-0. 1143, 0. 0320) similar to b. A 494 O 16. 1 (KIAA 0637) • EIIP: electron-ion interaction potential

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

Conclusion Homologous Bt and Df allergens contain unique and common Ig. E binding epitopes n Different sera have different crossreactivity profile n

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

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

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