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The use of gene-specific IgY
antibodies for drug target discovery
Wei-Wei Zhang
Genomics and gene expression data require interpretation at the protein
level to validate the biological or pathological findings. To match withthe efficiency and capacity of DNA microarray application, methods andapproaches of protein analysis in multiplex and high-throughput mannerare required for effectively discovering disease-related proteins. Twodimensional gel and MS-based technologies provide researchers withsuch approaches, but neither of them can directly and selectively detecttarget proteins in situ. Antibodies are one of the most crucial tools formeeting this need. Efficient generation of antibodies based on genomics
and gene-expression information is an important strategy for producing
gene-specific antibodies to link genome to proteome. Avian immunoglobulins
isolated from egg yolk (so-called IgY) have several attractive advantages
over conventional IgG antibodies.

Wei-Wei Zhang
GenWay Biotech
10130 Sorrento Valley Road
Suite C, San Diego
CA 92121, USAe-mail:wzhang@genwaybio.com

364▼ The completion of human genome sequencing and its initial annotation have greatly
facilitated the application of DNA microarraysfor gene expression studies, particularly todisease markers and potential drug targets.
Previous cytogenetics and current DNA chipbased methods have generated an enormous
quantity of data that require interpretationand validation at the protein level [1–3]. By
contrast, several approaches designed to study
diseases directly at the protein level haveconcurrently been developed [4–6]. Thesemethods include 2-D gels coupled with MS,
MS combined with liquid chromatography(LC–MS–MS), and surface enhanced laser desorption-ionization time-of-flight MS (SELDITOF MS) [7]. One of the common limitationsof these methods is that they do not have direct access to proteins in situ in cell and tissue
samples because proteins must be in solution
or in the form of cell lysates for analysis. The
development of protein and tissue microarrays
provides possibilities for measuring groups of
disease-related proteins directly in body fluids, tissue extracts and tissue biopsy samples

www.drugdiscoverytoday.com[8–11]. However, without specific antibodies
or other affinity reagents, the usefulness ofthese methods could also be limited.

Antibodies bridging genome toproteome
It has been recognized that the developmentof libraries of protein-specific antibodies isneeded to profile the human proteome and tounderstand proteins in groups and their networks of interactions (referred to as a ‘proteincompendium’). The ideal situation would be
to produce antibody libraries based on theavailable data of genomics and gene expression studies, so that gene expression profiles
can be linked directly to proteomic profiles orprotein compendia resulting from antibodybased detection and quantification of groups
of proteins.
In this sense, to use the information of
genomics and gene expression to generate antibodies will comprise a unique approach tolink genomic information to proteomic profiles. A gene-specific antibody means that theantibody is produced via immunization witha gene-expression vector that can produce a
corresponding antigen in the host animal
to induce antibodies [12,13]. This approach(Fig. 1) can make use of the vast amount ofdata that is being generated from genomics
and gene expression studies, particularly fromDNA microarray technology. The major advantage of this approach is to avoid the increased cost and longer times usually required
to prepare antigens by conventional methods;
to produce antigens via direct gene expression
in animals eliminates the intermediate stepsof recombinant protein expression and purification, thus simplifying the process and increasing its effectiveness. Antibodies producedby this approach carry gene sequence information; these so-called gene-specific antibodies

1359-6446/03/$ – see front matter ©2003 Elsevier Science Ltd. All rights reserved. PII: S1359-6446(03)02655-2

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can be used to detect and screen protein
Antibodies
targets in tissue or cell samples. The dataof identification and quantification oftarget proteins can effectively be traced
back to their encoding gene sequences
because each antibody contains intrinsic
corresponding gene sequence information.
Genetic
ImmunologicalIn this way, gene-specific antibodies have
immunization
detection
Relational
databasea crucial bridging function linking genomic profiling to proteomic mapping.
Relation between
gene, antibody, protein,
The gene-specific antibody approach istissue and disease
one of the methodologies of affinity-mediated protein discovery. The success of
Biomarkers or drug targets
Diagnostic or therapeutic
this approach depends on having the caproteins or antibodies
pability and throughput to produce large
numbers of antibodies with high specificity and affinity. Conventional methods
Data analyses
Genes
Proteins
primarily use rodents, rabbits or larger
Drug Discovery Today
mammals (i.e. goats) to produce antibodies,in either polyclonal or monoclonal forms.
Figure 1. Approach and application of gene-specific antibodies for protein discovery
These antibodies are IgG and have beenand validation. The strategy of this approach is to avoid current difficulties
used successfully in various immunoassays
encountered in interpreting gene expression data at the protein level and instead use
antibodies as the bridge to directly link the genome to the proteome. This is done by
and clinical applications for diagnosis
generating antibodies from genes and applying the antibodies to detect specific geneand therapy.
products in tissues or cells, as well as the target proteins’ relationship to diseases. The
There is another class of immunoglobproteins and the corresponding antibodies identified can be used for various
biomedical applications.
ulins called IgY, which can be isolated fromegg yolks of the lower vertebrates, such as
birds, reptiles and amphibia [14,15]. IgG
antibodies have undergone extensive development in many
There are several attractive advantages of using chickens
immunoassay applications; however, the usefulness of IgY
as the immunization host and their eggs as the sources for
antibodies is not well known, despite their initial reportantibody isolation, as described below.
110 years ago [16]. IgYs, distinct from IgGs in molecular
structure and biochemical features, have many attractive
Better immune responsiveness to mammalian antigens
advantages over IgGs and are suitable for further developOne of the important advantages of using IgY is an enment in immunoassays and clinical applications. This artihanced immunogenicity against conserved mammaliancle discusses the special characteristics of IgY antibodies
proteins because of the phylogenetic distance between
and their potential application as polyclonal gene-specificdonor and recipient organisms [19]. This makes the proantibodies for screening and validating biomarkers andduction of antibodies against conserved mammalian prodrug targets.
teins generally more successful in chickens than in othermammals. In addition, IgY antibodies tend to recognize
the same protein in several mammalian species, making
Overview of IgY antibodies
them more widely applicable.
IgY antibodies are the predominant serum immunoglobulin
in birds, reptiles and amphibia, and are transferred in thefemale from serum to egg yolk to confer passive immunity
High affinity antibodies with persistent titerto embryos and neonates [17]. This process corresponds to
Chicken IgY antibodies were found to have high affinity
placental IgG transfer in mammals, which confers passive(avidity): antibodies with high avidity against bacterial or
immunity to the fetus. The nomenclature of IgY was initiallyhuman proteins have been developed [20,21]. The avian
proposed by Leslie and Clem because of its enrichment in
immune response was also shown to be persistent: 20–30 µg
egg yolk [14]. IgY is the functional equivalent of IgG. Fromof a highly conserved mammalian antigen induced highan evolutionary perspective, IgY antibodies were considered
and long-lasting IgY titers in the yolk from immunized
to be the ancestor of mammalian IgG and IgE antibodies [18].
hens [19,22].

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Non-invasive collection of antibodies
Distinct from bleeding immunized animals each time forthe production of antiserum, collecting eggs from immunizedhens for isolation of IgY antibodies is a non-invasive, nonstressful process for the animal and for the human handler.
This is not only a much easier and more reliable procedure,
but also a method of biological production that is favorable
to animal welfare [23].

A simple and economical isolation process
IgY antibodies are concentrated in egg yolks. The isolation
process involves separation of yolks from egg whites, followed by the purification of antibodies in yolks from lipids
and other materials. Different materials have been usedand various methods were developed, including polyethylene glycol (PEG) precipitation, DEAE fractionation, chloroform extraction, water dilution, precipitation with dextran
sulphate or dextran blue or xanthan gums, separation in a
two-phase system (phosphate and Triton X-100), a freezethaw cycle coupled with gel filtration on Biogel P150, andso on [24–32]. The isolation procedures are generally efficientand economical, although the various methods generate
IgY antibodies with different yields, purity, stability andactivity.

Large yield and scalable production
A chicken usually lays ~280 eggs in a year and an egg yolk
contains 100–150 mg of IgY antibodies. This can result in
28–42 g of IgY per year from each chicken [33]. It was
shown that antigen-specific IgY antibodies were between
2% and 10% of the total IgY harvested [34]. The industrialized scale of hosting and caring for millions of chickens
has been well developed in industry, therefore, the production of IgY antibodies can be readily scalable.

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functions. The various parameters of the two classes ofantibodies are compared in Table 1. The major differences
observed between IgG and IgY are described below.

No binding to bacterial or mammalian Fc receptors
IgY antibodies do not bind to bacterial Fc receptors, suchas staphylococcal protein A or streptococcal protein G[37,38], indicating the immunological difference of the Fc
region from that of IgG. However, it has been shown thatthe protein A-reactive site was generated after IgY boundantigen [37].

No reaction to mammalian immunoglobulins andcomplement factors
Chicken egg-yolk immunoglobulins do not react withmammalian IgG or IgM [39], nor with human anti-mouse
IgG antibodies (HAMA) [40,41], nor binding to the rheumatoid factor (RF), which is an anti-immunoglobulin autoantibody found in many different diseases [42]. IgG molecules
often give false positive results by interaction with RF in
immunoassays [36]. The lack of cross-reactivity between
IgY and IgG can be used in many ways to reduce unwanted
reactions in assays using anti-IgG antibodies. IgY does not
activate mammalian complement factors [43], which also
helps to reduce the assay interference by complement factors
in mammalian serum samples [44].

Remarkable affinity and avidity
IgY antibodies were shown to have high avidity against
bacterial or human proteins [20,21,45]. This could result
from either the evolution divergence between mammal
and avian species, or from the different affinity maturation
process of IgY [18,38].

Different immunoprecipitation characteristics
Comparison of IgY with IgG
Despite the similarities between IgY and IgG antibodies,
there are some profound differences in their chemical
structures. The IgY heavy chain is 65–70 kDa, whereas themolecular mass of the mammalian IgG heavy chain is~50 kDa. The IgY light chain is 19–21 kDa; the IgG is
22–23 kDa [22,35]. The greater molecular mass of IgY is a
result of an increased number of heavy-chain constantdomains and an extra pair of carbohydrate chains. In addition, the hinge region of IgY is shorter and less flexible
than mammalian IgG. Recently, it has also been suggested
that IgY is a more hydrophobic molecule than IgG [36],which matches the lipid-rich environment of the egg yolk.
The structural and amino acid sequence differences
determine the differences between the two types of antibodies in their biochemical features and immunological366

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The immunoprecipitation characteristics of IgY are different from that of IgG, presumably because of the different
structure of their hinge regions [18].

Resistance to extreme pH and temperature
IgY is stable at pH 4–9 and up to 65°C in aqueous condition; this compares with IgG, which is stable at pH 3–10
and up to 70°C [22,46]. However, the resistance of IgY to
the more extreme pH ranges increases if high salt conditions or stabilizing reagents such as sorbitol are present[47]. IgY was reported to be stable at 4°C for an extended
period [25].

Suitable for surface applications
IgY was shown to have good stability after being applied tolatex microspheres because of its more hydrophobic surface

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Table 1. Comparison of IgY and IgG antibodies
Features of comparison

IgG

IgY

Refs

AnimalMammalBirds, reptiles, amphibia[16,18]Sources

Blood plasma

Egg yolk[14,17]

Molecular Weight(by SDS–PAGE)

Whole: 150 kDa

Whole: 180 kDa[22]Light chains: 22 kDa × 2

Light chains: 21 kDa × 2

Heavy chains: 50 kDa × 2

Heavy chains: 70 kDa × 2

Molecular weight(by MALDI-TOF MS)

Whole: 150 kDa

Whole: 167 kDa[35]

Light chains: 23 kDa × 2

Light chains: 19 kDa × 2

Heavy chains: 50 kDa × 2

Heavy chains: 65 kDa × 2

Basic structure differences

Flexible hinge region, shorter Fc stemwith 2 pairs of carbohydrate groups

Shorter and less flexible hinge, longer
Fc region with 3 pairs of carbohydrategroups[18]

Immune response to
mammalian antigens

Adversely affected by phylogenetic
homology

Enhanced by phylogenetic differences[19]

Affinity maturation mechanism Somatic hypermutation

Pseudo-V gene conversion[18,38]Comparable[20,21]Milligrams with 1–10% specificantibodies if mice or rabbits used

Grams with 2–10% specific antibodies[34]

Cross reactivity

Reaction to mammalian
immunoglobulins and complement
factors

No binding to mammalian
immunoglobulins and complement
factors[39,64]Non-affinity isolation

Need to remove various plasmacomponents

Need to mainly to remove the lipid
components in egg yolk

[24–32]Affinity purification

Proteins A or G, or antigen-based
purification

Limited to antigen-based purification

[33]

8

10

Affinity or avidity

Good (10 -10

Quantity (yield per month per
animal)M)

Stability

Good, stable at pH 3–10, up to 70°C

Good, stable at pH 4–9, up to 65°C

[22,46,47]

Hydrophobicity

Less hydrophobic than IgY

Fc region is hydrophobic[36]

Immunoprecipitation

Good

Less effective due to short hinge structure [18]Productivity

Limited in quantity if mice and rabbits High with greater quantity and long
are hosts
duration

Scalability

Relatively difficult

Feasible and practical[33]

Monoclonal antibodies

Have been well developed

Two cases reported, more development
needed[67,68]Immune suppression

Several products are underdevelopment

May be useful for xenotransplantation[52]

Diagnosis

Widely used, especially monoclonal
antibodies

Useful and practical for various
applications[41,53]

Therapeutics

Well developed

To be further developed such as in
antibiotic-alternative therapy[33,43,65,
66]

compared to mammalian antibodies [48]. Immobilized IgY
antibodies were also shown to improve the detection ofserum antigens with surface plasmon resonance [44].

Amenable for various applications
IgY has been successfully used in conventional immunoassays, such as ELISAs, western blots, immunoprecipitation,[19,22]

immunohistochemistry, immunocytochemistry, cell sorting, cell and tissue staining, [49–51]. The quality of theseassays using IgY antibodies is either comparable to, or better
than, using conventional IgG antibodies. IgY antibodies
have also been shown to have advantages in different applications, such as xenotransplantation [52], diagnostics[53] and antibiotic-alternative therapy [43].

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could be considered as mono-specificor pseudo-monoclonal antibodies. Depending upon the needs of application,
Gene-expression vectors
the specific antibodies in the total IgY
Protein
Genomic DNA
isolated from egg yolk might need toantigensor cDNA
undergo antigen-based affinity purifiChickens
and eggs
cation for highly-concentrated andmore-specific products (Fig. 2).
The antibodies produced this way
Gene-specific
can be used to screen and identify unpolyclonal IgY
known proteins in biological samples,
antibodies
Testing antigens
to localize their specific protein targets
QC assaysin tissues or cells, to determine their
Antibody
Tissue or cellpotential function and to analyze their
Ab chips
microspheres
arraysexpression profiles. Large quantities ofproteomic data can be generated andorganized into an integrated and relational database. The new proteins idenMonoclonal antibodies
Therapeutic proteinstified can be further developed as diagdiagnosis or therapy
drug targets or biomarkers
Integrated databasenostic markers or therapeutic targets
Drug Discovery Todayof important diseases, such as cancer.
Monoclonal or therapeutic antibodies
Figure 2. Gene-specific antibody approach and its potential applications. The approach
can also be further developed based
employs DNA or protein antigen-based immunization in chickens to generate genespecific IgY antibodies. The antibodies can be used for multiplex HTS of unknown
upon the IgY antibody and its target
proteins in tissue or cell samples, for example, through tissue arrays, antibody chips or cell
antigen information [12].
sorting. This process can localize protein targets in tissues or cells, determine their
DNA immunization-based gene-spepotential function and analyze their expression profiles. Large quantities of data can begenerated and organized into an integrated and relational database. The new proteinscific antibody production has also been
identified can be further developed as therapeutic targets or agents or as diagnostic
successfully applied for IgG antibodies
markers. Monoclonal or therapeutic antibodies can also be further developed based upon
[58]. Figure 2 indicates that the genethe IgY antibody and its target antigen information.
specific antibody approach does not
exclude using protein antigens. In fact,
the recombinant protein antigens are needed for immuGene-specific IgY antibodies
nization enhancement, in combination with DNA immuThe approach of gene-specific antibodies employs gene exnization, antibody testing and affinity purification. The
pression vectors to immunize animals for the production
approach described in Fig. 2 is more focused on using genof antibodies. Immunization with plasmid DNA encoding
omic or gene-expression information to make antibodies
target proteins efficiently induced both humoral and cellin chickens. This strategy has several practical benefits, as
mediated immune responses in host animals [54–56]. In
described below.
this method, the production of specific antibodies is initiated by the gene expression vector injected into the hostanimal. The gene product endogenously produced in the
Expedites the antibody production process
injected animal becomes the immunogen to induce the reBy using gene expression directly in the immunization
sponse of the host immune system. Polyclonal antibodies
host, the process will simplify the operations of antigen
produced by this method recognized both linear and conforpreparation by expressing target protein and purifying
mational antigens, as assayed by western blots and ELISAs,
in vitro (in Escherichia coli or other protein expression sysrespectively [57].
tems). This process is particularly good for avoiding the poGene-specific antibodies also take advantage of bioinfortential contaminations of unwanted antigens associatedmatics from genomic and immunogenicity analyses to sewith protein purification.
lect gene fragments for protein domains as the specificantigens. This process can largely eliminate the homologous
Avoids the need for protein antigens for priming
regions between proteins to make the antibodies specific
The success of using gene expression in the immunization
to the target proteins. Therefore, gene-specific antibodies
host largely depends on the protein expression level. Thus,368

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to ensure generation of high titer, recombinant protein
antigens can be used as boosters to further stimulate theimmune response. Using a gene expression vector as theprimary immunization step can replace using protein antigens to prime the animals, which avoids the delay in commencing immunization that can be caused by the preparation time of protein antigens. However, this process might
increase the cost and complexity of the immunization
process, which could make the process difficult to scale up
to high-throughput levels.

Meets special requirements: membrane protein antigens
Genetic immunization is useful for some special requirements where protein antigen preparation has proven difficult, such as membrane proteins and protein antigens that
require post-translational modifications [58]. The antigens
generated in host animals via gene expression are expected
to be close to their native form and to undergo certain
post-translational modifications. Those requirements are
difficult to achieve via in vitro or bacterial protein expression systems. The challenge is to determine if post-translational modifications in avian species are similar to those ofnative mammalian (murine) genetic immunization [58].

Uses available genomic and gene-expression information
In the post-genomic era, the tremendous amount of genomics and gene-expression information needs to be usedappropriately and effectively. It has been recognized byscientists in the field of genomics and proteomics thatgenomics and gene expression data can not be readily anddirectly interpreted at the protein level because of transcription and translation control, post-translational modifications
and the different half-lives between mRNA and proteins [59].
Using genomics and gene-expression data to make genespecific antibodies can overcome this difficulty and provide
scientists with tools or reagents to access proteins and analyze the vast number of gene products at the protein level.

reviews

Gene-specific polyclonal IgY antibodies have enormous
potential for various applications. By rapidly developing
the technology of affinity reagents for meeting the needsof proteomics and for screening and validating drug
targets and biomarkers, polyclonal IgY antibodies will become more popular. However, gene-specific polyclonal IgY
antibodies should be viewed as a complementary technology approach to other affinity reagent methodologies,
particularly the relatively mature technology of monoclonal
IgG antibodies.
Monoclonal IgG antibodies have the recognized advantages of single epitope-based specificity, selected high affinity and continual supply, making them good reagentsfor diagnosis and therapeutics, in addition to their wideuse in conventional immunoassays. The limitations of IgG
monoclonal antibodies, besides their non-specific crossreactivity to human immunoglobulins and complement
factors [40,60–63], are the time and effort requried to findgood pairs in multiplex assays, such as antibody chips or
microsphere arrays. The relatively higher cost and longer
time needed to prepare highly selected monoclonal antibodies make them less suitable for HTS.
By contrast, polyclonal gene-specific IgY antibodies canbe produced more quickly and at less cost. It should befeasible to use polyclonal antibodies both for capturing or
detecting antibodies and for self-pairing. Self pairing canbe used for releasing – to a certain extent – the rate-limiting bottleneck in developing paired antibodies for sandwich-based immunoassays. These features make polyclonal
antibodies a better fit for conducting initial HTS. Althoughpolyclonal antibodies do not provide an indefinitely sustainable supply – as monoclonal antibodies can – the large
yield of initial IgY antibody enables the production of large
batches of each individual antibody to form a libraryfor target screening purposes. The key is to develop an approach that is simple and fast, and that directly links gene
expression information to gene product (protein) status intissue or cell samples for protein expression profiling.

Provides biological linkage between genome and proteome
The gene-specific antibody approach provides antibodies
that are developed specifically with gene-coding information;
therefore, in principle any protein target discovered invarious types of assays or in different biological samples
can be traced back to their gene sources, which is particularly useful for identifying unknown proteins. This approachis called Antibody-Mediated Identification of Genes andProteins (AMIGAP), which uses antibodies to bridge thegap between genes and proteins. If a gene-specific antibody
library can be developed that corresponds to all the protein components of a given organism, the proteome andgenome can be linked.

Concluding remarks
Immunization of hens represents an excellent alternativefor efficiently generating polyclonal antibodies because:(1) chicken housing is inexpensive, (2) egg collection is
non-stressful to hens, (3) isolation and affinity purificationof IgY antibodies is extremely high-yielding, fast and simple, and (4) there are extensive applications of IgY. In addition, the yolk immunoglobulin has several intrinsic biochemical advantages, particularly no cross-reaction withmammalian immunoglobulins [64]. Because they do not
activate mammalian complement and show no interaction
with mammalian Fc receptors or other polyvalent antibodies

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that could mediate an inflammatory response in the gastrointestinal tract, IgY antibodies are attractive for peroralor aerosol immunotherapy [43,65,66]. Other direct clinicalapplications of IgY, such as inhibition of xenograft rejection[52], are also being developed. Considering the benefits ofIgY technology in polyclonal antibody production and theuniversal application of these antibodies in research andmedicine, IgY technology will have an increasing role inresearch, diagnostics and immunotherapy.
Here, the approach of gene-specific polyclonal IgY antibodies and their applications for unknown protein andantibody screening have been introduced and discussed.
Direct gene expression in host animals using DNA as theprimary antigen for immunization, known as genetic immunization, has the merit of simplifying the conventional
immunization process using protein antigens, although thisapproach does not necessarily exclude its combination
with protein antigens. For example, the primary immunization could be genetic, with booster immunizations
using recombinant protein antigens. The antibodies developed in such an approach will not only be useful in various types of immunoassays but will also prove helpful inlinking protein target information back to their cognate
genes. It is foreseen that gene-specific polyclonal IgY antibodies will be further developed and widely used forscreening and validating drug targets and biomarkers, aswell as for the production of novel antibodies.

Acknowledgements
I sincerely thank the staff of Genway Biotech for their supportin preparing this article.

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