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Prepared by: Chua Gek Huey

Study of IgE cross-reactivity of Blomia tropicalis (Bt) and Dermatophagoides farinae (Df) Grp 2, 3 and 5 recombinant allergen. Prepared by: Chua Gek Huey. Presentation. Part 1: Competitive inhibition Part 2: Manual Epitope prediction Part 3: Allergen Prediction.

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Prepared by: Chua Gek Huey

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

  2. Presentation • Part 1: Competitive inhibition • Part 2: Manual Epitope prediction • Part 3: Allergen Prediction

  3. Part 1: Competitive Inhibition Part 1: Competitive Inhibition

  4. Motivation • Allergy on the increase • 1 in 3 in UK has allergy related problem Link • 40 to 50 million people in the United States Link • 1 in 5 school children in Singapore asthmatic • Immunotherapy: hypoallergen • Allergen characterization • Extract diversity • Variability in patient response • House dust mite – a major source of allergen

  5. Hypothesis • Homologous allergens have • Some unique epitopes • Some common epitopes • Sera of different allergenic individuals have different level of cross-reactivity and affinity for homologous allergens

  6. Objective • Assess levels of IgE cross-reactivity between • rBlo t 2 and rDer f 2 • rBlo t 3 and rDer f 3 • rBlo t 5 and rDer f 5 • Clustering of patients base on cross-reactivity profile

  7. Method • Competitive inhibitionLink • Allergen composition • Patient immune responses • Recombinant allergen • pET32 in E. Coli (BL21) • Expressed as fusion protein with s-tag, his-tag, trxA (his-tag for protein purification using resin) • Italian and Singaporean sera Sera 1 Sera 2 Bt 2 Df 2

  8. Methods • Immuno-dot blot (least sera needed) • Fluorescent allergosorbent (FAST) • Cross-immunoelectrophoresis (CIE) • Cross enzyme-immunoelectrophoresis (CEIE) • Enzyme-linked immunosorbent assay (ELISA) • Western blot

  9. Immuno-dot blot • Alkaline phosphatase goat anti-human IgE (1:600) • Visualized with BCIP/NBT staining (1:1500) • Nitrocellulose membrane • Test allergen (liquid phase) – Urea free • Dectection allergen (solid phase)

  10. BCIP/NBT (contains a phosphate group) Colored Product Tagged with alkaline-phosphatase Goat anti-human Human anti-allergen Blocking Buffer Detection allergen BCIP/NBT Visualization

  11. s-tag his-tag trxA Allergen insert Controls • Internal negative control: pET vector • Ensure IgE not bound to pET vector • System negative control: non-allergenic sera • Internal positive control: IgE

  12. Test allergen or pET Serum Diluant inhibitor Inhibition

  13. s-tag s-tag his-tag his-tag trxA trxA Allergen insert Experimental set up Increasing concentration of test allergen No inhibition Pre-absorption Sera + pET vector Sera + test allergen

  14. Draw inhibition curve (1)

  15. Draw inhibition curve (2) 1. Change to red 2. Take down reading, I • Intensity = (255 – I)

  16. Inhibit with Bt x x 1 1 7 6 1 4 3 2 8 5 Calculation of % inhibition - 100% x Draw inhibition curve (3)

  17. Inhibition Curve • Percent inhibition plotted against concentration of allergen in diluted sera • Potency: concentration corresponding to 50% inhibition • Slope: allergen affinity • Always plot self inhibition and cross-inhibition • Always plot 2 separate graphs when comparing 2 allergens

  18. Inhibition Curve • Slope: affinity (high affinity => steep slope) • Potency: 50% inhibition (more potent => lower conc.) A: typical self inhibition AlaSTAT

  19. % inhibition % inhibition 100 100 rDf x rBt x 50 50 Conc. Of rDf x (ug/ul) Conc. Of rBt x (ug/ul) Inhibition Curve • rDf x contains epitopes not in rBt x • rBt x epitopes are in rDf x • * Only applicable to the sera used

  20. Problem 1 • Low reactivity of positive control • IgE conc. = 250IU • Cause: 2oab degradation

  21. Test allergen: rDf 2 Test allergen: rBt 2 rBt 2 rDf 2 Problem 2 (1) • Lower reactivity of 0 inhibition

  22. Problem 2 (2) • Lower reactivity of 0 inhibition Test allergen: rDf 2 Test allergen: rBt 2 rBt 2 rDf 2

  23. Solution to Problem 2 • Include pET pre-absorption for all sera • Use purified pET • Point to consider: • Total concentration of protein in diluted sera should be constant to eliminate reduced reactivity due to steric hinderance

  24. rDf 2 rBt 2 Result/Discussion • Some Italian sera reactive to only Df 2 • Some Singaporean sera reactive to only Bt 2 (CSW) • Bt 2 contains unique epitopes not found in Df 2 and vice versa • Some Singaporean sera reactive to both • Bt 2 and Df 2 have some common epitopes

  25. rBt 2 rDf 2 IgE rBt 2 rDf 2 pET Result/Discussion (Sera 2220) • rBt 2 can inhibit rDf 2 completely • rDf 2 can inhibit rBt 2 completely • Conclusion: • both rBt 2 and rDf 2 have • same specificity • * for this sera only

  26. rBt 2 rDf 2 Result/Discussion (Pool sera) • Conclusion: • rBt 2 is a sub set of rDf 2 • * for this sera only • rBt 2 can’t inhibit rDf 2 completely • rDf 2 can inhibit rBt 2 completely rBt 2 rDf 2 IgE rBt 2 rDf 2 pET

  27. No inhibition: Sera + test allergen + diluant Add test allergen +diluant Add diluant Proposal for change (1) Proposed change: Current protocol: Sera + pET (1:1) No inhibition: Sera + pET + diluant

  28. Proposal for change (1) • Minimize experimental deviation • Point to note: • Add BSA to get constant test allergen/BSA/pET concentration across a set of experiment

  29. IgE rBt x rDf x Bt Df pET IgE rBt x rDf x Bt Df pET Proposal for change (2) Proposed change: Current protocol:

  30. Proposal for change (2) • Ensure constant quantity of sera added for each dot • Volume of diluted sera added to each dot must be decided right from screening step

  31. Planning: • Use ELISA to measure concentration of purified recombinant protein and BSA • Set maximum test allergen concentration in diluted sera which is dependent on test allergen concentration • rBt 2 = 5ug/ul and rDf 2 = 10ug/ul • Set max allergen concentration in diluted sera = 0.5 x lower test allergen concentration = 0.5 x 5ug/ul = 2.5ug/ul

  32. Planning: 3. Back calculate dilution factor • 10ul of sera + 10ul of pET + 100ug of test allergen (20ul of rBt 2 or 10ul of rDf 2) Total volume of diluted sera = 40ul • Dilution factor = 1:3 (10ul sera + 30ul PBS/BSA) Note: Final BSA concentration in diluted sera = 2.5ug/ul • For detection allergen, ensure equal concentration of both detection allergens

  33. Conclusion • rBt 2 and rDf 2 their own unique epitopes • More sera have to be used in order to draw conclusions on their cross-reactivity

  34. Part 2: Manual Epitope prediction Part 2: Manual Epitope Prediction

  35. Bioinformatics Tools used • Emboss • Global alignment (Needle) • Local alignment (Water) • Dotplot (Dotmatcher) • Wordmatch • NCBI Blast • Search for short nearly exact match • ProtScale • Hydropathic plots (Kyte & Doolittle)

  36. Bt 2 and Df 2 hydropathic plot

  37. Bt 2 and Df 2 dotplot

  38. Grp 2 Allergen (Bt 2 & Df 2) • BT2_040 (141aa), BT046 (144aa), BT2_047 (144aa) • DF2 (140aa) • Global alignment (Needleman) • Identity: 53/149 (35.6%) • Similarity: 83/149 (55.7%) • Local alignment (Smith Waterman) • Identity: 50/116 (43.1%) • Similarity: 74/116 (63.8%)

  39. Example 1: Allergen (hydropathic value, EIIP) Der f 2 C I I H R G K (-0.0714, 0.035) Blo t 2 C I I H K G K (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 bA494O16.1 (KIAA0637) • EIIP: electron-ion interaction potential

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

  41. Part 3: Auto Epitope prediction Part 3: Allergen Prediction

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