Clinical Chemistry 48:1
121–130 (2002)
Antigen Microarrays for Serodiagnosis of
Infectious Diseases
Letizia Mezzasoma,1* Tito Bacarese-Hamilton,1* Manlio Di Cristina,1 Ruggero Rossi,2
Francesco Bistoni,2 and Andrea Crisanti1†
Background: Progress in robotic printing technology
has allowed the development of high-density nucleic
acid and protein arrays that have increased the through-
put of a variety of assays. We generated protein microar-
rays by printing microbial antigens to simultaneously
determine in human sera antibodies directed against
Toxoplasma gondii, rubella virus, cytomegalovirus
(CMV), and herpes simplex virus (HSV) types 1 and 2
(ToRCH antigens).
Methods: The antigens were printed on activated glass
slides with high-speed robotics. The slides were incu-
bated first with serum samples and subsequently with
fluorescently labeled secondary antibodies. Human IgG
and IgM bound to the printed antigens were detected by
confocal scanning microscopy and quantified with in-
ternal calibration curves. Both microarrays and commer-
cial ELISAs were utilized to detect serum antibodies
against the ToRCH antigens in a panel of characterized
human sera.
Results: The detection limit (mean ⴙ 2 SD) of the
microarray assay was 0.5 pg of IgG or IgM bound to the
slides. Within-slide, between-slide, and between-batch
precision profiles showed CVs of 1.7–18% for all anti-
gens. Overall, >80% concordance was obtained between
microarray assays and ELISAs in the classification of
sera; for T. gondii, CMV, and HSV1, concordance ex-
ceeded 90%.
Conclusions: The microarray is a suitable assay format
for the serodiagnosis of infectious diseases and can be
easily optimized for clinical use. The ToRCH assay
performs equivalently to ELISA and may have poten-
1
Department of Biology, Imperial College of Science, Technology and
Medicine, London SW7 2AZ, United Kingdom.
2
Dipartimento di Medicina Clinica e Sperimentale Università degli Studi
di Perugia, Via del Giochetto, 006100 Perugia, Italy.
*These authors contributed equally to this work.
†Address correspondence to this author at: Imperial College of Science,
Technology and Medicine, Department of Biology, Biomedical Sciences Build-
ing, 5th Floor, Imperial College Road, London SW7 2AZ, United Kingdom. Fax
44-207-5945439; e-mail acrs@ic.ac.uk.
Received August 24, 2001; accepted October 15, 2001.
121
Automation and
Analytical Techniques
tially important advantages in throughput, convenience,
and cost.
© 2002 American Association for Clinical Chemistry
Assays with the ability to detect in parallel antibodies
with different specificities have a wide range of potential
applications in epidemiologic research and vaccine devel-
opment as well as in the diagnosis of allergies and
autoimmune and infectious diseases. Although ELISA-
based tests are suitable for this purpose, available assays
can be time-consuming and require large quantities of
both sample and reagents, thus limiting their application
for mass screening (1, 2 ). The advent of automated, robust
microdeposition technologies has allowed the develop-
ment of high-density ordered arrays of molecules (mi-
croarrays) (3 ). This assay format incorporates key fea-
tures, such as true parallelism, miniaturization, and high
throughput, that could overcome most of the current
ELISA limitations (4 ). Studies using DNA microarrays
have shown that this assay format is ideal for studying the
expression profiles of thousands of genes simultaneously
in tissues and organs under different experimental condi-
tions (5–7 ). Currently, DNA microarrays are regarded as
one of the most powerful available tools for understand-
ing the functional relationships between the large reper-
toires of genes available in databases generated as a result
of genome sequencing projects (8, 9 ).
Ordered arrays of peptides have been used for unrav-
eling protein–protein and protein–nucleic acid interac-
tions (10, 11 ). Previous reports have shown that protein
arrays printed on membranes can be used to screen
expression libraries for binding to target molecules as well
as to unravel protein binding to either RNA or DNA
(12–15 ). More recently, arrays have been generated for
high-throughput screening of recombinant antibodies
(16 ). These antibody arrays contain thousands of bacterial
clones, each expressing a different single-chain antibody.
Arrays of proteins covalently bound to glass slides were
shown to retain their ability to interact specifically with
other proteins or with small molecules in solution (17 ).
Protein arrays have also been used to develop compara-
122 Mezzasoma et al.: Microarrays for Serodiagnosis
Fig. 1. Schematic representation of the array used in this study.
Colored circles indicate the positions where T. gondii, rubella virus, CMV, HSV1, and HSV2 antigen preparations were printed in replicate. The array was designed to
contain internal calibration curves generated by printing increasing amounts of purified human IgG and IgM. Rabbit myosin was printed as negative control. The
fluorophores Alexa 546 and Alexa 594 were also included in the array (white circle).
tive fluorescence assays to measure the concentrations of
several specific proteins and antibodies in complex solu-
tions (18 –21 ). These studies have shown that protein
microarrays could provide a practical means to quantify
thousands of different protein species in clinical or re-
search applications. In spite of these advances, the devel-
opment of protein arrays for research and clinical appli-
cations has lagged because of the poor stability of
proteins, complex coupling chemistry, and weak detec-
tion signals. High-density protein arrays remain difficult
to generate and to validate for clinical use.
We used a protein microarray to determine in human
sera the presence of specific antibodies directed against
parasite and viral antigens, including Toxoplasma gondii,
cytomegalovirus (CMV),3 rubella virus, and herpes sim-
plex virus (HSV) types 1 and 2. The clinical performance
of this assay was validated with a collection of sera
previously characterized with commercial ELISAs for
their reactivity against the selected microbial antigens.
Materials and Methods
preparation of microarrays
Microbial antigens as well as human IgG and IgM at
different concentrations were printed on silanized glass
microscope slides (CEL Associates), using computer-con-
trolled high-speed robotics (Total Array System; Biorobot-
ics) (22 ). Samples were transferred from 384 microtiter
plates to glass slides by use of stainless steel solid pins
(200-␮m diameter). Each pin was estimated to transfer ⬃1
nL of sample to the slide. Arrays consisted of a 7 ⫻ 7
matrix that included five microbial antigens printed in six
replicates, the IgG and IgM internal calibration curves in
duplicate, and rabbit muscle myosin as control. The
fluorophores Alexa 546 and/or Alexa 594 (50 pg) were
3
Nonstandard abbreviations: CMV, cytomegalovirus; HSV, herpes sim-
plex virus; PBS, phosphate-buffered saline; and EIA, enzyme immunoassay.
incorporated in the array as a reference signal. T. gondii,
CMV, and HSV1 and HSV2 preparations were supplied
by Radim S.p.A. Rubella virus antigen (grade K1S) was
purchased from Microbix Biosystems Inc. Human IgG
and IgM (reagent grade) were purchased from Sigma
Chemical Company. Antigens were dissolved in phos-
phate-buffered saline [PBS; 1⫻ (0.2 g/L KCl, 1.44 g/L
Na2HPO4, 0.24 g/L KH2PO4, 8 g/L NaCl, pH 7.4)] con-
taining Tween 20 (0.1 mL/L). Polyvinylpyrrolidone (10
g/L) was added to the human IgG and HSV1 antigen
solutions, sodium dodecyl sulfate (0.1 g/L) was added to
the human IgM solution, and sucrose (100 g/L) was
added to the CMV antigen solution. No additives were
present in the other antigen preparations. Printing was
performed in a cabinet at 25 °C and 55% humidity. These
conditions were constantly monitored by a thermohy-
grometer. Printed slides were stored for 12 h inside the
cabinet before removal and storage. Slides were stored in
boxes at room temperature in the presence of silica gel bags
as desiccant and used within 90 days of being printed.
serum samples and elisa methods
Human sera (n ⫽ 60) were supplied by BioMedical
Resources Inc. The sera were arranged in five panels on
the basis of their reactivity against T. gondii, rubella virus,
HSV1, HSV2, and CMV as measured by Abbott IMx
assay, Sigma enzyme immunoassay (EIA), and Zeus EIA.
The panels VP6627, VP6628, and VP6678 consisted of two
groups of five sera containing either IgG or IgM directed
against T. gondii, rubella virus, and CMV, respectively.
Each panel also contained five sera selected for their lack
of IgG and IgM reactivity against different ToRCH anti-
gens. Panel VP6680 contained five sera with IgM directed
against HSV1. Panel VP6679 consisted of five positive sera
with IgG against HSV1 and five negative sera.
All sera were analyzed for reactivity against T. gondii,
rubella virus, HSV1, HSV2, and CMV antigens by the
ELISAs (EIA Well) supplied by Radim S.p.A. The assays
 Clinical Chemistry 48, No. 1, 2002 123

Fig. 2. Fluorescent scans of array areas printed with increasing concentrations (top to bottom) of human IgG (A; 2, 10, 25, and 50 mg/L) and IgM
(C; 0.4, 2, 4, and 8 mg/L) and incubated with anti-IgG and -IgM secondary antibodies labeled with Alexa 546 and Alexa 594, respectively.
The fluorescence was visualized in a pseudo-color scale (dark blue to white) corresponding to increasing fluorescence. The mean values of duplicate photomultiplier
count (pmc) measurements were plotted as a function of the concentrations of IgG (B) and IgM (D) to generate dose–response curves. The equation and the coefficient
of determination are indicated for each curve.

were performed according to the manufacturer’s instruc-
tions.
processing of microarray slides
Printed slides were incubated for 1 h at room temperature
with a solution containing 10 g/L bovine albumin in PBS
to block nonspecific antibody binding. An adhesive tape
(Gene-Frame; Abgene Limited) was used to contain sam-
ples/reagents within the array area. Serum samples were
diluted 1:200 in 2⫻ PBS containing 10 g/L bovine serum
albumin and 0.1 mL/L Tween 20 and allowed to react
with the array for 15 min at room temperature in a humid
chamber. To reveal IgG bound to the printed antigens, the
slides were washed five times (1 mL each time) with PBS
containing 0.1 mL/L Tween 20 and subsequently incu-
bated for 5 min with Alexa 546-labeled anti-human IgG
monoclonal antibody 2F6 (Radim) suspended in a solu-
tion containing 2⫻ PBS, 10 g/L bovine serum albumin,
and 0.1 mL/L Tween 20. To reveal IgM, the slides were
incubated for 5 min with Alexa 594-labeled goat immu-
noglobulins directed against the anti-human IgM ␮ chain
(OEM Concepts Inc.). Before the fluorescence was read in
the scanner, the slides were washed and dried at 37 °C.
The secondary antibodies, monoclonal antibody 2F6 and
the goat immunoglobulins, were labeled with succinimi-
dyl Alexa 546 and 594, respectively, and purified accord-
ing to the instructions provided by the manufacturer
(Molecular Probes Inc.). The efficiency of the labeling
procedure was analyzed by measuring the molar ratio of
dye to protein.
124 Mezzasoma et al.: Microarrays for Serodiagnosis

Fig. 3. Fluorescent scans of antigen arrays incubated with samples from VP6678/1 (A), VP6678/15 (B), VP6628/2 (C), and VP6627/8 (D) serum
panels, which differ according to their reactivities in ELISA in the presence of IgG directed against the ToRCH antigens.
Serum antibodies were detected by incubating the slides with Alexa 546-labeled secondary antibody directed against human IgG. The intensity of fluorescence emitted
by the fluorophore was visualized in a pseudo-color scale (dark blue to white).

data collection and analysis
The slides were analyzed with a GSI 5000 scanner. Images
were generated with the ScanArrayTM software provided
by GSI Lumonics and quantified using the QuantArrayTM
software provided by the same company (23 ). Human
IgG and IgM dose–response curves were fitted with a
linear curve fit (Excel; Microsoft). The amounts of IgG and
IgM in the sera were determined by interpolating the
photomultiplier counts collected at the microbial antigen
spots with the IgG and IgM internal calibration curves.
Results
array design and analysis of IgG and IgM
calibration curves
Purified human IgG and IgM as well as T. gondii, CMV,
rubella virus, HSV1, and HSV2 antigen preparations were
printed on amino-silane-activated glass slides. This sur-
face was chosen on the basis of experiments indicating
that proteins, such as antibodies and albumin, bound with
high efficiency and reproducibility to amino-silane-acti-
vated glass slides (data not shown). Each antigen was
printed in six replicates within a 7 ⫻ 7 matrix (Fig. 1). The
array was designed to contain a negative control (rabbit
myosin) and the fluorophores Alexa 546 and Alexa 594 as
sources of reference signal. Internal calibration curves
were generated by printing, in duplicate, 1 nL of immu-
noglobulin solution containing increasing concentrations
of IgG or IgM (2–50 and 0.4 – 8 mg/L, respectively). The
reactivities of the secondary antibodies against the nega-
tive control (myosin) were used as the first point of the
calibration curves. Regression analysis demonstrated that
linear dose–response curves were obtained by plotting the
 Clinical Chemistry 48, No. 1, 2002 125

Fig. 4. Fluorescent scans of antigen arrays incubated with samples from VP6678/1 (A), VP6678/15 (B), VP6628/2 (C), and VP6627/8 (D) serum
panels.
Serum antibodies were detected by incubating the slides with Alexa 594-labeled secondary antibody directed against human IgM. The fluorescence emitted by the
fluorophore was visualized in a pseudo-color scale (dark blue to white).

photomultiplier counts against the amounts of immuno-
globulins printed on the slides (Fig. 2). Both IgG and IgM
calibration curves showed similar slopes and coefficients
of determination (r2) close to 1. These experiments also
revealed that the cross-reactivities of the anti-IgM goat
serum and monoclonal antibody 2F6 against human IgG
and IgM, respectively, were negligible.
analysis of serum reactivity against arrayed
antigens
Human sera from panels VP6627, VP6628, VP6678,
VP6680, and VP6679 were used to investigate the ability
of the microarray assay to reveal the presence or absence
of IgG and IgM directed against the different ToRCH
antigens. These sera were certified for the presence or
absence of antibodies directed against distinct ToRCH
antigens on the basis of the Abbott IMx assay, the Sigma
EIA, or the Zeus EIA. Radim ELISAs for ToRCH antigens
were used to confirm the reactivities of all sera. Fluores-
cent scans of microarray slides incubated with represen-
tative sera of the different panels showed that this assay
allows the detection of specific antibodies directed against
microbial antigens in accordance with ELISA reactivity
data (Figs. 3 and 4). In general, sera that failed to show a
positive reaction in ELISA did not react against the
corresponding microbial antigens in the microarray assay,
whereas fluorescent signals were usually associated with
positive reactions in the ELISAs (Figs. 3 and 4). No
126 Mezzasoma et al.: Microarrays for Serodiagnosis

Fig. 5. Mean photomultiplier count (pmc) measurements collected from the arrayed antigens (colored symbols), incubated with samples from
VP6678/1 (A), VP6678/15 (B), VP6628/2 (C), and VP6627/8 (D) serum panels, were interpolated to the dose–response curves generated by
plotting the fluorescent intensity as a function of printed amounts of IgG (blue diamonds).
The SE of replicate measurements was ⬍10%. The equation and the coefficient of determination are indicated for each curve.

reactivity was detected against the myosin negative con-
trol, thus demonstrating the absence of nonspecific inter-
ference in the assay. The r2 values for both the IgG and
IgM curves in these arrays were 0.97– 0.99, indicating that
the internal calibration curves generated under these
experimental conditions showed a high reproducibility
and a linear dose–response relationship (Figs. 5 and 6).
A set of human sera showing different reactivities in
the ELISA (low, medium, and high), was used to monitor
the performance of the microarray assay for each ToRCH
antigen. This analysis revealed that increasing concentra-
tions of IgG directed against the microbial antigens gen-
erated fluorescence signals with precision profiles that
showed in most cases a CV ⬍10% within and between
slides (Table 1). Slightly higher CV values were observed
when comparing slides from different batches (Table 1).
These values were similar to ELISA-generated data, as
stated in the instruction manual for the ELISA. The
detection limit of the microarray assay was determined by
assessing the reactivity of the sera against 20 replicates of
rabbit myosin printed on slides. The detection limit,
defined as mean photomultiplier counts of the negative
control plus 2 SD interpolated on the IgG calibration
curve, was 0.5 pg.
microarray assay for multianalyte
serodiagnosis
We compared the microarray assay and Radim ELISA for
their ability to determine in human sera the presence or
absence of IgG reactive against the ToRCH antigens. This
 Clinical Chemistry 48, No. 1, 2002 127

Fig. 6. Mean photomultiplier count (pmc) measurements collected from the arrayed antigens (colored symbols), incubated with samples from
VP6678/1 (A), VP6678/15 (B), VP6628/2 (C), and VP6627/8 (D) serum panels, were interpolated to the dose–response curves generated by
plotting the fluorescent intensity as a function of printed amounts of IgM (blue diamonds).
The SE of replicate measurements was ⬍10%. The equation and the coefficient of determination are indicated for each curve.

analysis could not be extended to serum IgM because we
lacked a sufficient number of characterized positive sera
for accurate comparison with the ELISA. Notably, the
antigen microarray and the Radim ELISA shared the same
antigen and antibody reagents, facilitating the compari-
son of the two diagnostic assays. The cutoff values of the
distinct serum reactivity determinations within the mi-
croarray assay were calculated using reference sera that,
according to information that accompanied the Abbot
IMx, Sigma EIA, and Radim ELISA, lacked antibodies
directed against T. gondii, rubella virus, CMV, HSV1, or
HSV2. These values incorporated the 95th percentile of
these reference sera. The reactivities of the serum samples
were classified as either positive or negative according to
the presence of IgG concentrations above or below the
cutoff value. Sera with IgG concentrations between ⫾ 10%
of the cutoff value were regarded as equivocal. According
to these criteria, the microarray assay classified 19.6% of
samples as positive, 76.8% as negative, and 3.6% as
equivocal for the presence of IgG directed against T.
gondii; corresponding ELISA data were 21.4% positive,
69.7% negative, and 8.9% equivocal (Table 2). For CMV,
the microarray classified 64.3% of samples as positive,
32.1% as negative, and 3.6% as equivocal; corresponding
ELISA data were 66.1% positive, 32.1% negative, and 1.8%
equivocal. For rubella virus the microarray classified
17.9% of samples as positive, 75% as negative, and 7.1% as
equivocal; ELISA classified 87.5% as positive, 1.8% as
negative, and 10.7% as equivocal (Table 2).
For HSV1, the microarray classified 80.3% of samples
as positive, 17.9% as negative, and 1.8% as equivocal;
corresponding ELISA data were 78.6% positive, 19.6%
negative, and 1.8% equivocal. For HSV2, the microarray
classified 22.2% of samples as positive, 66.7% as negative,
128 Mezzasoma et al.: Microarrays for Serodiagnosis

Table 1. Within-slide, between-slide, and between-batch precision profiles of the microarray assay.
IgG serum reactivity

T. gondii CMV Rubella virus HSV1 HSV2

Low Medium High Low Medium High Low Medium High Low Medium High Low Medium High
Within slide (n ⫽ 6)
pmca 30.0 61.0 111.5 8.1 27.5 73.2 16.7 30.0 50.7 6.3 77.0 124.5 4.9 14.8 49.3
SD 0.87 4.50 1.90 0.51 0.48 6.33 0.60 1.69 2.93 0.30 3.72 5.89 0.19 0.57 1.19
CV,b % 2.9 7.4 1.7 6.4 1.7 8.6 3.6 5.6 5.8 4.8 4.8 4.7 3.9 3.9 2.4
Between slides (n ⫽ 12)
pmc 28.3 66.3 101.7 7.5 25.6 76.8 16.8 26.7 52.4 6.0 79.6 128.7 4.6 14.5 51.8
SD 2.07 7.32 12.40 0.70 2.54 9.57 0.45 3.89 2.99 0.39 6.44 7.09 0.48 0.63 2.81
CV,% 7.3 11 12 9.3 9.9 12 2.6 15 5.7 6.5 8.1 5.5 10 4.4 5.4
Between batches (n ⫽ 18)
pmc 27.8 66.3 99.7 7.8 26.1 76.0 15.8 27.4 50.9 6.0 76.2 130.8 4.3 14.6 53.1
SD 2.44 7.39 10.48 0.76 2.72 9.38 1.74 5.06 3.50 0.31 7.70 11.31 0.64 0.61 3.04
CV,% 8.8 11 10 9.7 10 12 11 18 6.9 5.2 10 8.6 15 4.2 5.7
a
pmc, photomultiplier count.
b
CVs were determined for photomultiplier counts collected from the arrayed antigens, each incubated with three serum samples classified on the basis of their
reactivities in ELISA as negative, equivocal, or positive. The CVs were calculated as the SD of the photomultiplier counts divided by the mean.

and 11.1% as equivocal; corresponding ELISA data were
18.5% positive, 51.9% negative, and 29.6% equivocal (Ta-
ble 2).
comparison of test protocols
Comparison of microarray assay and ELISA test protocols
(Table 3) indicated that the major advantage of the mi-
croarray test is that regardless of the number of analytes
determined on a slide, the time-to-result and costs were
the same. With ELISA formats, each analyte is determined
in a separate assay, thereby increasing time and costs.
Discussion
Our data indicate that a protein microarray assay with
indirect fluorescence detection can be used to determine
the presence or absence of specific antibodies directed
against various microbial antigens in human sera. The
presence of internal dose–response IgG and IgM calibra-
tion curves allowed us to demonstrate that antibodies
from different serum samples bound to printed antigens
can be quantified with high reproducibility. In addition,
the calibration curves provided unique advantages over
current immunoassay protocols in which the calibration
curves and the samples are processed in separate tubes or
wells. In these assays, matrix problems (differences be-
tween the calibrator and sample matrices) occur fre-
quently and represent a known source of bias (24 ). In
contrast, in our microarray format any given signal de-
veloped within the arrayed antigens is interpolated to a
calibration curve processed under the same physicochem-
ical conditions (e.g., time, temperature, protein content,
fat content, and bilirubin concentration) as the antibody
reactivity being determined. Under these conditions, the
dose–response curve will compensate for variability in the
sample physicochemical conditions, thus minimizing ma-
trix effects.
The use of a dedicated microarray robot coupled with
optimized printing solutions and glass substrates allowed
us to generate high-quality fluorescence data suitable for
a study aimed at assessing the clinical performance of the
ToRCH microarray assay. For this purpose the microarray
assay and several commercially available ELISAs were
compared for their ability to reveal IgG directed against
the selected microbial antigens in a set of characterized
human sera. Notably, both the microarray assay and the
ELISAs used in the comparative study shared the same
antigen preparations and antibody reagents. This analysis
revealed that the microarray assay is able to identify

Table 2. Comparison of microarray and ELISA for their ability to detect IgG directed against ToRCH antigens in human sera.
Serum reactivity, n (%)

Microarray assay ELISA

Positive Negative Equivocal Positive Negative Equivocal

T. gondii 11/56 (19.6%) 43/56 (76.8%) 2/56 (3.6%) 12/56 (21.4%) 39/56 (69.7%) 5/56 (8.9%)
Rubella virus 10/56 (17.9%) 42/56 (75%) 4/56 (7.1%) 49/56 (87.5%) 1/56 (1.8%) 6/56 (10.7%)
CMV 36/56 (64.3%) 18/56 (32.1%) 2/56 (3.6%) 37/56 (66.1%) 18/56 (32.1%) 1/56 (1.8%)
HSV1 45/56 (80.3%) 10/56 (17.9%) 1/56 (1.8%) 44/56 (78.6%) 11/56 (19.6%) 1/56 (1.8%)
HSV2 12/54 (22.2%) 36/54 (66.7%) 6/54 (11.1%) 10/54 (18.5%) 28/54 (51.9%) 16/54 (29.6%)
 Clinical Chemistry 48, No. 1, 2002 129

L. Mezzasoma was supported by the University of Peru-

Table 3. Comparison of microarray and ELISA test
gia. This work was entirely funded by a grant by Radim
protocols.
S.p.A. (Rome, Italy). We thank Clare McBrearty and
ELISA Microarray
Stanislavo Marcolini for excellent technical assistance and
Sample
Luigi Sparano for encouragement and support.
Dilution 1:300 1:200
Volume, ␮L 100 80
Incubation, min 60 15
Temperature 37 °C Room temperature References
Wash, ␮L 1050 5000 1. Kricka L. Trends in immunoassay technologies. J Clin Immunoas-
say 1993;16:267–71.
Conjugate
Volume, ␮L 100 0 2. Silzel JW, Cercek B, Dodson C, Tsay T, Obremski RJ. Mass-
Incubation, min 30 1 sensing, multianalyte microarray immunoassay with imaging de-
tection. Clin Chem 1998;44:2036 – 43.
Temperature 37 °C Room temperature
Wash, ␮L 1050 5000 3. Schena M, Heller RA, Theriault TP, Konrad K, Lachenmeier E,
Davis RW. Microarrays: biotechnology’s discovery platform for
Substrate/Stop
functional genomics. Trends Biotechnol 1998;16:301– 6.
Volume, ␮L 100 ⫹ 100 None
4. Walter G, Bussow K, Cahill D, Lueking A, Lehrach H. Protein arrays
Incubation, min 10 None
for gene expression and molecular interaction screening. Curr
Temperature 37 °C NAa
Opin Microbiol 2000;3:298 –302.
Read time, min/sample 2 2
5. DeRisi JL, Iyer VR, Brown PO. Exploring the metabolic and genetic
Signal stability 15 min ⬎3 months
control of gene expression on a genomic scale. Science 1997;
Total time, min
278:680 – 6.
1 analyte 110 20
6. Duggan DJ, Bittner M, Chen Y, Meltzer P, Trent JM. Expression
10 analytes 1100 20
profiling using cDNA microarrays. Nat Genet 1999;21:10 – 4.
Estimated cost, US$
7. Liotta L, Petricoin E. Molecular profiling of human cancer. Nat Rev
1 analyte 1.5 5
Genet 2000;1:48 –56.
10 analytes 15 5
8. Watson SJ, Meng F, Thompson RC, Akil H. The “chip” as a specific
a
NA, not applicable. genetic tool. Biol Psychiatry 2000;48:1147–56.
9. Devaux F, Marc P, Jacq C. Transcriptomes, transcription activators
and microarrays. FEBS Lett 2001;498:140 – 4.
positive and negative sera with the same efficiency as the 10. Cahill DJ. Protein and antibody arrays and their medical applica-
ELISAs. For rubella, the Radim ELISA classified 49 sam- tions. J Immunol Methods 2001;250:81–91.
ples (87.5%) as positive, whereas only 10 (17.9%) of these 11. Emili AQ, Cagney G. Large-scale functional analysis using peptide
sera were shown to have specific antibodies in the mi- or protein arrays. Nat Biotechnol 2000;18:393–7.
croarray assay. A small number of these samples (n ⫽ 10) 12. Bussow K, Cahill D, Nietfeld W, Bancroft D, Scherzinger E,
Lehrach H, Walter G. A method for global protein expression and
were analyzed in an independent assay (Abbott IMx), and antibody screening on high-density filters of an arrayed cDNA
the results of this control experiment agreed with the library. Nucleic Acids Res 1998;26:5007– 8.
microarray assay rather than with the Radim ELISA data. 13. Lueking A, Horn M, Eickhoff H, Bussow K, Lehrach H, Walter G.
Our data also indicated that the microarray assay could be Protein microarrays for gene expression and antibody screening.
used to determine the presence or absence of specific Anal Biochem 1999;270:103–11.
serum IgM. The number of positive sera available was too 14. Bussow K, Nordhoff E, Lubbert C, Lehrach H, Walter G. A human
small to infer conclusions concerning the clinical perfor- cDNA library for high-throughput protein expression screening.
Genomics 2000;65:1– 8.
mance, but this preliminary analysis indicated that similar
15. Ge H. UPA, a universal protein array system for quantitative
results were obtained with ELISAs and the microarray
detection of protein-protein, protein-DNA, protein-RNA and protein-
assay. ligand interactions. Nucleic Acids Res 2000;28:e3(i–vii).
16. de Wildt RM, Mundy CR, Gorick BD, Tomlinson IM. Antibody arrays
In conclusion, our findings demonstrate that not only can for high-throughput screening of antibody-antigen interactions. Nat
good analytical and clinical data be obtained with mi- Biotechnol 2000;18:989 –94.
croarrays, but that this test format may have potentially 17. MacBeath G, Schreiber SL. Printing proteins as microarrays
important advantages in convenience and cost compared for high-throughput function determination. Science 2000;289:
with standard ELISA formats. The microarray test format 1760 –3.
can be incorporated in a fully automated random-access 18. Haab BB, Dunham MJ, Brown PO. Protein microarrays for highly
parallel detection and quantitation of specific proteins and anti-
immunoassay platform for routine use in clinical chemis-
bodies in complex solutions. Genome Biol 2001;2:1–13.
try laboratories. Protein microarrays have the potential to
19. Huang RP, Huang R, Fan Y, Lin Y. Simultaneous detection of
improve, in the near future, the diagnosis of infectious multiple cytokines from conditioned media and patient’s sera by
diseases and pathologic conditions such as allergies and an antibody-based protein array system. Anal Biochem 2001;294:
autoimmune diseases. 55– 62.
130 Mezzasoma et al.: Microarrays for Serodiagnosis

20. Wiese R, Belosludtsev Y, Powdrill T, Thompson P, Hogan M.
Simultaneous multianalyte ELISA performed on a microarray plat-
form. Clin Chem 2001;47:1451–7.
21. Wiltshire S, O’Malley S, Lambert J, Kukanskis K, Edgar D,
Kingsmore SF, Schweitzer B. Detection of multiple allergen-
specific IgEs on microarrays by immunoassay with rolling circle
amplification. Clin Chem 2000;46:1990 –3.
22. Schena M, Shalon D, Davis RW, Brown PO. Quantitative monitor-
ing of gene expression patterns with a complementary DNA
microarray. Science 1995;270:467–70.
23. ScanArrayTM and QuantArrayTM operating manuals, Ver. 2.0. Wa-
tertown, MA: GSI Lumonics, 2000.
24. Wood W. “Matrix effects” in immunoassays. Scand J Clin Lab
Invest 1991;51:105–12.