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ScienceDirect
Cross-reactivity in antibody microarrays and multiplexed
sandwich assays: shedding light on the dark side of multiplexing
David Juncker1,2,3, Sébastien Bergeron1,2, Veronique Laforte1,2,3 and
Huiyan Li1,2
Immunoassays are indispensable for research and clinical
analysis, and following the emergence of the omics paradigm,
multiplexing of immunoassays is more needed than ever.
Cross-reactivity (CR) in multiplexed immunoassays has been
unexpectedly difficult to mitigate, preventing scaling up of
multiplexing, limiting assay performance, and resulting in
inaccurate and even false results, and wrong conclusions.
Here, we review CR and its consequences in single and dual
antibody single-plex and multiplex assays. We establish a
distinction between sample-driven and reagent-driven CR, and
describe how it affects the performance of antibody
microarrays. Next, we review and evaluate various platforms
aimed at mitigating CR, including SOMAmers and protein
fractionation-bead assays, as well as dual Ab methods
including (i) conventional multiplex assays, (ii) proximity ligation
assays, (iii) immuno-mass spectrometry, (iv) sequential
multiplex analyte capture, (v) antibody colocalization
microarrays and (vi) force discrimination assays.
Addresses
1
McGill University & Genome Quebec Innovation Centre, McGill
University, 740 Dr. Penfield Avenue, Montreal, Quebec, Canada H3A
0G1
2
Department of Biomedical Engineering, McGill University,
740 Dr. Penfield Avenue, Montreal, Quebec, Canada H3A 0G1
3
Department of Neurology and Neurosurgery, McGill University,
740 Dr. Penfield Avenue, Montreal, Quebec, Canada H3A 2B4
Corresponding author: Juncker, David ([email protected])
Current Opinion in Chemical Biology 2014, 18:29–37
This review comes from a themed issue on Arrays
Edited by Robert S Matson and David F Smith
1367-5931/$ – see front matter, # 2013 Elsevier Ltd. All rights
reserved.
impediment, and hence only receives little attention
compared to that devoted to the developments of new
assay technologies and methods, such as antibody
microarrays and high sensitivity assays. Ironically, progress in the development and application of novel assay
technologies is often stumped by CR.
Immunoassays depend on an affinity binder — a polyclonal or a monoclonal Ab, a recombinant binder, an
aptamer, or a receptor — that binds a target protein with
high specificity and affinity. The binding is transduced
and amplified into a detectable signal, and in the ideal
scenario, the intensity of the signal is ratiometric with the
concentration of target analyte. Improving immunoassays is predicated on the availability and quality of the
affinity binders, and the need for more, better, and
cheaper binders is widely recognized [3,4]. CR of affinity
binders can be tested using random peptide arrays
for example, but CR to non-homologous amino-acid
sequences was found to be widespread [5]. The suppression of CR is further complicated by the large parameter
space of possible three dimensional conformations
adopted by proteins [6]. The Human Protein Atlas has
established a polyclonal Ab production pipeline with
rigorous quality control standards, and in a Herculean
effort, produced Abs against 15 000 of the 20 000
human proteins (Human Protein Atlas; URL: http://
www.proteinatlas.org/). Yet, when Schwenk et al. evaluated a preselection of 11,000 affinity-purified, monospecific Abs, only 531 Abs produced a single band on a
Western blot, indicating that 95% bound to proteins
outside of the expected band [7]. Whereas some of the
binding might be ascribed to protein isoforms, cleaved
proteins, or post-translational modifications, much is
likely caused by CR. Collectively, these studies underline that affinity binders often cross-react.
http://dx.doi.org/10.1016/j.cbpa.2013.11.012
Introduction
Cross-reactivity (CR) to non-target proteins is ubiquitous and widespread for antibodies (Abs) [1,2], and
together with a lack of Abs against many targets, arguably the biggest obstacle in establishing high performance and large scale multiplexed immunoassays. Unless
CR is adequately addressed and suppressed, or at least
mitigated, it can be devastating to the performance
and reliability of immunoassays. CR is not a mainstream
scientific area of research, and rather seen as an
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The challenge of scaling up assays and enhancing their
sensitivity may thus be formulated as follows: how to
produce a ‘perfect assay system’ while using ‘imperfect
building blocks’, that is, cross-reacting affinity binders?
Or, how should a multiplexed immunoassay with ultrahigh sensitivity while efficiently suppressing CR be
designed? What is the trade-off, i.e. [2,8], how does
multiplexing and CR affect assay performance and can
it be predicted? To provide some answers to these questions, we first review and define CR in single Ab and dual
Ab single-plex and multiplex assays. We review the
strategies developed to mitigate CR over the last decade
Current Opinion in Chemical Biology 2014, 18:29–37
30 Arrays
in multiplexed assays, and assess their robustness, scalability, and potential for ultrasensitive detection. Finally,
we provide some suggestion for future studies and development.
Direct detection of binding can be accomplished by
labeling the entire sample with biotin and incubating it
with fluorescently labeled streptavidin (Figure 1a) or
alternatively, by using label-free detection technologies
that record a change in refractive index, mass, or conductivity at the surface [9]. However, signal arising from CR
(and non-specific adsorption) is typically indistinguishable from the one arising from specific binding
(Figure 1b) thus limiting the performance of this assay
format. To clarify the language, we define this type of CR
as sample-driven CR.
CR and non-specific binding has been studied extensively for single-plex assays, and whereas it may not be
possible to eliminate it completely, it is fairly well understood and managed [10]. Dual Ab assays, also called
sandwich assays, and often simply referred to as enzyme
linked immunosorbent assays (ELISAs) embody an effective strategy to mitigate CR by binding two distinct epitopes on the same protein: a capture Ab (cAb) immobilizes
and concentrates the analyte, while the simultaneous
binding of a labeled detection Ab (dAb) transduces the
binding into a detectable signal (Figure 1c). The strength
of the sandwich assay stems from its tolerance to CR
because a single CR (or non-specific binding) does not
result in a detectable (false positive) signal (Figure 1d).
Indeed, two simultaneous spurious binding events are
required to lead to detectable CR, but the odds for it to
occur are very low. This point highlights the importance to
distinguish between CR that leads to false positive signals
and CR that does not lead to a signal, and which can be
tolerated, but should not be ignored. In all cases, CR can be
further minimized by seeking affinity binders with high
specificity and affinity, and by developing assays protocols
that minimize CR. For example, binders with low dissociation constants (and low off-binding rates) have long
been used, because they can withstand harsh wash steps in
ELISA and other assays, while weakly bound and crossreacting species are washed off [4].
Recently, the limit of detection (LOD) for sandwich
assays was extended to aM and even zM [11,12,13],
even outperforming nucleic acid tests with PCR amplification [14]. High performance Abs and high signal
amplification, which are sometimes combined with digital
assay formats that tally single binding events, are the key
to these advances. Sandwich assays have also been
adopted in multiplexed immunoassays — comprising
both antibody microarrays on chips and dispersed
bead-based assays (also called bead arrays), but as
described below, new types of CR arise as a consequence
of adding the dAbs as a mixture.
Multiplexed assays and cross-reactivity:
antibody microarrays and bead-based assays
Multiplexed sandwich assays (MSAs) were proposed in
1989 by Ekins et al. a few years before the introduction of
Figure 1
Single affinity binder immunoassay
(a) Ideal assay
Key:
(b) Assay with CR
Sandwich immunoassay
(c) Ideal assay
(d) Assay with CR
Capture Ab 1
Protein 1
Labeled Protein 1
Detection Ab1
CR Proteins
Labeled CR Proteins
Label
Current Opinion in Chemical Biology
Cross-reactivity and its effects in single-plex immunoassays with single-AB and dual ABs (sandwich assays). (a) An ideal single-Ab assay with two
specifically bound proteins that are fluorescently labeled for detection. (b) In case of non-specific adsorption (purple protein), CR (red protein), and
protein–protein complexes (blue protein) additional fluorescent labels are immobilized that generate increased or even false positive signals. (c) The
same assay in a sandwich format with a cAb and dAb yields the same assay result under ideal conditions and (d) following non-specific adsorption,
CR, and protein–protein complex formation, highlighting its greater tolerance to CR and other spurious binding events.
Current Opinion in Chemical Biology 2014, 18:29–37
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Cross-reactivity in multiplexed immunoassays Juncker et al. 31
DNA microarrays [15]. Since then multiplexing has progressed from 4 to 50 targets for MSAs, while DNA
microarrays were scaled from 400 to 6.4 million targets
within a decade. Results obtained with MSAs are not
always reproducible. In one study, a series of biomarkers
for early diagnosis of Alzheimer’s disease were identified
[16], but could not be validated subsequently. The failure
was attributed to the variability of the antibody arrays
[17]. Vulnerability to CR, which will be detailed below,
can account for the difficulty of scaling up MSAs and for
the lack of reproducibility.
Reagent-driven cross-reactivity in multiplexed sandwich
assays with reagent mixing
Conventional MSAs are performed by incubating a microarray or dispersed beads with a sample, followed by the
addition of dAbs as a mixture, with the expectation that
each dAb will bind to target analytes bound to the
corresponding cAb. Whereas thermal agitation ensures
that each reagent encounters its target, it also results in
combinatorial interaction of every dAb with all other (i)
analytes, (ii) cAbs and (iii) dAbs. The scenarios of possible
pair-wise CR between cAb, dAb and analytes is summarized in Figure 2. The number of pair-wise interactions is
defined as liability pairs, and for each scenario, it increases
proportionally to the square of the number of targets N
adding up to 4N(N 1) [18]. This sum neglects the
scenario cAb-to-cAb CR which may arise in dispersed
assay formats, but is less critical as the assay readout is
linked to the dAb. Thus, for a 14-plex assay, 728 liability
pairs exist, and for a 100-plex, 39 600; these numbers
represent the vulnerability to CR. The CR for an assay
with only 14 targets was determined experimentally
(Figure 2c) and the results highlight its severity. In the
most favorable case, CR contributes to increased background noise, compromising the LOD of the assay, but in
the worst case it generates a false positive signal. The
vulnerability is also expressed by the fact that a single
contaminated dAb, or an additive in the mixture, can
compromise all assays as it interacts with the entire array.
We define CR arising because of reagent mixing as
reagent-driven CR.
In spite of these issues, MSAs with reagent mixing have
become standard both in microarray and bead-based
formats. However, researchers and vendors alike devote
enormous efforts in establishing and optimizing working
combinations of Ab pairs, which are however limited to
between one and a few tens depending on the targets
and the Abs [19]. But regardless of optimization, as
N increases, the vulnerability increases as 4N2, and
spurious CR can arise by an idiosyncratic property of
the sample, or a bad reagent. The technical capability for
scaling up and making larger arrays has long been available, and for example Luminex Inc. advertised 100-plex
assays for over a decade, but they were not realized as
MSA. A residual CR signal often remains (one vendor
admitted that 10% is tolerated [20]) and is harder to
minimize the larger the array is, thus imposing suboptimal
assay conditions while increasing the background signal.
Indeed, some Abs such as the EGF and CEA cAbs
in Figure 2c broadly cross-react and cannot be used in
MSAs. To minimize CR, diluting the sample 100-fold
while using better signal amplification was proposed [21],
Figure 2
(a) Ideal assay
(b) Cross-reactivity scenarios
(ii)
(iii)
(c) Cross-reactivity in a 14-plex assay
(iv)
(v)
>0.2
FGF
EGFR
OPN
EGF
ENG
LEP
uPA
CEA
HER2
IL–6
IL–8
ANG2
uPAR
GM–CSF
0.1
Analyte
(i)
N(N-1)
N(N-1)
N(N-1)/2
Capture Ab 1
Protein 1
Detection Ab 1
Capture Ab 2
CR Proteins
CR Detection Abs
Key:
N(N-1)
N(N-1)/2
Label
FGF
EGFR
OPN
EGF
ENG
LEP
uPA
CEA
HER2
IL-6
IL-8
ANG2
uPAR
GM-CSF
Number of liability
pairs for N targets
0
Capture antibody (cAb)
Current Opinion in Chemical Biology
Scenarios for reagent-driven CR arising because of reagent mixing in multiplexed sandwich assays as well as experimentally measured CR. (a) An
ideal assay while (b) shows possible pair-wise cross-reactivity scenarios along with the number of combinatorial liability pairs for an assay with N
targets. Cross-reactive binding of (i) dAb to target protein, (ii) dAb to cAb, (iii) dAb to dAb, (iv) protein measured on the array to a cAb, and (v) protein–
protein interactions of two proteins targeted on the array. These combinations apply to both microarray and bead-based assay formats and can occur
simultaneously. (c) Experimental results for cross-reactivity in a 14-plex assay. 14 arrays were each incubated with one of the analytes, followed by a
mixture of the 14 dAbs. The color code is shown on the right and the CR signal is shown relative to the signal of 32 ng/mL of the target protein; red
indicates a CR > 20%. Reproduced with permission from Ref. [18]. In an actual assay, all analytes and dAb are mixed, and whereas the analyte
concentrations may be either lower or higher, there are additional combinations that can give rise to CR.
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Current Opinion in Chemical Biology 2014, 18:29–37
32 Arrays
and whereas it helps, the dilution of already low concentration analytes can make them undetectable.
End-users of commercial MSAs conducted numerous
studies evaluating the performance of various kits, and
initial studies with limited multiplexing found good correlations and concluded MSAs to be reproducible [22].
More recently, studies with higher number of targets and
a more critical analysis found a lack of reproducibility and
correlation between kits from the same [20] and from
different vendors [23–26]. In addition, one study found
significant differences depending on whether an assay
was run in single-plex or in a multiplex format, indicating
that MSAs may not be accurate [27]. Because of these
findings and additional issues [28], the use of MSAs with
reagent mixing is often not recommended for quantitative
analysis and clinical studies [20,23–27,28]. The authors
of these studies are seemingly unaware of the vulnerability to reagent-driven CR, which could explain the
observed variability and contradictory conclusions.
their performance to classical Abs for a variety of applications. SOMAmers are now being routinely used to
identify candidate biomarkers for various diseases [31].
Another approach to mitigate CR is based on protein
fractionation according to mass followed by large scale
multiplexed assays using home-made beads (Figure 3b)
[33,34]. While target proteins are concentrated in
particular fractions, cross-reacting proteins are expected
to be diluted, thus increasing the signal-to-noise ratio.
Furthermore, bound proteins can be cross-validated for
the accurate mass. The shortcomings of this approach are
the complexity of the protocol, the difficulty to quantify
proteins due to the many steps, and uncertainty about
contributions of residual CR to the signal. Conversely,
this approach scales well and experiments with 1725 Abs
were conducted making it the largest multiplexed assay
reported to date [34].
New strategies to contain cross-reactivity in
multiplexed sandwich assays
New strategies to contain cross-reactivity in
multiplexed single-antibody assays
Proximity-induced, pair-wise recognition of specific
binding only
Single-Ab arrays suffer from sample-driven CR, but
unlike reagent-driven CR in MSAs, vulnerability of each
spot of an array is independent of the size of the array.
Simultaneously, only a single Ab is needed per target (and
no matching required), and hence single-Ab arrays (and
bead assays) with over 1000 Abs and sensitivities in the
ng/mL have been developed. Such arrays were used for
biomarker discovery studies for cancer by comparing the
signal between healthy controls and patient samples
[7,29,30]. But based on the results of the study by
Schwenk et al. [7], CR is expected to contribute to the
signal, however, an increase in CR may also be a reflection
of disease progression, and an increase of signal bears a
biomarker value regardless of its origin. It will be important to evaluate the robustness of these assays and whether
they can be reproduced by different laboratories.
Proximity ligation assay (PLA) [35], and more recently
proximity elongation assay (PEA) [36] are two clever
approaches that molecularly discriminate specific binding
from CR. Each Ab pair of a sandwich is tagged with a
DNA recognition barcode, and upon simultaneous binding to a target, the two DNAs overlap and get joined,
either by DNA ligation assisted by a connector oligonucleotide [35], or directly hybridized to each other by
complementary sequences [36]. After elongation, the
newly formed DNA strands are amplified by PCR and
transduced into a measurable signal (Figure 3c). To
prevent random linkage in the solution of complementary
Abs, the sample is diluted before completing the linkage
reaction. Sandwich assays of up to 24-plex were demonstrated while requiring 1 mL of sample and minute
amounts of reagents. The assay protocol is however
somewhat lengthy, the DNA barcodes need to be
selected carefully to avoid CR, and to enable robust
read-out with high multiplexing depends on a microfluidic platform that physically isolates each reaction to
eliminate CR during PCR [35]. Conversely, the low
volumes allow for multiple reactions to be run in parallel,
the concept of PLA and PEA is flexible and PCR amplification could be made conditional on the binding of three
Abs, while new applications such as the recognition of
protein complexes, or of posttranslational modifications
(PTM), as well as high sensitivity assays with single
molecule detection were shown [37].
Mitigation of cross-reactivity in single affinity-binder
assays
In an attempt to overcome the limitations of Abs, DNAbased binders called aptamers were developed. To
further enhance their affinity, Gold and colleagues developed so-called SOMAmers that integrate bases with
chemical side groups reminiscent of amino acids to generate a greater binding diversity [31]. To date, over 1000
SOMAmers have been selected for both high affinity and
low dissociation rate constants, and a solution-phase assay
protocol compatible with plasma while minimizing CR
was established (Figure 3a). The main distinction between SOMAmers and antibody arrays is the dispersed
assay format with two purification steps, and the distributed binding interaction of the oligomers with the ligand
[32]. The CR of SOMAmers has been studied in a limited
range of conditions, and it will be interesting to compare
Current Opinion in Chemical Biology 2014, 18:29–37
Temporal separation of reagents minimizes reagentdriven cross-reactivity
A more radical approach to avoiding reagent driven CR is
to not mix the reagents and two strategies have been
proposed: temporal and spatial separation of reagents.
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Cross-reactivity in multiplexed immunoassays Juncker et al. 33
Figure 3
(a) SOMAmers
(b) Fractionation-bead assay
L
(1) Bind
(2) Catch 1
and wash
L
Si PCB
Pi
Si PCB
SB
B
Si
(2) Incubate labeled
antigens with the
beads
hv
I
II
III
IV
(2)
(2) Ligation via
connector
SB
(3) Cleave
Pi Si
(4) Catch 2
(3)
(3) Measure signal
by flow cytometry
Pi Si
SB
(5) Elute
(3) PCR Pre-amplification
Si
(4) Analyze data
(6) Amplify by
PCR and
quantify
II
III
IV
Signals
I
(4) Quantification by qPCR
Proteins
(e) ACM
(d) SMAC
I
Incubate cAbbeads with:
(1) Mix samples and
proximity probes
(1)
SB
Pi Si
(c) PLA
(1) Fractionate the
proteins by size
II
III
(1) Sample preparation
(1) Spotting
of cAbs
same sample same sample
(1) sample
(f) Immuno-MS
Spot 1
(1)
Spot 2
Spot 3
(1) reference array
Spike isotopelabeled peptides
+
etc. (1)
(2) Incubation
with sample
(2) dAbs
Enzyme
digestion
(g) Force discrimination
receptor oligo
unzip oligo with
Cy3 and biotin
NeutrAvidin
biotinylated dAb
(2) Affinity capture & elution
target antigen
cAb
capture array
(2)
(2)
Spot 1
Spot 2
Spot 3
(2)
(3) fluorescent
labels
(4) Quantify
binding
(3)
(3)
.
(4)
(4)
(3)
(3) Spotting
of dAbs
Spot 1
Spot 2
Spot 3
Spot 1
Spot 2
Spot 3
(3) Mass Spectrometry
(4) Signal
readout
Current Opinion in Chemical Biology
Six multiplexed assay formats that mitigate CR comprising two single-Ab and four sandwich assays. Purple triangles represent a CR event. (a) Cy3labeled and biotin (B)-labeled SOMAmers (S) in a suspended assay format bind to cognate proteins (Pi) followed by multiple washing rounds involving
streptavidin-coated beads (SB), biotinylation and photocleavage (PC) to eliminate CR (purple triangle protein). (b) Pre-fractionation concentrates and
partitions proteins to enhance signal-to-noise ratio of single-Ab binding on beads. The ‘green’, ‘red’ and ‘blue’ proteins are detected in fraction II, and
IV respectively, while the ‘purple (CR)’ protein is sequestered in fraction I. (c) Proximity ligation assay (PLA) uses pairs of Abs tagged with unique
sequence specific reporter fragments. Signal amplification only occurs when a matched pair of Abs bind the same target analyte to create an PCRamplifiable DNA strand, and is unaffected by cross-reacting Abs. (d) Sequential multiplex analyte capturing (SMAC) sequentially adds and retrieves
sets of Ab-coated magnetic beads that are subsequently incubated with matching dAbs. (e) The antibody colocalization microarray (ACM) spatially
addresses each dAb to the matched cAb spot only, thus avoiding mixing altogether, and reproducing the conditions of classical ELISA at the
microscale. (f) Immuno-MS combines Ab-based purification and MS analysis. Proteins are trypsinized into peptides, isotope labeled peptides spiked
in, and both captured on beads using peptide specific Abs, followed by elution and MS analysis. (g) Force-discrimination arrays use soft stamps to
mechanically colocalize each dAb to the matched cAb spot, followed by dissociation. A DNA zip-probe only ruptures in the event of specific binding,
but not CR, and is recorded by the transfer of the Cy3 fluorophore to the cAb spot.
In sequential multiplex analyte capturing (SMAC),
batches beads each coated with different cAb are sequentially added to the sample, retrieved, and incubated in
tubes with the respective dAb in solution, followed by
quantification (Figure 3d) [38]. Analytes of interest are
captured one-by-one; alternatively, different beads with
different cAb, can be added, and multiple targets captured simultaneously. SMAC is versatile and has been
used to map PTMs by incubating a mixture of beads
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coated with distinct Abs targeting multiple distinct
PTMs, followed by incubation with a single dAb against
EGFR. The complementary scenario where cAbs targeting different proteins are incubated with the sample,
followed by incubation with a single dAb against a phospho-tyrosine was also shown. Whereas the number of
sequential steps that can be performed with SMAC
will be limited by practical considerations, the versatility
and the ease of concatenating various assays in one
Current Opinion in Chemical Biology 2014, 18:29–37
34 Arrays
experimental process flow may open up many applications for SMAC.
Spatial separation of reagents eliminates reagent-driven
cross-reactivity
Reagent induced CR can be eliminated by reverting to
the same conditions as in ELISAs where each cAb is only
exposed to a single dAb. Haab and colleagues formed
multiple replicate arrays with a variety of cAbs, and
applied a single dAb or affinity binder to each of them.
Whereas this strategy is not useful to multiplex protein
quantification, is can be utilized to measure multiple
PTMs as well as to uncover protein complexes [39,40].
This approach requires a separate array for each parameter
measured, and somewhat larger sample volumes, but it
is notably well suited for biomarker discovery studies in
blood as large volumes can be obtained.
MSAs with spatial separation of dAb were introduced by
our group and implemented by delivering each dAb to a
single microarray spot with the cognate cAb. We named
this technology antibody colocalization microarray (ACM)
(Figure 3e) [18]. The ACM can be seen as a microarray of
single-plex microscale assays, and hence the CR is
expected to be identical to the one found in a conventional sandwich ELISA. The ACM requires high precision microarray spotters and alignment to deliver both
cAb and dAb solutions within <30 mm when using
100 mm-wide spots. Whereas the initial assay protocol
required spotting during the experiment, we developed
a technology called snap-chip for the registered transfer of
reagent droplets from microarray-to-microarray [41]. cAbs
and dAbs are thus pre-spotted on two distinct slides and
stored. To conduct an assay, the cAb slide is incubated
with the sample, then aligned and snapped with the dAb
slide to transfer all dAbs simultaneously to the cAb spots.
The ACM can be multiplexed and run at high-density
with minute sample and reagent consumption while
being scalable.
Immuno-mass spectrometry assays identify the target
analyte
Mass spectrometry (MS) is biased toward high abundance
molecules, but conversely has the ability to fingerprint
proteins via the peptides it detects, offering a powerful
tool to distinguish actual binding from CR. To detect
lower abundance proteins, Ab-based enrichment was
combined with MS analysis by a method called SISCAPA
[42]. The sample is first digested by trypsinization, followed by spiking with synthetic, isotope-labeled proteotypic peptides that serve as reference. Next, the sample is
incubated with beads functionalized with peptidespecific Abs, followed by elution and liquid chromatography–MS (Figure 3f). The sensitivity of SISCAPA can
reach pg/mL, but only when using large sample volumes
and the protocol is complex. A variant that uses matrix
assisted laser desorption ionization is simpler and faster
Current Opinion in Chemical Biology 2014, 18:29–37
but at the cost of lower sensitivity [43]. The scaling up of
either method is not trivial and is limited by the availability of peptide-specific Abs [42].
Whereas all methods discussed to this point aimed at
reducing CR, one group leveraged CR to their advantage.
Broadly cross-reacting Abs were generated using short
peptide sequences (shared by many proteins, while avoiding sequences present in high abundant proteins) as the
antigen. Then, using a similar protocol as SISCAPA,
peptides from trypsinized samples are enriched, and
the sequence of each peptide, and hence the corresponding protein, identified by MS [44]. The accuracy of this
method remains to be validated with more complex
samples with large differences in protein concentration,
but using this approach, large numbers of proteins can be
targeted simultaneously.
Force-based discrimination immunoassays distinguish
specific binding and cross-reactivity
A nanoscale force spectroscopy sensor using programmable DNA linkers has been developed to detect and
quantify binding in a tug-of-war test. A dAb conjugated
to a DNA-force sensor that is set to rupture only in the
event of strong, specific binding of the dAb to a target
analyte (but not CR) is mechanically pulled away, and the
transfer of the labeled DNA probes reveals captured
analytes (Figure 3g) [45,46]. This approach has already
been used to probe 7 targets simultaneously. Using the
snap-chip method developed for the antibody colocalization microarray, it might be possible to miniaturize and
further multiplex this concept, and based on the results
obtained with assays using acoustic or magnetic stringency tests [12], it might be possible to extend the LOD
by several orders of magnitude.
Discussion
CR is hard to eliminate from immunoassays as Abs are
imperfect and often a ‘black box’, yet assays with ever
more multiplexing and higher sensitivities are sought
after. Much of the discussion in this opinion is based
on incidental observations of CR and reasoning. Indeed,
systematic studies of CR are rare [5,18] and the source of
CR or assay interference has been difficult to identify. A
synopsis of the various methods developed to mitigate
CR presented in this opinion is shown in Table 1.
Whereas, the discussion was largely focused on Abs,
the conclusions drawn here are applicable many other
types of affinity binders.
There are two fundamental routes to multiplexing, one
being single Ab assays and the other dual Ab assays.
Single Ab assays are easy to scale up, but are susceptible
to sample-driven CR [7]. Efforts to mitigate CR include
longer washing and the use of non-antibody binders [31],
as well as sample pre-fractionation [34]. If immuno-MS
methods could be adapted to work with full length
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[45,46]
High
Yes
7
4 hours
No blood test
Yes
[18,41]
Moderate
Yes
50 (80)
4–24 hours
20 mL
Yes
[38]
High
Maybe
Moderate
23
72 hours
No blood test
Semiquantitative
fg/mL
Very strong
pg/mL
Strong
Strong
8–24 hours
8–24 hours
10–35 mL
1–45 mL
proteins rather than peptides, it will be possible to shed
light on the identity of proteins and protein complexes
captured by single Ab assays and on the source of differences [7,29,30].The tried and tested strategy to mitigate
CR and improve specificity is to use two distinct
parameters to capture and quantify an analyte in a sample.
The two parameters are for example two distinct Abs as in
the sandwich assay, or a chromatographic separation
followed by affinity binding, or affinity binding followed
by MS. However, one needs to consider whether these
assays are scalable, and dual Ab, sandwich assays with
dAbs mixtures introduce reagent-driven CR. The vulnerability to CR was shown to scale with the number of
targets N as 4N2 and to be severe for a 14-plex assay. It
may give us pause before deploying conventional MSAs
in a clinical context with therapeutic decisions and
patients at stake. A better understanding of CR and
how it plays out in multiplex assays will help improve
the performance of multiplexed immunoassays. Among
the various methods listed in Table 1, the most promising
strategies operate by differentiating specific and CR
binding, as for example PLA [35], or by identifying
the bound species using MS [44], or by eliminating
mixing altogether as with the ACM [18]. Many of these
methods might be enhanced further, for example by
introducing additional stringency steps, such as forcebased discrimination [46] along with greater signal amplification [11,12,13,14]. Reliable and sensitive multiplexed immunoassays will accelerate life science
research, and help discover novel candidate protein biomarkers at ever lower concentrations for earlier and more
accurate disease diagnosis, and also support clinical translation.
Acknowledgements
We wish to thank NSERC, CIHR and the CCS for funding, and Andy Ng
for reading the manuscript. DJ acknowledges a CRC, and VL the NSERCCREATE ISS program for support.
References and recommended reading
Immuno-MS
Proximity ligation
assay (PLA)
Sequential multiplex
analyte capturing (SMAC)
Antibody colocalization
microarray (ACM)
Force discrimination
Very strong
Strong
pg/mL
ag/mL
89
23 (92)
Yes
Moderate
No
Demonstrated
Moderate–high
Moderate
[16,17,18,19,
21,22–27,28]
[42,43,44]
[35,36,37]
Moderate (kits)
Limited
pg/mL
Variable-weak
1–.50
4–24 hours
10–100 mL
No
[7,29,30]
[31,47]
[33,34]
Low
Moderate
High
No
No
No
Yes
Yes
Yes
810 (>1100)
>1000
1725
4–24 hours
24–48 hours
8–24 hours
3–200 mL
20–100 mL
–
ng/mL
pg/mL
Semiquantitative
Weak
Moderate
Moderate
Single antibody array
Dispersed aptamers
Fractionation and
single Ab beads
MSA with mixing
Sample volume
(plasma/serum)
Limit of
detection
Containment
of CR
Technology
Comparison of multiplex antibody array and bead-based assay technologies
Table 1
Time to
result
Level of multiplexing
(non-published info)
Scalable
Potential for high
sensitivity assays
Complexity
Pertinent
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Cross-reactivity in multiplexed immunoassays Juncker et al. 35
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