© 2011 Expert Reviews Ltd
Protein biomarker discovery
Analysis of the proteome in human samples
promises a deep insight into the mechanisms
involved in the manifestation of human dis-
eases, and also offers the possibility of discover-
ing new biomarkers for the early diagnosis of
disease. This guides and monitors molecularly
targeted and effective treatment strategies for
which proteins are the main therapeutic drug
targets [1–3]. A number of strategies for disease-
related protein biomarker discovery have been
employed to date. So far, mass spectrometry
(MS), in various combinations with electropho-
retic and chromatographic separation methods,
has emerged as the dominant discovery tool
for the comparative proteomic characterization
of specimens obtained from disease-affected
donors and healthy controls [4–7]. Even though
several technical and practical limitations have
been associated with MS-based proteomic meth-
ods, there are a multitude of examples of recent
important achievements, such as that compara-
tive protein quantification has been enabled by
various labeling methods . Even so, the dif-
ficulty of analyzing extended patient cohorts, of
detecting low abundant proteins in body fluids
and how to generate highly reproducible patterns
using MS-based methods needs to be further
addressed. Some of these issues can be overcome
by preprocessing the samples via multistep frac-
tionations, but possible variations along these
fractionations can add an additional ana lysis
layer  and these time- and labor-intensive steps
prohibit a routine use of MS-based methods for
the high-throughput ana lysis of large patient
cohorts that is needed in clinical studies .
Affinity proteomics, which is based on the
use of capture reagents such as antibodies, has
emerged as a complement for MS-based meth-
ods. Now also being implemented into miniatur-
ized and parallelized assays, affinity arrays are
being applied more widely to offer a powerful
biomarker discovery approach.
Regardless of the method chosen and despite
the growing interest and efforts in biomarker
discovery, only a small number of protein bio-
markers have so far been translated into clinical
practice [11,12]. Furthermore, the specificity and
sensitivity limitations of the clinically utilized
single-protein biomarkers, such as prostate spe-
cific antigen (PSA) [13,14], gave rise to a consen-
sus that the discovery of biomarker panels of
Jochen M Schwenk1
and Peter Nilsson†1
1Science for Life Laboratory,
Department of Proteomics, School of
Biotechnology, KTH Royal Institute of
Technology, Stockholm, Sweden
†Author for correspondence:
Current approaches within affinity-based proteomics are driven both by the accessibility and
availability of antigens and capture reagents, and by suitable multiplexed technologies onto
which these are implemented. By combining planar microarrays and other multiparallel systems
with sets of reagents, possibilities to discover new and unpredicted protein–disease associations,
either via directed hypothesis-driven or via undirected hypothesis-generating target selection,
can be created. In the following stages, the discoveries made during these screening phases
have to be verified for potential clinical relevance based on both technical and biological aspects.
The use of affinity tools throughout discovery and verification has the potential to streamline
the introduction of new markers, as transition into clinically required assay formats appears
straightforward. In this article, we summarize some of the current building blocks within array-
and affinity-based proteomic profiling with a focus on body fluids, by giving a perspective on
how current and upcoming developments in this bioscience could enable a path of pursuit for
Keywords: affinity reagents • antibodies • antigens • biomarker discovery • body fluids • protein microarrays
• proteomic protein profiling • suspension bead arrays
Systematic antibody and
profiling with microarrays
Expert Rev. Mol. Diagn. 11(2), 219–234 (2011)
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Expert Rev. Mol. Diagn. 11(2), (2011)
proteins rather than single markers will be required for improved
accuracy in disease diagnosis [15,16]. However, the realization of
this paradigm shift could potentially be complemented by a suc-
cessive transition to systematic biomarker discovery strategies
using affinity arrays.
Microarrays as tools in protein biosciences
By the late 1980s, the principle of sensitive miniaturized and
parallelized immunoassays had already been described . The
theory herein suggested that the miniaturization of immuno-
assays provide an increase in detection sensitivity by improving
the signal-to-noise ratios without consuming effective amounts of
target from the analyzed solution. This solid-phase assay system
thereby enables the ana lysis of multiple analytes and parameters
in a single experiment, as the accurate and ordered positioning of
capture reagents onto a solid support was facilitated with robotics.
Ever since, the development and wide application of microar-
ray technologies in the field of genomics paved the way for the
advancement of protein arrays . Today, the use of arrayed sets
of proteins likewise allows for the rapid and simultaneous screen-
ing of very small volumes of specimen for multiple analytes, such
as antigens or antibodies. For this purpose, planar microarrays,
which are built on functionalized glass slides, or color-coded bead
systems forming suspension bead arrays, are used .
The latter format is an attractive array alternative to pla-
nar systems, with combinations of fluorescent dyes giving rise
to several hundred unique bead identities. Both commercially
established  and homemade protocols  are available for the
creation of such bead arrays. Each antibody or antigen can then
be coupled to specific microsphere identities, and once a batch
of coupled beads has been created, the very same can be used
throughout subsequent studies. After mixing microspheres of sep-
arate identities, an array in suspension is available for the desired
assays. After the assay, the amount of captured target molecules
is determined using a flow cytometry-based instrument. As both
workflow and read-out of such arrays utilize microtiter plates, the
complete assay procedure can be performed in a standard labora-
tory format and this opens the system for adaptation and imple-
mentation into (semi-) automated liquid handling. Furthermore,
flexibility is added by the possibility of creating new bead array
compositions for a given ana lysis . The bead arrays hold great
potential to become an important tool for screening material
stored in biobanks, as they offer the possibility of screening many
clinical samples simultaneously .
The challenges and possibilities within the coming proteomic
era will now put a focus on the systematic use of affinity reagents
for protein profiling. In the following sections, we will present and
discuss the currently available tools in order to compose an ana lysis
platform to decipher the biological complexity across diseases.
Systematic proteomic profiling with affinity arrays
In comparison to MS-based discoveries, affinity arrays require
first a target selection to be made in order to perform protein-
profiling experiments with affinity reagents. The most commonly
employed strategy is therefore through directed approaches, which
are referred to as hypothesis-driven, biased, targeted, supervised,
candidate-based or knowledge-driven. This approach involves
the study of subsets of prioritized target candidates related to a
particular disease context. These subsets can be defined based
on existing biological and clinical knowledge about the path-
ways involved in disease pathogenesis, based on results from, for
example, mutation screening, gene-expression profiling or pro-
tein-interaction network databases. Other sources could include
the affinity libraries built in-house, and alternative test systems
in which affinity reagents have been used (Figure 1).
In contrast to the above, undirected capture reagent selections
for biomarker studies can enable an unbiased and hypothesis-
generating ana lysis to help discover new connections and correla-
tions between protein levels in clinical samples of a particular con-
text. This approach is designed without any prior disease-related
selection and thus is not ‘limited’ to any existing knowledge. The
only limitation is which antibodies or antigens are available. Thus
far, MS-based methods have been considered as one of the cor-
nerstones of undirected, discovery-based screening approaches, as
disease–protein connections are being made with the determina-
tion of profiling experiments and not upfront. Affinity-based pro-
teomics can offer emerging tools for similar, undirected screening
approaches, but the major obstacle for this proteomic discipline
has been the limiting number of reagents for assaying proteome-
wide collections of clinical material in an unbiased manner. Given
this and the lack of access to high-throughput and multiplexed
technology platforms in settings close to clinical studies, the num-
ber of diagnostic or prognostic analytes being included is limited
to the range of hundreds [12,24,25]. Therefore, systematic efforts are
needed to stratify the protein profiling of large numbers of targets
and samples in a routine flow, allowing for the implementation of a
new set of antibodies or antigens as soon as they become available
for proteomic-profiling laboratories (Figure 2).
The concept of antibody arrays is built on capture molecules
being immobilized for a parallel detection of multiple targets
in a given sample . Arrays of this design are generally uti-
lized for two purposes: either measuring the abundance of a
target protein or conducting functionality studies . So far,
full-length antibodies, antibody fragments such as single-chain
variable fragments (scFvs) and fragment antigen-binding (Fab)-
fragments, alternative protein scaffolds and nucleic acid-derived
aptamers have been implemented as capture agents. To detect
interactions between the immobilized agents and the target
molecules, either direct labeling of the sample can be chosen
or a labeled secondary antibody can be used, thus creating a
sandwich assay . The most common platform for these so-
called forward-phase arrays are planar arrays, which are suitable
for simultaneously screening a large number of targets in one
sample. In bead-based arrays, the bead surface is the location
for immobilization and the method has been shown to offer
beneficial features when it comes to the parallel ana lysis of many
samples in profiling studies [29–31].
Ayoglu, Häggmark, Neiman et al.
When considering the two assay formats
introduced earlier, multi plexed sandwich
assays have the advantage of being built
on a pair of antibodies to detect target
proteins in a specific and sensitive man-
ner, used for low abundant proteins, such
as cytokines . However, it is currently
not possible to utilize pairs of antibodies in
any given combination as cross-reactivity
between the detection antibodies interferes
with target ana lysis . Therefore, the
number of markers being studied with a
sample can be extended by analyzing the
same sample sequentially with different
panels of compatible sandwich pairs .
When it comes to validating and translat-
ing new biomarker candidates discovered
from different disciplines into clinical stud-
ies, the sandwich set up is the assay format
of interest as the secondary affinity molecule adds specificity to the
test . Emerging modifications to the sandwich design include
the introduction of multiple detection reagents and connecting
these by DNA ligation and polymerization . Here a signal is
created upon the ligation of oligonucleotide-modified antibodies
being in proximity owing to binding to a nearby location. This
has also been shown to allow three antibodies to recognize and
detect target molecules , which could become of greater value
for studying protein complexes and subtyping protein families
or splice variants. Beside such efforts, technological advances are
being introduced to improve sensitivity down to sub-femtomolar
ranges, using single-molecule arrays . These arrays are built
on the detection of bead-captured complexes in femtoliter-sized
reaction chambers with custom-built imaging systems.
In order to facilitate the introduction of growing numbers of new
targets and binders for different biomedical disciplines, alternative
and more flexible first-line strategies must be considered. This could
involve different means of detecting interactions and one appealing
strategy for this purpose is the direct labeling approach . In this
assay, fluorescence- or biotin-based labeling can be employed for
the direct modification of proteins . Alternatively, dual-labeling
strategies involving two different fluorophores for samples can be
combined on one array . The direct-labeling approach currently
offers the only procedure to drive the discovery of novel target
molecules via affinity arrays because it appears not to be limited
to the number or compatibility of involved antibodies. Although
sandwich assays often provide superior sensitivity and specific-
ity, updates to the direct-labeling approach show the potential
to further improve the single-antibody-based analysis. Recently,
experimental protocols were described that allow the omission of
the purification of excess dye molecules after labeling owing to
extended quenching , and the retrieval of epitopes in complex
samples via heating , show the potential to further improve
the single-antibody-based ana lysis. With the growing number
of affinity reagents, their usage in direct-sample labeling and
microsphere-based assays appears to tackle the requirements of
a high-throughput proteomic profiling; covering the ana lysis of
large sample collections with any given set of affinity reagents. The
findings made during such initiatives demand both biological and
technical verification, and a translation into sandwich immunoas-
says to investigate whether new biomarkers will be introduced into
the clinic through affinity proteomics on arrays.
Systematic generation of capture reagents
The major bottleneck for affinity-based high-throughput ana-
lysis is the generation and availability of high-quality and vali-
dated affinity tools directed against every protein of interest .
To overcome this and to determine global protein profiles of a
given sample, large numbers of antibodies, antibody fragments or
other capture reagents have to be available. Currently, different
programs have the aim of creating and providing future research
with the tools needed for proteome-wide ana lysis. In 2007, the
European consortium, Proteome Binders [45,301], was initiated to
collect and combine a range of affinity reagents from different
laboratories. Such efforts in generating affinity tools and apply-
ing them is coordinated to build up an infrastructure, includ-
ing features such as the Antibodypedia [46,302], a database listing
available antibodies against human proteins with correspond-
ing validation data. Other initiatives have been described for the
generation of antibodies towards a whole-proteome level, such as
the Human Protein Atlas [47,303], where the current version hosts
validated antibodies towards proteins from 8400 human genes,
corresponding to 42% of the protein-encoding genes in humans.
Several other efforts, such as a genome-wide monoclonal antibody
generation , the Src Homology 2 (SH2)-consortium  and
the National Cancer Institute (NCI) affinity capture project ,
have also been set up with a similar aim.
For antibody arrays, full-length IgG antibodies are most com-
monly used for implemention into antibody array applications.
Today a broad range of monoclonal or polyclonal IgG-derived
e.g., transcriptome analysis
In-house binder libraries
e.g., scFv arrays
e.g., HER2 and breast cancer
e.g., insulin and
Figure 1. Capture reagent selection strategies for protein array applications.
scFv: Single-chain variable fragment.
Systematic antibody & antigen-based proteomic profiling with microarrays
Expert Rev. Mol. Diagn. 11(2), (2011)
binders can be obtained from a range of immunization and puri-
fication protocols. The production of extended and renewable
collections of monoclonal binders is still hampered by a lower
throughput, but efforts are being made to address this issue .
The aims of such are to enable production and screening in a high-
throughput manner, such as by spotting the hybridoma superna-
tants in an array format . One alternative to monoclonal initia-
tives is the large-scale antibody generation by the Human Protein
Atlas project . Here, systematic generation of affinity-purified
polyclonal antibodies, also referred to as monospecific antibodies,
and their detailed validation is combined with protein profiling in
a variety of tissues and cells . The starting point for this produc-
tion pipeline is the in silico selection of antigens, the so-called pro-
tein epitope signature tags (PrESTs), consisting of approximately
75–150 amino acids of the target protein’s sequence, which are
chosen based on criteria such as low homology to other human
proteins . The PrESTs are expressed in Escherichia coli as recom-
binant protein fragments and used for immunization in rabbits.
The sera are then collected and affinity purified using the same
PrEST as for the immunization. The eluted monospecific antibod-
ies are validated by methods such as Western blotting with plasma,
cell and tissue lysates. In addition, the specificity is evaluated by
protein microarrays composed of 384 PrEST proteins [55,56]. A
feature of these monospecific polyclonal
antibodies is that they are oligo-epitopic 
and therefore offer the possibility that some
of them could recognize epitopes both in
linear and structural states .
Besides full-length antibodies, antibody
fragments such as scFv or Fab can be uti-
lized as affinity molecules for array-based
applications. Both antibody variants are
also generated from libraries constructed
by random combinations of antibody frag-
ment genes [59,60]. In such systems, the
complementary-determining regions of an
antibody, which define its specificity, can be
randomized by various techniques, creat-
ing semi- or fully-synthetic libraries [61,62].
For such selection, maturation and expres-
sion approaches such as phage display ,
ribosome display , cell surface display on
Staphylococci , and yeast display  can
Alternative scaffolds based on other
natural or synthetic affinity probes have
also been developed and can be used in
array-based applications. One example
of such an alternative scaffold is the affi-
body molecule , which originates from
the immunoglobulin-binding region of
staphylococcal protein A and consists of
58 amino acids. By randomizing a num-
ber of positions in the protein, a library of
affinity molecules can be created and used
to screen for the desired binding properties . Nucleic acid-
derived aptamers constitute another type of capture agent, where
a sequence of nucleic acids bind their target with affinities compa-
rable to antibodies . They can be produced by systematic evo-
lution of ligands by exponential enrichment, a method that selects
sequences in oligonucleotide libraries in an iterative manner ,
and have been applied in studies within diagnostics, therapeutics,
purification and drug development [70,71]. Other alternatives are
offered by scaffold proteins such as DARPins , or formats such
as molecularly imprinted polymers . In addition, by utiliz-
ing lectins as carbohydrate-binding proteins, the glycosylation
pattern of modified proteins, rather than the protein backbone,
can be explored . Furthermore, small-molecule microarrays
can be used for the interrogation of organic compounds and for
the identification of molecule–protein interactions, as in drug
development and in receptor-ligand studies . All binding mol-
ecules certainly have intrinsic advantages and disadvantages in
the application of interest, but the choice for a certain capture
agent also depends on factors such as their affinity, selectivity,
specificity and, most importantly, availability.
Alongside the technological advances being made on the plat-
forms and assay systems, a variety of biomolecules are emerg-
ing. The value and optimal use of these components and their
Antigen based Antibody based
Cell free Cell basedImmunization Libraries
Protein on chip Protein in solution
Antibody derivedAffinity scaffold
Expert Rev. Mol. Diagn. © Future Science Group (2011)
Figure 2. Systematic generation and profiling of antigens and antibodies for
protein array applications.
Ayoglu, Häggmark, Neiman et al.
combinations is to be determined based on their anticipated appli-
cation, but with the range of broadening resources and techno-
logies, current research points towards the future aim of having
renewable affinity reagents at hand for all human proteins and
on arrays .
Systematic generation of antigens
Systematic antigen-based approaches for proteomic profiling are
likewise dependent on access to a large amount of protein targets.
Generally, protein targets for these arrays could be represented
either by purified full-length proteins, protein fragments or pep-
tides, but also by overexpressing cells or as a naturally occurring
component of complex samples, such as cells, tissue lysates or
plasma and serum.
The parallel expression and purification of proteins, with the
intention to use them either directly as antigens or for antibody
production as a downstream application, is a challenging task
considering the highly diverse nature of proteins. To this aim,
proteins are conventionally generated using cell-based expres-
sion methods, which involve in vivo cloning and expression of
proteins in suitable heterologous expression systems and the sub-
sequent purification of these proteins. There are various possible
approaches to obtain high-quality proteins and these approaches
differ in certain aspects, such as cloning method, choice for the
expression promoter and affinity tag, cultivation method, puri-
fication strategy and protein characterization for the analytical
testing of the expressed proteins. Bacterial expression systems,
such as E. coli, have been extensively used and optimized for the
production of human proteins over past years . Moreover,
eukaryotic alternatives to bacterial expression systems, such as
the yeasts Pichia pastoris and Saccharomyces cerevisiae, insect cells
and mammalian cells, can also be employed . In general, cell-
based generation methods are mature and are also widely avail-
able commercially. The labor-intensiveness is typically still an
associated drawback with the cell-based methods. Likewise, the
limitations in maintaining protein stability, integrity and func-
tionality in the course of purification are still problematic. Cell-
free protein-generating methods provide an attractive alternative
to conventional cell-based methods, allowing in situ expression
of the proteins to be employed on a microarray surface, prior to
Cell-free protein generation methods involve the use of crude
cell lysates containing all the vital components required for the
direct in vitro synthesis of a protein from a DNA template in the
form of plasmid DNA or PCR product. Frequently, extracts from
E. coli, wheat germ and rabbit reticulocyte are used and commer-
cially available lysate-expression systems are on the market [78–81].
A greater high-throughput, a greater ability to manipulate pro-
teins by altering the lysate environment, as well as the possibility
to synthesize toxic or membrane proteins that are challenging to
express in living cells [82,83], are counted as advantages of the cell-
free protein generation methods, whereas the relative variability
and inadequacy of protein yield and increased cost compared
with the cell-based methods are the main drawbacks .
Antigen arrays have exhibited great potential in being powerful tools
for the parallel characterization of serological immune-response
repertoire to both pathogen and human protein (auto-)antigens .
The possibility of identifying the immuno dominant pathogen
causing various infectious diseases using antigen arrays [86–91]
contributes to a better understanding of host–pathogen immu-
nity. It promises not only a more rapid but also a more targeted
identification of novel candidates for the development of effec-
tive vaccines and monitoring the outcome of vaccine trials [92,93].
Likewise, antigen arrays have proven to be important tools for the
investigation of self-reactive immune processes manifested during
the course of both autoimmune disorders [94–100] and various types
of cancer [101–107]. A fundamental feature of autoimmune diseases
and certain cancer types is the presence of autoantibodies in the sys-
temic circulation that recognize self- or tumor-associated antigens,
respectively. The persistence and intrinsic stability of antibodies
in blood makes the autoantibodies clinically relevant serological
targets and diagnostically valuable hallmarks of both cancer [108–
112] and autoimmune diseases . Consequently, the potential of
serological ana lysis of antigen arrays displaying human proteome
to measure disease-related antibody responses and, therefore, to
assist antigen-specific therapies are of greater interest [114–117]. Apart
from their disease-oriented applications, antigen arrays are also
utilized for the ana lysis of the natural autoantibody repertoire in
healthy individuals to gain insight into the formation of immune-
state organizational motifs . Besides the aforementioned uses,
antigens arrays are also applied as a platform for the verification of
selectivity of antibodies .
There are two main strategies for producing antigen arrays: the
most conventional approach is to recombinantly express and purify
antigens, followed by spotting or coupling them onto solid sup-
ports, such as glass slides or beads. The second approach is to use
in situ expression of the antigens ‘on-chip’, directly before use on
the array by exploiting the cell-free protein generation methods .
Within the last decade, various on-chip protein production
methods have been developed, enabling highly parallel protein-
expression possibilities. By combining the lysate expression with
the capture of produced protein there are intrinsic advantages and
drawbacks. In a protein in situ array, being the first method of its
kind, the slide surface is prepared to immediately capture the pro-
duced proteins and the template DNA is dissolved in droplets of
lysate, which are added to the array slide . For the nucleic acid
programmable protein array, the template DNA is spotted on the
slide together with a capture agent, such as an antibody. During
incubation with lysate, the agents capture the synthesized proteins
and as a result, the DNA stays colocalized to the protein . In the
DNA array to protein array method, DNA is spotted onto a slide
in a manner similar to the nucleic acid programmable protein array
procedure. A protein-capture slide is prepared as for the protein
in situ array and the two slides are assembled in a sandwich format
separated by a membrane soaked in lysate. Proteins are expressed
when the DNA slide comes in contact with the lysate and diffuse
from the DNA slide through the membrane and are captured by the
second slide, creating a pure protein array. In this way, the generated
Systematic antibody & antigen-based proteomic profiling with microarrays
Expert Rev. Mol. Diagn. 11(2), (2011)
protein array is separate from the DNA and the DNA slide can be
reused . Proteins produced by cell-free expression have, among
other things, been used within the field of immunological stud-
ies and vaccine development, where the protein arrays have been
utilized for the screening of antibodies in patient sera [89,92,103,123].
Interaction studies have also been performed in which the produced
proteins serve as baits, as reviewed by Chandra et al. .
Using synthetic peptides for array applications as capture agents
instead of proteins offers an interesting alternative within antigen-
based arrays. Peptides can be synthesized in large quantities, with
different types of modifications and immobilization features to
adapt to the downstream ana lysis. Possible disadvantages of pep-
tides compared with full-size proteins include a lower affinity, a
structurally limited representation of the protein they originate
from, and an incomplete coverage of binding regions that affect
the specificity of the interaction. For these reasons, peptide arrays
are still generally used as a complement to protein arrays [31,124].
Peptide arrays can be produced either by a parallel peptide syn-
thesis in situ, namely directly on array surfaces, or by spotting the
peptides as already-synthesized molecules . Peptides of around
10–15 residues were initially synthesized on cellulose membranes
in a macroarray format . Yet, peptide macroarrays have been
further developed into microarrays, enabling screening in a more
high-throughput manner  and overlapping peptide fragments
covering the entire protein sequence of interest can simultane-
ously be synthesized on the membrane and then incubated with
an antibody-containing sample. By synthesizing peptides and
sequentially introducing them to array formats, antibody–peptide
interaction can be monitored in order to identify epitopes on pla-
nar [127–131] and bead-based assays [132,133]. Enzyme interactions
with their peptide substrates or inhibitors have also been stud-
ied together with enzyme profiling using peptide arrays [134,135].
Furthermore, peptide arrays are being exploited for the serologic
diagnosis of infectious diseases , which supports the potential
of these arrays as important tools for profiling and diagnostic
applications . High-density peptide microarrays are still wait-
ing to be fully explored, but they are now emerging as potentially
very attractive future tools with at least the theoretical possibility
of synthesizing millions of peptides on an array and therefore
enabling a proteome-wide coverage [137,138].
Reverse-phase protein arrays are constructed by immobilizing com-
plex specimens, such as cell or tissue lysates or total serum/plasma,
in an orderly fashion. These samples are simultaneously probed
with specific antibodies targeting different proteins of poten-
tial interest. Reverse-phase configurations are well suited for
screening large sample cohorts all in one experiment and under
identical experimental conditions, and for studying any disease-
related changes in the expression of specific proteins and in pro-
tein modifications . This approach had been praised mainly
for its potential to utilize minimal amounts of precious clinical
material obtained through a biopsy. Reverse-phase arrays were
therefore initially designed for the immobilization of microdis-
sected tissue lysates in order to study specific tissue cell sub-
populations  and functioned as miniature equivalents to the
classical immunohistochemistry assays . Likewise, they have
originally been employed to study human cancer cell lines by
immobilizing cultured cell lines . The possibility of studying
post-translationally modified proteins in cancer tissues and cell
lines have made reverse-phase arrays a powerful tool for profiling
signal transduction pathways in cancer [143–145] and thus within
application-oriented cancer research.
A posterior proof-of-principle study also demonstrated that the
reverse-phase array format allows for the immobilization of thou-
sands of serum samples and thus large-scale screening of protein
levels as well . In that sense, serum arrays shifted the original
focus of the application of reverse-phase arrays : array setups
producing results comparable to that of ELISA offered a rapid
alternative to evaluate and experimentally verify putative blood
biomarkers [148,149]. Serum arrays can therefore serve as comple-
mentary verification platforms to support the findings from for-
ward-phase screening approaches such as antibody microarrays 
and the possibility of including and testing multiple replicates and
sample dilution series simultaneously increases the assay’s robust-
ness for such purposes . Serum arrays have also been success-
fully employed in identifying deficiencies of specific serum pro-
teins within immunodeficiency screening from dried blood spot
samples of newborns  or for the determination of IgA levels in
children . These arrays therefore represent a flexible tool both
for the large-scale identification and verification of certain serum
protein profiles, owing to their highly parallel nature and require-
ment for very small volumes of serum. Just like antibody arrays,
the full feasibility and success of reverse-phase array setups strongly
depends on the availability of high-quality affinity reagents [154,155].
Nonetheless, there is an intrinsic read-out sensitivity issue associ-
ated with reverse-phase array applications, simply due to the fact
that very small volumes of samples are deposited on the array sur-
faces. This can be exemplified by the fact that if 400 pL of plasma
are spotted on an array and the protein of interest has a molecular
weight of 50 kDa and it is present at a concentration of 10 pg/ml
(0.2 pM), the theoretical number of molecules that have been spot-
ted is 80. Presumably, only a fraction of these 80 molecules will
be available for detection, as epitopes could be masked by other
proteins or alongside immobilization. As a result of this, the reverse-
phase protein microarrays should be regarded as suitable for the
ana lysis of medium to highly abundant protein targets, but still
with the unique possibility to analyze thousands of samples in par-
allel. In earlier studies, sensitivity was obtained in the lower µg/ml
range for serum microarrays targeting IgA . Advances using pla-
nar waveguide-based evanescent field fluor escence excitation 
have significantly increased the read-out sensitivity [157–161].
Proteomic profiling considerations
Blood is, in most cases, the preferred sample source in clinical
diagnostics, and a multitude of methods have been established
for studying its cell and cell-free subproteomes . Besides the
Ayoglu, Häggmark, Neiman et al.
hematopoietic components, blood is under continuous exposure
from cells outside the blood stream owing to turnover, secretion,
and disease-related leakage of proteins from tissue into blood
vessels. This leads to the assumption that all proteins are likely to
be present in blood at a certain point in time and under certain
(disease) conditions  and this has incited technological devel-
opment. While the stated assumption hampered efforts to define
plasma proteins [164–166], this dynamic fluid is also known to differ
significantly between nondiseased individuals from different gen-
der and age groups. Even though the process of collecting blood is
favorable over tissue, owing to a less invasive sampling procedure,
the different blood collection procedures and the protocols for the
preparation of serum or plasma do have an impact on the study
outcome and are not currently standardized [167–169]. Within these
blood-derived samples, their unique composition and the broad
concentration range of different proteins poses a challenge to any
Other than blood-derived samples, cerebrospinal fluid (CSF),
urine, saliva, gastric juice and tear fluid [41,170–174] offer interesting
sources for biomarker discovery. These may be advantageous in the
context of some diseases, for example, if the affected area is known
to be separated from blood by cellular barriers (e.g., blood–brain
barrier) or encapsulated from direct blood supply (e.g., inner wall
of the bladder). Proteins present in the brain passively diffuse
into the CSF with a total protein concentration of approximately
0.5 mg/ml and thereby 100-fold less than in plasma. The whole
CSF volume is replaced approximately four times a day [175,176]
and it has been of great interest in the context of neurological
diseases such as amyotrophic lateral sclerosis, Alzheimer’s disease,
Parkinson’s disease and multiple sclerosis [170,177,178].
Another interesting body fluid for clinical diagnosis is urine.
The total protein secretion is approximately 150 mg per day and
the concentration in urine varies with food and water intake and
the time it stays inside the bladder. This poses a challenge when
it comes to consistency of sample collection, and a high con-
centration of salts and small peptides could obstruct proteomic
profiling. Compared with other body fluids such as blood and
CSF, urine has the advantage of being collected by a noninvasive
procedure and it has been used mainly in studies of the kidney and
bladder-related diseases such as diabetic nephropathy , blad-
der cancer [180,181] or even prostate cancer , but also to study
diseases at other locations owing to the possibility of detecting
plasma-originating proteins [183,184].
Cells & tissues
Besides more accessible body fluids as a source for proteomic
profiling, samples derived from cells and tissue do often directly
represent the affected organ and cells. For a cell culture-based
ana lysis, profiling by affinity array methods has been established
to resolve the protein composition via size fractionation of lysed
cells , from ex vivo culture techniques  or arrays with immo-
bilized whole cells [185–187] and tissue microarrays . Tissue-
derived samples obtained from surgical biopsies certainly offer the
most direct insight into disease and allow a comparison between
affected and nondiseased proximal tissue of the same origin, but
their availability is often limited. Furthermore, the ana lysis of tis-
sue or cellular samples with multiparallel affinity tools demands
sample lysis to create soluble representations. This solution pres-
ents a mixture of different cells and cell types in comparison
with tissue sections where specific cells and areas can be defined.
In order to allow lysate-based methods to be competitive with
immunohistochemistry approaches, such as tissue microarrays,
additional procedures, such as laser capture micro-dissection ,
need to be inserted upstream of multiplexed read-out systems .
In proteomic studies with different clinical material, consider-
ations regarding cohort design, sample-collection strategies and
storage are of utmost importance but go beyond the analytical
focus of this article. However, the pre-analytical preparations are
necessary to transform and adapt the state of the specimen to
the analytical platform . In most commonly used proteomic
efforts with MS tools, extensive efforts have been dedicated to
sample prefractionation by chromatographic methods or protein
depletion in order to obtain a more sensitive detection [192–199].
Currently, these upstream procedures cannot be circumvented
since factors such as sample complexity, high-abundant compo-
nents and sample composition pose a challenge for detection and
reproducibility [164,198]. However, MS methods are still on the fore-
front in cell- and tissue-based approaches  and for the ana lysis
of post-translational modifications . In affinity proteomics
where reverse-phase arrays are used with lysed cells and tissues, the
impact of lysis protocols on sample immobilization and read-out
strategies has to be carefully considered . In antibody array
proteomics, protocols for protein labeling are widely applied and
generally require the removal of unincorporated dye molecules to
reduce background levels, but no fractionation or depletion steps
are needed to achieve sensitivity in the lower ng/ml range. Recent
developments in the field offer both planar  and antibody sus-
pension bead array protocols  that circumvent any purification
steps and hence allow the analysis of diluted copies of the sample
without altering its original composition.
Tools for statistical ana lysis and biomarker classification of protein
array data are evolving alongside the technical developments in
the field and thus, further efforts have to be made to develop,
compare, evaluate and standardize them. With a growing interest
in using protein microarrays as analytical platforms, data inte-
gration and ana lysis are being implemented, originating from
established DNA microarray ana lysis [203,204]. This can include
performance controls that are being integrated into microarray
experiments in order to examine the quality of a large micro-
array data set before any subsequent and more detailed statistical
ana lysis is performed [203,205]. Such controls encompass replicate
ana lysis, various types of positive, negative, normalization and
cross-reactivity controls , spike-in ana lysis, implementation of
dual-color detection assays, preparation of common references ,
and incorporation of assays with randomized arrangements of
samples and analytes .
Systematic antibody & antigen-based proteomic profiling with microarrays
Expert Rev. Mol. Diagn. 11(2), (2011)
To identify potential outliers, data pretreatment can be
used [207–209]. This could include data centering, scaling [210,211]
and normalization  to filter and reduce the noise within a
high-dimensional dataset , and to account for possible sys-
tematic experimental variation. Considering the underlying inter-
actions in any protein microarray data, the determined signal
intensities depend on several factors, such as binding affinities and
target abundance, as well as epitope accessibility, all with regard
to sample and assay environment. The use of sample-dependent
scaling factors, such as in probabilistic quotient normalization
method , takes into account technical dilution artifacts and
differences in total protein content of different samples, without
interfering with data dynamics .
In order to obtain a high probability of declaring a protein profile
to represent a biological difference related to a certain physiological
state, stringent statistical ana lysis is needed to separate random,
technically biased and individual patient variation. Compared with
classical single-plex ELISA assays, where the optimization for every
target protein and affinity reagent can be addressed individually, the
settings of high-throughput multiplexed assays cannot be adjusted
to suit all included targets. Thus, the ana lysis of protein microar-
ray data has at least two groups of variance parameters to take into
consideration: first, the technical variance introduced by different
affinity reagents targeting different proteins with different charac-
teristics and second, the biological variance in the protein composi-
tion at a given time of sampling. In comparative studies involving
case and control sample groups, both the within- and across-group
variance has to be considered for identifying differences. When
investigating variance effects, analyses of linear models [41,215,216]
are common tools. Technical artifacts, such as position-based bias
or time drift in the ana lysis, need to be addressed , as well as
artifacts introduced by sample collection procedures and storage .
Significance is commonly stated by p-values, in order to define
contributors and classifiers for disease identification. Even though
the null-hypothesis significance test is the most frequently used sta-
tistical method within biomedical science, resulting p-values should
be handled with care. Criticism has been raised regarding the usage
of p-values as the sole decision criterion for hypothesis testing .
One of the points raised is the direct link between sample cohort
size and the resulting p-value, which means that lower p-values
will always be achieved in larger study cohorts. Nevertheless, null-
hypothesis testing is convenient to narrow down the list of target
proteins to a smaller subset and could be performed by parametrical
t-tests for normally distributed data, or by nonparametrical rank
sum tests for data following a non-normal distribution.
Data ana lysis strategies that include multiple testing corrections
can comprise false-discovery rate criteria [219,220] or hypothesis tests
that account for these multiple corrections simultaneously .
Every decision on whether to include or discontinue with one or
several candidates is also closely dependent on the downstream
strategies. It is therefore important to balance statistical cutoff
thresholds and their combination with false-discovery rate correc-
tions with the capacities of the verification methods . Hence,
multiplexed procedures included in secondary stages facilitate to
involve more than just few of the top candidates.
When interesting potential biomarker candidates have been
identified, the target’s diagnostic, prognostic or therapeutic value
needs to be assessed. Diagnostic accuracy has been judged with
receiver operator characteristics that allows the examination of
a biomarker in terms of sensitivity and specificity, and the area
under the curve is often utilized as a comparative parameter
between different biomarkers or combinations of such . To
evaluate the combined potential of panels of biomarkers, multi-
variate prediction models , principle-component ana lysis and
support vector machines [222–224] are the tools now being applied
with growing interest.
Verification of affinity array discoveries
The technical verification of protein markers discovered via anti-
gen or antibody-based protein microarrays aims to ensure that the
obtained results were not made as a result of artifacts. By re-ana-
lyzing the samples using the initially employed set up, reproduc-
ibility of the results can be evaluated based on the variation within
and across multiple experiments. In addition, related proteomic
platforms could serve as a technical verification for each other to
exclude platform bias . To evaluate the sensitivity and specificity
of interesting interactions, spike-in experiments and competitive
assays may be employed , both preferring antigens represent-
ing the native protein to be available. Other important strategies
include applying and comparing profiles from multiple antibod-
ies generated towards the same target protein, potentially also by
different providers or methods and preferably targeting different
epitopes of the protein. Binders raised towards separate epitopes
of the same target protein could potentially help elucidate the
conformational stage and the degree of modification of the pro-
tein. Orthogonal array methods and Western blots can be used
to determine the molecular mass of a protein target detected in
a given specimen and indicate differences in protein abundance
when comparing different samples. To identify proteins captured
by a specific, immobilized reagent, the occurring interaction can
be displayed via bead-based immunoprecipitation, whereupon the
captured binding partners can be eluted and monitored on Western
blot ana lysis or MS [226–228].
In most studies, a sandwich assay is regarded as the optimal tech-
nical verification assay as it is closest to clinical practice and it com-
bines specificity with sensitivity in combination with the possibility
of quantitative ana lysis. As discussed earlier, these assays are the
least flexible to multiplex and are difficult to establish. Since there
are already a number of methods allowing a technical verification
of potential findings, a combination of at least two of the presented
approaches should be included before the consideration of finding
a candidate worth entering a biological verification phase.
To reproduce technically verified findings from a discovery phase,
it is necessary to assess the biological relevance of a biomarker
candidate in independent sample cohorts. This would extend the
possibility of drawing disease-related conclusions from the initial
findings, as it takes into consideration dissociating discoveries
Ayoglu, Häggmark, Neiman et al.
made solely because of a certain cohort provider, collection pro-
cedure, specimen preparation, storage or handling. Thus, larger
sets of samples from one or several cohorts should be consid-
ered as another initial step of biological verification, because the
cohort size, determined on the basis of a power ana lysis, would
address technical errors associated with the discovery phase [24,229].
Any later phase of biological verification should include initially
employed sample cohort(s), thus making use of a sample cohort
overlap for a further technical interpretation. In addition to an
increased size, a higher degree of diversity and definition of the
verification cohort is required to accelerate the biological confir-
mation, to achieve further disease-specific subclassifications or
to account for other unrelated benign pathologies . As the
biomarkers advance through the verification phases, there is a
growing need for increasing sample numbers from unrelated col-
lections. This task has to be co-orientated together by biobanks
in order to serve the scientific community with appropriate clini-
cal material and collections [231–233]. There is a growing initiative
towards a more standardized sample collection and storage in bio-
banks [234,235] and also to provide samples in formats that would
allow faster processing in research laboratories. In parallel to these
standardization efforts, affinity-based array platforms are being
introduced to meet high-throughput requirements and to study
clinical material stored in biobanks in order to discover, verify and
qualify potential biomarker targets [24,236].
Performing systematic proteomic profiling with the use of
antigen or antibody-based methods and by disconnecting the
reagent selection from disease associations opens a gateway to
previously unexpected discoveries within the biomarker context.
The improving accessibility and availability of reagents from a
variety of sources increases the prospect that the implementa-
tion of antigens or antibodies into highly multiplexed and high-
throughput array-based technologies will open possibilities for
new biomarkers to emerge. To meet the needs for improved diag-
nostics and patient care, screening larger patient cohorts with
high-throughput and multiparallel tools should ultimately aim
at translating discoveries into clinical ana lysis formats.
A first coverage by antibodies and antigens of the human pro-
teome is estimated to be achieved within 5 years by efforts such
as the Human Protein Atlas . As a result of these initiatives,
the massive parallelism of ana lysis of samples and targets will fol-
low and populate the newly conquered grounds with systematic
and nonbiased profiling endeavors. Among the growing number
of new findings that are made during this phase, the benefit of
multiparallel methods will also have to manage the verification
of large panels of biomarkers in order to reach the next level
towards personalized medicine, which is the stepwise translation
and introduction of biomarkers into the clinic.
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial conflict with
the subject matter or materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
• Enhanced adaption and implementation of protein array-based approaches will open the technology to more researchers and
• The accessibility and availability of antibodies and antigens is increasing.
• Ongoing developments of microarray and multiplexing technologies will enable more samples to be profiled on more targets with
• The prospect for systematic and nondirected selection of targets holds great potential to identify new protein–disease connections.
• Infrastructure to access biobank material will enable more streamlined, improved and broader studies to be performed on many
different sample collections.
• The transition from affinity-based biomarker discovery offers new opportunities to go beyond biomarker verification.
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