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
Outline
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Background
Hypothesis
Objectives
Methods
Trouble shooting
Results/ Discussion
Future Work
Acknowledgement
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

Symptoms
Asthma
 Rhinitis (nose)
 Dermatitis (skin)
 Conjunctivitis (eyes)
 Anaphylaxis

Overview of allergic immune
response
Source: The HOPGENE Program for Genomic Applications (copyright of John Hopkins University)
What is an allergen?
Protein
 Criteria:
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Stimulate IgE production
bind to at least one IgE
release mediators such as histamine and
leukotriene
Sources of allergens
Food
 Pollen
 Mould
 Insects
 Dust mites
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Treatment
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Pharmacotherapy
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Antihistamine
Steroids
Immunotherapy (Allergy shots)
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Injection of tiny amts of allergen in
increasing doses
Increase in IgG
Decrease in IgE
Allergen characterization
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Complete characterization of allergen extracts
difficult:
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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 IgE binding in different serum
Objective
Blomia tropicalis vs Dermatophagoides
farinae
 To evaluate the degree of cross-reactivity
between
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Blo t 2 vs Der f 2
Blo t 3 vs Der f 3
Blo t 5 vs Der f 5
Methods
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Competitive Inhibition
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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
 Recombinant allergen

(Bt & Df Grp 2, 3 & 5)
• pET32
• E. coli (BL21)
• Expressed as a fusion protein with his-tag, s-tag
& trxA
Immuno-dot blot
• System negative control : serum from non-allergenic individual
• alkaline phosphatase anti-human IgE , alkaline phosphatase substrate
IgE
Positive ctrl
Bt x
Df x
Bt
Df
pET
Negative ctrl
Competitive inhibition
Test allergen
Serum
Diluant
inhibitor
•Inhibitor replaced by pET vector for zero-inhibition
Competitive inhibition
Detect
allergens
No
inhibition
Inhibit with Bt x
Inhibit with Df x
Increasing conc.
Increasing conc.
IgE
Bt x
Df x
Bt
Df
pET
• concentration of inhibitor: 2g/l , 4g/l , 6g/l
Problem 1
Low reactivity of positive control
 IgE conc. = 250IU
 Cause: 2oab
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Problem 2
• 0 inhibition has lower reactivity than those with inhibition
40
Inhibit with Bt x
35
Increasing conc.
rBt 2
rDf 2
Intensity
30
25
Blo t 2
20
Der f 2
15
10
5
0
0
10
Concentration of allergen
(ug/ul)
20
Results/Discussion
rBt 2
rDf 2
•Serum=Singaporean Sera
(2220)
IgE
rBt 2
rDf 2
pET
• Complete inhibition for
both allergens
•Both allergens contain same
epitopes which bind to IgE
of this sera
Results/Discussion
rBt 2
rDf 2
•Serum=pool
(I221, I86, 230, 22523, I146)
IgE
rBt 2
rDf 2
pET
• rBT2 unable to inhibit
rDF2
• rDF2 is able to inhibit
rBT2 completely
• rDF2 contains some unique
epitopes not in rBT2
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
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Proposal
Standardize volume of diluted serum
used
 Separate every dot into different well
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Bioinformatics
Hydropathic plots
 Dotplots
 Blastp
 unique short amino-acid sequences
 conserved within mites
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Hydropatic plots (Group 2)
Dot plot (Group 2)
Bioinformatics (Group 2)
Allergen
Der f 2
Blo t 2
Der p 2
Gly d 2
Eur m 2
allergen A precursor
Lep D 1
Tyr p 2
CI IHRGK
CI IHKGK
CI IHRGK
CV I H R G K
CV I H R G T
CV I H K G K
CV I H R G E
CV I H K S K
Homo sapiens
CI INRGK
similar to bA494O16.1 (KIAA0637)
(hydropathic value, EIIP)
(-0.0714, 0.035)
(0.0143, 0.0266)
(-0.0714, 0.035)
(-0.1143, 0.0358)
(0.3429, 0.0440)
(-0.0286, 0.0274) [Psoroptes ovis]
(-0.0571, 0.0314)
(-0.0857, 0.0386)
(-0.1143, 0.0320)
• EIIP: electron-ion interaction potential
Bioinformatics
Der f 2
Blo t 2
Lep d 2
Lep D 1
Gly d 2
LVGDNGVLAC
LVGDHGVVAC
LVGDHGVMAC
L IGDHGVMAC
L IGEHGVLAC
Streptococcus
pneumoniae
LVGDHGLVAN
Conclusion
Homologous Bt and Df allergens contain
unique and common IgE binding
epitopes
 Different sera have different crossreactivity profile
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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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