Antibody microarrays for allergen standardization

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How do we measure potency? Total protein (hymenoptera) Overall allergen (grasses, mites)

Pooled human antibody Specific allergen (cat, ragweed)

Sheep antibody

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Radial immunodiffusion assay with monospecific antiserum Examples: cat,

ragweed Advantages

quantitative monospecific

Disadvantages need to identify

relevant allergen(s)

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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

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Specific loss of a single allergen

0

0.1

0.2

0.3

0.4

0.5

-6-4-20

log dilution

resp

onse

(a

bsor

banc

e)

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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

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Two possible solutions: Divide the signal by

Separating the allergens, or

Separating the antibodies

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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

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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

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Aims To develop an antibody microarray

method for profiling complex allergen mixture

Apply this technique to German cockroach allergen standardization

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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.

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…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.

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Nitrocellulose coated chip

Antibody microarray plan

Spotted clonal antibodies…

…which bind to specific allergens Allergen mixture

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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

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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.

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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:

1. They often respond strongly to mammalian antigens which generate a weak response in mammals (cat/dog allergens).

2. They do not require bleeding. IgY collected from egg yolk.

3. The chicken genome encodes only two immunoglobulin variable domains! This means a very small primer set, reduction of primer bias in library construction.

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Use of Fabs versus scFv in phage-display

scFv = single chain Fragment variable

Fab = Fragment antibody binding.

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Heavy chainsLight chains

AAAAAAAAAAAAAAAATTTTTTTTTTTTTTTTTTT

Oligo dT Priming

mRNA polyA tail

cDNA synthesis

PCR

VL + CL

Heavy Chain (Fd)

1. First-Round PCR

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+

CL

VL

2. Second-Round PCR κ λ

~750 bp

3. Third-Round PCR

+

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Complete Fab product

VL VHCL CH

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Fab fragment display on phage surface protein p3

P3 coat protein

VL CL

VH CH

Fab Fragment

M13 filamentous phage

pCOMB3X vector

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Methods used for panning phage libraries.

HA tag

PHAGE

Non-specific scFv

Release bound phagewith trypsin

specificscFv

Bound allergen

incubate 1011 scFv -

expressing phage per well

Remove unbound

phage with PBSt

Infect E. coli andexpand phage overnight

Isolate phage

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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.

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Animal libraries built so far.

Chicken Rabbit AllergensC160 R91 rFel d 1

Amb a 1yellow jacket venom

C161 R90 rBla g 1rBla g 2rBla g 4rBla g 5

C162 R92 German CRC163 (nd) American CRC165 (nd) rBla g 2C168 (nd) rBla g 4

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Animal libraries built so far.

Chicken Rabbit AllergensC160 R91 rFel d 1

Amb a 1yellow jacket venom

C161 R90 rBla g 1rBla g 2rBla g 4rBla g 5

C162 R92 German CRC163 (nd) American CRC165 (nd) rBla g 2C168 (nd) rBla g 4

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C160 pooled anti-Fel d1 scFv Vs. YJV, Fel d1 and Amb a1

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1

1.2

1.4

1.6

1.8

2

-1 -2 -3 -4 -5 -6 R92serum

R91serumscFv Dilution log10

OD

450

nm

YJV Fel d1 Amb a1

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C160 pooled anti-Amb a1 scFv Vs. YJV, Fel d1 and Amb a1

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1

1.2

1.4

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1.8

-1 -2 -3 -4 -5 -6 R92serum

R91serumscFv Dilution log10

OD

450

nm

YJV Fel d1 Amb a1

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C160 pooled anti-YJV scFv Vs. YJV, Fel d1 and Amb a1

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-1 -2 -3 -4 -5 -6 R92serum

R91serumscFv Dilution log10

OD

450

nm

YJV Fel d1 Amb a1

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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.

Gel stained anti-Fel d 1 anti-Amb a 1 anti-YJV

250 kD75 kD50 kD

30 kD

15 kD10 kD

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Conclusions

1. We can raise chicken antibodies against multiple antigens from a single immune library.

2. Panning against multiple proteins at once can work effectively.

3. Antibodies raised against rFel d 1 recognize nFel d 1.

4. Chicken scFv molecules secrete well in E. coli.

5. His-purified scFv functions effectively in ELISA and Western blot.

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Animal libraries built so far.

Chicken Rabbit AllergensC160 R91 rFel d 1

Amb a 1yellow jacket venom

C161 R90 rBla g 1rBla g 2rBla g 4rBla g 5

C162 R92 German CRC163 (nd) American CRC165 (nd) rBla g 2C168 (nd) rBla g 4

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Nitrocellulose coated chip

Spotted antibodies bind specific proteins

AntigenFluorophore labeling

Dense arraysSimple arrays

Fab

Antigen

scFv

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Simple arrays

Fab

Antigen

scFv

1. Apply scFv anti-Amb a 1 OR scFV anti-Fel d 1 to nitrocellulose slide

2. Block with ovalbumin

3. Incubate with

a. ragweed extract or

b. cat hair extract or

c. negative control (ova)

4. Polyvalent rabbit serum R91 (anti-Amb a 1 + anti-Fel d 1 + anti-YJV)

5. Anti-rabbit conjugate

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Animal libraries built so far.

Chicken Rabbit AllergensC160 R91 rFel d 1

Amb a 1yellow jacket venom

C161 R90 rBla g 1rBla g 2rBla g 4rBla g 5

C162 R92 German CRC163 (nd) American CRC165 (nd) rBla g 2C168 (nd) rBla g 4

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Anti-Amb a 1

Anti-Fel d 1

Cat hair 1:20 ragweed 1:20 1% ovalbumin

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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

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Key players Jonny Finlay, PhD Nicolette deVore, PhD