Review TRENDS in Biotechnology Vol.21 No.
6 June 2003 275
Antibodies in proteomics I:
generating antibodies
Andrew Bradbury1, Nileena Velappan1, Vittorio Verzillo2, Milan Ovecka1,
Leslie Chasteen1, Daniele Sblattero3, Roberto Marzari3, Jianlong Lou4, Robert Siegel5
and Peter Pavlik1
1
B Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA
2
Institute for Neurodegenerative Diseases, UCSF, San Francisco, CA 94143, USA
3
Dipt. Biologia, University of Trieste, Trieste, Italy
4
Department Anesthiology, University of California, San Francisco, CA 94110, USA
5
Mailstop P7-56, Pacific Northwest National Lab, Richland, WA 99352, USA
The explosion in genome sequencing, and in sub-
sequent DNA array experiments, has provided exten-
sive information on gene sequence, organization and
expression. This has resulted in a desire to perform
similarly broad experiments on all the proteins encoded
by a genome. Panels of specific antibodies, or other
binding ligands, will be essential tools in this endea-
vour. Because traditional immunization will be unlikely
to generate antibodies in sufficient quantity, and of the
required quality and reproducibility, in vitro selection
methods will probably be used. This review – the first
of two – examines the strategies available for in vitro
antibody selection. The second review discusses the
adaptation of these methods to high throughput and
the uses to which antibodies, once derived, can be put.
Full or draft genome sequences are available for increas-
ing numbers of organisms, including human [1,2], yeast [3]
and many others (see www.tigr.org/tigr-scripts/CMR2/
CMRHomePage.spl for a list of microbial genome
sequences). This has allowed the implementation of
genome-wide studies, the most extensive of which have
been carried out in yeast, with individual gene knockouts
[4], overexpression and proteome chips [5], intracellular
localization by tagging [6], protein – protein interaction
studies by phage display [7], yeast two-hybrid [8,9], and
widespread mass spectrometric (MS) analysis of purified
complexes [10,11] providing large amounts of information.
One of the reasons for using yeast so extensively is the
availability of homologous recombination, which permits
the replacement of endogenous genes by modified copies.
In fact, most of the above-mentioned studies would not
have been possible without this technique, which often
involves the genetic fusion of a translated tag to each gene
product. However, this powerful technology is not avail-
able for most genomes and the only alternative to the
fusion of a general tag is the derivation of specific binding
ligands for all gene products that can be used for techniques
in which antibodies have been traditionally used
(e.g. Western blotting flow cytometry, immunoprecipitation,
Corresponding author: Andrew Bradbury (amb@lanl.gov).
http://tibtec.trends.com 0167-7799/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0167-7799(03)00112-4
immunofluorescence, immunohistochemistry and purifi-
cation) but within a proteomic context rather than on a
single gene scale. In addition to traditional uses, such
binding ligands will also be useful in new proteomic
techniques still under development, such as antibody
chips, and – potentially – in applications such as
biosensors. It is probable that greater understanding of
protein function at a genomic level will come when such
banks of binding ligands are derived and made generally
available, as has been done virtually for DNA chips by the
publication of genomic sequence.
The traditional binding ligand is the antibody, and
polyclonal antibodies are usually produced by immuniz-
ation with proteins, conjugated peptides [12] or DNA
expression vectors [13]. The fact that multiple different
antibodies recognize a single target in polyclonal sera is
both a strength (polyclonals can be used in all experimen-
tal formats) and a weakness (each polyclonal serum, even
from the same animal, is unique and not reproducible).
With the introduction of hybridoma technology [14], it
became possible to avoid polyclonal antibodies, which also
have problems of cross-reactivity and background, and
produce large amounts of monoclonal antibodies of defined
specificity. However, although extremely useful, this
technology is not easily amenable to high throughput
and is unable to overcome the problems of toxicity or poor
immunogenicity. It is hoped that the adoption of a new
suite of technologies, generically termed the ‘biomolecular
diversity’ or ‘display’ technologies will be useful. In
general, these technologies involve the selection of specific
binders from large libraries of binding ligands and usually
involve several selection cycles, each of which has several
common features (Table 1). These cycles are usually
carried out two or three times before binding ligands can
be screened directly and, if the library is of good quality,
binding ligands can usually be obtained against all targets.
The first examples of biomolecular diversity focused on
peptides [15– 17] and used phage display – in which
displayed peptides are fused to one of the coat proteins of
filamentous phage – as the platform to identify mono-
clonal antibody binding epitopes. Specific binders have
also been isolated from large naı̈ve antibody libraries
276 Review TRENDS in Biotechnology
Table 1. Key elements of the biomolecular diversity
technologies
Step Feature
1 The generation of diversity at the nucleic acid level to create
the ‘library’, usually of binding ligands. For example, natural
or synthetic V genes, V genes with mutations
2 The coupling of genotype to phenotype, alternatively
translated protein with encoding gene, or information to
function. This is done using living organisms or in vitro
methods
3 The application of selective pressure. For example, binding to
a specific target
4 Amplification of selected clones after selection, by growth,
infection or PCR
[18 – 24] and the display of hormones [25] and many other
proteins (see [26 – 28] for reviews) has been carried out
with the predominant goal of optimizing binding affinity.
In addition to phage display, bacteria [29] and yeast [30]
have been developed as microorganismal display methods,
whereas ribosome display [31] and puromycin display [32],
in which mRNA (or cDNA) is coupled directly to the
polypeptides they encode, have been developed as in vitro
methods. All of these are physical selection methods and
require significant quantities of the selector to perform
selection and screening. Several genetic methods for the
selection of binding ligands have also been developed: such
methods do not require the physical selector during the
selection procedure and rely on the in situ synthesis of, and
subsequent interaction between, binding ligand and
target, to confer a selectable phenotype. Both the yeast
two-hybrid system [33] and the protein complementation
assay [34] have been adapted to scFv selections [35 – 37] in
model systems, and offer the possibility of selection
without the need for antigen synthesis and purification.
Different genome projects are in different phases of
development and therefore no single method will be
appropriate to all genomes. If purified proteins are available,
physical selection methods will probably be most effective. If
only the sequenced genome is available, genetic methods –
in which interaction between binding ligand and target
confers survival on the cell containing the interacting pair, or
the use of synthetic peptides as selectors – are likely to prove
more effective, avoiding the need to produce purified protein.
Whichever methods are used, however, validation of the
specificity of selected antibodies will be an important and
difficult component of the complete process.
This review will concentrate on the processes of
selecting binding ligands and not on the alternative
binding ligands themselves, which were reviewed recently
[38]. Because much of this technology was developed
within the context of antibody fragments, and of scFvs and
Fabs in particular, these will be the most commonly
Table 2. Differences between scFvs and Fabs
ScFv Fab
Single protein molecule Two protein molecules,
chains must find each other
Less stable More stable
Better tolerated by bacteria More difficult to synthesize
Can form dimers (diabodies) No dimerization
DNA insert 700 bp DNA insert .1500 bp
More intellectual property constraints Fewer intellectual property constraints
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Vol.21 No.6 June 2003
Interact library
with selector
Wash Amplify
Elute
TRENDS in Biotechnology
Fig. 1. Physical selection methods: phage antibodies, or other selectable element,
are represented by ‘Cs’. Each ‘C’ can bind only to a target of the same color. After a
library of phage antibodies is interacted with a target in solid phase, the non-
specific binders are washed away and the specific binders are eluted and sub-
sequently amplified.
described binding ligands and the differences between
them are summarized in Table 2. However, most of the
technology described is also directly applicable to alterna-
tive scaffolds, and these are mentioned when they might be
more appropriate.
Physical selection methods
To select binding ligands against protein targets using
physical selection methods (Fig. 1; Table 3), a selector
needs to be available in a purified, synthetic or recombi-
nant form. Depending on the method of selection used,
200– 1000 mg is usually required to carry out selection and
screening. There are two general methods of physical
selection. In the first, antigen is fixed to a solid support,
such as a polystyrene tube or pin, and incubated with the
library of antibodies. Those that recognize the antigen
bind and can be eluted after non-binding antibodies are
washed away. In the second method, antigen is labeled,
usually with biotin or fluorescein, and used to separate
antibodies that bind from those that do not. In the case of
biotin, this is carried out using streptavidin-coated
magnetic beads (MACS) [39], whereas with fluorescein,
fluorescence-activated cell sorting (FACS) is used [40]. In
phage, bacterial and yeast display, living organisms are
responsible for amplification, display and the coupling of
phenotype and genotype, whereas ribosome- and puromy-
cin-based mRNA display systems rely on PCR for
amplification, and in vitro translation of RNA to produce
the binding ligand, which is then attached to the encoding
RNA (or cDNA) either covalently (puromycin display) or
non-covalently (ribosome display). More than one round of
selection is usually required for all these methods,
although screening thousands of clones can yield a greater
diversity of binders after a single round [41], albeit with a
far lower percentage of positives.
 Review TRENDS in Biotechnology
Table 3. Display formats and their use
Display method Selection method Recloning
required
for expression
Phage Solid support, MACS, in vivo Noa
Bacteria Solid support, MACS, FACS Yes
Yeast MACS, FACS Yes
Ribosome Solid support, MACS Yes
Puromycin Solid support, MACS Yes
(Profusione)
a
Recloning is not required for quick experiments but it is recommended to produce large amounts of antibody. FACS, fluorescence-activated cell sorting; MACS, streptavidin-
coated magnetic beads.
Binders have been isolated from large naı̈ve phage and
in vitro display libraries, whereas bacterial [42] and yeast
display [43] libraries have been used predominantly for
affinity maturation, with yeast display also having been
used for the improvement of antibody expression and
folding [44]. Recently, a large naı̈ve yeast display library
has also been produced, with antibodies against several
targets being selected with affinities as good as those from
phage libraries [45].
The availability of sufficient quantities of antigen for
selection and screening is one of the major bottlenecks in
the use of physical selection methods, with screening being
the more antigen-intensive process. There are two general
approaches to selection and screening: gene based and
proteome based. In gene-based selection, the identity of
the selector is known in advance and, as a result, the
specificity of selected antibodies is known. Synthetic
peptides [46,47], polypeptide fragments or recombinant
full-length proteins would be examples of suitable selec-
tors. Proteome-based selection uses fractionated natural
sources (e.g. tissue or cell extracts). Once selected, the
identity of the antigen recognized by such selected
antibodies has to be determined subsequently. Selection
of phage antibodies on proteins transferred onto PVDF
membranes after separation by 2D gel electrophoresis has
been described [48]. Although this might be a solution to
the use of natural protein sources for selection, the
screening and identification of appropriate binders in
high throughput remain key issues, and the problems of
sufficient amounts of selector remain. For example, some
proteins are present in serum at 1 pg ml21, which would
require purification of at least 1000 litres to yield 1 mg. In
general, the big advantage of gene-based selection
methods is that antigen identity is known in advance
and sufficient quantities can be produced, whereas
proteome-based selection methods have the advantage
that the antigen is in its most natural state and will
include appropriate post-translational modifications.
Phage and yeast display
Phage display has been by far the most commonly used
method to select antibodies. Most phage antibody libraries
have been created by cloning large numbers of different
antibody genes upstream of the gene 3 coat protein gene
and using phage [19] or phagemid [20 – 24] as the display
vehicle. Most of these use scFvs as the antibody format,
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Vol.21 No.6 June 2003 277
Ab libraries Predominant uses and notes
published
Yes Selection from naı̈ve libraries, affinity maturation
No Affinity maturation
Yes Affinity/stability maturation. Affinity can be determined from
FACS analysis. Selection from naı̈ve libraries
Yes Selection from naı̈ve libraries, affinity maturation can also be
built into naı̈ve selection
No Selection from naı̈ve libraries, affinity maturation can also be
built into naı̈ve selection
with one large Fab library also published [22]. The
antibody genes are derived either from natural sources
(e.g. human peripheral blood lymphocytes) or created
synthetically by introducing diversity using oligonucleo-
tides into frameworks with desirable properties. When an
antibody gene is cloned upstream of gene 3, the antibody is
displayed as a fusion protein with the gene 3 coat protein.
A library of such phage antibodies theoretically consists of
as many as 1011 different members (the diversity is
generally measured by counting the number of indepen-
dent colonies), with each different specificity being
represented by a relatively small number of phage in a
library. In general, diversity is limited by the transfection
efficiency of bacteria. However, recombinatorial methods
of library creation [21,49,50], in which VH and VL genes
are shuffled using cre recombinase after an initial cloning
step, are capable of creating far larger libraries and –
because recombination is associated with amplification –
almost unlimited supplies of such libraries, which is
important in a genomic context.
Although antibodies with subnanomolar affinities
can be selected directly [20,23], or affinity matured
from these libraries [51 – 53], this usually requires a
considerable degree of effort, which would be difficult
to marshall at a genomic scale. As a result, the usual
affinity range of antibodies selected directly from such
libraries is 10 – 1000 nM. However, the effective affinity
can be increased significantly by genetically fusing multi-
merization domains, such as jun/fos for dimerization, to
the ends of such selected scFvs [54].
Selection of phage antibodies is usually carried out
against single antigens. A potentially high-throughput
method using antigen immobilized on polystyrene pins in a
microtiter format has recently been described [55] and it is
likely that similar methods can also be developed for
biotinylated antigens [39] using robotic washing and
elution systems. However, selection is usually the least
difficult part of the procedure, with the identification of
different positive clones being far more time- and antigen-
intensive (see Screening in the second review of this
series).
Antibodies selected from naı̈ve phage antibody libraries
have very variable expression levels (ranging from 10 mg
to 20 mg per liter), and can be evolved to be expressed at
higher levels [56– 58]. An alternative to the evolution of
individual antibodies is to create libraries in which most
278 Review TRENDS in Biotechnology
antibodies are well expressed. Several promising libraries
[24,59 – 61] have been constructed with one or more stable
scaffolds and synthetically introduced diversity. It has
been shown [62] – at least in the case of yeast – that scFv
stability, expression and display are related, indicating
that improvement in any one of these parameters is likely
simultaneously to improve the others, and suggesting that
the adoption of any of the strategies described below to
increase the intracellular stability of scFvs will also
increase expression levels. The recent description of a
naı̈ve yeast scFv library [45] is an encouraging addition to
the repertoire of selection technologies. Antibodies with
good affinities were selected from this library using
biotinylated antigens and either magnetic or fluorescent
selection procedures. It will be interesting to compare the
ease of use of this technology with phage display – it is
likely that both will have specific advantages and
disadvantages.
In vitro display systems
In in vitro display systems, genes are coupled to the
proteins they encode after translation in an in vitro
translation mix. In RNA display (or Profusione) [32,63],
puromycin covalently links the RNA to the encoded
protein, whereas in ribosome display [31,64] the ribosome
itself acts as a non-covalent linker between gene and
encoded protein. The affinities of scFvs isolated from
primary selections are similar to those from phage
antibody libraries, although theoretical library sizes are
much larger than most phage libraries (no transfection is
required). Whereas positive binders can be selected after
two or three rounds using phage display, in vitro display
systems tend to require many more cycles. An advantage of
in vitro display systems is the possibility of incorporating
in-built affinity maturation [65,66] by using rounds of
error-prone PCR or DNA shuffling [67] between selection
rounds. Whereas this requires even more selection rounds,
antibodies with picomolar affinities have been achieved
using this method, and it is more likely to be amenable to
automation for high-throughput selection. Once selected,
antibodies or other binding ligands selected by in vitro
display systems are usually cloned into bacterial
expression systems; in vitro translation systems [68] are
a currently unused alternative. This represents a bottle-
neck in the procedure because not all antibodies selected
by in vitro display are well expressed in bacteria. Within
this context, other scaffolds, such as the tenth fibronectin
type III domain [69], have also been used in ribosome
display with the isolation of high-affinity binders against
several targets.
Genetic selection methods
Physical selection methods are appropriate for the selec-
tion of binding ligands in cases where a physical selector is
available. Although this field is advancing rapidly, with a
few groups producing small amounts of proteins on a
genomic scale [5,70,71], in general, sufficient quantities of
selectors are not available for most proteome projects. One
way around this is to avoid the use of physical selectors
altogether and to develop genetic selection methods (Fig. 2)
that use DNA encoding either the whole or part of the gene
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Vol.21 No.6 June 2003
of interest. The selection of binders from libraries is
essentially a protein – protein interaction problem, with
the yeast two-hybrid system [33] being the most widely
used genetic selection method to identify such inter-
actions. Under ideal circumstances, it would be possible to
clone the gene of interest as the bait and to transfect an
antibody library as the prey each time a selection was
carried out. However, most current antibody libraries
[20 –23] contain more than five billion clones, which far
exceeds the transfection capability of yeast. This problem
has been overcome by carrying-out a single round of
selection on a protein of interest and cloning the selection
output into a yeast two-hybrid vector [35,72,73]. Although
this reduces the diversity of the library to 105 – 106
(amenable to yeast transfection), permitting the selection
of several different scFvs, it suffers from the need for a
physical selection before performing the genetic selection,
so eliminating some of the advantages of a genetic
approach. Furthermore, most scFvs are not functional
under the intracellular conditions used in this genetic
approach because they contain disulfide bonds, which are
required for their stability. These cannot be formed in the
reducing environment of the cytoplasm.
It might be possible to overcome the need to carry-out
physical selection before genetic selection by using either
bacterial genetic systems [74–77], relying on transcriptional
activation, or ‘protein complementation assays’ [34,78,79],
in which an enzyme required for cell survival (e.g.
dihydrofolate reductase or b-lactamase) is divided into
two parts in such a way that enzyme activity is
reconstituted only when the two parts are brought
together, by virtue of the presence of two interacting
species, such as antigen– antibody. Preliminary exper-
iments [37] using model antibodies and DHFR, showed
that specific antigen– antibody pairs conferred survival
Survival Death
Functional transcription Non-functional
factor, antibiotic transcription factor,
resistance or essential antibiotic resistance
enzyme or essential enzyme
TRENDS in Biotechnology
Fig. 2. Genetic selection methods: an antibody fragment (or other binding ligand)
is attached to half of a selection protein; the target to be recognized is attached to
the other half of the selection protein. The two halves can come together only
when antibody and target interact, so stabilizing the selection protein. Under
appropriate selective conditions, the cell cannot survive unless the selection pro-
tein is functional. Examples of appropriate selection proteins are given in the
figure.
 Review TRENDS in Biotechnology
more than 107 times more effectively than non-specific
pairs. This, coupled with the high transfection efficiency of
Escherichia coli, might make this method useful for
proteomic-scale antibody selections.
Most of these bacterial systems also require antibodies
to be stable in the cytoplasm because, with the exception of
b-lactamase, they all involve cytoplasmic interactions.
Although the stability of any single antibody can be
increased significantly by appropriate mutation and
selection [44,80 – 83], it would be more appropriate to
develop libraries of binding ligands, in which a high
proportion of binders are functional under intracellular
conditions, rather than to optimize individual scFvs. Such
libraries are not yet available but a diverse body of work
[72,84 – 88] indicates that frameworks based on antibody
consensus sequences are generally more stable. By using
either stable antibodies or consensus frameworks such as
scaffolds, with synthetic oligonucleotides providing diver-
sity, it should be possible to select stable scFvs without the
need for further manipulation. Such libraries of antibodies
would also be extremely useful as intracellular antibodies
[89] in downstream target investigation. However, recent
work has shown that many scFvs can be stabilized in the
cytoplasm, as well as being expressed at high levels, by
fusion to the C-terminus of maltose binding protein [90],
perhaps avoiding the need for cytoplasmically stable
antibody frameworks altogether.
The selection for antibody– antigen interactions using
b-lactamase complementation [79] in a fashion similar to
that used for DHFR would avoid the need for libraries of
antibodies that were stable intracellularly, because this
enzyme is active in the periplasm. However, it does require
antigens to be secreted into the periplasm, and it is
expected that cytoplasmic antigens might not be stable
under these conditions. As a result, it is possible that
genetic selection approaches might require the use of more
than one method to be effective, depending on the identity
of the antigen. This caveat also applies to selection by
avidity capture [91], an approach that can be best
described as a combined genetic/physical method, which
relies on co-expression of antigen and phage antibody in
the periplasm. After antigen and antibody have interacted
in the periplasm, positive clones are screened using a
filter-based approach [41] that identifies many different
specific antibodies, albeit after one round of traditional
phage display selection.
Conclusion
At present, several different methods are available to
select antibodies against gene products in high through-
put. Determining which method should be used for a
particular genome will depend on whether purified
selectors (proteins or protein fragments) are available. If
so, physical selection methods (phage display, yeast
display and ribosome display) are most suitable, although
it is not yet known which of these is most effective. In the
absence of purified selectors either peptides or genetic
selection methods can be used, although both of these lag
far behind physical selection methods in maturity and ease
of use. The second review in this series will deal with
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Vol.21 No.6 June 2003 279
screening and the adoption of these technologies to high
throughput.
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