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DOI 10.1002/pmic.200500721
Proteomics 2006, 6, 2972–2981
RESEARCH ARTICLE
A simple and rapid protein array based method for the
simultaneous detection of biowarfare agents
Birgit Huelseweh1, Ralf Ehricht2 and Hans-Juergen Marschall1
1
2
German Armed Forces Scientific Institute for Protection Technologies–NBC Protection, Munster, Germany
CLONDIAG Chip Technologies, Jena, Germany
A protein chip has been developed that allows the simultaneous detection of a multitude of different biowarfare agents. The chip was developed for the ArrayTube platform providing a cheap
and easy to handle technology solution that combines a microtube-integrated protein chip with
the classical procedure of a sandwich-enzyme-linked immunosorbent assay and signal amplification by streptavidin-poly-horseradish peroxidase. Specific immunoassays for Staphylococcus
enterotoxin B, ricin, Venezuelan equine encephalitis virus, St. Louis encephalitis virus, West Nile
virus, Yellow fever virus, Orthopox virus species, Francisella tularensis, Yersinia pestis, Brucella
melitensis, Burkholderia mallei and Escherichia coli EHEC O157:H7 were developed and optimized.
All assays could be completed within 1 to 1 1/2 h and detection levels were demonstrated to be as
low as in well established ELISAs. Most interesting, as a result of careful antibody screening and
testing, it is currently possible to analyse at least five of the “dirty dozen” agents on one single
protein chip in parallel.
Received: October 4, 2005
Revised: January 10, 2006
Accepted: January 13, 2006
Keywords:
ArrayTube / Biochip / Biodetection / Biowarfare agents / Protein array
1
Introduction
A growing demand for methods to detect the presence of
biological warfare (BW) agents and other pathogens in samples from the environment, the battlefield, and food is currently driving a need for new detection technologies. Especially for biodefense and risk evaluation, an early and definite
Correspondence: Dr. B. Huelseweh, German Armed Forces Scientific Institute for Protection Technologies–NBC Protection,
POB 1142-D-29623 Munster, Germany
E-mail: [email protected]
Fax: 149-5192-136-355
Abbreviations: AT, ArrayTube; BW, biological warfare; 4G2, D14G2-4-15 Dengue 2 virus antibody; QSV, quantitative staining
value; RaVac, Vaccinia virus; SA-Poly-HRP, streptavidine-polyhorseradish peroxidase; SEB, Staphylococcus enterotoxin B;
SLEV, St. Louis encephalitis virus; TCID, tissue culture infectious
dose; VEEV, Venezuelan equine encephalitis virus; WIS, ArmedForces Scientific Institute for Protection Technologies-NBC Protection; WNV, West Nile virus; YFV, Yellow fever virus
ª 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
identification of bacteria, viruses and toxins is of enormous
importance. Apart from a fast and safe evaluation, a high
degree of automation and robustness is demanded, since the
analysis should be feasible in the field. Handling of a system
should be easy and should be accomplished by untrained
staff with little laboratory and technical experience.
Following the progress of DNA chip technology, protein
microarrays have emerged for these applications since they
are not restricted to the detection of DNA or RNA carrying
microbes, spores and viruses. Antibody arrays can also be
applied to detect toxins. Furthermore, antibodies can detect
molecules on microbial surfaces, so that in comparison to
nucleic acid detection technologies and devices no additional
time is needed to break open the target cells. However, the
protein chip technology and its applications are still in its
infancy. Protein chips are used in research applications but
not for routine microorganism identification and routine
diagnostics [1–5].
Compared to DNA arrays, designing and producing an
antibody array of high quality is a delicate and critical affair.
Functionality and long time stability of antibodies after
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Protein Arrays
Proteomics 2006, 6, 2972–2981
immobilization has to be ensured. Furthermore, the production of a reliable antibody chip for the identification of
microorganisms requires a careful screening and tuning of
capture and detection antibodies. Cross reactivities have to be
minimized and the antibody’s affinity is as important as its
specificity.
Compared to other array solutions the recently developed
ArrayTube (AT) platform from CLONDIAG chip technologies, Jena, represents a less expensive and easy to handle
system for the development of protein and DNA arrays of
different formats [1, 6–9].
The heart of the device is a chemically modified glass
surface assembled to form the bottom of a 1.5 mL plastic
polystyrol microtube. The chip is 363 mm in size with an
active area of 2.462.4 mm. Antibodies are deposited on the
glass surface by contact spotting. Handling and analysis of
the protein chip is easy and rapid and involves the simple
steps of an ELISA in a sandwich format, including a signal
amplification step by streptavidine-poly-horseradish peroxidase (SA-Poly–HRP). Specific interactions of antibody and
antigen are simply revealed by colorimetric detection. Read
out of processed ATs is done by simple optical transmission
microscopy in combination with an image analysis software.
Endpoint detection is possible as well as dynamic monitoring of the colorimetric precipitation.
In the following, we report the development of an antibody protein chip for the AT platform especially for the rapid
detection of potential biowarfare agents. The identification
was focused on microorganisms and pathogens that were
classified by the Atlanta-based Centers for Disease Control
and listed either as Category A or B agents (http://www.bt.
cdc.gov/agent/agentlist-category.asp).
Whereas agents of highest priority like Yersinia pestis,
Variola major virus (smallpox) or Francisella tularensis are
highly infectious, easily transmitted and cause a high mortality, agents of category B have the potential for illness and
transmission, but are of only moderate morbidity and only
cause a low mortality.
We established specific assays for the reliable identification of Staphylococcus enterotoxin B (SEB), Ricin, Venezuelan equine encephalitis virus (VEEV), St. Louis encephalitis
virus (SLEV), West Nile virus (WNV), Yellow fever virus
(YFV), Orthopox virus species, Francisella tularensis, Yersinia
pestis, Brucella melitensis, Burkholderia mallei and Escherichia
coli EHEC O157:H7. Each assay was carefully optimized and
validated and can now be completed within 1 to 1 1/2 h.
Detection sensitivities were demonstrated to be as low as in
well established ELISAs that need several hours of reaction
time [10, 11; Marschall, Armed Forces Scientific Institute for
Protection Technologies–NBC Protection (WIS), Munster,
personal communication 1992–2003; Niederwhrmeier,
WIS, Munster, personal communication, 2003]. Most interesting, as a result of a careful antibody screening and testing,
it has been demonstrated for the first time that at least five
BW agents classified to category A or B, can be analysed in
parallel on one single microarray.
ª 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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The screened antibodies in combination with the technological platform provide the basis for a rapid virus-, bacteria- and toxin-specific Protein chip, not only useful for the
easy detection of biological warfare agents but also for purposes like monitoring food and water supplies.
In contrast to other immuno- or biosensors it easily
allows to monitor unspecific binding and cross reactions of
different antibodies and antigens.
2
Materials and methods
2.1 Antigen preparation
2.1.1 Viruses
Alpha- and Flaviviruses were either grown in Vero or
BHK 21 cells in the biosafety level 2 and 3 facilities. Virus
titers were determined by the 50% tissue culture infectious
dose (TCID50/mL) method [12, 13]. All viruses used in this
study represent models for BW relevant Flavi- or Alphavirus
species and are part of the collection of WIS. Except YFV 17D
and VacM1, all viruses were inactivated before use. Inactivation of viral antigens was performed by 0.1% b-propionolactone for 1 h at 47C and 4 h at 377C if necessary.
2.1.2 Bacteria
Bacterial strains were cultured according to standard cultivation procedures. Most strains are part of the collection of
WIS: Francisella tularensis WIS 140, Yersinia pestis WIS 412,
Burkholderia pseudomallei WIS 203, Burkholderia mallei
WIS 205, Brucella melitensis WIS 163 and Escherichia coli
O157:H7 (last from Dunn, Germany). Inactivation of bacteria was achieved either by formaldehyde, heat incubation
or a combination of both.
2.1.3 Toxins
Staphylococcal enterotoxins were purchased from Toxin
Technology, Inc., USA. Crude extracts of Ricin were prepared
from castor beans (Ricinus communis) by aqueous extraction
and ammonium sulfate precipitation [14], using a modified
protocol (Binder, ZInst SanBw Mnchen, personal communication, 2003).
2.2 Antibodies
Monoclonal antibodies against viruses and bacteria were
prepared from WIS-owned hybridoma celllines [10, 11, 15–
17]. Purification was done by immunoaffinity chromatography on goat anti-mouse or protein G affinity sepharose.
Antibodies MAB 984, 8150, 8151, 8152, 8741, 8744, 8745 and
8747 were purchased from Chemicon International, Inc.,
USA.
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B. Huelseweh et al.
Polyclonal antibodies against Vaccinia virus (RaVac),
Br. melitensis (163/098) and B. pseudomallei (205/102) were
raised according to standard procedures [18].
Rabbit anti-SEB polyclonal antibody and SEB antigen
were purchased from Toxin Technology (Sarasota, FL,
USA), goat anti-E. coli O157:H7 was a product of Kirkegard & Perry Laboratories, USA. Rabbit anti-Ricin polyclonal antibody (RCA60) was a product from Sigma, Germany, Mouse anti-Ricin monoclonal antibody 2R1 (Clone
CP23) was purchased from Hytest, Finland and mouse
anti-Ricin monoclonal antibodies 1RK1 and CH1 were a
generous gift of Spiez-Laboratory the Swiss Institute for
NBC Protection.
D1-4G2-4-15 4g2 is an antibody originally raised specifically against Dengue 2 virus [19]. All detection antibodies
were coupled to biotin-NHS ester (Vector Laboratories, USA
or Sigma-Aldrich Chemie, Germany) for 2 h at room
temperature according to the manufacturer’s direction.
Unincorporated biotin was removed by gel filtration on
PD-10 columns (Amersham Biosciences, USA). Positive
detection reactions were reported by streptavidine-HRP
conjugates (Amersham Biosciences Europe, Germany)
2.3 Protein array preparation
Antibody arrays were spotted using either a Microgrid II
spotting machine (BioRobotics Inc., Cambridge, UK) or a
capillary spotting device (CLONDIAG, Jena, Germany). The
antibodies were applied in a final concentration of 0.2 to
0.5 mg/mL in 16PBS spotting buffer containing 10 to
20 mM trehalose for preservation. The average spot size was
80–100 mm. Negative controls contained PBS buffer without
antibodies.
Proteomics 2006, 6, 2972–2981
used in a 1 to 10 000 dilution. Online read-out of washed
ATs was performed in an AT reader (atr01, CLONDIAG)
for 6 min at 257C, recording one image per 10 seconds.
Data analysis was done with the manufacturer’s specifications and a software called IconoClust. This software is
based on a transparency analysis method and results can
either be plotted as transmission value, 1/transmission
value or quantitative staining value (QSV). Like the transmission value, the QSV is a value for precipitation but it
integrates all data points along the time axis for each spot.
Therefore, the QSV value represents the slope of a linearized curve fit of the precipitation kinetic. Signal intensity
and background measurements were recorded for each
spot on the array. Extinctions of local backgrounds were
subtracted from spot extinctions.
3
Results and discussion
Antibody array analysis was applied to the rapid identification of different BW agents using the AT platform. A general
protocol for detection was established and so far, 27 different
monoclonal or polyclonal antibodies or antibody combinations have been tested for the specific binding of their cognate antigen. Three main chip layouts (layout 1, 2 and 3) had
been produced (Fig. 1) and were tested for the sensitivity and
specificity of their cognate BW antigens.
2.4 Array analysis
We tested surface-modified glass slides coated with either a
special epoxy-, aldehyde- or amino layer (CLONDIAG).
Before starting a protein array analysis all ATs were conditioned by washing them twice with 500 mL 16PBS 1 0.01%
Tween 20 for 2 min. All incubations of the analysis were carried out on a horizontal tube shaker (Thermomixer, Eppendorf, Germany) at 350 rpm at 257C. In order to block unspecific binding sites, the ATs were incubated in 16PBS 1 0.1%
FCS 1 0.01% Tween 20 1 1% fat free milk-powder or
1% FCS for 15 min, followed by three washing steps with
5006L 16PBS 1 0.01% Tween 20 for 2 min each. Antigen
binding was allowed to proceed in 16PBS 1 0.1%
FCS 1 0.01% Tween 20 for 30 min, followed by three washing steps as above.
Incubation of ATs with specific biotinylated secondary
antibodies was performed in 16PBS 1 0.1% FCS 1 0.01%
Tween 20 for 30 min. After washing, specific binding of
the secondary antibody was reported by SA-Poly-HRP
(Pierce Biotechnology, Inc., USA) and TrueBlue (KPL Inc.,
USA) or TMB (CLONDIAG) staining. SA-Poly-HRP was
ª 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Chip layout of three typical AT protein array prototypes.
Specific monoclonal and polyclonal antibodies were contact
spotted in a 3-or 6-fold repetition. The spotting concentration
was either 0.2 mg/mL or 0.5 mg/mL.
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Proteomics 2006, 6, 2972–2981
3.1 BW agents detected on protein chips: Production
and optimization of individual BW-immunoarrays
Since the efficient immobilization of biomolecules is a key
factor for the success of a microarray experiment, we tested
surface-modified glass slides coated with either a special
epoxy-, aldehyde- or amino layer . With aldehyde slides,
smearing was a particular problem. The spot profile was
inhomogeneous and single spots flattened out (data not
shown). Antibody arrays printed on amine slides showed a
better performance than aldehyde slides but retained protein
samples with a lower efficiency. Furthermore, they exhibited
a higher overall background. The most reliable and consistent AT results, however, were obtained with epoxy-modified surface layers. Uniform spot profiles and a low background were the main and convincing reason to use epoxyslides as standard slides for AT arrays.
Capture antibody function was preserved by depositing
the antibodies in low-salt printing buffer containing trehalose, a well known protein stabilizer [20–24].
Manufactured antibody arrays were stored under an
argon atmosphere at 47C in order to extend the shelf life.
Long term stability of capture probes was analyzed for a few
selected antibodies, namely 4G2, pAKanti-E. coli, pAKanti-
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Ricin, 5B4, 12.2, 13.2, 3.4 and asialofetuin. Most of the tested
capture antibodies were stable for more than half a year. A
significant signal decrease over time was not noticed for any
of the tested sandwich assays (data not shown).
3.2 Detection of individual BW agents and
determination of their detection limits
In order to investigate the performance of the designed antibody arrays, the detection specificity and sensitivity of individual analytes was examined. The specific antigen- antibody
reaction was dynamically monitored by transmission microscopy recording one image per ten seconds. As demonstrated in
Fig. 2, the read out can be presented either in form of a bar
graph or curve while each data point represents the mean value
from a triplicate spot measurement. Endpoint images are
shown for each assay in order to give a clear and precise idea of
the experimental results. Detection limits of individual BW
analytes are noted in the text and are summarized in Table 2.
For the identification of E. coli O157:H7 a specific affinity
purified antibody was used as capture antibody. Cross-reactivity to other E. coli strains was minimized through preadsorption to non E. coli O157:H7 serotypes. As detetection
antibody we used a polyclonal antibody broadly reactive with
Figure 2. Shown is the analysis
of a typical AT experiment,
where E. coli O157:H7 was used
as specific antigen. A polyclonal
antibody broadly reactive with
all “O” and “K” antigenic serotypes of E. coli was used as
detection antibody in a 1:2000
dilution. Each data point represents the mean value of 3 spots;
the QSV of negative controls
was below 0.1. Since dynamic
data were acquired, image series can be either plotted as bar
graph (A) or as curve (B). In
addition the endpoint image
(the 60th image) of the analysis is
shown in Fig. 2A and Fig. 2B.
Curve a in Fig. 2B demonstrates
a clear positive result for the
detection of E. coli O157:H7,
whereas b (the concentration of
lanes) summarizes all negative
results. In Fig. 2C the mean
QSV 6 SD at different E. coli
O157:H7 concentrations is given
for three independent array
experiments and protein chips.
Capture antibodies were at least
stable for more than 6 months.
ª 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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B. Huelseweh et al.
most of the “O” and “K” antigenic serotypes of E. coli. The
experimentally calculated limit of detection for E. coli
O157:H7 was 56103 cfu/mL. These results are consistent
with other published data on biosensors [25, 26, Feller, WIS,
Munster, personal communication, 2003]. In general, intraand interspecific assay variance was below 10%. The bar
graph in Fig. 2 was generated by plotting the mean
intensity 6 SD of three independent experiments. For each
experiment the mean value from triplicate or sixplicate spots
was calculated.
A complete AT analysis can be performed in 1 h. However, incubation times can be shortened without extensive
loss off sensitivity. For E. coli O157:H7 it was demonstrated
that reducing the incubation period of antigen and biotinylated secondary antibody from 30 to 5 min only drops the
limit of detection for one order of magnitude from
56103 cfu/mL to 56104 cfu/mL. Similar results were
obtained when the cognate target antigen and the detection
antibody were incubated simultaneously. However, a combined incubation of secondary antibody and SA-Poly-HRP
conjugate can not be recommended in this specific assay as
it drastically reduced the assays’ sensitivity (data not
shown).
The BW agent Ricin is a component of the common castor oil seed (Ricinus communis) and its significance as a
potential biological warfare relates to its wide availability, its
stability and its extremely low LD50 of 3 mg/kg body weight in
mice [27]. Ricin is toxic by several routes of exposure,
including the respiratory and gastrointestinal route. Figure 3
shows the specific binding of 0.5 ng/mL Ricin to the monoclonal antibody CP23 and asialofetuin, a glycoprotein lectin
that has long been known as affinity adsorbens for ricin and
Ricinus agglutinin [28].
The AT immunoassays showed excellent performance
when the biotinylated monoclonal antibody RK1 was used
for detection. A ricin detection limit below 1 ng/mL was
manifested. However, the immunoassays were of only minor
quality when the polyclonal antibody RCA60 was used for
detection. A high extent of cross-reactivity and unspecificity
was noted. The polyclonal antibody was also unsuitable to
capture ricin from solution, since it exhibited unspecific
binding with many other tested bacteria and viruses.
Similar sandwich microarray assays as for Ricin were
developed for the detection of SEB. SEB is one of seven
structurally and biologically related toxins. It causes classical
food poisoning and is considered as BW agent of category B.
Commercially available antibodies were used as capture and
detection antibody. In an AT analysis, SEB had a detection
limit down to 0.2 ng/mL.
VEEV belongs to a group of positive-stranded RNA viruses of the genus Alphaviruses, that cause a disease with
clinical signs of fever, headache, myalgia and malaise and
represents a class A agent. In order to identify VEEV, a combination of three earlier developed monoclonal antibodies [9]
and different commercially available monoclonal antibodies
were tested as capture antibodies. Whereas the monoclonal
ª 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2006, 6, 2972–2981
antibodies developed for our institute were highly specific
[9], we noted lots of unspecific cross reactivities for most of
the commercially purchased VEEV antibodies. Additional
purification of these antibodies by affinity chromatography
could not reduce the problem of unspecificity. Only antibody
MAB 8747 was reasonably specific for VEEV binding as
indicated in Fig. 3. Antibody MAB 8742, actually specific for
Western equine encephalitis virus, showed reliable results
and the expected cross reactivities with VEE strain TC83. Although VEEV and Western equine encephalitis virus were
dedicated to different antigenic complexes, their E1 and E2
envelope proteins and the nucleocapsid protein share a high
percentage of amino acid homology [29, 30].
VEE virus TC83 could be detected below a TCID50/mL of
26106 and the results were consistent for different tested
virus preparations.
For YFV detection, a positive-stranded RNA virus of the
genus Flavivirus, we used the monoclonal antibody 4G2 as
capture molecule, an antibody originally raised specifically
for Dengue 2 virus [19]. This monoclonal antibody displays a
broad cross reactivity among Flaviviruses and therefore is
often used as a group reactive antibody. It recognizes a conserved epitope in the envelope protein of this genus. YFV
detection in an AT analysis was performed with either the
YFV specific antibody MAB 984 or the group specific
mAb 4G2 as biotinylated detection antibody. YFV strain 17D,
the classical vaccination strain, could be detected below a
TCID50/mL of 16105 (see Table 1).
Like YFV, WNV belongs to the family Flaviviridae and the
genus Flavivirus. In contrast to YFV, however, WNV is a
member of the Japanese encephalitis serocomplex, which
also includes Japanese encephalitis virus , SLEV, Murray
Valley encephalitis and Kunjin virus. As demonstrated in
Fig. 3C, WNV is recognized by the group reactive Flavivirus
antibody 4G2 and shows a strong cross reactivity with SLEV
specific antibodies MAB 8741 and 8744. However, WNV does
not interfere with anti-SLEV-antibody MAB 8745, an antibody specific for Group A SLEV. A TCID50/mL of 66102 was
manifested as detection limit for WNV in different AT assays
(see Table 1).
Three specific monoclonal antibodies were tested on an
AT chip as capture antibodies for SLEV. All antibodies
(MAB 8741, MAB 8744 and MAB 8745) identify antigenic
epitopes on the 53 000 dalton envelope (E) glycoprotein.
MAB 8745 was specific for strain MSI-7, a virus isolated in
Mississippi in 1975 belonging to lineage Group A of SLEVs
[31]. MAB 8741 and MAB 8744 however displayed cross
reactivity to SLEV strain MSI-7, but also cross react with
Japanese encephalitis virus, WNV, Murray Valley encephalitis, YFV and Dengue virus 1 to 4. The lowest virus titer
for which AT-SLEV- detection was demonstrated was a
TCID50/mL of 56106. Further dilutions of the antigen have
to be evaluated.
As shown in Fig. 3D cross reactivities of SLEV with 4G2
were obvious and expected, however, no cross reactivity of
SLEV with WNV specific antibodies MAB 8741, MAB 8744
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Protein Arrays
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Figure 3. Representative microarrays for the detection of single
BW agents. For array A, B, C, D,
E, F, H, I, J and L, a chip with
layout 3 is shown. For array F
and G a chip with layout 2 and
for array K a chip with layout 1 is
shown. For each AT analysis the
60th (endpoint) picture is presented. Concentrations of antigens and the used detection
antibodies were indicated on
each image. The dilutions of
detection antibodies varied and
were optimized by titration for
each biotinylation.
and MAB 8745 occurred. Using a combination of supposed
specific monoclonal antibodies for the identification of SLEV
as specific detection molecules was not successful and only
caused a higher unspecific background. The most specific
detection antibody for SLEV was MAB 8744.
Detection of orthopoxvirus species by an AT immunoassay was achieved by using the monoclonal antibody 5B4 as capture antibody and 5B1 as detection antibody.
Both antibodies have been described earlier and were validated for a set of Orthopox strains [17]. As shown in Table 1,
the calculated limit of detection for RaVac was a TCID50/mL
of 56103. Consistent with earlier results the established AT
ª 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
assay was able to identify cow- and camel pox at similar
TCID50/mL values (data not shown).
Besides the monoclonal antibody 5B1 we also tested a
recently raised polyclonal antibody, pAKanti-RaVac, as
detection antibody and obtained similar results and detection
limits, except that the biotinylated polyclonal antibody produced a higher unspecific background.
Also, the organism Yersinia pestis, responsible for the
plague, is classified by the Center for Disease Control as a
potential biological weapon of category A. Pathogenic
Y. pestis strains produce two antiphagocytic components,
F1 antigen and the VW antigens, that both are required for
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Proteomics 2006, 6, 2972–2981
Table 1. Summary of experimentally tested capture and biotinylateddetection antibodiesa).
Analyte
Inactivation
Tested Capture
Antibodies
Tested Detection
Antibodies
Detection limit
Vaccinia
Not inactivated
5B4/2F2; 5B1
5B1b)
pAKanti-Vacb) (RaVac)
56103 TCID50/mL
YFV 17D
Not inactivated
D1-4G2-4-15;
WNV MAB 984
D1-4G2-4-15b)
WNV MAB 984b)
16105 TCID50/mL
SLEV
b-Propionolactone
SLEV MAB 8741;
SLEV MAB8744;
SLEV MAB 8744;
SLEV MAB8745
SLEV MAB 8745b);
SLEV MAB 8744b);
SLEV MAB8745b);
SLE MAB 8744b)
56106 TCID50/mL
WNV NY
b-Propionolactone
WNV MAB 8150;
WNV MAB 8151;
WNV MAB 8152
15R4b);
WNV MAB 8151b)
66102 TCID50/mL
VEEV TC83
b-Propionolactone
12.2/13.2/3.4;
VEEV MAB 8747
VEEV MAB 8747b);
8.6b)/b42.2/12.2b)/b13.2
26106 TCID50/mL
E. coli 0157:H7
Heat
anti-E. coli 0157:H7
anti-E.colib)
56103 cfu/mL
b)
Y. pestis
Heat & formaldehyde
Yp G20, YPF19
YPF19
56105 cfu/mL
F. tularensis
Heat & formaldehyde
FT140/11/1/06
FT140b)/11/1/06
26106 cfu/mL
B. mallei
B. pseudomallei
Heat & formaldehyde
PS6F6, 3PM15
PS6F6 ; b205/102
26106 cfu/mL
B. melitensis
Heat & formaldehyde
BM040/01
BM040b)/01;
163b)/098
16106 cfu/mL
SEB
Not inactivated
anti-SEB
anti-SEBb)
Ricin
Not inactivated
RCA60; Clone 23,
Asialofetuin
b)
b)
0.2 ng/mL
b)
RCH1 , RK1 ;
RCA60b)
0.1 ng/mL
a) For each antigen the best antibody combination is underlined and the corresponding detection limit is given.
b) Biotinylated detection antibodies
virulence. In our AT assays, antigen was captured by the
F1 specific monoclonal antibody YpG20. Specific antigen
detection was performed with the Mab YPF19, an antibody
that as well recognizes Y. pestis F1 capsular antigen.
Mab YPF19 is unspecific for Y. pseudotuberculosis and
Y. enterocolitica. As noted in Table 1 the limit of detection was
below 56105 cfu/mL. A comparable limit of detection for
Y. pestis was reported for classical sandwich-ELISA (Niederwhrmeier, WIS, Munster, personal communication,
2003).
Insignificant cross-reactivity of the detection antibody
with the mouse anti-F. tularensis antibody and the polyclonal
anti-Ricin antibody RCA60 was observed (see Fig. 3).
The specific identification of Francisella tularensis in an
AT-assay was achieved by using the monoclonal antibody
FT140/11/1/06 as capture and as detection antibody. FT140/
11/1/06 was originally raised against F. tularensis ATCC 6223
var. tularensis. It is directed to epitopes on the core moiety of
the lipopolysaccharide molecule and has been demonstrated
to be specific for the lipopolysaccharide molecule of all
ª 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
F. tularensis subspecies [10]. In an AT assay the lowest concentration detected above unspecific background was
26106 cfu/mL. For the AT limits of detection for F. tularensis
were similar or identical to those obtained with confirmatory
ELISAs (Niederwhrmeier, WIS, Munster, personal communication, 2003).
Burkholderia pseudomallei and B. mallei, two highly
pathogenic and closely related bacteria, responsible for
melioidosis and glanders, were identified by using the
monoclonal antibody PS6F6 to capture the cognate target
antigen. Sofar, the polysaccharide capsule is the only described determinant of virulence.The antibody PS6F6 was
raised specifically against the exopolysaccharide of B. pseudomallei. However the monoclonal antibody also recognizes
B. mallei. By using the same monoclonal antibody as detection antibody, the lowest concentration for which AT detection was demonstrated was 26106 cfu/mL for both species.
Use of a specific polyclonal antibody as detection antibody
(pAK205/102) resulted in a loss of sensitivity down to
26107 cfu/mL.
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Protein Arrays
Proteomics 2006, 6, 2972–2981
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Figure 4. Shown are representative AT results obtained for a protein chip
with layout 3 after performing a parallel analysis of two, three, four and
five different BW agents. From each analysis the 60th picture is presented.
Concentrations of analytes in experiment A to I were as follows: A: SEB
(5 ng/mL) and ricin (2 ng/mL); B: YFV 17 D (26105 TCID50/mL) and
WNV NY (103 TCID50/mL); C: E. coli O157:H7 (56104 cfu/mL) and Y. pestis
(56106 cfu/mL); D: YFV 17 D (26105 TCID50/mL), WNV NY (103 TCID50/mL)
and Vac (105 TCID50/mL); E: VacV (105 TCID50/mL), F. tularensis (107 cfu/mL)
and SEB (2 ng/mL); F: VacV (105 TCID50/mL), E. coli O157:H7 (56104 cfu/
mL) and SEB (2 ng/mL); G: VacV (105 TCID50/mL), E. coli O157:H7
(56104 cfu/mL), F. tularensis (107 cfu/mL) and SEB (5 ng/mL); H: YFV 17 D
(105 TCID50/mL), WNV NY (103 TCID50/mL), SLEV (26107 TCID50/mL) and
VacV (26105 TCID50/mL); I: YFV 17 D (105 TCID50/mL), WNV NY (103 TCID50/
mL), SLEV (26107 TCID50/mL), VacV (105 TCID50/mL) and ricin (2 ng/mL).
The following biotinylated (b) detection antibodies were used: A: bRCH1,
b
anti-SEB1; B: b4G2, bWNV MAB8151; C: banti-E.coli, bYPF19; D: b4G2,
b
WNV MAB8151, b5B1; E: b5B1, bantiSEB, bFT140/11/1/06; F: b5B1, bantiE. coli, banti-SEB; G: b5B1, banti-E. coli, banti-SEB, bFT140/11/1/06; H: b4G2,
b
WNV MAB8151, b5B1, bSLEV MAB 8744; I: b4G2, bWNV MAB8151, b5B1,
b
SLEV MAB 8744, bRCH1. Also in a simultaneously AT analysis most of the
signals can already be detected after 60 to 90 s. The signals clearly
increase over time. Shown in the diagram as a representative example are
the dynamic data that were aquired for 4G. Curve a clearly demonstrates
the positive detection of Vaccinia, curve b corresponds to the detection of
E. coli O157:H7. Curve c presents the positive result for YFV 17 D and
curve d demonstrates the positive detection of F. tularensis.
A further priorized and critical threat agent is Brucella
melitensis, one of three closely related species of the genus
Brucella that are pathogenic for man. The bacterial disease is
characterized by intermittent fever, chills and weakness. As
demonstrated in Fig. 3, identification of Br. melitensis was
achieved by using the monoclonal antibody BM040 as capture antibody. For colorimetric detection the polyclonal antibody 163/098 was used. Br. melitensis could be ascertained
down to 106 cfu/mL (see Table 1). The results are consistent
with in-house ELISA results. (Niederwhrmeier, WIS, Munster, personal communication).
ª 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.3 Parallel analysis of BW agents
In order to characterize the ability of the AT technology to
detect multiple analytes simultaneously, we combined toxins, viruses and bacteria in a single sample and used specific biotinylated antibody cocktails for the detection. Antigen concentrations in samples used for the simultaneous
analysis were approximately 5–20 fold higher than for the
determination of the limits of detection in individual AT
experiments. As demonstrated in Fig. 4, up to five different
BW agents can be assayed on a single microarray without
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2980
B. Huelseweh et al.
difficulties and most of the signals are already detected after
60 to 90 s. The kinetic diagram shown in Figure 4 corresponds to Fig. 4G and clearly proves how the signals
increase over time. As a result of a careful antibody screening and optimization background problems did not arise.
Detection limits for individual BW analytes in a parallel
assay were either identical to or about factor 2 to 5 above
limits in single assays (data not shown).
4
Concluding remarks
Sensitive and rapid AT assays enable the detection of diverse
BW agents with a limited number of antibodies. Detection
limits for viruses, bacteria and toxins were similar or identical to those obtained with ELISAs but in general are less
time consuming. Even the presence of irrelevant bacteria in
10 to 100-fold excess, did not interfere with specific detection.
AT assays are suitable for parallel analysis and so far between two to five different BW agents can be assayed on a
single microarray without difficulties. This is clearly in contrast to other recent reports that describe a method for multianalyte sensing but demonstrate the detection of only one
or two to three single analytes [32, 33].
The quality of an AT immunoarray is still depending on
the individual affinities/avidities of the immobilized antibodies as well as the applied detection antibodies. In general,
we prefer the use of monoclonal antibodies because the continuous culture of hybridoma cells offers us the potential of
an unlimited supply.
The present analysis should be considered as an alternative or additional analysis to other immuno- or biosensors.
Compared to other solutions for the detection of pathogens
only a few micrograms of each antibody are sufficient to
produce thousands of microarrays.
With further optimization the current antibody chip
may evolve into a generally accepted screening and analysis
tool for NBC Defense. Test automation, restriction to endpoint detection and the possibility of assay transfer to fully
automated solutions could improve the practicability of AT
assays for staff with little laboratory and technical experience.
Proteomics 2006, 6, 2972–2981
5
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