Antibody microarrays for allergen standardization

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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
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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 Mar32(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 Oct33(10)1443-9.  
• Beyer. Characterization of allergenic food
  proteins for improved diagnostic methods. Curr
  Opin Allergy Clin Immunol. 2003 Jun3(3)189-97.
  Review.  
• Fall et al. Microarrays for the screening of
  allergen-specific IgE in human serum. Anal Chem.
  2003 Feb 175(3)556-62.  
• Harwanegg et al. Microarrayed recombinant
  allergens for diagnosis of allergy. Clin Exp
  Allergy. 2003 Jan33(1)7-13. Review.  
• Kim et al. Quantitative measurement of serum
  allergen-specific IgE on protein chip. Exp Mol
  Med. 2002 May 3134(2)152-8.  
• Hiller et al. Microarrayed allergen molecules
  diagnostic gatekeepers for allergy treatment.
  FASEB J. 2002 Mar16(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
  25797(1-2)229-40.  
• Haab. Methods and applications of antibody
  microarrays in cancer research. Proteomics. 2003
  Nov3(11)2116-22.  
• Hallborn and Carlsson. Automated screening
  procedure for high-throughput generation of
  antibody fragments. Biotechniques. 2002
  DecSuppl30-7. Review.  
• Schweitzer and Kingsmore. Measuring proteins on
  microarrays.Curr Opin Biotechnol. 2002
  Feb13(1)14-9. Review.  
• Borrebaeck. Antibodies in diagnostics - from
  immunoassays to protein chips. Immunol Today.
  2000 Aug21(8)379-82. Review.

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Antibody microarray plan
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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
• 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.

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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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Complete Fab product
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Fab fragment display on phage surface protein p3
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Methods used for panning phage libraries.
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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.
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Animal libraries built so far.
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Chicken scFv exhibits excellent specificity and
sensitivity in Western Blot.
In each blot (left to right) Markers, Yellow
Jacket Venom, Ragweed extract, Cat hair extract.
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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.

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Animal libraries built so far.
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• 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

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Animal libraries built so far.
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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