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

4. Motivation Allergy on the increase 1 in 3 in UK has allergy related problem Link Link 40 to 50 million people in the United States Link 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 inhibition LinkLink Allergen composition Patient immune responses Recombinant allergen pET32 in E. Coli (BL21) pET32 Expressed as fusion protein with s-tag, his-tag, trxA (his-tag for protein purification using resin) trxA Italian and Singaporean sera Bt 2Df 2 Sera 1Sera 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 Visualization Blocking Buffer Detection allergen Human anti-allergen Goat anti-human Tagged with alkaline- phosphatase BCIP/NBT (contains a phosphate group) Colored Product

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

12. Inhibition SeruminhibitorDiluant Test allergen or pET

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

14. Draw inhibition curve (1)

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

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

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

19. Inhibition Curve % inhibition Conc. Of rDf x (ug/ul) 100 50 % inhibition Conc. Of rBt x (ug/ul) 100 50 rDf x rBt x 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: 2 o ab degradation

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

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

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. 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 rBt 2 rDf 2

25. Result/Discussion (Sera 2220) IgE rBt 2 rDf 2 pET rBt 2 rDf 2 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. Result/Discussion (Pool sera) IgE rBt 2 rDf 2 pET rBt 2 rDf 2 rBt 2 can’t inhibit rDf 2 completely rDf 2 can inhibit rBt 2 completely Conclusion: rBt 2 is a sub set of rDf 2 * for this sera only rBt 2 rDf 2

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

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

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: 1. Use ELISA to measure concentration of purified recombinant protein and BSA 2. Set maximum test allergen concentration in diluted sera which is dependent on test allergen concentration 1. rBt 2 = 5ug/ul and rDf 2 = 10ug/ul 2. 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 1. 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 2. 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

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: 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)

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

41. Part 3: Auto Epitope prediction

51. Proposed method: Resonant Recognition Model (RRM) Protein sequences transformed into numerical sequences using electron-ion interaction potential (EIIP) EIIP: average energy states of all valence electrons Calculated using a general model pseudopotential (parameters: change in momemtum, delocalized electron in interaction with potential, w)

52. Digital Signal Processing Numerical series analyzed by digital signal analysis (Fourier transformation, wavelet transformation) Findings: One peak only exists for a group of protein sequences sharing same biological function No significant peak exists for biologically unrelated protein sequences Peak frequencies are different for different biological functions

53. 1 st Objective Replace MEME by RRM method for motif detection Improve the current allergen prediction algorithm May replace EIIP values by hydropathic values

54. 2 nd Objective Epitope prediction through observation of hydropathic plots Make use of mapped epitopes Observe hydropathicity and freq spectrum of these epitopes May have to interpolate if window length is small Collect as much information to find epitopes unique characteristics

55. Problems to be solved No standardized database for testing

56. Acknowledgement Dr Chew Fook Tim Dr Shang Huishen Ms Ong Seow Theng Ms Ong Tan Ching All lab 3 and 1 labmates not forgetting Ms Lim Puay Ann