Antibody microarrays for allergen standardization

1. Antibody microarrays for allergen standardization

2. How do we measure potency? • Total protein (hymenoptera) • Overall allergen (grasses, mites) • Pooled human antibody • Specific allergen (cat, ragweed) • Sheep antibody

3. Radial immunodiffusion assay with monospecific antiserum • Examples: cat, ragweed • Advantages • quantitative • monospecific • Disadvantages • need to identify relevant allergen(s)

4. Competition ELISA with pooled allergic human sera • Examples: mites, grasses • Advantages • quantitative • reflects spectrum of allergen recognition • does not require identification of relevant allergens • Disadvantages • use of pooled sera • effects of fluctuations in individual allergens difficult to measure

5. Specific loss of a single allergen

6. The dilemma of these potency measures: • In order to measure specific allergens, we need to know which allergens are relevant • If we measure overall allergenicity, we are unable to detect the absence of specific (and potentially important) allergens

7. Two possible solutions: • Divide the signal by • Separating the allergens, or • Separating the antibodies

8. Western blot with pooled allergic human sera • Advantages • identification of individual protein bands • reflects spectrum of allergen recognition • does not require identification of relevant allergens • Disadvantages • qualitative • use of pooled sera • no definite identification of allergens • cross-reactivity

9. Antibody microarray • Advantages • quantitative • reflects spectrum of allergen recognition • does not require identification of relevant allergens • Disadvantage • New technology; initial development will be labor intensive and expensive

10. Aims • To develop an antibody microarray method for profiling complex allergen mixture • Apply this technique to German cockroach allergen standardization

11. We are NOT developing allergen microarrays… Deinhofer et al. Microarrayed allergens for IgE profiling. Methods. 2004 Mar;32(3):249-54. Jahn-Schmid et al. Allergen microarray: comparison of microarray using recombinant allergens withconventional diagnostic methods to detect allergen-specific serum immunoglobulin E. Clin Exp Allergy. 2003 Oct;33(10):1443-9. Beyer. Characterization of allergenic food proteins for improved diagnostic methods. Curr Opin Allergy Clin Immunol. 2003 Jun;3(3):189-97. Review. Fall et al. Microarrays for the screening of allergen-specific IgE in human serum. Anal Chem. 2003 Feb 1;75(3):556-62. Harwanegg et al. Microarrayed recombinant allergens for diagnosis of allergy. Clin Exp Allergy. 2003 Jan;33(1):7-13. Review. Kim et al. Quantitative measurement of serum allergen-specific IgE on protein chip. Exp Mol Med. 2002 May 31;34(2):152-8. Hiller et al. Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment. FASEB J. 2002 Mar;16(3):414-6.

12. …but antibody microarrays Glokler and Angenendt. Protein and antibody microarray technology.J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Nov 25;797(1-2):229-40. Haab. Methods and applications of antibody microarrays in cancer research. Proteomics. 2003 Nov;3(11):2116-22. Hallborn and Carlsson. Automated screening procedure for high-throughput generation of antibody fragments. Biotechniques. 2002 Dec;Suppl:30-7. Review. Schweitzer and Kingsmore. Measuring proteins on microarrays.Curr Opin Biotechnol. 2002 Feb;13(1):14-9. Review. Borrebaeck. Antibodies in diagnostics - from immunoassays to protein chips. Immunol Today. 2000 Aug;21(8):379-82. Review.

13. Allergen mixture …which bind to specific allergens Spotted clonal antibodies… Nitrocellulose coated chip Antibody microarray plan

14. Need clonal antibodies • Standard monoclonal antibody generation, or • Phage-display technology • Hyperimmunize chickens or rabbits • RNA from spleens/bone marrow • RT-PCR and PCR to generate cDNA libraries with expression of antibody on phage surface • Select and enrich for allergen-specific cDNA using phage panning • Express selected antibodies

15. Advantages of phage-display technology • Theoretically cloning the entire immunoglobulin repertoire • Large quantities of allergen-specific antibodies are potentially available for: • Structural studies • Allergen purification • Definition of the B-cell epitopes of major allergens • Analysis of complex protein mixtures by antibody microarray • Retrieved antibodies can be expressed in E. coli or yeast • The use of several species can mean the generation of fragments with considerably different specificities.

16. Which animal models and why? Why not mice? • We decided on two animal models: rabbit and chicken. • Mice provide small samples and have a large number of V-region families (complex PCRs to construct libraries). • Rabbits provide large quantities of sera, and have a small set of V-region families (simpler) and are a well-established model for recombinant antibody generation. • Chickens are useful as: • They often respond strongly to mammalian antigens which generate a weak response in mammals (cat/dog allergens). • They do not require bleeding. IgY collected from egg yolk. • The chicken genome encodes only two immunoglobulin variable domains! This means a very small primer set, reduction of primer bias in library construction.

17. Use of Fabs versus scFv in phage-display scFv = single chain Fragment variable Fab = Fragment antibody binding.

18. Oligo dT Priming cDNA synthesis TTTTTTTTTTTTTTTTTTT AAAAAAAAAAAAAAAA mRNA polyA tail PCR 1. First-Round PCR Light chains Heavy chains VL + CL Heavy Chain (Fd)

19. 2. Second-Round PCR κ λ VL + CL ~750 bp 3. Third-Round PCR +

20. VL CL VH CH Complete Fab product

21. VL CL VH CH M13 filamentous phage Fab Fragment pCOMB3X vector P3 coat protein Fab fragment display on phage surface protein p3

22. Remove unbound phage with PBSt incubate 1011 scFv - expressing phage per well Bound allergen P H A G E Release bound phage with trypsin Non-specific scFv HA tag specificscFv Isolate phage Infect E. coli and expand phage overnight Methods used for panning phage libraries.

23. Current directions for antibody phage display in LIB • Generation of mono-specific recombinant antibody fragments from rabbit and chicken to allergenic proteins Fel d 1, Amb a 1 and whole yellow jacket venom. • Generation of antibody fragments from rabbit and chicken to the allergenic proteins of German and American cockroaches.

24. Animal libraries built so far.

25. Animal libraries built so far.

29. Chicken scFv exhibits excellent specificity and sensitivity in Western Blot. Gel stained anti-Fel d 1 anti-Amb a 1 anti-YJV 250 kD 75 kD 50 kD 30 kD 15 kD 10 kD In each blot (left to right): Markers, Yellow Jacket Venom, Ragweed extract, Cat hair extract.

30. Conclusions • We can raise chicken antibodies against multiple antigens from a single immune library. • Panning against multiple proteins at once can work effectively. • Antibodies raised against rFel d 1 recognize nFel d 1. • Chicken scFv molecules secrete well in E. coli. • His-purified scFv functions effectively in ELISA and Western blot.

31. Animal libraries built so far.

32. Simple arrays Dense arrays Antigen Fluorophore labeling Fab Antigen scFv Spotted antibodies bind specific proteins Nitrocellulose coated chip

33. Simple arrays Fab Antigen scFv • Apply scFv anti-Amb a 1 OR scFV anti-Fel d 1 to nitrocellulose slide • Block with ovalbumin • Incubate with • ragweed extract or • cat hair extract or • negative control (ova) • Polyvalent rabbit serum R91 (anti-Amb a 1 + anti-Fel d 1 + anti-YJV) • Anti-rabbit conjugate

34. Animal libraries built so far.

35. ragweed 1:20 Cat hair 1:20 1% ovalbumin Anti-Amb a 1 Anti-Fel d 1

36. Microarray development plan • Develop a quantifiable “fingerprint” of complex allergen mixtures using clonal allergen-specific scFvs and polyvalent sera • Advance to more complex allergen extracts • Yellow jacket venom • German cockroach • American cockroach • How are we going to assure that our arrays recognize diverse allergens/epitopes? • BstOI and Western analyses • cluster analyses

37. Key players • Jonny Finlay, PhD • Nicolette deVore, PhD