# Presentation: 330121

## Antibody microarrays for allergen standardization

## How do we measure potency?

- Total protein (hymenoptera)
- Overall allergen (grasses, mites)
- Pooled human antibody
- Specific allergen (cat, ragweed)
- Sheep antibody

## Radial immunodiffusion assay with monospecific antiserum

- Examples: cat, ragweed
- Advantages
- quantitative
- monospecific
- Disadvantages
- need to identify relevant allergen(s)

## 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

## Specific loss of a single allergen

## 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

## Two possible solutions:

- Divide the signal by
- Separating the allergens, or
- Separating the antibodies

## 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

## 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

## Aims

- To develop an antibody microarray method for profiling complex allergen mixture
- Apply this technique to German cockroach allergen standardization

## 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.

## ...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.

## Antibody microarray plan

## 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

## 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.

## 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.

## Use of Fabs versus scFv in phage-display

- scFv = single chain Fragment variable
- Fab = Fragment antibody binding.

## Slide 18

## Slide 19

## Complete Fab product

## Fab fragment display on phage surface protein p3

## Methods used for panning phage libraries.

## 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.

## Animal libraries built so far.

**Animal libraries built so far.**

## Animal libraries built so far.

**Animal libraries built so far.**

## Slide 26

## Slide 27

## Slide 28

## In each blot (left to right): Markers, Yellow Jacket Venom, Ragweed extract, Cat hair extract. 

**Chicken scFv exhibits excellent specificity and sensitivity in Western Blot.**

## 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.

## Animal libraries built so far.

**Animal libraries built so far.**

## Slide 32

- 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

## Animal libraries built so far.

**Animal libraries built so far.**

## 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?
*Bst*OI and Western analyses
- cluster analyses

## Key players

- Jonny Finlay, PhD
- Nicolette deVore, PhD