Interdisciplinary detection science
www.rsc.org/analyst
ISSN 0003-2654
CRITICAL REVIEW
Christof M. Niemeyer et al.
Sensitivity by combination: immuno-
PCR and related technologies
Volume 133  |  Number 6  |  June 2008  |  Pages 685–824
PAPER
Ganesh D. Sockalingum et al.
Detection of pathological aortic
tissues by infrared multispectral
imaging and chemometrics 0003-2654(2008)133:6;1-G
i-SECTION: CRITICAL REVIEW www.rsc.org/analyst | The Analyst

Sensitivity by combination: immuno-PCR

and related technologies
Michael Adler,a Ron Wacker*a and Christof M. Niemeyer*b,c
DOI: 10.1039/b718587c

The versatility of immunoassays for the detection of antigens can be combined with the
signal amplification power of nucleic acid amplification techniques in a broad range of
innovative detection strategies. This review summarizes the spectrum of both,
DNA-modification techniques used for assay enhancement and the resulting key
applications. In particular, it focuses on the highly sensitive immuno-PCR (IPCR) method.
This technique is based on chimeric conjugates of specific antibodies and nucleic acid
molecules, the latter of which are used as markers to be amplified by PCR or related
techniques for signal generation and read-out. Various strategies for the combination of
antigen detection and nucleic acid amplification are discussed with regard to their
laboratory analytic performance, including novel approaches to the conjugation of
antibodies with DNA, and alternative pathways for signal amplification and detection. A
critical assessment of advantages and drawbacks of these methods for a number of
applications in clinical diagnostics and research is conducted. The examples include the
detection of viral and bacterial antigens, tumor markers, toxins, pathogens, cytokines and
other targets in different biological sample materials.

1. Introduction: the power of polyclonal antibodies, as well as from our

a
Chimera Biotec GmbH, Emil-Figge-Str. 76 A,
D-44227, Dortmund, Germany.
E-mail: wacker@chimera-biotec.com; Fax: +49
(0)231-9742-844
b
Technische Universität Dortmund, Fachbereich
Chemie, Lehrstuhl für Biologisch-Chemische
Mikrostrukturtechnik, Otto-Hahn-Str. 6,
D-44227, Dortmund, Germany. E-mail: christof.
niemeyer@tu-dortmund.de; Fax: +49
(0)231-755-7081
c
ISAS – Institute for Analytical Sciences,
Bunsen-Kirchhoff-Strasse 11, 44139 Dortmund,
Germany
DNA
Our increasing knowledge of the composi-
tion, structure and properties of biological
molecules allows us to take advantage
of the functionality of these molecules,
to be implemented as versatile tools in
a broad variety of novel analytical as-
says. As an example, the development
of immunological detection methods has
largely benefited from the elucidation of
structure and functionality of antibodies,
knowledge of how to raise mono- and
understanding of enzymatic amplification
systems. Our ability to link antibodies and
enzymes to generate bio-molecular devices
has led to the variety of specific and sensi-
tive assays, such as the so-called enzyme-
linked-immuno-sorbent-assay (ELISA).1 In
ELISA, an antibody specifically recog-
nizes the target (antigen) structure and
the connected enzyme usually converts a
low molecular weight chemical substrate
into a colored, fluorescent or lumines-
cent product. Due to the catalytic nature
of enzyme-mediated substrate conversion,

Dr Michael Adler (chemist and head of laboratory) and Dr

Ron Wacker (biologist and product manager) are employees at
Chimera Biotec GmbH, a company commercializing analytical
applications of DNA–protein conjugates. The company was
founded in 2000 by Dr Adler and Dr Christof M. Niemeyer.
Since 2002, Dr Niemeyer has been Professor of Chemistry at the
Technical University of Dortmund (Germany) where he holds
the chair of Biological and Chemical Microstructuring. Since
2005, he has held an additional appointment as a group leader
at the ISAS – Institute for Analytical Sciences, Dortmund. His
research interests concern the chemistry of semisynthetic DNA–
protein- and nanoparticle-conjugates and their applications in
life-sciences, catalysis and molecular nanotechnology.
Dr Ron Wacker, Dr Christof M. Niemeyer and
Dr Michael Adler (left to right)

702 | Analyst, 2008, 133, 702–718 This journal is © The Royal Society of Chemistry 2008
the binding event of the antibody oc-
curring at the level of few molecules is
amplified, thus becoming detectable at
the macroscopic level. The utilization of
enzymatic activity for detection of im-
munological binding events, however, is
no longer limited to the typical small-
molecule enzyme substrates. Nowadays,
an increasing number of research and
commercial applications concern the use
of macromolecular substrates, in partic-
ular, nucleic acid molecules. The combi-
nation of DNA-processing enzymes with
the general principle of ELISA method-
ology results in a range of novel ana-
lytic techniques which are based on the
unique properties of DNA as a functional
molecule (Fig. 1, 2, 3). Considering the
various different strategies used for DNA
processing as well as the broad field of
applications, it is helpful to provide an
overview on this rapidly growing field of
assay development (Fig. 4) to assist the
non-expert in selecting a suitable tech-
nique which meets the demands of a given
analytical challenge.
The idea of combining an antibody-
based binding event with the signal am-
plification power of PCR goes back to
the 1992’s with the initial description of
Fig. 1 Key steps of DNA-enhanced immunoassays. Note that additional details regarding
immuno-PCR (IPCR).2 The polymerase antibody–DNA coupling are summarized in (Fig. 3) while aspects regarding signal-generation
chain reaction (PCR)3,4 is a prime tool and DNA detection are further illustrated in (Fig. 6 and 7).
for taking advantage of the intrinsic prop-
erties of the DNA molecule to be used
as the substrate for signal amplification5
(Fig. 3A). The exponential amplifica-
tion of a DNA molecule and, there-
fore, the detection of even single target
molecules,6 is nowadays routinely con-
ducted by PCR, this level of sensitivity
is inaccessible for conventional ELISA
methods (Fig. 5). Furthermore, the infor-
mation stored within the DNA sequence,
the physicochemical stability of DNA
molecules and the number of enzymes and
techniques available for selective manipu-
lation and labeling significantly enhances
the applicability of DNA-based assays.
Fig. 2 The intrinsic properties of DNA molecules can be used to improve almost all fields
However, there are several major chal- of immunoassays. The scheme summarizes the range of different targets studied by exemplary
lenges to be solved in order to real- applications including IPCR with protein chimeras,20,21 Universal-IPCR,13 immuno-RCA,58
ize practical and routinely applicable im- PD-IPCR,44 ImperacerTM ,37,55 competitive assays for small molecules134 or proximity ligation.62
munoassays with the implementation of
the exponential nucleic-acid amplification
by PCR or related techniques (Fig. 1): and drawbacks and appropriate tech- tion leads to novel requirements for assay
(1) Coupling of immunoassay reagents niques have to be chosen according to the design, as well as new options in terms
and DNA markers: several strategies are demands of the intended application. of the scientific or diagnostic questions
available for linking antibody recognition (2) Assay adaptation, modification and which can be addressed by the assay. While
with nucleic acid amplification (Fig. 3B). application: the enormous sensitivity due technically the combination of antibody
Each alternative has its own advantages to the exponential nucleic acid amplifica- binding and enzymatic DNA amplification

This journal is © The Royal Society of Chemistry 2008 Analyst, 2008, 133, 702–718 | 703
 critical assessment of the three aforemen-
tioned aspects to provide the reader with
expert knowledge regarding practical as-
say performance in this rapidly expanding
field of fundamental and applied research.
In the first part we briefly introduce dif-
ferent types of immunoassays which are
based on specific processing of the DNA
marker. While some approaches are still in
a proof-of-concept phase, other methods
have already reached the stage of routine
laboratory techniques. All techniques have
in common, a significant increase in the
sensitivity of the assay. In the second part,
we critically evaluate the potential advan-
tages and drawbacks of the established
methods, including a detailed discussion
and comparison of various different DNA
detection techniques. In the third part, we
spotlight selected applications from the
broad range of analytical targets (Fig. 2)
to discuss how advantages, in particular
applications, have been realized and how
problems have been circumvented, thus
illuminating the actual practical perfor-
mance of DNA-enhanced immunoassays.

2. Techniques: a journey
through the art of DNA-
enhanced immunoassays
Fig. 3 The evolution of immuno-PCR (IPCR): (A) the set-up of ELISA and IPCR is similar. The pioneering work on the use of DNA
Instead of an enzyme marker, such as alkaline phosphatase (left), IPCR uses amplification of
as an amplifiable label in immunoassays
attached DNA for signal generation (right). (B) Different strategies for coupling antibodies and
was presented in 1992 by Sano, Smith and
DNA: in the initial version of IPCR2 a Streptavidin (STV)–protein A chimeric fusion protein
was used for tagging the detection antibody with biotinylated DNA (I). In the universal
Cantor, who established an initial protocol
IPCR protocol,8–10,13,23 the signal generating complex is assembled in situ by subsequent for the IPCR method2 (Fig. 3B I). The key
incubation steps of biotinylated detection antibody, (strept-)avidin and biotinylated DNA; to IPCR was an inversion of conventional
either using a non-biotinylated primary and a species specific secondary antibody (II) or a ELISA protocols. ELISA uses antibody–
directly biotinylated primary antibody (III). The introduction of pre-assembled antibody– enzyme conjugates while the enzyme’s
DNA conjugates takes advantage of either species- or marker-specific secondary conjugates92 substrate is added subsequently as a freely
(IV) or direct conjugation of target-specific antibodies and DNA14 (V). Approaches such diffusing species. In IPCR the enzyme’s
as phage display mediated IPCR,44 tadpoles of antibodies and DNA,38 or native chemical substrate, i.e., a DNA template, is linked
ligation41–43 introduce elegant ways of coupling antibodies and DNA by circumventing artificial to the antibody while the enzyme is added
modifications such as biotin and complex crosslinking chemistries (VI). The linkage of multiple
subsequently (Fig. 3A). By amplifying the
antibodies and DNA molecules with particles, as used in the bio-barcode technology92 has led
DNA marker through PCR, the limit-of-
to polyvalent reagents, which allow one to connect single antibody–antigen binding events to
a larger number of DNA markers (VII). (C) Comparison of the multiple steps required for the detection (LOD) of a given ELISA is, in
in situ stepwise reagent assembly of the classical universal IPCR approach with the simplicity general, enhanced 100–10 000-fold (Fig. 5
of specific antibody–DNA conjugates. Note that each coupling step requires optimization of and 8). This simple and elegant approach
reaction parameters and leads to a loss in sensitivity. is still the most popular strategy for ul-
trasensitive protein detection by means of
DNA amplification, and nowadays it has
is still a modification of ELISA, the differ- to be coupled with suitable techniques to been established as a routine technique for
ences of both methods are the key for the allow fast, convenient and robust quantifi- applications in both fundamental research
proper choice and application of a suitable cation of the DNA amplification products and applied immunological diagnostics.8,10
assay. (Fig. 7). The true challenges for the practical ana-
(3) Signal amplification and assay read- A number of excellent review articles7–11 lyst, however, are found in the variety of
out: the products of ELISA signal am- have already addressed topics of the different techniques for reagent synthesis,
plification are typically easily detectable. emerging field of DNA-coupled im- signal generation and adaptation to the
Nucleic acid amplification, however, has munoassays. This article focuses on the analytical target under investigation.

704 | Analyst, 2008, 133, 702–718 This journal is © The Royal Society of Chemistry 2008
 Fig. 4 The immuno-PCR “family tree”: historical development of the different immuno-PCR techniques during the past 15 years. While the
number of applications was limited during the initial decade of method refinement (approx. 100 different assays were reported—see Fig. 8
for a statistical analysis), the broad range of today’s applications (see Fig. 2 and 10) now underlines the potential of this method. As the first
milestones in IPCR development focused on the basic questions of antigen–DNA coupling and DNA detection (see also section 2.1 and 2.2),
recent innovations are aiming towards the improvement of assay handling and homogenous assay design, thereby rendering the initial research
method to a routine analytical technique.

2.1 First challenge: coupling of
antibodies with DNA
2.1.1 Modular approach/in-situ cou-
pling. In order to physically link an-
tibodies with DNA for IPCR appli-
cations, various strategies have been
investigated.8,10,12 In general, two different
approaches were used in the majority of
examples. These (i) either use a linker
protein, such as streptavidin (STV) for
sequential coupling of (biotinylated) anti-
bodies and (biotinylated) DNA in a multi-
step protocol13 (Fig. 3B II & III and sec-
tion 2, below) or (ii) employ pre-assembled
and purified antibody–DNA conjugates
in a single-step protocol14 (Fig. 3B IV, V
& VII and following paragraph). Sano’s
initial IPCR was carried out following
the first strategy. The linker protein was
a genetic fusion of STV and protein A,
thus allowing for the binding of the Fc
part of an IgG and subsequent tagging
with biotinylated dsDNA2 (Fig. 3B I).
While the use of this linker allows for
simple labeling and high flexibility, the
multi-step assembly implies the disadvan-
tage of time consuming adjustments of
reagent concentrations for each individual
incubation step.15–17 Moreover, the protein
A-STV fusion is unsuitable for two-sided
immunoassays because it is impossible to
completely suppress its binding to plate-
immobilized capture antibodies. Nonethe-
less, this reagent is suitable for applica-
tions which concern the detection of di-
rectly immobilized target molecules, such
as single blastocytes in pre-implantation
diagnostics.18–21 In a related approach, a
bispecific fusion protein comprised of the
polypeptide chains of carcinoembryonic
antigen (CEA) binding protein and strep-
tavidin was recently used in the detection
of circulating CEA in human sera.22 The
“Universal IPCR” protocol of Zhou et al.,
the so far most popular and most reviewed
approach for model applications8–10,13,23
also follows the first strategy.
2.1.2 Chemical crosslinking. Start-
ing from the “classical” IPCR proto-
col, the potential of DNA as an ana-
lytic tool was exploited since the early
1990’s (Fig. 4). Nowadays, the use of
pre-assembled antibody–DNA conjugates
(Fig. 3B IV & V) is steadily increasing its
foothold in the design of IPCR assays.
One major advantage of this class of
reagents is the significant shortening of
the assay protocol and thus a reduction
of incomplete and/or non-specific binding
events which usually occur in each incu-
bation step.24–26 It has been demonstrated
that the performance of pre-assembled
antibody–DNA conjugates exceeds that of
the stepwise assembled complexes in the
modular approach26 (Fig. 3C). However,
the general drawbacks of these reagents
stem from their complex syntheses and pu-
rification procedures which require expert
knowledge in protein chemistry.14,25,27–33
With the development of refined protocols
for the synthesis as well as the availabil-
ity of universal species-specific antibody–
DNA conjugates, which can be used
similar to the common species-specific
antibody–enzyme conjugates in ELISA,34
the complex chemistry typically realized
by heterobifunctional crosslinkers14 is no
longer an issue for application of the
IPCR method.23,34,35 Application of dis-
tinct ready-to-use IPCR-reagents together
with optimized protocols allow even inex-
perienced users to facilitate highly sensi-
tive quantitative immuno-PCR (qIPCR)23
assays with average routine LOD of sev-
eral 1000 molecules (corresponding to

This journal is © The Royal Society of Chemistry 2008 Analyst, 2008, 133, 702–718 | 705

Fig. 5 Typical results of immuno-PCR (IPCR) experiments. (A) Comparison of IPCR, the
analogous conventional ELISA for the detection of Rotavirus antigens.108 Note the high
linearity and broad dynamic range of IPCR. (B) Comparison of different IPCR assay
techniques for the detection of human TNFa: the use of target-specific antibody–DNA
conjugates enables an increased sensitivity. The dark and light blue bars represent signals
obtained by sequential IPCR (see Fig. 3B III) and direct IPCR with pre-assembled antibody–
DNA conjugates (see Fig. 3B V), respectively. The red curve represents signals obtained in the
analogous ELISA.
sub-picogram amounts of typical pro-
teins) and a broad dynamic range of up to
six orders of magnitude8,10 (Fig. 5), utilized
e.g. in different pharmacokinetic clinical
studies.36,37 Further advantages and draw-
backs of this approach are discussed below
in section 2.
2.1.3 Native/biomimetic strategies.
In parallel to these application-oriented
developments, the properties of DNA
also were the key to novel approaches
in conjugate synthesis. As an example,
Burbulis et al.38 followed the pioneering
work of Niemeyer’s group39 by employing
the biological protein splicing system
for the coupling of DNA molecules
with intein-tagged binding proteins. The
resulting so-called “tadpole”-chimeras
revealed full functionality in IPCR
assays,38 thus demonstrating how direct
706 | Analyst, 2008, 133, 702–718
coupling of recognition proteins can be
achieved without further modification.40
Using the tadpoles, 32 000 molecules of
the model protein PA (protective antigen
subunit of B. anthracis) were detected
in spiked human serum, purified by
gel-fractionation and further extracted
with magnetic beads. The fractionation
was found to be necessary because
the beads bound the tadpoles non-
specifically, regardless of the protein
head.38 These initial problems suggested
that the complexity of the assay was
shifted from the actual coupling step to
the generation of molecules and assay
conditions suitable for this approach.
Recent advances in the field of intein-
mediated protein–DNA-coupling41–43 will
certainly support the general concept
of merging coupling strategies adopted
from biological processes with the
This journal is © The Royal Society of Chemistry 2008
targeted synthesis of functional IPCR
reagents.
Another biological inspired concept for
the conjugation of antibodies with DNA
was recently introduced by Guo et al. who
described a phage display-mediated IPCR
assay44 (Fig. 3 VI). Single chain variable
fragments (scFv) of IgG antibodies were
cloned and expressed on the surface of
phages45 and the complete phage was
used for binding to the target antigen.
Following the release of the DNA from the
immobilized phage, PCR amplification
enabled high sensitivity of this assay. This
was illustrated by detecting 10 pg ml−1
of purified Hantaan virus antigen NP or
1 ng ml−1 of the prion protein. This work
also indicated that IPCR is still feasible
even with low-affinity binders (i.e., the
scFv chains) thereby opening the door to
novel applications. In an recent continua-
tion, the usefulness of this technique for
the selection of recombinant antibodies
and their binding proteins in plasma cells
of multiple sclerosis CSF was demon-
strated by Yu et al.46 There, IPCR was used
as a screening tool with a sensitivity of as
few as 100 phage particles compared to a
detection limit of more than 104 particles
in conventional ELISA.
In a “minimalist” approach, target–
protein binding nucleic acid aptamer
sequences47,48 can substitute antibodies,
thereby using oligonucleotides for both,
target recognition and signal detection.
This strategy, which is elegant in its
simplicity as well as challenging in the
design of the aptamers, has been reviewed
recently.9
2.2 Second challenge: signal
generation and detection
From “classical” nucleic acid analysis, a
broad number of well-established quan-
tification techniques are already available
for application in IPCR-based assays. The
practical use of these techniques is there-
fore, discussed in detail in section 3.2,
below. For the purpose of evaluating the
special properties of DNA as a marker
molecule in immunoanalytics, however, a
number of unique detection strategies are
also especially interesting.
2.2.1 Transcription. In the “fluores-
cent amplification catalyzed by T7-poly-
merase technique” (short: FACTT,
which was initially termed “immuno-
detection amplified by T7 RNA
 for the firefly luciferase protein. Forma-
tion of the immunocomplex was followed
by a one-step, cell-free translation expres-
sion step, which enabled the detection
of 3000 DNA molecules or 50 000 anti-
gen molecules, respectively. Therefore, this
method not only represents an additional
example how nature’s functional elements
can be utilized for in vitro diagnostics
but it also underlines the versatility of
DNA strands as a tool for innovative
immunoassay design.
2.2.3 Phosphorylation/dephosphory-
lation. Protein detection by DNA ampli-
fication is also possible by addressing
the functionality of the proteins directly.
Banin et al.53 introduced an alternative
approach to IPCR for the detection
of the enzyme alkaline phosphatase
(AP) using phosphorylated DNA as the
substrate. In the presence of AP, the
DNA substrate was dephosphorylated
and, thus, no longer a target for a k
exonuclease which otherwise degrades
the phosphorylated DNA. Therefore, the
presence of AP is confirmed when samples
incubated with phosphorylated DNA and
Fig. 6 Semi-homogenous DNA-enhanced immunoassays carried out in liquid phase reac- k exonuclease still yield signals in PCR
tions. (A) Bio-Barcode technology: magnetic beads coupled with antigen-specific capture amplification of this DNA substrate. This
antibodies (1) and gold particles135,136 coated with antigen-specific detection antibodies as assay was highly sensitive, allowing for the
well as a DNA-marker sequence (Bio-Barcodes) (2) are incubated with the target antigen detection of a few thousand molecules of
(3). Antigen-linked particles are isolated by magnetic separation (4) and the DNA marker AP. The approach was implemented into
is subsequently released by denaturation (5). Since each particle is coated with a multitude immunoassays by using specific antibody–
of DNA markers, many Bio-Barcodes are released for each positive binding event. In the
AP conjugates, thereby enabling the
final step (6), the released DNA marker is detected, e.g., by sequence-specific hybridization,90
detection of Chlamydia antigens with
colorimetric changes by hybridization with additional gold nanoparticle probes,72 or for
maximum sensitivity down to a few molecules, a subsequent conventional PCR step74 (B)
about a 1000-fold increase in sensitivity,
Magneto-IPCR: magnetosome particles functionalized with antigen-specific antibodies (7) as compared to conventional colorimetric
are incubated with the target antigen as well as an antigen-specific antibody–DNA conjugate ELISA-detection of the antibody–AP
(8). Following magnetic separation, magnetosome particles containing DNA are detected conjugate.53
directly by PCR (9). (C) DDI-IPCR: capture-antibodies, coupled with an oligonucleotide (10)
2.2.4 Nucleotide encoded informa-
as well as an antibody–DNA detection conjugate (8) are simultaneously incubated with the
target antigen. Conjugates of all three compounds are isolated by hybridization with a solid tion. The information content of the
phase-bound capture oligonucleotide (11) and the DNA marker is detected by PCR (9). DNA’s base sequence and/or the length
of the DNA marker allow for a multiplex
detection of several antibody–DNA con-
polymerase-IDAT”), DNA-labeled anti- breast cancer tumor markers in tissue jugates within a single experiment when
bodies were coupled to their target anti- samples.49,50 A potential drawback of the several antibodies are labeled with indi-
gens and the DNA marker was amplified technique results from special precautions vidual DNA markers (Fig. 9). The feasi-
by transcription using T7 polymerase. The which have to be taken into account bility of this approach was demonstrated
amplified RNA was then quantified by to prevent degradation of the inherently already in 1994 by Ebersole’s group in
addition of the RNA-intercalating dye Ri- unstable RNA by the ubiquitous RNases. the simultaneous detection of three dif-
boGreen and subsequent fluorescence de- ferent antigens (b-galactosidase, human
tection. Thus, enzymatic transcription was 2.2.2 Expression. The genetic infor- thyroid-stimulating hormone and human
used for signal amplification. Although mation stored in the DNA marker has chorionic gonadatropin).14,54 An approxi-
this assay lacks the typical exponential also been harnessed to express a detection mately 100-fold enhancement in sensitiv-
amplification characteristics of PCR, an enzyme as a means for signal genera- ity, as compared to conventional ELISA,
impressive 1000-fold increase in sensitivity tion. This approach, termed Expression was observed. Interestingly, despite the
was reported, e.g. in the detection of Immunoassay51,52 took advantage of a bi- striking possibilities of such a multiplex
prion aggregates in serum or her2/neu otinylated DNA marker which encoded approach, no further applications of

This journal is © The Royal Society of Chemistry 2008 Analyst, 2008, 133, 702–718 | 707
 multiplex IPCR have been reported so far.
Possible explanations certainly include the
incompatibility with the widespread mod-
ular IPCR approach (Fig. 3B I–III), which
makes it impossible to individually label
certain antibodies with a distinct DNA
marker. Moreover, the general problem
of conventional multiplex PCR obviously
also applies to multiplexed IPCR assays.
The design of a set of DNA markers which
can be amplified simultaneously without
interference inevitably limits the amount
of simultaneously detectable targets.
The interference of simultaneously am-
plified DNA markers, however, can pur-
posefully be utilized for standardization
of IPCR assays when a second DNA
fragment is added to the assay as an
internal competitor. The calculation of
the ratio of products originating from
either the IPCR marker or the competi-
tor fragment was found to enhance the
signal-to-background ratio of the assay.
This approach had been used for quan-
titative antigen detection in plasma sam-
ples during a clinical phase I pharma-
cokinetic study of the anti-tumor drug
Aviscumine.8,55 As an additional advan-
tage, the generation of competitor signals,
which are inversely proportional to those
of DNA marker, simplifies the avoidance
of false-negative signals because correct
performance of the DNA-amplification
can be judged conveniently from the pres-
ence of competitor signals, even in the
absence of the target antigen. The use
of internal competitors, however, requires
methodologies for the independent de-
tection of the two PCR amplicons. To
this end, gel-electrophoresis8 as well as
sequence specific hybridization55 has been
Fig. 7 Comparison of the most prominent methods for the detection and quantification of used (Fig. 7).
DNA amplicons generated in DNA-enhanced immunoassays. (A) Intercalation fluorescence With respect to multiplexed DNA-
markers with increased specificity for double-stranded DNA, such as ethidiumbromide or enhanced immunoassays, the problems
SYBR greenTM , are used in gel-electrophoresis or real-time PCR analyses. Note that for
well-known for standard multiplex PCR
multiplex IPCR, it is necessary to separate amplicons of different length by gel-electrophoresis
have been effectively circumvented by
while multiplex real-time detection can not be performed using intercalation marks. (B)
Different types of sequence-specific fluorophore-labeled nucleic acid probes, e.g. TaqManTM or
a different type of DNA amplification,
ScorpionTM are typically used for real-time quantitative PCR. During elongation of primers, namely the rolling-circle amplification
the probes are modified and thereby, a fluorescent signal is generated. (C & D) Hybridization (RCA) of immobilized nucleic acids. In
assays for sequence-specific DNA-detection. Sensitivity can be increased by binding of multiple the immuno-RCA technology, an oligonu-
fluorophores to the amplified DNA by means of hybridization of fluorophore-labeled probes to cleotide primer, covalently attached to
products of RCA (C) or PCR (D) processes. In the case of immuno-RCA, the antibody–DNA– the detection antibody, is enzymatically
conjugate remains intact during DNA amplification and thus, a multitude of hybridization elongated by adding a circular single-
probes can bind to spots of microarrays, containing the immobilized antigen. In PCR-ELOSA strand DNA as the template56–59 (Fig. 7C).
(D), hapten-labeled amplicons, generated during PCR, are immobilized by means of surface- This method, recently reviewed in this
bound capture oligonucleotides and subsequent detection is carried out by using hapten-
journal,9 has been shown to be partic-
specific antibody–enzyme conjugates.
ularly well suited for multiplex analyses,
impressively demonstrated by the profiling
of 75 cytokines on microarrays,59 or the

708 | Analyst, 2008, 133, 702–718 This journal is © The Royal Society of Chemistry 2008
 to an enzyme-amplified assay59 with the
advantage of the localized DNA amplifi-
cation product at the site of the antibody.
The approach is therefore well suited for
protein microarray applications.

2.3 Third challenge: assay

performance in the liquid phase
The standard platform for performing im-
munoassays is so far the solid phase, either
by applying typical ELISA microplates
or using microarrays. While this assay
format suffers from inefficient interphase
binding steps, a different strategy takes ad-
vantage of semi-homogenous (involving
a surface/bead for subsequent separation
of bound/no-bound compounds) or true
homogenous assay conditions. There, cou-
pling steps are carried out in the liquid
phase which leads to improved binding
kinetics and thus more efficient labeling
of antigens with antibodies and markers
(Fig. 6).
2.3.1 Simultaneous binding of more
than one antibody–DNA conjugate. In
the proximity ligation method61,62 two an-
tibodies, each of which binds to a specific
epitope of the antigen, are tagged with
individual short oligonucleotides. Enzy-
matic ligation of the two tags in the
presence of a third DNA oligomer leads to
the formation of a longer DNA which sub-
sequently functions as the target for PCR
or RCA amplification. A detailed review
of this technique was published recently.9
In the related approach of nucleic acid de-
tection immunoassay (NADIA),63 a short
Fig. 8 Statistical analysis of references reporting DNA-enhanced immunoassays: (A) self-priming overlap of two antibody-
summary of detection limits reported. The majority of examples revealed a maximum linked oligonucleotide sequences is used
sensitivity in the 0.016 amol–16 amol range (1000–100 000 molecules, respectively), thus
to initialize PCR amplification.
defining the standard performance of the method. Note that the broad detection range
A major challenge for proximity lig-
of about ≤10 molecules2,74,102 or single cells18 up to 1010 molecules in all cases involves a
significant improvement of the analogous ELISA.137,138 A typical LOD is found at approx. ation and NADIA concerns the careful
1000 molecules/sample, which is in accordance with the expected theoretical kinetics of selection of specific antibody pairs, which
immunoassays.13 (B) Overview of the N-fold improvement of conventional ELISA by the bind to a target in an appropriate distance
analogous IPCR. The sensitivity enhancement varies from 5-fold31 to up to 1 000 000 000- for the intended ligation. As a solution
fold82,86 depending on the design and optimization state of the assay as well as the performance to this intrinsic design problem, the use
of the antibodies. The majority of studies reported a 100–1000-fold improvement in LOD. of a population of polyclonal antibodies
(C) Overview of the linear dynamic range of IPCR applications. While conventional ELISA against a given target was suggested.64
typically reveals a dynamic range of two orders of magnitude in antigen concentration, IPCR This population is divided into two parts,
shows a significantly broader dynamic range (see also Fig. 5). In the majority of IPCR
each of which should include suitable
applications, the dynamic range comprised about four orders of magnitude.
binding partners for different epitopes due
to the diversity of binders in a polyclonal
serum. Indeed, this approach proved to
simultaneous detection of 11 cytokines in occurs in a linear fashion, which is by far be strikingly effective in the detection of
a bead-based assay.58 Drawbacks of this not as efficient as the almost exponential cytokines in a homogenous test format
approach concern the necessity of specif- amplification occurring in PCR. How- with femtomolar detection sensitivities.64
ically designed circular DNA templates60 ever, the gain in sensitivity obtained in However, the variability of polyclonal
as well as the fact that primer elongation immuno-RCA was found to be similar antibodies may also be a drawback for

This journal is © The Royal Society of Chemistry 2008 Analyst, 2008, 133, 702–718 | 709
routine applications because slight vari-
ations in affinity and specificity may not
always warrant long-term assay repro-
ducibility. Nevertheless, the linkage of
immunological reactivity and spatial in-
formation makes this technique certainly
unique, especially for studying protein–
protein interactions61,65–67 or monitoring
changes in protein conformation, for in-
stance as a consequence of splicing or
post-translational modifications.62
While the above described techniques
take advantage of DNA as a marker
for signal amplification, the sequence-
specific recognition properties of DNA
can also be the harnessed to realize novel
immunoassay designs. By DNA-directed
immobilization (DDI), conjugates of an-
tibodies and short artificial DNA se-
quences were side-selectively immobilized
at surfaces decorated with complemen-
tary capture sequences.68,69 The combi-
nation of DDI and IPCR compatible
antibody–DNA conjugates enabled an el-
egant one-step assay for the detection
of antigens by simultaneously incubating
the target-containing sample with DNA-
labeled capture- and detection-reagents70
(Fig. 6C).
2.3.2 Immuno-PCR and bead tech-
nology. A combination of capture-
antibody-coated magnetic beads and
detection-antibody/detection-DNA coa-
ted gold nanoparticles71 (Fig. 3B VII) was
recently introduced as the so-called Bio-
Barcode technology,72–75 summarized in
Fig. 6A, and this method was also recently
reviewed in more detail.9,10 Although the
Bio-Barcode assays require specialized
equipment and tailored reagents, the po-
tential of the nanoparticle-conjugates, e.g.
for multiplex detection76 or highly sensi-
tive protein detection77 certainly justifies
this approach to homogenous assay tech-
nology.
As a biogenic alternative to the artifi-
cial magnetic particles used in the Bio-
Barcode assays, antibody-functionalized
biogenic magnetosome nanoparticles78
have recently been applied in the magneto-
immuno-PCR (M-IPCR) by Wacker
et al.79 (Fig. 6B). The appeal of this
approach stems from the versatility of
the native particles, which can be pro-
duced by fermentation of the mag-
netotactic bacterium Magnetospirillum
gryphiswaldense,80 and subsequently mod-
ified with antibodies by synthetic chemical
710 | Analyst, 2008, 133, 702–718
means.78 The genetically controlled bio-
mineralization process ensures uniform
particle shape, high magnetic susceptibil-
ity, and a narrow size distribution which
can be regulated by choice of the organism
facilitating the magnetosome production.
It is clear from the aforementioned
examples that almost all intrinsic prop-
erties of the DNA molecule have already
been harnessed by IPCR and related tech-
niques to improve the performance of
conventional immunoassays. While some
examples are obviously more fascinating
from a methodical point of view, quite
a number of innovative assay strategies
promise novel solutions to the analytical
challenges faced in the daily laboratory
routine. For a critical evaluation of the
properties of theses assays, advantages
and disadvantages of the various strate-
gies and applications are discussed in the
following section.
3. Advantages and disadvan-
tages: the price of sensitivity
A number of variations of the IPCR
method have been introduced to show
the 1992’s initial protocol is applicable
to today’s requirements of commercially
available reagent kits and routine diagnos-
tics. Many of these variations have led to
a general increase in assay sensitivity, and
in some cases dramatic improvements of
greater than a 100 000-fold enhancement
in ELISA LOD have been reported.73,81–83
In other cases, the IPCR principle was
supplemented with additional beneficial
features, such as multiplex detection or
compatibility with automation. Here, we
will discuss some general considerations
which users might wish to take into ac-
count when they strive to establish DNA-
based immunoassays.
3.1 General considerations regarding
assay design and complexity
To apply DNA-based immunoassays to an
analytical question, one central point of
consideration should concern the choice
of the appropriate method, which brings
with it the ideal balance between com-
plexity of the assay and performance in
the intended target detection. One partic-
ularly attractive aspect of combining an
immunoassay with PCR results from the
fact that the equipment and knowledge
required for both techniques is usually
This journal is © The Royal Society of Chemistry 2008
available in typical biochemical or clinical
laboratories. Therefore, combining these
two techniques in IPCR applications is
usually possible without the need for ad-
ditional instrumentation, unlike the case
for many other highly sensitive techniques,
such as electro-chemiluminescence.84
As pointed out above, the availability
of pre-conjugated antibody–DNA conju-
gates largely improves performance and
handling issues of IPCR assays (Fig. 3C).
Fortunately, problems which often arose
in the past from the chemical proce-
dures to conjugate proteins with DNA,
can nowadays be circumvented by taking
advantage of services and ready-to-use
reagents available from a growing number
of suppliers. Therefore, users who intend
to set-up an IPCR assay, should critically
evaluate whether it is worth the time and
cost effort to develop their own conjuga-
tion approach or rather adopt reagents
and assays which have already proven their
functional performance.
3.2 Various ways of analyte detection:
the key to quantitative IPCR
A drawback of the IPCR protocols, as
compared to conventional ELISA, may
result from the assay’s handling effort
and duration. In particular, the post-
assay quantification of the DNA ampli-
cons, which is mandatory for quantita-
tive antigen detection, is often considered
a major hurdle for switching from an
established ELISA to IPCR. ELISA is
essentially finished after the addition of
the substrate because the enzymatic sig-
nal generation is conveniently detectable
and measured after a given time with
an appropriate reader. In IPCR, instead,
the DNA amplicons are essentially invis-
ible, and thus they require specific means
for detection and quantification. Gel-
electrophoresis with DNA-intercalation
labels (Fig. 7A) can be used for this
task because it is available in almost
any (bio)chemical laboratory. Thus, this
technique has been used in many “clas-
sical” applications of IPCR and related
techniques.2,8,10,13,15,16,54,85–88 However, this
approach has intrinsic disadvantages. It is
hardly suitable for quantification purposes
because band intensity quantification usu-
ally does not allow one to quantify DNA
over ranges spanning more than two or-
ders of magnitude, unless time-consuming
dilution series or radiolabels are applied.
Additionally, gel-electrophoresis is very
cumbersome when it comes to large sam-
ple numbers, because it is difficult and
expensive to automate.27
Due to these problems, alternative ways
of DNA quantification, initially devel-
oped in the course of PCR, have gained
acceptance in DNA-based immunoassays.
For example, labeled probes can be hy-
bridized with the PCR products, thus
leading to their sequence-specific tagging
with an appropriate detection molecule
(Fig. 7B–D). This principle has been
demonstrated for a variety of techniques,
including silver-staining, colorimetric,72,75
scanometric,89 fluorescent56,61 or electrical
detection.90 Similarly, the PCR product
can be labeled with a hapten molecule ei-
ther during amplification (PCR-ELISA)27
or subsequent to PCR by hybridization
(PCR-ELOSA).91,92 The hapten-labeled
DNA is immobilized on a surface, often
microplates, and then coupled with an
antibody–enzyme conjugate which specifi-
cally binds to the hapten and allows DNA
detection by enzymatic signal amplifica-
tion. It should be noted that such labeling
by hybridization is mandatory in specific
cases which require sequence-specific de-
tection of the PCR amplicons, as is the
case in microarray multiplex assays58 or in
situ analyses.61 Although the throughput
of these labeling-based assays is much
higher than that of electrophoresis, and
they often reveal improved sensitivity and
precision, they still have the drawback
of being essentially two-step protocols.
Therefore, a simpler one-step detection
is needed to reduce assay duration and
to minimize error prone handling and
pipetting steps.
Real-time PCR, also often termed as
qPCR (quantitative PCR), is today’s dom-
inating technique for precise quantifica-
tion of DNA detection. In qPCR a flu-
orescent probe is added directly to the
amplification reaction and it emits flu-
orescent signals upon the generation of
PCR products93–95 (Fig. 7B). This tech-
nique makes any post-PCR quantification
steps obsolete and it minimizes the risk
of contamination of lab space by han-
dling PCR amplicons. Moreover, recent
advances in qPCR probes enable its use for
highly precise quantification of template
molecules. It is therefore straightforward
to combine IPCR with a qPCR-based
read-out.31 Consequently, IPCR can now
be performed as conveniently as an ELISA
This journal is © The Royal Society of Chemistry 2008
by simply adding a PCR-mix containing
the real-time probe, subsequently to the
immunoassay.
It is essential that the microplate materi-
als used in such qIPCR assays are compat-
ible with the real-time PCR instrument.
This means that, besides physical compati-
bility, the microplate materials must have a
certain degree of protein binding capacity,
which is usually not the case for typical
PCR microplates. Although the transfer
of the signal-generating immunocomplex
into PCR cycler-compatible vessels is
possible,29,30,96 the additional handling of
the contamination-susceptible PCR-mix
is not recommended because it often leads
to high background signals and a decrease
in precision of the assay. A number of
first-generation real-time cyclers (e.g. early
versions of the light-cycler) are capil-
lary or single-vessel based instruments,
which therefore require the transfer step.
Nowadays, microplates and modules in
the standard 96-well format, specifically
designed for IPCR with high protein
binding capacities and a cycler-compatible
format are available,92,97 and so are modern
microplate-compatible cyclers. The use of
these products is recommended, in par-
ticular, for routine and high-throughput
applications of IPCR. Fortunately, mod-
ern real-time cyclers are widely available
due to the enormous popularity of real-
time PCR for DNA analytics,98 and thus,
no additional equipment needs to be
purchased when users wish to establish
the real-time IPCR technique. The
Fig. 9 Multiplex and polyplex assays for the detection of several antigens in a single sample:
in multiplex assays, different antigens (a and b) are tagged with different DNA sequences. In
polyplex assays the sample is divided into small aliquots, each of which is analyzed individually
by a target specific assay.
variety of filters available for modern
real-time cyclers also allows recording,
typically, four different fluorescent signals
simultaneously, thus enabling small scale
multiplex detection14 (Fig. 9) or internal
standardization55 to be conveniently facil-
itated.
Due to the aforementioned reasons, it
is not surprising that real-time PCR is
nowadays routinely used in DNA-based
immunoassays.26,29–31,36,66,83,92,99–101 After
solving initial challenges with respect to
reproducibility and sensitivity, today’s
performance of qIPCR is well beyond
that obtained by conventional read-out
systems. In particular, qIPCR reveals low
errors and even an increase in sensitivity,
when compared to conventional end-
point-based IPCR read-out.27,70,92,26,92
Therefore, it seems appropriate to
postulate that real-time detection truly is
the key to make the initial research-based
IPCR into a routine laboratory method.
The resulting protocols for qIPCR23 are
similarly convenient to those of routine
ELISA protocols with respect to assay
handling, simplicity and robustness.
3.3 From research to routine
applications: assay robustness and
precision
While we have already discussed that
assay precision depends on the read-out
technique, a more in-depth discussion
of assay precision, robustness and back-
ground seemed helpful to provide deeper
Analyst, 2008, 133, 702–718 | 711
insight into potential pitfalls of the IPCR
method.
A major source of increased error rates
stems from the number of handling steps
involved in the assay protocol. With each
additional step the coupling efficiency
decreases27 and thus the possibility for
errors increases. Multi-step protocols have
been reported as highly sensitive but they
require careful, laborious adaptation to
each variation in target antigen, biologi-
cal sample, or antibodies.15,102–104 This ap-
proach, therefore, certainly benefits from
expert knowledge of IPCR operation
and, therefore, it is not recommended
for the inexperienced user who needs a
fast solution to a given analytical prob-
lem. With each deviation from the well
proven three-step standard ELISA proto-
col, “add target to microplate—incubate—
add detection conjugate—incubate—add
substrate—measure”, potential advan-
tages in sensitivity are inevitably linked to
increasing complexity, costs and possible
error sources.
The advantage of simplified assay pro-
tocols is repeatedly confirmed by com-
parison of different assay protocols for
a given target. Using the exemplary de-
tection of prostate specific antigen (PSA),
Lind et al. compared the performance
of IPCR-assays, carried out with either
pre-conjugated antibody–DNA reagents
or the sequential stepwise in situ cou-
pling using STV to bridge biotinylated
antibodies with biotinylated DNA.26 The
tumor marker PSA is an especially in-
teresting target for ultrasensitive diagnos-
tics due to its properties as a biomarker
for early detection of prostate cancer.105
Therefore, a number of studies used this
antigen to investigate the performance
of DNA-enhanced immunoassays.9 Uni-
formly, all methods revealed a significant
(35–200 000-fold) increase in sensitivity, as
compared to conventional ELISA. Small
sample volumes as well as short assay
duration were also typical for most as-
says. However, sequential IPCR and the
expression immunoassay both required
significantly more time and handling ef-
fort. The increased number of incuba-
tion steps also led to lower sensitivities
in these assays. In contrast, the most
sensitive detection was obtained by us-
ing a standardized ImperacerTM IPCR
kit106 and the nanoparticle-based Bio-
Barcode.107 The outstanding performance
of tailored reagents was also verified in a
712 | Analyst, 2008, 133, 702–718
recent study of different assay strategies
for the detection of human interleukin 6
(hIL6),23 which included detailed proto-
cols and comparison of sequential incuba-
tion, indirect and direct qIPCR (Fig. 3).
Sequential incubation as well as indirect
sandwich assay showed an LOD of 10
pg ml−1 hIL6 in human serum (100-fold
improvement compared to ELISA), while
the use of tailored pre-conjugated reagents
led to an LOD of 100 fg ml−1 (10 000-fold
improvement compared to ELISA).23
For selection of the appropriate assay
strategy, the main question, therefore, is
indeed the intended application. While
sequential coupling protocols have the
drawback of being time consuming, their
flexibility and the ready availability of
reagents make them attractive and well
suited for research applications. How-
ever, when maximum sensitivity, easy and
robust handling as well as fast routine
applications become a major issue, op-
timized protocols based on tailored pre-
conjugated detection reagents are clearly
advantageous.5
As the most prominent obstacle of
IPCR, high background signals often pro-
hibit meaningful results. Typically, highly
sensitive assays amplify both, specific sig-
nals and non-specific background noise.
An increase in the signal-to-background
ratio is therefore most important to gen-
erate meaningful results. Various points
need to be optimized to reach this goal:
(i) Choice of highly specific antibod-
ies. The quality of the antibody is in-
deed an imperative for any successful im-
munoassay. Although the highly sensitive
IPCR has been demonstrated to function
even with weak binders which are usually
not well suited for ELISA,44 the specificity
and affinity of the antibodies are the
key for the performance of the assay.27,92
Therefore, IPCR assays do perform only
as well as the specificity of the antibodies
involved permits.23
(ii) Blocking of non-specific interac-
tions. By the choice of an appropri-
ate blocking reagent for coating surface
materials and spiking of buffers, unspe-
cific binding of assay reagents can be
minimized, thereby reducing the overall
background signal.8,23,85 Owing to the ex-
tremely high sensitivity of PCR, it is
impossible to reduce background signal
completely, because single non-specifically
bound DNA molecules are sufficient for
This journal is © The Royal Society of Chemistry 2008
generating a certain level of background
signal. It was found useful to increase
the specific coupling efficiency by using
tailored antibody–DNA conjugates,14,23,26
thus supplementing the blocking step with
increased specific signals.
(iii) Dilution of the biological matrix.
Since the background signal is often
caused by the presence of interfering com-
pounds in the sample to be analyzed,
minimization of these disturbances was
achieved by dilution of the sample in
appropriate buffers, thus reducing the
influence of the biological matrix8,23,55,108
(Fig. 10). Furthermore, it is recommended
to use a biological matrix identical to,
or as similar as possible to, the sample
material for the preparation of calibration
curves in quantitative analyses. This can
also help to compensate for background
effects.23,55
(iv) Standardization and simplifica-
tion of assay handling. Finally, back-
ground signals can result from handling
steps with potentially contaminating ma-
terial, such as the transfer of DNA
containing solutions30 or highly concen-
trated DNA stock preparations for in situ
coupling of antibody–DNA conjugates.13
Moreover, low signals and high back-
ground values occur in IPCR assays when
the protein–DNA conjugate reagents are
not properly prepared and/or contami-
nated with free binding proteins and/or
marker DNA. To avoid these problems,
single-well solutions with plate materials
compatible with PCR cyclers as well as
low-concentrated, highly active antibody–
DNA conjugates should be used to reduce
background signals.
With the increase in kit-solutions and
service providers, the robustness and ac-
cessibility of IPCR technology has sig-
nificantly increased and diversified in the
past few years (Fig. 4). To estimate what
one might expect from the developments
of DNA-enhanced immunoassays typical
results from the literature have been sum-
marized (Fig. 8). Analysis of the reported
data suggests that the average detection
limit of IPCR is in the range of approx.
1000 molecules/sample. This corresponds
to an on average 100–1000-fold increase
in the LOD of the corresponding con-
ventional ELISA. In addition to the im-
provement of LOD, IPCR-based methods
usually reveal a significantly broader dy-
namic range for the detection of analytes
 its ability to work with very small sample
volumes.

4.1 Improvement of sensitivity as the

key to early detection of
neurodegenerative diseases

The potential of largely improved sensi-

tivity becomes a major issue when well-
established assays with conventional sen-
sitivity require sample materials which
can only be accessed by stressful inva-
sive techniques or post-mortem sample
drawing procedures. In these cases, the
increase in assay sensitivity can open the
door to the analysis of alternative sample
materials which can be collected with less
effort. With the increasing societal im-
pact of neurodegenerative diseases, such
as scrapie, CJD/vCJD and Alzheimer’s
disease, the research on infectious agents
and biomarkers as prime targets for ultra-
sensitive detection come into focus. The
relevant biomarkers of neurodegenerative
diseases are typically present in the brain,
and their concentration is very low at early
stages of the disease.109 For obvious rea-
sons, a more easily accessible target matrix
than brain homogenate, such as blood
samples, would allow for in vivo/ante
mortem detection. Although the presence
of infectious agents for vCJD in blood has
already been confirmed,110,111 the LOD of
conventional analytical techniques (e.g. lu-
Fig. 10 Immuno-PCR combines the versatility of ELISA with increased sensitivity for a
minescence assays, western blots, capillary
broad range of analytical targets: the robust method allows access to almost all biological electrophoresis) were found to be insuffi-
matrices, including e.g. single cells20 and cell culture,139 tissue samples,140 saliva,141 stool,100,108 cient for the detection of these makers in
brain material,83 food99,100,120 and plant extracts, serum and blood50,55,88,142 from human or non-brain derived samples.112,113
animal organisms. The combination of this robustness and sensitivity with the almost unlimited The detection of 10–100 infectious units
number of targets detectable by immunoanalytics (see also Fig. 2) marks the potential of the of PrPSc by IPCR in infected hamster
IPCR method for the analytical scientist. brain homogenate by Constantine et al.83
in 2005 as well as specificity tests in
human tissue samples carried out by
(see Fig. 5 for typical results and Fig. 8C plications. Therefore, in the final part of Gofflot et al.97,114 in the same year rep-
for a statistical evaluation). Therefore, the this review, we will discuss selected appli- resent very important initial steps to-
nearly 15 years of method developments cations in detail. While general overviews wards the early detection and improved
clearly indicate that DNA-enhanced im- of IPCR applications and related tech- analysis of transmissible spongiform en-
munoassays are superior to conventional niques have been published elsewhere,8,10,12 cephalopathy. In addition, non-infectious
enzyme-amplified detection methods and the applications discussed here have been PrP was chosen as the target for the
hold the potential to help unravel impor- chosen to particularly demonstrate the demonstration of phage-display moder-
tant questions of health, clinical diagnos- diversity of fields where the combination ated IPCR.44 Moreover, Chang et al. ob-
tics, and fundamental research. of protein detection and DNA-based sig- served a 50% sensitivity and 100% speci-
nal amplification is being applied today ficity of illness-associated PrPSc in blood
4. Applications: putting (see Fig. 2). As discussed below, three for early stage and 100% sensitivity for
features of these assays are particularly im- late-stage asymptomatic infections in mice
innovative assay strategies to
portant for practical applications. These by using their so-called “Am-A-FACTT”
the test
include (i) the increase in sensitivity, (ii) the method,49 which combined PrP amplifi-
The touchstone for novel analytical meth- assay’s robustness against the disturbing cation and aggregation with a fluorescent
ods is their performance in practical ap- effects of the biological matrix, and (iii) detection method based on amplification

This journal is © The Royal Society of Chemistry 2008 Analyst, 2008, 133, 702–718 | 713
of antibody–DNA conjugates by T7-RNA
polymerase (see section 2.2.1).
It is somewhat surprising that the de-
tection of prion proteins by IPCR was not
attempted earlier in spite of the fact that
abnormal PrPSc as a potential target was
already identified in 1991.115 However, as
for conventional immunoanalytics, IPCR
strongly depends on the performance of
the antibodies. For prion proteins, the key
detection antibody specific for PrPSc is still
missing, and therefore a proteinase K di-
gestion is required to distinguish between
native PrP and PrPSc . Obviously, if this
digestion is incomplete, high background
signals will occur, especially when highly
sensitive assays are used.83 Therefore, the
usefulness of IPCR in these applications
strongly depends on further development
of high quality antibodies. Nonetheless,
the implementation of real-time detection
has significantly increased the potential
of quantitative PrP-IPCR,83,97 including
assays for PrPSc with novel antibodies.116
4.2 Robust performance enables
monitoring of rare biomarkers
Another prime target for highly sensi-
tive IPCR assays are marker proteins
indicative for virus infections either at
the early (low virus load) stage of the
infection or else from complex matrices
which contain the marker in only very
small amounts. Recent studies on viral
antigen detection concerned, for instance,
the detection of rotavirus antigen in pa-
tient stool samples,108 norovirus antigen in
food samples,100 or HIV antigen in plasma
samples.117 All these applications have in
common, an on average 1000-fold increase
in sensitivity despite the fact that the IPCR
assay was carried out in the presence of the
biological matrices. Although IPCR is of-
ten expected to be highly susceptible to the
disturbing effects of various compounds
within complex matrices, these three ex-
amples clearly indicate the robustness
of this method in practical applications.
Along this line, similar robust behavior
of IPCR in routine applications was ob-
served in clinical phase I studies concern-
ing a new anti-tumor drug including more
than 40 patients,37,55 or in a recent phar-
macokinetic study with 15 individuals.36
These examples demonstrate nicely, that
the introduction of DNA as a signal-
generating marker in immunoassays does
not deteriorate robustness and stable per-
714 | Analyst, 2008, 133, 702–718
formance of immunoassays, which have
made ELISA the routine technology of
today’s analytical laboratories.
A recent work by Xiang et al. em-
ployed IPCR for screening of blad-
der cancer patients. The highly sensi-
tive detection of mutated p53 protein in
serum of workers exposed to hazardous
substances,118 demonstrated the usefulness
of the method as a tool for disease moni-
toring as well as for drug development.
Due to the high sensitivity of IPCR
and related methods, background effects
caused by interfering matrix components
can often be simply diluted away using ap-
propriate buffers, without significant de-
crease in the overall assay sensitivity.55,108
In addition to this purposeful dilution
of sample materials, there are also cases
where small amounts of antigen are
present in a very large sample volume.
Chang et al. have impressively demon-
strated that the sensitivity of IPCR is
sufficient to detect a single E. coli cell in a
volume of 10 l by using a cell-released en-
zyme as the target for immunodetection.86
This result underlines the appeal of IPCR
for the detection of low-level contamina-
tion, which is particularly important for
applications in the monitoring of food
allergens33 or toxins released by Clostrid-
ium botulinum,32,119 Bacillus thuringien-
sis120 or Staphylococcus aureus.121
It is noteworthy that in these examples
method development was not the prime
issue. Instead, well established IPCR pro-
tocols were used to provide answers to
analytical questions, such as the time-
and temperature-dependent relationship
of toxin secretion by the microorgan-
isms, because an outbreak of intoxica-
tion in a general population can be in-
duced by small numbers of microorgan-
isms not detectable with classical ana-
lytical techniques,121 IPCR and related
techniques can help to warrant a new level
of environmental and food safety.
4.3 New assay strategies for small
sample volumes
The high sensitivity of IPCR can also be
utilized for analyzing very small sample
volumes. While typical ELISA requires
about 50–100 ll of sample material, the
increased sensitivity of DNA-enhanced
immunoassays allows one to significantly
reduce the assay volume. For highly opti-
mized methods like ImperacerTM or Bio-
This journal is © The Royal Society of Chemistry 2008
Barcode, only 1–30 ll sample material is
required.74,108 This is especially important
in applications where only small amounts
of the sample are available and/or acces-
sible, such as in biopsy, pediatrics, animal
studies, or even single cell analytics.
Another great potential for applications
of the IPCR method results from the pos-
sibility to split given samples into parts to
increase the amount of information acces-
sible by conventional immunoanalytics.
This “polyplexing” differs from “multi-
plexing” such that polyplexing describes
a strategy in which a sample, normally
following to dilution, is split into sev-
eral individual assays for different targets,
while in multiplexing several analytes are
detected directly in a single experiment
(Fig. 9). Owing to the high sensitivity of
DNA-enhanced immunoassays, 1 ll or
less of a given sample is often sufficient
for analysis and, thus, the IPCR method
is perfectly well suited for polyplexing
analyses.
Polyplexing circumvents the general
problems of multiplex assays, which
usually result from non-specific interac-
tions of the various detection reagents
employed. Antibodies as well as DNA-
markers and their detection probes have to
be carefully selected, designed, evaluated
and modified to warrant for their non-
interfering performance in the assay.
In this process the challenge increases
exponentially with an increasing number
of simultaneous assays.122–124 In particular,
the generation and selection of highly
specific and non interfering antibodies
with similar binding efficiencies and
conditions for simultaneous application
in multiplex assays is a major problem
which severely complicates design
and applications of multiplex assays.
Furthermore, it has been discussed above
that the simultaneous detection of several
DNA-markers by PCR-based assays
brings with it additional complications. To
circumvent the problems of multiplexing
IPCR, an example of a polyplex assay was
reported by Case et al.102 which concerned
the parallel detection of the promutagenic
DNA base adduct O-6-methylguanosine,
its monomeric repair enzyme, O-6-
methylguanine-DNA methyltransferase
(MGMT), as well as a single peptide from
the N-terminus of MGMT. An optimized
sequential IPCR protocol was used for
the detection of the several structurally
diverse antigens from the catalytic cycle
of the cell protecting DNA-repair enzyme
MGMT, thus allowing the studying of
complex interaction effects within small
sample volumes. Similar detection of
several antigens with a standardized
IPCR method have been reported by Guo
et al.44 and Adler et al.92 These examples
clearly demonstrate the potential of
polyplex IPCR assays for the analysis of
various targets in a given sample.
An additional important field of ap-
plication for the combined detection of
entire sets of antigens concerns the family
of cytokines, because cytokine profiling
has been demonstrated to help under-
stand the inflammatory response in pa-
tients with trauma, shock, and sepsis by
studying specific cell-mediated immune
responses in blood.125 Typically, multi-
plexed microarray-based techniques have
been used for organ failure assessment126
and mortality prediction.127 Comprehen-
sive cytokine libraries have already been
analyzed by conventional multiplex ana-
lytics, in particular using microarrays, and
the performance of immuno-RCA was
impressively demonstrated by an 11-plex
cytokine assay in serum.58 For this appli-
cation, however, extensive careful testing
of the cross-reactivity of more than 130
analytes as well as systematic adaptation
of the assay procedure was necessary.
A polyplex assay of cytokines Il-2, Il-
4, VGEF, PDGF-BB and insulin was
demonstrated by Gullberg et al. using
the proximity ligation method64 while a
number of other cytokines had already
been studied in individual IPCR applica-
tions, such as TNFa,16,96,104,128 IL-18,17 IL-
6.23 In combination, these assays represent
a solid basis for the emerging field of
multiple protein analyses from a single
sample. Therefore, the realization of poly-
plex IPCR assays again nicely illustrates
how the sensitivity of DNA-enhanced
immunoassays can be used as a powerful
tool for creative assay design in novel
applications.
5. Summary and outlook
The use of DNA as a signal enhancing
tool in immunological diagnostics has
found a broad variety of applications in
recent years, demonstrating the versatility
of the molecule as well as the creativity
of the researchers who invented novel
strategies of assay design. Almost the
entire repertoire of natural DNA process-
ing, including replication, transcription,
translation, digestion and hybridization
has already been harnessed for the im-
provement of immunoassays (section 2). A
thorough review of the literature suggests,
however, that the old truth of Occam’s
razor that the best approach is typically
the simplest one, also applies to DNA-
enhanced immunoassays.
With real-time PCR, a powerful tool for
quantitative DNA amplification and de-
tection is nowadays implemented in DNA-
enhanced immunoassays. This enables an
easy, robust and highly precise signal read-
out of these assays. The second major chal-
lenge of DNA-enhanced immunoassays,
the actual conjugation of DNA marker
with detection antibodies, has found sev-
eral different solutions. Moreover, from a
user’s point-of-view, it is nowadays solved
by service of commercial providers, as is
the case for conventional ELISA reagents.
While the majority of challenges in general
method development have been addressed
by adjustment of assay strategy, three
major trends can be foreseen for future
applications:
(1) Homogenous single-phase assays
are becoming increasingly popular be-
cause this type of assay improves reagent
coupling, allows for single-step assays and
is well suited for automation. Neverthe-
less, one major advantage is lost in this
type of assay, that is, they are no longer
conveniently compatible with established
ELISA protocols, reagents and instru-
mentation. While certainly well suited for
stand-alone applications, it remains to be
seen whether the limitation to specialized
equipment will be a hindrance for broad
flexibility and distribution in research labs
and industry.
(2) Ultrasensitive multiplex and poly-
plex applications for the detection and
quantification of minute amounts of target
antigens. With the increasing knowledge
gained in proteomic research, monitoring
of protein families and comprehensive sets
of antigens will become a major challenge
for research. Moreover, the monitoring of
complex medical relevant response pat-
terns for individual treatment also calls for
large-scale protein detection, preferably
with ultimate sensitivity.129
(3) Tailored reagents and standardized
kit solutions for specific analytical chal-
lenges are likely to be the major applica-
tion of the ultrasensitive DNA-enhanced
immunoassays. New low abundant tar-
gets will become increasingly important
due to the progress in research and the
requirements of efficient diagnostics and
analytics. Another area which also calls
for ultrasensitive analyses concerns the
monitoring of biological compounds such
as contaminants in food, either by toxins
or genetically altered protein components.
Purity control is also of paramount im-
portance for clinical applications, such as
quality control of blood donations130 or
biological material for implantation. In
this respect, IPCR was already proven
to be most valuable for the detection of
low levels of viruses and infectious pro-
teins (see above). In view of the growing
importance of single cell analytics,131 the
application of IPCR to analyze targets in
single blastocytes132 suggests that this area
of research will also benefit from DNA-
enhanced immunoassays. Furthermore,
environmental monitoring and homeland
security issues provide additional chal-
lenging tasks for future applications.
Another rewarding field for DNA-
enhanced immunoassays is their combina-
tion with assays aimed at DNA/RNA an-
alytes, the latter of which are already avail-
able in a variety of commercial products.
The detection of the genetic information
or transcription products of a pathogenic
organism only allows for indirect con-
clusions on the actual distribution and
status of the organism. The most inter-
esting parts of it, that is shell particles for
immunization or disease-inducing toxins,
are not detectable by nucleic acid analysis
alone. Conventional immunoassays would
be helpful, but because their sensitivity is
not nearly in the range of PCR/RT-PCR,
they are typically not sensitive enough for
early stage or low-level infections. With
the aid of DNA-enhanced immunoassays,
protein detection can be linked to nu-
cleic acid analysis, in order to get the
whole picture of a given disease pattern
or to monitor artificially induced protein
expression.133 This should allow for more
precise diagnosis and therapy. Indeed,
initial progress towards this goal indi-
cated that both protein- and nucleic acid
analyses can supplement each other in a
meaningful way.81,117 Since, both protein
and DNA detection can be carried out
using the same real-time PCR instrument,
one can foresee that IPCR assays will be
capable of linking information on proteins
conveniently to that of nucleic acid target
molecules.

This journal is © The Royal Society of Chemistry 2008 Analyst, 2008, 133, 702–718 | 715
 IPCR and related DNA-enhanced im-
munoassays should therefore not be con-
sidered only as a better performing alter-
native to existing assay technologies. In-
stead, if correctly optimized and adapted,
they can be used as powerful tools to fill
the gap between ultrasensitive nucleic acid
detection and robust protein detection.
This can supplement microarray tech-
niques with high sensitivity detection, and
also help to obtain significantly more in-
formation from a single biological sample.
In the coming years, we anticipate that
IPCR technology will further expand into
a wide variety of scientific fields, enabling
fundamentally new insights into biologi-
cal systems as well as novel products for
many areas of human society.
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