Equivalence of ELISpot Assays Demonstrated between
Major HIV Network Laboratories
Dilbinder K. Gill1*., Yunda Huang2., Gail L. Levine3, Anna Sambor3, Donald K. Carter4, Alicia Sato2,
Jakub Kopycinski1, Peter Hayes1, Bridget Hahn4, Josephine Birungi5, Tony Tarragona-Fiol1, Hong Wan6,
Mark Randles6, Andrew Raxworthy Cooper1, Aloysius Ssemaganda5, Lorna Clark1, Pontiano Kaleebu5,
Steven G. Self2, Richard Koup7, Blake Wood2, M. Juliana McElrath4, Josephine H. Cox6, John Hural4, Jill
1International AIDS Vaccine Initiative Human Immunology Laboratory, Imperial College, London, United Kingdom, 2Statistical Center for HIV/AIDS Research and
Prevention, Seattle, Washington, United States of America, 3Foundation for the National Institutes of Health, Bethesda, Maryland, United States of America, 4Fred
Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 5Uganda Virus Research Institute, Entebbe, Uganda, 6International AIDS Vaccine
Initiative, New York, New York, United States of America, 7Vaccine Research Center, Bethesda, Maryland, United States of America
Background: The Comprehensive T Cell Vaccine Immune Monitoring Consortium (CTC-VIMC) was created to provide
standardized immunogenicity monitoring services for HIV vaccine trials. The ex vivo interferon-gamma (IFN-c) ELISpot is
used extensively as a primary immunogenicity assay to assess T cell-based vaccine candidates in trials for infectious diseases
and cancer. Two independent, GCLP-accredited central laboratories of CTC-VIMC routinely use their own standard operating
procedures (SOPs) for ELISpot within two major networks of HIV vaccine trials. Studies are imperatively needed to assess the
comparability of ELISpot measurements across laboratories to benefit optimal advancement of vaccine candidates.
Methods: We describe an equivalence study of the two independently qualified IFN-g ELISpot SOPs. The study design, data
collection and subsequent analysis were managed by independent statisticians to avoid subjectivity. The equivalence of
both response rates and positivity calls to a given stimulus was assessed based on pre-specified acceptance criteria derived
from a separate pilot study.
Findings: Detection of positive responses was found to be equivalent between both laboratories. The 95% C.I. on the
difference in response rates, for CMV (21.5%, 1.5%) and CEF (20.4%, 7.8%) responses, were both contained in the pre-
specified equivalence margin of interval [215%, 15%]. The lower bound of the 95% C.I. on the proportion of concordant
positivity calls for CMV (97.2%) and CEF (89.5%) were both greater than the pre-specified margin of 70%. A third CTC-VIMC
central laboratory already using one of the two SOPs also showed comparability when tested in a smaller sub-study.
Interpretation: The described study procedure provides a prototypical example for the comparison of bioanalytical
methods in HIV vaccine and other disease fields. This study also provides valuable and unprecedented information for
future vaccine candidate evaluations on the comparison and pooling of ELISpot results generated by the CTC-VIMC central
Citation: Gill DK, Huang Y, Levine GL, Sambor A, Carter DK, et al. (2010) Equivalence of ELISpot Assays Demonstrated between Major HIV Network
Laboratories. PLoS ONE 5(12): e14330. doi:10.1371/journal.pone.0014330
Editor: Rupert Kaul, University of Toronto, Canada
Received June 11, 2010; Accepted November 22, 2010; Published December 14, 2010
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This work was supported by the Collaboration for AIDS Vaccine Discovery (CAVD) Grant 38650 from the Bill & Melinda Gates Foundation. The Bill &
Melinda Gates Foundation had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
In support of the Global HIV/AIDS Vaccine Enterprise
(GHAVE), the Bill & Melinda Gates Foundation funded the
Collaboration for AIDS Vaccine Discovery (CAVD), an interna-
tional network of 17 Vaccine Discovery Consortia with five Central
Service Facilities (CSF) that provide immunology and statistical
support [1,2,3]. As one of the CSF of the CAVD, the overall goal of
the Comprehensive T Cell Vaccine Immune Monitoring Consor-
tium (CTC-VIMC) is to provide standardized immunogenicity
monitoring services in CAVD and GHAVE sponsored clinical trials
of HIV vaccine candidates. To this end, the CTC-VIMC
established a core of four cellular clinical immunogenicity testing
laboratories, all of which are accredited to good clinical laboratory
practice (GCLP) certification . Core laboratories include the
International AIDS Vaccine Initiative (IAVI) Human Immunology
Laboratory (London, UK), the Uganda Virus Research Institute
(UVRI; Entebbe, Uganda), the HIV Vaccine Trials Network
Laboratory (HVTN; Seattle, US) and NVITAL, core laboratory for
the Vaccine Research Center (Gaithersburg, MD).
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The Enzyme-linked immunosorbent spot (ELISpot) assay is a
commonly used bioanalytical method for monitoring cellular
immune responses in humans and animals. While being a
relatively simple assay, the ELISpot has been shown to be highly
specific, sensitive with good precision and stable over time .
ELISpot assays were originally developed to enumerate B-cells
secreting antigen-specific antibodies , and have since been
widely used as a screening tool to assess the T- cell immunoge-
nicity of, among others, candidate HIV vaccines [5,7,8,9,10,11].
IFN-g secretion, as assessed by the ELISpot, occurs as a result of
the recognition of cognate peptides or mitogenic stimuli by CD4
and/or CD8 T -cells. Secreted IFN-g is captured on IFN-g
antibody-coated membranes and detected through subsequent
recognition by further biotinylated IFN-g-specific antibodies,
which in turn complex with streptavidin-conjugated enzymes that
react with chromogenic substrates. The chromogenic reaction
causes a spot to form where the reacting cells released their IFN-g;
these spot forming units (SFUs) are then enumerated per number
of stimulated Peripheral Blood Mononuclear Cells (PBMC).
Typical stimulants used in such an assay are pools of overlapping
synthetic peptides that correspond to sequences incorporated into
vaccines. These pools consist of 8 to 15meric peptides overlapping
in sequence to ensure maximal coverage of potential CD4 and
Although the principal techniques underlying the assay remain
constant, the use of differing SOPs for the ELISpot assay may
result in variability of enumerated data between laboratories
[12,13]. Within the CTC-VIMC, both IAVI and UVRI core
laboratories use the IAVI IFN-g ELISpot SOP, whereas the
HVTN uses the HVTN IFN-g ELISpot SOP; both SOPs have
been qualified in-house and across collaborating sites and are now
routinely used to assess HIV vaccine candidates [14,15,16,17].
Early plans for the CTC-VIMC were to utilize a commercially
available ELISpot kit for all CAVD ELISpot tests. Unfortunately,
concerns regarding reagent stability mitigated against use of these
kits by the CAVD. Because significant time, effort and financial
resources had been invested by IAVI and HVTN to qualify and
propagate the use of SOPs across their respective laboratory
networks, there was an understandable reluctance from either
laboratory to use an alternative SOP when running specimens for
the CAVD initiative. This study was therefore designed to
generate sufficient statistical evidence to rigorously evaluate the
results of the IAVI and HVTN SOPs for concordance and to
guide the prospective comparison or pooling of ELISpot
immunogenicity assessments across laboratories within the CAVD
initiative. The CAVD Vaccine Immunology Statistical Center
(VISC, Seattle, WA) assisted in the design of this comparison study
and provided unbiased data management and analysis to assess
this objective. The over-arching strategy for the CTC-VIMC assay
comparison study, along with delineation of appropriate follow-up
procedures (e.g., assay transfer and ongoing performance
monitoring) is summarized in the flow chart presented in
Figure 1. Use of this systematic approach resulted in the adoption
of an appropriate study design. With a common set of specimens
and centrally prepared stimuli and controls, the findings from this
evaluation have justified the continued use of the two different
SOPs within the core laboratories of the CTC-VIMC.
Because the ELISpot assay readout is often dichotomized into
positive or negative responses based on pre-specified positivity
criteria, this study focuses on the comparison of the two IFN-c
ELISpot SOPs with respect to the percentage of positive responses
(i.e., response rates) from the tested samples and positivity call of
each individual sample. Acceptance criteria on the equivalence of
response rates and positivity calls were pre-specified prior to data
analysis, thereby avoiding subjectivity of study results and inferred
Furthermore, it was crucial to assess the false positive rates of
the ELISpot assay, in addition to comparing the distribution of
both background (i.e., responses from negative control wells with
no antigen stimulation) and background-subtracted responses from
antigen-stimulated wells. Responses to a Gag peptide pool from
HIV-negative samples were used to assess the false positive rate of
each assay. These assessments not only characterize the properties
of the assay, but can also be used to identify sources of
disagreement, if any, in the assay results.
To inform the design of the inter-laboratory comparison study,
a pilot study using a small set of common samples and peptides
was conducted at the IAVI and HVTN laboratories. Encouraged
by evidence of concordance from data collected from the pilot
study, the inter-laboratory comparison study was then designed
and conducted with the appropriate sample size required to
achieve statistical power in establishing equivalence between the
dichotomized outcomes of the two assays.
Materials and Methods
All specimens provided to the laboratories were anonymized
and could not be traced by to original donors. Donors provided
written informed consent and study protocols had been reviewed
and approved by the appropriate local Institutional Review
Boards: the WP Blood Transfusion Service, Johannesburg, SA;
the Seattle HIV Vaccine Trials Unit, Seattle USA; Duke
University, Durham, USA; and BRT Laboratories, Baltimore
PBMC samples isolated from HIV-1 seronegative individuals
with previously-characterized IFN-c ELISpot responses to CMV
pp65 peptides were selected by VISC to give evenly distributed
ELISpot responses to test the low-mid dynamic range of the assay.
PBMC were provided by IAVI from blood packs obtained from
the South African National Blood Transfusion Services, the
HVTN repository and the CTC-VIMC Proficiency Testing Core
(PTC) PBMC Repository at Duke University Human Vaccine
Institute, and from SeraCare Biosciences, Gaithersburg, MD. At
each of these laboratories PBMC were isolated within eight hours
of collection using Ficoll gradient centrifugation. Upon isolation,
PBMC were frozen in a controlled stepwise manner and stored in
the vapor phase liquid nitrogen. Three of the four laboratories
used freeze media containing 90% FBS and 10% DMSO and
SeraCare used 22% FBS, 7.5% DMSO and 70.5% RPMI (Table
S1). To ensure the cold chain was maintained all freezers were
constantly monitored for fluctuations in temperature. Shipments of
PBMC were conducted using dry shippers (Taylor Wharton,
MVE) allowing samples to be shipped to collaborating laboratories
in vapor phase.
Peptide pools used in the study were: a pool of 32 8–10mer
peptides representing immunodominant CD8+ T-cell epitopes
within cytomegalovirus, Epstein Barr virus and influenza (CEF
; a pool of 138 15-mer peptides overlapping by 11 amino acids
spanning the entire human cytomegalovirus (CMV) pp65 protein
and an IAVI HIV-1 clade A Gag pool (Gag) also comprised of 90
15-mer peptides overlapping by 11 amino acids (Anaspec Inc, San
Jose, CA). Peptides were used at a final concentration of 1.5 mg/
mL. Phytohemagglutinin (PHA; Sigma, Dorset, UK) was used as a
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Figure 1. CTC-VIMC/VISC ELISpot Standardization Methodology.
Equivalence of ELISpot Assays
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positive control at a final concentration of 10 mg/mL and 0.045%
(final concentration) DMSO (v/v) in PBS was used as a Mock
(negative control). Peptides were prepared centrally at 1006final
concentration to ensure that when each site diluted their peptides
to working concentration, a potential source of variation was
IAVI and HVTN independently developed their in-house
ELISpot assays using different cell counters and ELISpot readers.
Furthermore, each required the use of their own SOPs for cell
thawing, counting and ELISpot. Both ELISpot SOPs employed
the same anti-IFN-c capture (1-D1K), biotinylated anti IFN-c
detection (7-B6-1) monoclonal antibodies (Mabtech, Nacka,
Sweden) as well as Immobilon-P membrane ELISpot plates.
Different ELISpot readers were used; HVTN used white plates
(Millipore; MSIPS4W10) compatible with the CTL reader
(Cellular Technologies, Cleveland, Ohio), while IAVI (and UVRI)
used clear plates (Millipore; MAIPS4510) for spot enumeration by
the AID ELISpot reader (AutoImmun Diagnostika, Germany).
IAVI had previously compared the use of pre-coated versus self-
coated ELISpot plates and found no significant difference in
performance between the two . For this study pre-coated
plates were used exclusively by IAVI and self-coated plates were
used by HVTN.
IAVI Method Overview
Mabtech pre-coated IFN-c ELISpot 96-well plates were washed
3 times in 200 ml PBS (Sigma, Dorset, UK) per well prior to
blocking with 200 mL R10 media (RPMI 1640) supplemented with
10% (v/v) foetal bovine serum (FBS) 2 mM L-glutamine, 100 units
penicillin, 0.1 mg/mL streptomycin, 10 mM HEPES buffer and
1 mM sodium pyruvate (all from Sigma) and incubated at 37uC
for at least 2 hours. Cryopreserved PBMC were thawed, washed
and resuspended in 5 mL R20 (R10 with 20% FBS) and incubated
overnight in a humidified incubator at 37uC with 5% CO2in air.
On the day of assay, cells were counted (Vi-cell counter, Beckman
Coulter) and resuspended in R10 at 46106viable cells/mL.
Samples with viability of less than 80% following overnight
incubation were discarded and a fresh vial tested. Blocking R10
media was decanted and 100 mL of peptide (1.5 mg/mL final
concentration), PHA or Mock were added followed by 50 mL of
cells to give a density of 200,000 cells/well. Plates were incubated
as above for 16–24 hours.
Plates were subsequently washed manually, once with 200 mL
0.05% (v/v) PBS/tween, then a further 5 times in 0.05% PBS/
tween using a M384 Atlas automated plate washer (Titertek;
Biological Instrumentation Services Ltd, Kirkham UK). All
subsequent washes were automated. 100 mL of 0.22 mm filtered
biotinylated anti IFN-c 7-B6-1 monoclonal antibody were added
at 1 mg/mL in 0.5% BSA/PBS. Plates were incubated for a
further 2–4 hours at room temperature, washed 6 times with
200 mL per well with 0.05% PBS/tween, and incubated with
100 mL of avidin-biotin peroxidise complex (ABC complex; Vector
labs) for 1 hour at room temperature. Plates were washed 3 times
with 200 mL 0.05% PBS/tween followed by a further 3 washes
with PBS prior to addition of 100 mL 3-Amino-9-ethylcarbazole
(AEC) chromagen (Sigma) for 4 minutes before the reaction was
stopped by rinsing under running tap water. The protective plastic
backing was removed immediately and plates were left to dry
overnight in the dark. SFU were enumerated using an automated
AID ELISpot reader (AutoImmun Diagnostika, Germany). The
pass/fail criteria set by the IAVI protocol states that the Mock
wells should have less than 10 spots per well and those wells
containing only R10 (no cells) should have less than 5 spots per
well. For positive controls (PHA) there should be greater than 10
spots per well. If any of these criteria were not met, then the plate
was failed and repeated.
HVTN Method Overview
The HVTN method is based on the validated Merck ELISpot
assay , 96-well hydrophobic polyvinylidene difluoride-backed
plates (Millipore, Bedford, MA, US) were coated with anti-IFN-c
monoclonal antibody 1-D1K at a concentration of 10 mg/mL in
PBS, overnight at 4uC. On the following day the plates were
washed 4 times with 250 mL of PBS per well. 200 mL of R10
(RPMI supplemented with 10% FBS v/v (Gemini Bio-products),
2 mM L-Glutamine; 25 mM HEPES; 5 units Penicillin strepto-
mycin (all Gibco BRL Life Technologies, Carlsbad, CA, US) were
added to each well and incubated at 37uC, 5% CO2for at least
2 hours. Cryopreserved PBMC were thawed, washed and
resuspended in 5 mL R20 (R10 with 20% FBS) and incubated
overnight in a humidified incubator at 37uC with 5% CO2in air.
On the day of assay set up, cell count and viability were
determined using a Guava Cell Counter. Samples with ,66%
viability were discarded. Cells were re-suspended at 26106cells/
mL. 100 mL of cells (200,000 cells per well) and 25 mL of peptide
(1.5 mg/mL final concentration), Mock or PHA were added and
plates incubated at 37uC, 5% CO2for 18–22 hours. The Mock
normally used by HVTN is R10 (with no DMSO), but for the
purpose of this study blinded stimuli included 0.45% DMSO in
Immediately prior to the end of incubation, biotinylated mouse
anti-human IFN-c 7-B6-1 solution was prepared to 1 mg/mL in
0.5% BSA/PBS diluent. Plates were washed seven times with
250 mL per well of 0.05% PBS/tween using an automated Elx405
plate washer (BIOTEK Instruments Inc, Winooski VT, US) after
which 100 mL of biotinylated antibody were added and plates left
at room temperature for 2–3 hours.
Following 4 washes with 250 mL/well with 0.05% PBS/tween,
100 mL of Alkaline Phosphotase-conjugated anti-biotin antibody
(AP-anti biotin; diluted 1:750 in 0.5% BSA/PBS; Vector
Laboratories, Burlingame, CA, US) were added and incubated
for 2–3 hours at room temperature. Plates were washed 4 times
with 250 ml per well with 0.05% PBS/tween. Finally, 100 mL of
BCIP/NBT (pre-filtered through 47mm Whatman filter paper;
Pierce, Rockford, IL, US) were added for 7 minutes before the
reaction was stopped by rinsing the plate three times with 250 mL
per well of deionised water. The blue colored spots formed by
IFN-c -secreting cells were counted with an automated CTL
ImmunoSpot plate reader (Cellular Technologies, Cleveland,
The pass/fail criteria set by the HVTN protocol states that the
average of the Mock wells should have less than 20 spots per well
and those wells containing only R10 (no cells) should have an
average of less than 6 spots. For positive controls (PHA) there
should be greater than 400 spots per well. If any of these criteria
are not met, then the plate is deemed to have failed and is
Potentially significant differences in method are detailed in
Existing data were not available to help to design an efficient
design for the proposed inter-laboratory equivalence study. We
therefore conducted a pilot study using centrally prepared peptide
pools and 30 specimen samples selected by VISC based on
background-subtracted responses to CMV in the range of 0–1500
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SFU /10‘6 PBMC. Both IAVI and HVTN measured responses of
these 30 specimens to CMV, CEF and Gag and repeated the assay
twice within their own laboratories. Data collected were used to
estimate parameters needed for the design of the inter-laboratory
comparison study, such as the overall response rate, the difference
in response rates and the proportion of positivity calls. Data from
each laboratory were selected to estimate inter-laboratory
difference and duplicates within each laboratory were used to
estimate the intra-laboratory difference and served as a basic
assumption in deriving acceptance criteria of equivalence for the
inter-laboratory comparison. Based on responses to CMV, CEF
and Gag from the pilot study, a total of 155 samples from the
specimen repositories were randomly selected for testing in the
inter-laboratory comparison study. As data derived from the 30
pilot study samples were used to derive the acceptance criteria of
equivalence, these were re-tested in the inter-laboratory compar-
ison study to avoid the risk of over-estimating the concordance
measures. Antigen responses to CMV, CEF and Gag were
assessed separately in these evaluations.
A specific plate layout was adopted by both laboratories in order
to reduce the impact of plate layouts on the comparison and to
fulfil pass/fail and quality control criteria for both laboratories. To
this end, antigen and control wells were plated in triplicate with
two rows of triplicate Mock wells to incorporate IAVI’s
quadruplicate pass/fail criteria. For robust assessment of technique
in each laboratory, all stimuli and pre-characterized PBMC were
blinded. One set of instructions designed to complement both
SOPs was included to ensure that plate layouts and procedures
were carried out in a specified manner.
Considering the extensive experience of the UVRI laboratory
with the IAVI SOP , the inter-laboratory reproducibility of the
ELISpot assay between IAVI, HVTN and UVRI was assessed in a
smaller sub-study which included 28 of the original 30 samples
from the pilot.
Unreliable plates/samples from each lab were filtered out based
on each lab’s own pass/fail criteria. An assay result from the IAVI
lab was included in the analysis if the mean response from Mock
was ,55/million PBMC. A result from the HVTN lab was
included if the mean response from Mock was #100/million
PBMC, the mean response from positive control wells was
$2,000/million PBMC, and the variance of the three replicates
divided by (median+1) was ,25.
The proportion of responders (i.e., response rate) was
determined based on each lab’s own positivity criteria. An IAVI
sample was positive for a given antigen if the mean response was
.46background (or greater than 0 if the background was 0), the
coefficient of variation across the wells was ,70%, and the
background subtracted SFU was .38 SFU per million PBMC.
The HVTN adopts positivity criteria described by Moodie et al.
2006 . Because responses were examined separately, no
multiplicity adjustment for multiple antigens was made in
determining the positivity of responses to CMV, CEF or Gag.
The adjusted Wald interval for difference of proportions with
matched pairs  was used to establish equivalence based on the
95% confidence interval (CI) between the difference in response
rates being contained in the 215% and 15% interval. To evaluate
the proportion of concordant positive responses, score-based CIs
for proportions were employed. Equivalence was established if the
lower bound of the 95% confidence interval on the proportion of
concordant positive responses was greater than or equal to 70%.
Comparison of the background and background-subtracted
responses were displayed with boxplots where the box indicates
the median and interquartile range; whiskers extend to the furthest
point within 1.5 times the interquartile range from the upper or
lower quartile. Wilcoxon signed rank tests were used to compare
the rank ordering of the responses. The Concordance Correlation
Coefficient (CCC; ) was calculated to assess agreement and to
Table 1. Differences between SOPs.
IAVI SOPHVTN SOP
Plate PreparationPlates pre-coated with primary antibody MABTECH 1-DIKSelf-coated with primary antibody MABTECH 1-DIK
*PBMC CountingVi-CellGuava PCA
PBMC ConcentrationAdded 50 mL of 4.06106/mL (200,000/well final)Added 100 mL of 2m PBMC/mL (200,000/well final)
Spot enumerationAID Reader CTL reader
Pass CriteriaPHA control, .10 spots per well; Mock negative control,
,10 spots per well R10/CEF only ,5 spots per well
PHA control, mean of 3 wells .400 spots per well, Mock
negative control ,20 spots per well R10 only wells,
average of ,6 spots per well
*Median cell recovery 74% (93% viable) at HVTN and 77% (95% viable) at IAVI.
Table 2. Statistics achieved in the pilot study.
Observed differences in response rates (95% C.I.)
95% lower bound of the observed proportion of concordant
StimuliInter-lab Intra–lab 1Intra–lab 2Inter-labIntra–lab 1Intra–lab 2
CMV0% (6%,6%) 0% (6%, 6%)3.3% (12%, 5%) 89%89%83%
CEF 0% (14%, 14%)0% (11%, 11%) 6.7% (24%, 5%)70%79%66%
Gag 0% (6%, 6%)0% (6%, 6%) 0% (6%, 6%)89% 83% 89%
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identify any sources of disagreement of background and
background-subtracted responses between results from different
laboratories. The CCC is a combined measure of precision and
accuracy that measures deviation from the 45-degree identity line.
A concordance coefficient with value of 1 indicates a perfect
agreement, 21 indicates a perfect disagreement, and 0 indicates
Raw data from both laboratories were submitted to VISC via a
secure web upload to the Atlas Portal (https://atlas.scharp.org).
Pilot Study and Design of the Inter-Laboratory
Data from the pilot study showed that inter-lab differences in
response rates were 0% for both CMV and CEF responses while
the width of the 95% CI varied due to different numbers of
samples being filtered for each stimulus. Intra-lab differences of the
response rates between duplicate runs were in the range of 0% to
Table 3. Results on the comparison of response rates and positivity calls in the inter-laboratory comparison study.
Antigen IAVI response rateHVTN response rateDifference in response rate (95% CI) Concordance (LB 95% CI)
CMV 98/146=67.1% (59.1%,
0/131=0.0% (21.5%, 1.5%)131/131=100% (97.2%, 100%)
5/132=3.8% (20.4%, 7.8%) 125/132=94.7% (89.5%, 97.4%)
Gag 6/146=4.1% (1.9%, 8.7%) 7/137=5.1% (2.5%, 10.2%)3/132=2.3% (21.9%, 6.4%)125/132=94.7% (89.5%, 97.4%)
Figure 2. Boxplots of net IFN-g ELISpot responses from the inter-laboratory comparison study. Boxes represent the inter-quartile range
of 25–75thpercentiles, the whiskers extend to the most extreme data point which is no more than 1.5 times the inter-quartile range from the box. The
color of the dots indicate the positivity of the actual responses (red for positive and black for negative) determined by the positivity criteria described
in section 4.5. SFU=spot forming units.
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6.7% with the width of the 95% CI all being smaller than 30%.
The 95% lower bound of the observed proportion of concordant
pairs was in the range of 70% to 89% for inter-lab differences and
66% to 89% for intra-lab differences. See Table 2 for details. In
addition, response rates of the 30 samples from the pilot were
observed to be in the upper range of 73% to 83% (data not
shown). These statistics observed in the pilot study served as a
reference for the true values of the parameters in the sample size
calculations and a gauge in determining the pre-specified
acceptance criteria of equivalence for the inter-laboratory
Given the preliminary evidence of comparability between the
two SOPs above, the design of the inter-laboratory comparison
study proceeded. The acceptance criteria on the difference in the
response rates and the proportion of concordant calls were set as
the 95% CI being within the interval of [215%, 15%] and the
95% confidence limit being greater than 70%, respectively. Based
on these acceptance criteria, power calculations were conducted
assuming the true response rate of 70% and 80% with sample sizes
of 90, 120 and 150. 5000 datasets were simulated to assess the
empirical statistical power with both acceptance criteria satisfied
with a type I error rate of 0.05. With a sample size of 150,
reasonable power (,80%) can be achieved if the true proportion
of concordance is at least 90% when the true difference in
response rates is no more than 6%. In total, 155 samples were
selected for the inter-laboratory comparison study allowing for a
3% possible assay failure rate as observed in the pilot study.
Data from the inter-laboratory comparison study demonstrated
that both response rates and positivity calls passed the pre-
specified acceptance criteria for equivalence (Table 3). Specifically,
for CMV responses, the 95% C.I. on the difference in response
rates was (21.5%, 1.5%) and the lower bound of the 95% score
confidence interval on the proportion of concordant positivity calls
was 97.2%. For CEF responses, the 95% C.I. on the difference in
response rates was (20.4%, 7.8%); the lower bound of the 95%
score confidence interval on the proportion of concordant
positivity calls was 89.5%. The 95% C.I. on the difference in
response rates was contained in the pre-specified equivalence
interval of [215%, 15%] for both CMV and CEF responses; the
lower bound of the 95% confidence interval on the proportion of
concordant positivity calls was also greater than the pre-specified
equivalence margin of 70%. Note that not all 155 samples
contributed evaluable data: based on pre-specified filtering
criteria, 5 CMV responses, 5 CEF responses and 5 HIV A Gag
responses were excluded in the IAVI dataset; 15 CMV responses,
14 CEF responses and 14 Gag responses were excluded in the
HVTN dataset. Consequently, the numbers of evaluable samples
for each antigen from each lab are smaller than the total number
of samples (n=155) that were tested at each laboratory.
This study demonstrates the ability of two central laboratories,
IAVI and HVTN, using their respective validated IFN-c ELISpot
SOPs, to produce highly comparable results. As detailed in
Table 3, the response rates from the two labs were: 98/146
(67.1%) vs. 87/136 (64%) for CMV and 118/146 (80.8%) vs.
114/137 (83.2%) for CEF (IAVI and HVTN respectively). In
addition, very low false positive rates assessed on Gag were
achieved in both laboratories with 6/146 (4.1%) from the IAVI
laboratory and 7/137 (5.1%) from the HVTN laboratory.
Secondary comparisons on the quantitative responses show that
magnitudes of the raw CMV and CEF response were similar
between the two laboratories. As shown in Figure 2, the median
CMV responses were 542 SFUs/106PBMC (range: 0 to 5193)
and 513 SFUs/106PBMC (range: 3 to 4000) for IAVI and
HVTN respectively; the median CEF responses were 423 SFUs/
Figure 3. Scatter plot of all net CMV IFN-g ELISpot responses between the IAVI & HVTN. The dotted line is an identity line indicating
perfect concordance. The CCC’s were calculated based on methods referred in section 4.5. SFU=spot forming units.
Equivalence of ELISpot Assays
PLoS ONE | www.plosone.org7December 2010 | Volume 5 | Issue 12 | e14330
106PBMC (range: 0 to 5713) and 646 SFUs/106PBMC (range:
15 to 4000) for IAVI and HVTN respectively. In addition, the
rank ordering of the net responses were tested comparable
between the two laboratories for CMV and CEF (p=0.74 and
0.93, respectively). Overall, magnitudes of the net CMV and CEF
responses between IAVI and HVTN also showed high concor-
dance with CCC of 0.95 for all responses and CCC of 0.8 for
positive responders; Figure 3).
similarity of responses between the IAVI and UVRI had been
previously established through proficiency testing (15), the
availability replicate samples from the pilot study enabled a
comparison of data generated by UVRI to that from IAVI and
HVTN. Due to the small sample size of this comparison, no
formal hypotheses testing procedures were carried out for these
data. Nevertheless, the observed response rates and response
magnitudes across thethree
comparability. Specifically, the response rates to CMV, CEF
and Gag between the three laboratories were; for CMV 80%, 80%
and 79%; for CEF 83%, 83%, 79%; for Gag 0%, 0% and 3% (for
labs showed encouraging
Figure 4. Boxplots of net CMV IFN-g ELISpot responses from the 30 pilot study samples measured in duplicates within each of the 3
laboratories. Inter- and intra-laboratory analysis was performed between the UVRI, IAVI Core laboratory and HVTN laboratory results. 28 of the 30
pilot study samples were also tested by UVRI. Boxes represent the interquartile range of 25–75thpercentile; bars represent the 95thpercentile of IFN-g
ELISpot responses. SFU=spot forming units.
Equivalence of ELISpot Assays
PLoS ONE | www.plosone.org8December 2010 | Volume 5 | Issue 12 | e14330
HVTN, IAVI and UVRI, respectively). In addition, Figure 4
shows boxplots of net responses to CMV and CEF for 30 samples
(28 of 30 for UVRI) tested on two runs each at HVTN, IAVI and
UVRI laboratories. Responses to Gag were all below 50 SFUs/106
PBMC (data not shown). The concordance correlation coefficients
between IAVI and UVRI for CMV and CEF are 0.95 and 0.8,
respectively (data not shown).
The IFN-c ELISpot assay has been used routinely to evaluate
immune responses to vaccine candidates for HIV, other
infectious diseases and cancer. It is often used as a first line-
screening assay due to speed, ease of use, sensitivity and cost
effectiveness. Indeed, the increased use of automated ELISpot
readers  and plate washers have allowed the semi-
automated quantification of antigen-specific cells . With
achievement of the pre-specified criteria, this study has
determined that different SOPs developed for the same assay
can yield comparable results. Moreover, what were considered
major differences in methodology did not affect the overall
sensitivity of the assay. It was also speculated at the study’s
outset that the two cell counters might be a source of discordant
responses, but cell recoveries and viabilities were comparable
for both laboratories.
This comparability was indeed a fortuitous outcome; had
equivalence not been demonstrated, the CTC-VIMC would have
adopted one SOP for use by the four core laboratories when
performing immunogenicity assays for the CAVD. Further, these
positive findings have supported an assessment of the compara-
bility of ELISpot results from a third central laboratory (UVRI),
follow-up transfer of one of the SOPs to a fourth central laboratory
(NVITAL), and the implementation of a longitudinal performance
monitoring program. Previous studies have raised concerns
regarding variability in ELISpot assays between laboratories
. However, when transferred using stringent training pro-
grams, in addition to standardized equipment and reagents, assay
reproducibility has been demonstrated across multiple GCLP
accredited laboratories [14,15,17,24] supporting the role of core
Contributions of this study are three-fold: first, a prototypic
example is provided of the process needed to conduct similar types
of studies to evaluate other bioanalytical methods in the fields of
vaccines and other diseases; second, the paired-data collected in
such a comparison study provide information on appropriate
calibration factors to apply to future studies when IFN-c ELISpot
assay results assessing the same vaccine product from the two labs
are pooled; and lastly rigorous statistical evidence shows that the
dichotomized IFN-c ELISpot assay data generated from the IAVI
and HVTN laboratories are comparable and assures possible
comparisons of results on independent samples between these two
major HIV network laboratories.
Combined with data from UVRI, our study supports the
transfer of these qualified SOPs across a laboratory network and
has enabled the fourth CTC-VIMC core laboratory, NVITAL, to
select and complete a rigorous technology transfer of one of the
In implementing the GHAVE model, the CAVD forged new
collaborations to leverage of the expertise of scientists devoted to
HIV/AIDS vaccine research. This was especially true for the
central laboratories of the CTC-VIMC whose members represent
major stakeholders in HIV vaccine development: IAVI, HVTN
and NIAID. With resources provided to standardize T cell assays
for CAVD and GHAVE sponsored trials, our core clinical
laboratories were given an unprecedented opportunity to
objectively assess the equivalence of their respective ELISpot
assays. What began as an effort to identify a single standardized
assay for monitoring CAVD sponsored vaccine has already
yielded greater impact on the HIV/AIDS vaccine field. It is now
established that the results of studies with ELISpot measures
performed by the IAVI or HVTN networks are suitable for
comparison, and that rational conclusions can be based on such
The CTC-VIMC central repository was able to supply PBMC
obtained in sufficiently large numbers through leukapheresis. The
importance of this resource can not be over emphasized: the
availability of replicate samples allowed two and three way inter-
laboratory comparisons, as well as subsequent technology transfer
of the ELISpot assay to the fourth CTC-VIMC core laboratory.
After an inter-lab assay comparability study is concluded and
formal assay transfers have been completed, regular monitoring of
the performance and robustness of the assay is recommended (See
Figure 1). The PBMC supplied by the CTC-VIMC repository has
also permitted implementation of a quality assessment program to
monitor ELISpot performance over time using common reagents
and a panel of specimens. Such a program of follow-up testing is
designed to rapidly detect any performance deviations if they
occur [13,15,25]. Now completing its fourth quarterly assessment,
the CTC-VIMC’s monitoring program has generated data that
demonstrate consistent performance of the ELISpot assay
overtime in all four central laboratories.
While HIV researchers have the definite advantages of a single
disease focus, a somewhat common set of reagents (HIV peptides),
and adequate funding, this study offers hope to other researchers
who rely on the IFNc ELISpot that in GCLP laboratories, sources
of variability can in fact be controlled and systematically evaluated
. Other disease specific collaborations can benefit by
incorporating similar design, methods and material considerations
as they approach standardising their T cell immune monitoring
Found at: doi:10.1371/journal.pone.0014330.s001 (0.03 MB
Details of PBMC processing and subsequent viability
We gratefully acknowledge Ambrosia Garcia-Louzao, Project Manager for
the CTC-VIMC sponsored PBMC Repository at the Duke University
Human Vaccine Institute, for coordinating specimen shipments. We would
also like to thank Patricia D’Souza at the DAIDS, NIH for providing
additional PBMC specimens from SeraCare.
Conceived and designed the experiments: DKG YH RAK JG. Performed
the experiments: ARC AS. Analyzed the data: DKG YH AS MJM JH.
Contributed reagents/materials/analysis tools: DKG PJH JC JG. Wrote
the paper: DKG YH GL JTK JC. Scientific Program Manager FNIH
CTC-VIMC: GL. Program Manager FNIH: AS. HVTN Laboratory
Manager: DC. Principle Scientist IAVI HIL: PJH. HVTN QC Manager:
BH. UVRI laboratory Manager: JB. IAVI HIL Program manager: TT-F.
Program Manager IAVI: HW. IAVI HIL QA Manager: MR. PI of UVRI
Laboratory: PK. PI SCHARP: SS. PI CTC-VIMC: RAK. SCHARP
Program manager: BW. PI HVTN: MJM. PI IAVI HIL: JG.
Equivalence of ELISpot Assays
PLoS ONE | www.plosone.org9December 2010 | Volume 5 | Issue 12 | e14330
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