Vol.2, No.1, 32-37 (2010)
Copyright © 2010 Openly accessible at http://www.scirp.org/journal/HEALTH/
Development of a high-throughput cell based 384-well
influenza A quantification assay for interpandemic and
highly pathogenic avian strains
Melicia R. Gainey, Ann M. Wasko, Jennifer N. Garver, David J. Guistino, Eric M. Vela*, John E.
Battelle Biomedical Research Center West Jefferson, OH 43162, USA; velae@battelle.org
Received 25 November 2009; revised 1 December 2009; accepted 2 December 2009.
Influenza remains a world wide health threat,
thus the need for a high-throughput and robust
assay to quantify both seasonal and avian in-
fluenza A strains. Therefore, a 384-well plate
format was developed for the median tissue
culture infectious dose assay (TCID50) utilizing
the detection of nucleoprotein by an in situ en-
zyme linked immunosorbent assay (ELISA)
which was optimized for sensitivity in this assay.
Highly pathogenic avian influenza, A/Vietnam/
1203/04 (H5N1), and interpandemic strains, A/
New Caledonia/20/99 (H1N1) and A/Brisbane/
10/07 (H3N2), were quantified using this high-
throughput assay. Each 384-well plate can be
used to analyze ten viral samples in quadrupli-
cate, eight dilutions per sample, including all
necessary assay controls. The results obtained
from 384-well plates were comparable to tradi-
tional 96-well plates and also demonstrate re-
peatability, intermediate precision, and assay
linearity. Further, the use of 384-well plates in-
creased the throughput of sample analysis and
the precision and accuracy of the resulting titer.
Keywords: Avian Influenza; ELISA;
High-Throughput Assay; Interpandemic Influenza A;
Interpandemic, or seasonal, influenza takes a toll on
public health resources, economic productivity, and
causes up to 500,000 deaths annually world-wide [1].
Highly pathogenic avian influenza (HPAI) strains cause
heavy economic losses in countries dependent upon
poultry revenues and have caused more than 250 deaths
globally [2]. In addition, HPAI strains could potentially
give rise to a pandemic. Given the economic and health
implications of these viral strains, many new vaccines
and therapeutics are being developed to counter this
threat. In order to determine efficacy of these novel
products, it is necessary to quantify antibody responses
and influenza A viruses in various matrices, ideally in a
high-throughput format. Currently, several high throu-
ghput assays to detect influenza virus exist which utilize
reverse transcription polymerase chain reaction (RT-PCR)
alone [3], and coupled with flow cytometery [4] or utilize
a latex turbidimetric immunoassay [5]. There are also a
few high-throughput screening methods to determine
putative antiviral compounds [6] and influenza antago-
nists [7]. Although these methods are able to analyze
many samples for the presence of influenza or screen
potential therapeutics, these assays do not quantify in-
fluenza virus. For high-throughput quantification, a
reverse-phased high performance liquid chromatography
has been used to enumerate the amount of hemagglutinin
in several types of viral stocks [8], but does not provide
an infectious titer. A branched DNA technology can be
used for quantification of influenza A viruses, but this
assay was developed for use as an antiviral assay [9] and
not for viral quantitation. Median tissue culture infectious
dose assays (TCID50 assay) utilizing Alamar blue in
96-well plates have also been described [10]. Analysis by
this assay provides an infectious titer by indirect means;
determining the metabolism of uninfected healthy cells
instead of detecting the presence of viral infection. To
date, 384-well plate assays have been described for the
detection of influenza via RT-PCR [11] and for the
screening of antivirals utilizing Flash plate technology
[12] or cell based luminescence assays [13]. Thus,
384-well plates have not been harnessed to provide as-
sessment of infectious titers of influenza viruses.
As no world wide standard exists for the quantification
of influenza viruses, the World Health Organization’s
(WHO) Manual on Animal Influenza Diagnosis and
Surveillance [14] was chosen as the basis for this work.
The manual recommends that viral samples be analyzed
M. R. Gainey et al. / HEALTH 2 (2010) 32-37
SciRes Copyright © 2010 http://www.scirp.org/journal/HEALTH/Openly accessible at
in quadruplicate by the TCID50 assay in a 96-well mi-
croplate format. These assays may be interpreted by the
visual observation of cytopathic effect (CPE) or the de-
tection of influenza nucleoprotein by an in situ ELISA. To
increase the assay throughput and efficiency, a 384-well
microplate assay with an ELISA readout was developed
based on recommended WHO manual TCID50 procedures.
Titration of HPAI and interpandemic influenza A viruses
in a 384-well microplate was compared to the traditional
96-well format. Based on preliminary qualification data,
the 384-well format measures accurate and precise titers
with less variability and higher assay linearity than the
96-well plates, suggesting this method provides more
reliable titer data. Further, the use of 384-well plates
reduces the time and effort required for analysis to 24
minutes per sample, a reduction of 80%. This assay for-
mat has also been adapted for micro-neutralization assays
and is amenable for use with manual or robotic liquid
2.1. Metabolic Assay
384-well plates were seeded with MDCK cells at 2.5 x
105 or 3.0 x 105 cells/mL and incubated overnight at 37°C
and 5.0% CO2. A 5% (w/v) Tetrazolium salt, 3- [4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) solution was prepared in 1X phosphate buffered
saline (PBS), added to each well and incubated 2 hr at
37°C. The reduced MTT was released from the cells by
adding Solubilization Buffer (50% dimethyl formamide
solution containing 20% w/v sodium dodecyl sulfate) and
incubating for 2 hr at 37°C. The plates were read at 550
nm with a 690 nm reference. All subsequent experiments
utilized a seeding density of 3.0 x 105 cells/mL due to
lower variability, which was analyzed by calculating the
coefficient of variance according to the location on the
2.2. Viruses and Cells
Each viral strain was received from the Centers for Dis-
ease Control (Atlanta, GA) and propagated in 8-10 day
old specific-pathogen free chicken embryos (Charles
River Franklin, CT) at 37°C for 24 hr for A/ Vietnam/
1203/04, 37°C for 72 hr for A/New Caledonia/20/99, and
35°C for 48 hr for A/Brisbane/10/07. The harvested al-
lantoic fluid was stored at -70°C. Madin-Darby canine
kidney (MDCK) cells (Salisbury lineage, Sigma St. Louis,
MO), were maintained in complete Eagle’s minimal es-
sential media (EMEM) containing 1% Penicillin- Strep-
tomycin and 10% fetal bovine serum. MDCK plates
seeded at 70% confluency were purchased from Diag-
nostic Hybrids, Inc. (Athens, OH).
2.3. Viral Quantification
Viral stocks were quantified by inoculating serial dilu-
tions in quintuplicate on 96-well plates or quadruplicate
on 384-well plates onto confluent MDCK monolayers.
TPCK-treated trypsin at 2.0 μg/mL (US Biological
Swampscott, MA) was added to the inoculation media for
the interpandemic strains. Each plate contained cell cul-
ture control (CC) wells containing inoculation media
alone. The inoculated plates were incubated at 37°C and
5.0% CO2 in a humidified incubator for 20 ± 1 hr prior to
2.4. In Situ Influenza A Nucleoprotein ELISA
The ELISA was performed as previously described [14]
with the following noted changes. Plates were fixed by
the addition of 1 ½ volumes of 80% cold acetone in water
directly to the inoculum and incubated at room tempera-
ture (RT) for 30 min. All subsequent incubations were
increased from the recommended RT to 37°C in a hu-
midified incubator to increase the sensitivity of the assay.
For the primary incubation, an equal mixture of mouse
anti-influenza A nucleoprotein monoclonal antibody
clone MAB8257 and MAB8258 (Chemicon International;
Bellirica, MA) was used. The ABTS Microwell Peroxi-
dase Substrate System (Kirkegaard and Perry Laborato-
ries, prepared according to manufacturer’s instructions)
substrate and stop solution was used according to the
manufacturer’s instructions. The plates were read at 405
nm with a 490 nm reference. The positive threshold was
calculated as the average optical density (OD) of the CC
wells plus two standard deviations. Each sample well was
scored as positive for infection if the OD was above the
positive threshold and negative for infection if the OD
was less than or equal to the positive threshold. The me-
dian infectious dose for all samples was calculated using
the Spearman Kärber formula [15].
3.1. Optimization of the 384-Well Plate
To assess the suitability of 384-well plates for a cell-
based infectivity assay, MDCK cells were seeded at two
concentrations and the metabolic activity was determined
by reduction of MTT. The metabolism of cells seeded in
the outer wells was identical to the cells seeded in the
inner wells (see Table 1). The variability was analyzed by
calculating the coefficient of variance (CV) by location
on the plate and found to be less than 13% for inner and
outer wells as well as the entire plate. Therefore, 384-well
plates were found suitable for this assay due to the low
variability demonstrated between inner and outer wells.
Further, seeding the plate at 3 x 105 cells/mL provided
less variability (CV < 8%, see Table 1).
M. R. Gainey et al. / HEALTH 2 (2010) 32-37
SciRes Copyright © 2010 http://www.scirp.org/journal/HEALTH/
Openly accessible at
Table 1. 384-well plate variability measured by metabolism. 384-well plates were seeded with two concentrations of MDCK cells and
metabolism was determined by the reduction of MTT. The delta OD (difference between OD at 550 nm and 690 nm) was averaged for
the outer and inner wells in comparison to the entire plate. The standard deviation (SD) and coefficient of variance (CV) were deter-
mined for each location.
2.5 x 105 cells/mL 3.0 x 105 cells/mL
Outer Wells Inner Wells Entire Plate Outer Wells Inner Wells Entire Plate
Average ΔOD 0.28 0.29 0.29 0.35 0.35 0.35
SD 0.04 0.03 0.03 0.03 0.03 0.03
CV 13% 11% 11% 8% 7% 8%
Table 2. In situ nucleoprotein ELISA threshold calculation. Changes made to the WHO animal influenza diagnosis and surveillance
ELISA threshold obtained titers comparable to visual determination of CPE.
(AVG CC + 2SD)
5.7 5.0 5.6
Different plate Same plate analyzed with different thresholds
The in situ nucleoprotein ELISA was adapted for this
plate format and optimized for sensitivity, efficiency, and
safety (see Materials and Methods 2.4). Removing the
inoculum and washing the monolayer prior to fixation
was not desirable for the 384-well plate due to the in-
volvement of multiple pipetting steps and manipulation
of potentially infectious materials. Thus, the critical step
for making this assay feasible and safe is the addition of
the fixative directly to the inoculated wells. Tests dem-
onstrated no adverse effects to the reported titer or in-
crease in the background of the assay by changing the
fixation step (data not shown). To further optimize the
ELISA, the incubation temperature was raised which
reduced the concentration of conjugate required thus
increasing the sensitivity of the assay. The use of the
ABTS microwell peroxidase substrate system improved
the environmental impact of this assay by eliminating the
waste generated from the o-phenylenediamine dihydro-
chloride (OPD) substrate, which is both toxic to humans
and dangerous to aquatic systems according to the mate-
rial safety data sheet. Use of the ABTS substrate in-
creased the positive signal without significantly increas-
ing the background of negative wells (data not shown).
Unlike OPD, the ABTS substrate does not change color
upon the addition of the stop solution thus eliminating
concerns about the timing of stopping the reaction. Fi-
nally, the positive threshold was defined as the average of
optical density (OD) of the CC wells plus two standard
deviations which provides titers consistent with side by
side CPE readout experiments (Table 2).
Samples and controls are diluted in a standard 96-tube
microtiter box (see Figure 1A). Inoculation of the
384-well plate is efficient and simple when utilizing a
24-channel pipette (see Figure 1B). The content of each
microtiter tube is inoculated into four wells on the
384-well plate. This process can be adapted easily to
ES1-5S2-5S3-5S4-5S5-5S6-5S7-5S8-5S9-5S10-5PC-5 CC
FS1-6S2-6S3-6S4-6S5-6S6-6S7-6S8-6S9-6S10-6PC-6 CC
GS1-7S2-7S3-7S4-7S5-7S6-7S7-7S8-7S9-7S10-7PC-7 CC
HS1-8S2-8S3-8S4-8S5-8S6-8S7-8S8-8S9-8S10-8PC-8 CC
A1111111111 11 111 11 11 11 111
B1111111111 11 111 11 11 11 111
C2222222222 22 222 22 22 22 22 2
D2222222222 22 222 22 22 22 22 2
E3333333333 33 333 33 33 33 333
F3333333333 33 33 333 33 33 33 3
G44444444444 44 44 444 44 44 44
H4444444444 44 444 44 44 44 44 4
I5555555555 55 55 555 55 55 5CCCC
J55555555555 55 55 555 55 55CCCC
K6666666666 66 666 66 66 66 6CCCC
L6666666666 66 66 666 66 66 6CCCC
M7777777777 77 77 777 77 77 7CCCC
N7777777777 77 777 77 77 77 7CCCC
O88888888888 88 88 888 88 88CCCC
P8888888888 88 888 88 88 88 8CCCC
S9S10PC NC/CCS5S6S7S8S1 S2 S3 S4
(a) TITER-BOX. Samples and controls are diluted in a standard 96-tube
microtiter box. Each color represents a different viral sample or control,
thus ten samples (S1 through S10) with a positive control virus (PC),
sample matrix matched negative control (NC), and cell culture control
(CC) are prepared. Eight dilutions of each sample and PC are prepared
with four dilutions for the NC.
(b) 384-WELL PLATE: Each tube is inoculated into four wells in the
384-well plate. The number in each well represents the sample/control
dilution inoculated into that location
Figure 1. 384-well plate set up and layout.
M. R. Gainey et al. / HEALTH 2 (2010) 32-37
SciRes Copyright © 2010 http://www.scirp.org/journal/HEALTH/Openly accessible at
Table 3. Intra-assay and inter-assay variability data for TCID50 assays. The repeatability (intra-assay variability) and intermediate
precision (inter-assay variability) of each plate format was assessed for three strains of influenza A.
(Intra-Assay Variability)1
Intermediate Precision
(Inter-Assay Variability)2
384-well 96-well 384-well 96-well
Expected Titer3
A/Vietnam/1203/04 (H5N1)
A/New Caledonia/20/99 (H1N1)
A/Brisbane/10/07 (H3N2)
1) Value reported is the geometric mean of the titer of three iterations by one operator on one testing day
2) Value reported is the geometric mean of the titer of three iterations by two operators on two testing days
3) The expected titer was based on prior titer certification on 96-well plates by two operators over three testing days using nine randomly selected
aliquots of the stock
robotic or manual liquid handler capabilities and has been
used in our facility.
3.2. Assay Acceptability
To compare the new 384-well plate with the traditional
96-well plate, the repeatability (intra-assay variability)
and intermediate precision (inter-assay variability) were
evaluated (see Table 3) for three influenza strains (H1N1,
H3N2, and H5N1). Use of three different influenza vi-
ruses demonstrated the robustness of the assay for quan-
tification of any influenza A strain. The expected titer of
each strain was established using 96-well plates prior to
the experiment. To establish repeatability and precision,
the mean titer obtained must be within 0.5 log of the ex-
pected titer.
The repeatability was assessed by three iterations of the
same viral lot by one operator on a single day of testing.
The geometric mean and standard deviation were calcu-
lated for each plate type and viral strain (see Table 3). For
the 384-well plates, all mean titers were within 0.4 log of
the expected titer and were deemed repeatable for all three
strains. Further, the standard deviations were less than or
equal to 0.2 log TCID50/mL which demonstrates low
variability regardless of the influenza strain quantified on
the 384-well plate. The 96-well plates produced titers
within 0.6 log of the expected titer, indicating less re-
peatability with this plate format.
The intermediate precision for each assay format and
viral strain was assessed by three iterations of the same
viral lot by two operators on two testing days. The geo-
metric mean and standard deviation of the resulting 12
data points were calculated (see Table 3). All of the mean
titers obtained with 384-well plates were within 0.2 log of
the expected titer, thus establishing the intermediate pre-
cision of the 384-well plate. The 96-well plate demon-
strated less precision with all mean titers falling within 0.6
log of the expected titer. In addition, the standard devia-
tions for the 384-well plates were calculated to be 0.3 log
TCID50/mL or less, compared with 0.8 log TCID50/mL or
less for 96-well plates, indicating lower variability be-
tween assays, operators, and testing days for the high-
throughput plate format. All strains performed as ex-
pected in comparison to titers established using 96-well
plates, thus verifying the robustness of the 384-well plate.
The linearity of an assay measures the ability to obtain
results proportional to the concentration of the sample.
For each assay format, the linearity was assessed by pre-
paring four serial dilutions of the viral stock. Each dilu-
tion was then quantified in each plate type. The predicted
titer for each dilution was calculated based on the dilution
from the certified stock titer. The observed titer was
plotted against the predicted titer and a linear regression
analysis was performed (see Figure 2). For the purposes
of this study, an r2 to 0.85 was considered linear. Al-
though the 96-well plate for both the A/ Vietnam/1203/04
and A/New Caledonia/20/99 strains demonstrated linear-
ity, r2 >0.96, the 384-well plate for both strains demon-
strated greater linearity, r2 >0.99 which suggests the use of
a 384-well plate format provides more reliable data.
However, both 96-well and 384-well plates are expected
to produce results proportional to the concentration of the
sample quantified.
M. R. Gainey et al. / HEALTH 2 (2010) 32-37
SciRes Copyright © 2010 http://www.scirp.org/journal/HEALTH/
Openly accessible at
r2 = 0.9942
r2 = 0.9865
r2 = 0.9959
r2 = 0.9640
Predicted log Titer (TCID50/mL)
Observed log Titer (TCID
A/Vietnam/1203/04 384-wellA/Vietnam/1203/04 96-well
A/New Caledonia/20/99 384-wellA/New Caledonia/20/99 96-well
Four serial dilutions of each strain were prepared and then quantified in each assay format. The observed log titer was compared
with the predicted titer for each dilution. The linear regression for each format was determined. An r2 >0.85 was considered linear
for each assay format.
Figure 2. Assay linearity for influenza A (H5N1 and H1N1).
3.3. Advantage of High-Throughput 384-Well
To compare efficiency of the new 384-well plate format
with the standard 96-well plate, the average time required
to analyze one sample in a TCID50 assay from seeding the
plate to analysis of the data was determined. The average
time for the 96-well assay (plates seeded in house) was 2
hr per sample from start to finish. For the most efficient
384-well assay, commercially available seeded plates
were used, removing the need for cell culture mainte-
nance. An additional advantage of these pre-seeded plates
is that more assays can be conducted each week without
the need of multiple cell lines being maintained for har-
vest. The average time for the analysis of one sample on a
pre-seeded 384-well plate was 0.4 hr. Thus, using pur-
chased 384-well plate increases efficiency of the TCID50
assay by 80%, thereby reducing cost significantly.
HPAI infections in animals cause economic losses in
countries dependent on poultry revenues [16]. Further,
HPAI and interpandemic (seasonal) influenza infections
in humans burden public health resources [17]. A myriad
of vaccines and therapeutics are under development;
however, assays that test influenza virus titers and screen
the efficacy of vaccines and therapeutics cannot be used
to quantify influenza virus and those that do measure
infectious virus, do so by indirect methods [9, 10]. The
TCID50 assay directly quantifies infectious influenza A
viruses by utilizing an in situ nucleoprotein ELISA that
detects the influenza A nucleoprotein in infected mon-
olayers [14]. Therefore, a 384-well plate TCID50 assay
with an ELISA readout was developed and tested for
robustness, accuracy, precision, and reliability. The time
and effort required for sample testing was also deter-
mined. For this study, two plate formats were examined: a
traditional 96-well plate and a high throughput 384-well
plate. To demonstrate the robustness of the assays, three
influenza A strains were quantified: A/ Vietnam/1203/04
(H5N1), A/New Caledonia/20/99 (H1N1), and A/ Bri-
sbane/10/07 (H3N2). All viral strains performed as ex-
pected without modification of the procedure, demon-
strating the robustness of the new 384-well plate format
when compared with the traditional 96-well plate. Addi-
M. R. Gainey et al. / HEALTH 2 (2010) 32-37
SciRes Copyright © 2010 http://www.scirp.org/journal/HEALTH/Openly accessible at
tionally, each assay format was tested for intra- and in-
ter-assay variability to ascertain the precision of the assay
and for linearity to verify the overall performance of the
plate format. Both 96-well and 384-well plate formats
tested were found to be repeatable, precise, and linear.
The 384-well plate appears to be more accurate, precise
and linear than the traditional 96-well plate and demon-
strated less overall variability. Further, the efficiency
afforded by using purchased pre-seeded 384-well plates
was substantial. The increase in efficiency greatly lowers
the cost and time required to obtain an infectious titer for
a variety of viral samples without sacrificing precision.
The 384-well plate format with ELISA readout offers a
high-throughput, more efficient alternative for the de-
termination of infectious influenza A titers in compliance
with WHO recommendations for the assay. The 384-well
plate has also been adapted for automation via liquid
handling, considerably increasing the throughput of
sample analysis in order to determine the efficacy of
novel vaccines and therapeutics.
The authors would like to acknowledge Dr. Hank Lockman and Mr.
Aaron Martin for their assistance in the production of this manuscript. In
addition, the authors would like to thank Drs. Herb Bresler, Catherine
Smith, and Carol Sabourin for providing their support and technical
assistance. This work was funded through the Battelle Independent
Research and Development Program.
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