BACKGROUND
human cytomegalovirus  is endemic in all human populations, with a seroprevalence ranging from  <dig> to 100% depending on age, gender and location  <cit> .
in healthy individuals, cmv replication is efficiently controlled by cell-mediated immunity .
in immunocompromized individuals however, the reduced frequency and functionality of cmv-reactive effector cells is associated with severe clinical complications due to uncontrolled virus replication .
quantitative assessment of functional cmv-reactive effector cells in immunocompromized individuals might help to identify patients at increased risk for cmv-mediated clinical complications and to adjust antiviral and immunosuppressive therapy  <cit> .
reliable monitoring of cmv-specific cmi in immunocompromized individuals, such as solid-organ or allogeneic stem cell transplant recipients, requires a specific, standardized but also highly sensitive assay capable of detecting low numbers of cmv-reactive cells.
such sensitivity might be achieved by using highly immunogenic stimulants and via the reactivation of a broad spectrum of physiological effector cells involved in the protection against cmv replication in vivo, notably cd4+ , cd8+ , but also natural killer  and natural killer t  cells .
current diagnostic methods to detect and monitor cmv-specific cmi are mostly based on the restimulation of cd4+ and/or cd8+ effector cells with pools of overlapping peptides, cocktails of preselected immunodominant cmv-peptides or lysates of cmv-infected cells and the subsequent measurement of induced cytokine production  or cell proliferation, by flow cytometry, enzyme-linked immunosorbent assay  or enzyme-linked immunospot assay .
a different approach consists in the direct staining with cmv-peptide-loaded multimers and enumeration of cmv-specific cd8+ t cells by flow cytometry.
however, this method is lacking a functional readout and is restricted to certain hla types, impeding its use in routine diagnostics.
elisa-based assays, such as quantiferon®-cmv are advantageous in that they require low blood volumes and are easy to perform  <cit> .
however, they are restricted to the detection of ifn-γ-producing cd8+ t cells and do not allow single-cell-level measurement.
due to analyte dilution, elisa-based assays usually result in reduced sensitivity  <cit>  and often yield indeterminate results, especially in immunocompromized patients .
intracellular cytokine staining  and subsequent flow cytometric analysis is usually more sensitive than elisa  <cit>  and allows the assessment of both the functionality and the phenotype of cmv-reactivated cytokine-producing cells.
however, this method is difficult to standardize and only detects the intracellular analyte, not the biologically active cytokine secreted over the stimulation period.
elispot-based assays identify and enumerate biologically active, cytokine-secreting cells from isolated peripheral blood mononuclear cells , at the single-cell level both qualitatively and quantitatively  <cit> .
elispot represents the most sensitive read-out system and thus is most appropriate for the detection of low-level responses  <cit> .
in particular, elispot is more sensitive than ics for the detection of antigen-specific cells, such as in vitro-reactivated memory t cells, which produce only low amounts of cytokines  <cit> .
elispot was recently successfully employed over elisa in the detection of congenital cmv infection .
conventional cmv-specific elispot assays are based on the detection of ifn-γ-producing cd4+ and/or cd8+ t cells, depending on the antigens used for pbmc stimulation  <cit> .
the careful selection and formulation of antigens is crucial to ensure specificity, sensitivity and thus a diagnostic value to the assay.
we have previously shown that urea-formulated recombinant  proteins are processed by the exogenous and endogenous antigen processing machinery, resulting in the presentation of naturally generated peptides by mhc class ii and class i molecules  <cit> .
stimulation of pbmc by t-activated® proteins thus mimics more closely a natural infection, resulting not only in the specific activation of memory t  and nk cells, but also possibly in the bystander activation of nk and nkt-like cells present in the pbmc population  <cit> .
we describe here the establishment, optimization and standardization of a highly sensitive elispot assay that takes advantage of both the ifn-γ elispot readout and the immunodominant and highly immunogenic t-activated® ie- <dig> and pp <dig> cmv proteins as stimulants.
we also compared the performance of the optimized assay to that of intracellular staining and flow cytometry in healthy cmv-seropositive donors, and demonstrated the ability of t-activated® ie- <dig> and pp <dig> antigens to activate a broad spectrum of cmv-reactive cells, including cd3+cd4+ , cd3+cd8+ , cd3−cd56+  and cd3+cd56+  cells.
therefore, the combined use of t-activated® ie- <dig> and pp <dig> cmv proteins with our optimized ifn-γ elispot defines a highly sensitive assay meeting all conditions for a reliable and standardized immune monitoring diagnostic tool.
methods
proteins, elispot plate precoating and detection conjugate
urea-formulated t-activated® proteins were prepared as previously described  <cit> .
the t-activated® immunodominant region of cmv pp <dig>  was provided by mikrogen .
full length ie- <dig>  was kindly provided by christina paulus and michael nevels .
t-activation® of ie- <dig> was performed by lophius biosciences.
staphylococcal enterotoxin b  and phytohemagglutinin  were used as positive controls for pbmc stimulation.
96-well elispot plates  and 8-well elispot strips  each pre-coated with anti-human-ifn-γ mab 1-d1k  as well as the detection conjugate mab-ap   coupled with alkaline phosphatase  were obtained from microcoat biotechnologie gmbh.
blood collection and pbmc preparation
blood samples were collected in lithium heparin tubes  from healthy individuals with known cmv serostatus by venipuncture and stored for up to 8 hours at room temperature  until further processing.
isolation of peripheral blood mononuclear cells  was performed using standard ficoll-paque density centrifugation as specified by the manufacturer .
pbmc were finally suspended in serum-free aim-v® medium  and counted either manually in a neubauer’s chamber or using the hem-o-test  <dig> cell-counting device .
automated cell counting was performed in the whole blood venous mode of the calibrated analyzer.
elispot assay
ifn-γ elispot assays were performed as previously described  <cit>  unless specified otherwise in the results section.
briefly,  <dig> ×  <dig> freshly isolated pbmc were plated in four replicates into 96-well elispot plates or 8-well elispot strips precoated with anti-human-ifn-γ mab 1-d1k  and stimulated for 19 h at 37 °c under 5% co <dig> with either 3 μg/ml t-activated® pp <dig> antigen or 15 μg/ml t-activated® ie- <dig> protein.
as unstimulated control , cells were incubated for 19 h in cell culture medium.
after cell removal, plates were developed for 2 h at room temperature  in the presence of  <dig>  u/ml ifn-γ-specific alkaline phosphatase-coupled mab 7-b6- <dig>  spot detection was performed following incubation for 6 min in the dark with a 1-step nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate substrate .
ifn-γ-specific spot-forming cells  were counted using a bioreader®  <dig> eα .
of note, comparable results were obtained on two other readers .
intracellular cytokine staining
intracellular cytokine staining was performed as previously described  <cit> , with the following modifications.
pbmc from six healthy cmv-seropositive donors were isolated and four replicates each were stimulated for 6 h at 37 °c and 5% co <dig> with 3 μg/ml t-activated® pp <dig> antigen or with 15 μg/ml t-activated® ie- <dig> protein in the presence of co-stimulatory anti-cd <dig> and anti-cd49d monoclonal antibodies .
the unstimulated pbmc control cells were incubated with co-stimulatory anti-cd <dig> and anti-cd49d molecules only.
stimulation with seb and anti-cd28 + anti-cd49d served as positive control.
after the first two hours of incubation, 1 μg/ml brefeldin a  was added to prevent cytokine secretion from activated cells.
surface markers  were stained for 30 min at 4 °c using the following conjugated antibodies : anti-cd <dig> apc-cy <dig>  anti-cd <dig> brilliant violet  <dig>  anti-cd <dig> fitc, anti-cd <dig> percp.
cells were fixed and permeabilized for 30 min at 4 °c in bd cytofix/cytoperm .
intracellular staining of ifn-γ was performed using anti-ifn-γ apc  in bd perm/wash buffer  for 45 min at 4 °c.
samples were analyzed on a bd facscanto™ ii flow cytometer .
live-gating of cells was performed during acquisition.
mean  number of event acquisition was  <dig>  .
results were reported as percentage of the gated population producing ifn-γ  and as the number of ifn-γ+ cells/ <dig>  lymphocytes .
serology
the cmv serological testing of blood donors was performed using the fully automated cmv immunoglobulin m  and igg tests on the architect instrument  or the bep® iii system  by the diagnostics department of the institute for medical microbiology and hygiene .
cmv igg-serology was used as primary reference measurement procedure .
statistics
statistics applied to assay development was performed using sigmaplot version  <dig>  and graphpad prism  <dig> .
at the start of this study, a positive elispot test result was defined by a statistically significant difference between quadruplicate sfc values of non-stimulated and specifically stimulated approaches of at least one of the two antigens used, calculated using the mann–whitney u-test  .
two-sided exact p values are reported.
p values <  <dig>  were considered statistically significant.
statistical analyses to assess variations between two or more settings were performed by generating arithmetic means for all replicates of each given setting.
those means were grouped to generate an overall arithmetic mean and standard deviation.
the coefficient of variation in %  was calculated in microsoft excel.
the curve fitting for sigmoidal curves of the titration experiment  was performed using the four parameter logistic function of graphpad prism  <dig> .
for the assessment of the elispot assay sensitivity in seropositive healthy blood donor collectives, the non-parametric mann–whitney u-test  was performed to determine if a significant difference exists between the groups of negative control and stimulation quadruplicates.
multiple-group comparisons were performed using a non-parametric one-way anova  with dunn’s multiple comparison post test.
sfc values were depicted using graphpad prism  <dig>  either as tukey box plots showing median values , interquartile ranges , lower and upper whiskers  and outliers , or as histograms.
for assay validation of optimized elispot, positivity cut-off was calculated on ifn-γ elispot results obtained from a collective of  <dig> healthy donors using sas  <dig>  software and vfp  version  <dig> .fig.
<dig> determination of optimal concentrations of t-activated® pp <dig> and ie- <dig> for the stimulation of pbmc.
pbmc were isolated from one cmv-seropositive healthy donor blood sample and stimulated for 19 h with increasing concentrations of t-activated® pp <dig>  or ie- <dig> .
ifn-γ elispot results of four replicates are expressed as mean sfc/ <dig>  pbmc.
error bars represent standard deviations.
the y-axis scale was adjusted to pp65-  and ie-1-  specific values to optimize data resolution.
curve fitting was made using the four parameter logistic function of graphpad prism  <dig>
RESULTS
ifn-γ elispot assay optimization following pbmc stimulation with t-activated® ie- <dig> and pp <dig> cmv antigens
freshly isolated pbmc were used for the elispot assay.
to prevent loss in t cell functionality, for instance due to activated granulocytes  <cit> , heparinized blood samples were processed with no further additives within 8 h. a total number of  <dig> ×  <dig> pbmc per well was chosen for the development of the elispot assay protocol as this cell count is below confluency and can usually be obtained from samples of less than 15 ml whole blood.
the cmv immediate-early protein ie- <dig> and the late tegument protein pp <dig> represent well-characterized immunodominant t cell antigens  <cit> .
full-length ie- <dig> and a  <dig> amino-acid c-terminal fragment of pp <dig> were produced and formulated in the presence of urea  to increase their stimulatory capacity for different types of cmv-reactive effector cells of cell-mediated immunity  <cit> .
optimal t-activated® antigen concentration was first determined by performing dose–response experiments.
freshly isolated pbmc of one healthy cmv-seropositive donor were stimulated with  <dig>  fg/ml to  <dig>  μg/ml t-activated® pp <dig> or with  <dig>  to  <dig>  μg/ml t-activated® ie- <dig>  and the number of ifn-γ secreting cells was determined by ifn-γ elispot.
t-activated® pp <dig> revealed a much stronger capacity to stimulate ifn-γ secreting effector cells than t-activated® ie- <dig>  reaching a plateau of responsiveness between  <dig>  and  <dig>  ng/ml pp <dig> vs. approximately  <dig>  μg/ml for ie- <dig> .
accordingly, t-activated® antigen concentrations of 3 μg/ml pp <dig> and 15 μg/ml ie- <dig> were selected for further pbmc stimulations and elispot assays.
assay sensitivity and specificity were determined by stimulating pbmc isolated from  <dig> each cmv-seropositive and cmv-seronegative healthy donors with the defined pp <dig> and ie-1 t-activated® antigen concentrations.
the number of reactive effector cells was quantified by ifn-γ elispot.
significant stimulation was defined using a mann–whitney u-test as a statistically significant difference between sfc values of non-stimulated and cmv antigen-stimulated conditions .
t-activated® pp <dig> and ie- <dig> induced a significant activation of responsive effector cells in  <dig> out of  <dig> and  <dig> out of  <dig> pbmc preparations from individual cmv-seropositive donors, respectively .
in this collective, t-activated® pp <dig> showed an overall greater capacity to activate responsive cells with a median of  <dig> sfc/ <dig>  pbmc , compared to t-activated® ie- <dig> with a median of  <dig> sfc/ <dig>  pbmc .
nevertheless, substantial response of up to  <dig> sfc/ <dig>  pbmc was detected in response to t-activated® ie- <dig> in individual samples of cmv-seropositive donors .
all  <dig> pbmc samples  from different cmv-seronegative donors showed negative test results after stimulation with pp <dig> , while  <dig> out of  <dig>  pbmc samples from cmv-seronegative individuals were negative after stimulation with ie- <dig> .
spot count within cmv-seronegative ie-1-stimulated pbmc was higher than in cmv-seronegative pp65-stimulated pbmc but did not exceed  <dig> sfc/ <dig>  pbmc .fig.
<dig> determination of assay sensitivity and specificity.
pbmc from  <dig> each cmv-seropositive and cmv-negative healthy blood donors were left unstimulated  or were stimulated with t-activated® pp <dig> or ie- <dig> and ifn-γ elispot assays were conducted as before.
mean sfc values per  <dig>  pbmc of  <dig> replicates are shown as box plots.
median values  are indicated in brackets.
y-axis scales were adjusted in each graph for better resolution of sfc counts.
median age and range of cmv-seronegative and cmv-seropositive subjects was  <dig>  and  <dig>  years.
gender distribution in cmv-seronegative  and cmv-seropositive  groups was comparable.
differences between unstimulated and stimulated conditions were tested using the non-parametric two-sided mann–whitney u  test.
p-values <  <dig>  were considered statistically significant
ifn-γ has been shown to be secreted continuously during antigen stimulation.
thus, signal intensity in ifn-γ elispot is dependent on the duration of stimulation  <cit> .
the duration of antigen stimulation in ifn-γ elispot reported in the literature usually ranges from  <dig> to 24 h .
to address the influence of the incubation time on the test results, ifn-γ elispot were performed on pbmc from  <dig> independent cmv-seropositive healthy donors following stimulation with t-activated® pp <dig> and ie- <dig> antigens for  <dig>   <dig> and 21 h. no statistically significant differences in sfc numbers were detected between the  <dig> conditions , demonstrating signal stability in this time range.
thus, an incubation time of 19 h was chosen for the optimized elispot assay.fig.
<dig> effect of duration of antigen stimulation on ifn-γ elispot test results.
sfc counts  in ifn-γ elispot following stimulation of pbmc samples from three cmv-seropositive healthy donors  with t-activated® ie- <dig> or pp <dig> for  <dig>   <dig> and 21 h. unstimulated pbmc  were used as a negative control.
differences between stimulation durations were tested using the non-parametric two-sided one-way anova kruskal-wallis test .
p-values <  <dig>  were considered statistically significant
numerous protocol variables can affect elispot test results.
for instance, the medium used for primary cell culture often includes serum which contains various batch-dependent non-characterized bioactive molecules in different concentrations  <cit> .
in order to define standardized cell culture conditions, the impact on the assay performance of different serum-containing media  and of serum-free media  was investigated.
serum-free media yielded the best effector cell responses, comparable to that of rpmi  <dig> supplemented with 5% fcs .
in addition, aim-v® exhibited lowest background signals in unstimulated conditions , thus maximizing signal-to-noise ratio.
consequently, the elispot protocol was further established using aim-v® serum-free medium.fig.
<dig> evaluation of different cell culture media on the performance of ifn-γ elispot.
a boxplot diagram showing elispot results upon stimulation of pbmc of  <dig> cmv-seropositive healthy blood donors  years; 40% male and 60% female) with t-activated® pp <dig> in various cell culture media.
median values  are indicated in brackets.
differences between medium conditions among stimulated conditions were tested using the non-parametric two-sided one-way anova kruskal-wallis test .
b elispot results of the non-stimulated cells incubated in aim-v or in ultraculture  serum-free media are shown separately with an expanded y-axis scale.
differences between both conditions were tested using the non-parametric two-sided mwu test .
p-values <  <dig>  were considered statistically significant.
serum-containing media were composed of rpmi  <dig> supplemented with 5% serum : fcs, nta, nts or human ab
elispot assays can be performed with various membrane materials, including nitrocellulose , mixed cellulose ester  and polyvinylidene fluoride .
we compared ifn-γ elispot results from pbmc samples of six healthy individuals  employing the most commonly used mce plates, pvdf plates and pvdf strips from millipore .
pvdf membranes require an activation step with ethanol prior to binding of the capture antibody.
in addition, more stringent washing steps prior to spot development were needed using pvdf-based plates compared to mce plates, to avoid undesirable background staining of membranes.
nevertheless, the resolution of the detected spots in terms of sharpness and homogeneity was improved on pvdf membranes , resulting in higher spot counts compared to mce membranes .
since pvdf 8-well strips were more performant and because their use might allow reduced costs, in particular when single patient samples are tested in clinical routine, the pvdf 8-well-strip format was chosen for the optimized assay development.fig.
<dig> comparison of ifn-γ elispot performance using different microtiter plates.
elispot were performed using pbmc from three cmv-seropositive healthy donors  on various microtiter plate materials .
age  of donors was  <dig> ,  <dig>  and  <dig> .
sfc values per  <dig>  pbmc are shown as box plots for non-stimulated pbmc , and for t-activated® ie-1- and pp65-stimulated pbmc.
median values  are indicated in brackets.
differences between microtiter plate materials were tested using the non-parametric two-sided one-way anova kruskal-wallis test
optimal coating of microtiter plates with capture antibody is crucial for a robust assay performance.
the density of ifn-γ capture antibody bound to the pvdf membrane should not be limiting and, in particular, should allow the reliable detection of high spot counts.
pvdf strips were coated with increasing concentrations  of anti-ifn-γ antibody.
pbmc from five cmv-seropositive donors were seeded into the coated wells and either left unstimulated or stimulated with t-activated® ie- <dig> or pp <dig>  in the range of  <dig>  to  <dig>  μg/ml, increasing antibody concentrations had no effect on background staining in unstimulated pbmc .
similarly, ie-1-specific low-to-moderate sfc levels  were comparable in all coating-antibody conditions .
the same was true for pp65-specific responses up to ~ <dig> sfc/ <dig>  pbmc .
in contrast, in individual donors with higher spot counts , detection of pp65-reactive cells increased in a dose-dependent manner with the concentration of ifn-γ capture antibody used for coating, especially between  <dig>  and 5 μg/ml antibody.
at antibody concentrations of 5 μg/ml and beyond, spot counts remained stable .
based on these results, a concentration of 5 μg/ml ifn-γ capture antibody was chosen for coating in the optimized elispot protocol.fig.
<dig> titration of the ifn-γ capture antibody.
pbmc  from five cmv-seropositive healthy donors  years;  <dig> male and  <dig> female) were seeded on pvdf microtiter strips coated with increasing concentrations  of ifn-γ capture antibody.
cells were left unstimulated   or were stimulated with t-activated® ie- <dig>  or pp <dig>  cmv antigens, as before.
sfc mean values per  <dig>  pbmc are shown as box plots for the collective of  <dig> donors   and as histograms  for pp65-stimulated pbmc of individuals donors .
differences between coating conditions were tested using the non-parametric two-sided one-way anova kruskal-wallis test
elispot assays rely on the detection of captured cytokine by cytokine-specific antibodies, which can be either directly coupled to a reporter enzyme , like alkaline phosphatase , or a combination of a biotinylated secondary antibody and a streptavidin-conjugated reporter enzyme .
a one-step assay development saves handling time and prevents operator errors.
ap-conjugated mab   was used as detection conjugate for the standardized elispot protocol.
different incubation parameters  were tested.
spot counts were comparable in all conditions.
however, spot morphology and thus proper detection was best upon incubation with ap-conjugate for 2 h at rt, compared to other conditions .
increase in the concentration of the detection conjugate from  <dig>  to  <dig>  u/ml resulted in slightly elevated sfc median values for pp <dig>  and the maximum spot counts exceeded those generated by the two-step assay development .
therefore, a one-step assay development for 2 h at rt with  <dig>  u/ml detection conjugate was chosen.
finally, standardization of the sfc staining reaction was addressed to complete assay optimization.
duration of incubation with the ap substrate affects spot size and/or background level, and is thus critical for reliable spot enumeration.
a one-step chromogenic alkaline phosphatase substrate nbt/bcip  was used as staining solution, and incubation times ranging from  <dig> to 13 min in the dark were evaluated.
sfc counts were comparable in all conditions.
however, shorter incubation times resulted in smaller spot diameter, while longer incubation times yielded enhanced background levels .
therefore, a staining duration of six minutes in the dark was defined for the optimized elispot protocol.
linearity and precision of the optimized cmv elispot assay
the optimized elispot protocol was used to determine the working range of pbmc that ensure assay linearity.
pbmc from one healthy cmv-seropositive donor were seeded at a density ranging from  <dig> ×  <dig> to  <dig>  ×  <dig> pbmc per well.
for cell numbers between  <dig> ×  <dig> and  <dig> ×  <dig> pbmc per well, elispot counts were directly proportional to the number of pbmc seeded, following stimulation with either ie- <dig>  or pp <dig>  .
because of the usually lower spot count resulting from ie- <dig> stimulation, the use of  <dig> ×  <dig> pbmc per well was chosen for the standardized elispot assay, to ensure sufficient sfc values.fig.
<dig> ifn-γ elispot assay linearity.
a working range of pbmc per elispot assay.
increasing number of pbmc from one cmv-seropositive healthy donor were seeded per well and elispot assays were performed as described, following stimulation with t-activated® ie- <dig> or pp <dig>  mean sfc values and standard deviation obtained for  <dig> - <dig>  pbmc per well are depicted.
scale of the y-axes was adjusted for pp <dig>  and ie- <dig>  for a better data resolution.
b linearity between the number of cmv-reactive pbmc and enumerated sfc.
the indicated numbers of pbmc from one cmv-seropositive healthy donor were mixed with pbmc from one cmv-seronegative donor  and stimulated with t-activated® pp <dig> antigen according to the optimized protocol.
mean sfc values and standard deviation of quadruplicate measurements are shown for two donor pairs .
in both panels, regression lines and corresponding coefficient of determination r <dig> were generated using the regression line tool of graphpad prism  <dig>
to further verify the linearity between the number of cmv-reactive effector cells and enumerated sfc, increasing numbers of pbmc from one cmv-seropositive healthy donor were seeded and the total number of pbmc was adjusted to  <dig> x  <dig> per well using pbmc from one cmv-seronegative donor.
two donor pairs  that showed no allo-reactivity in a 19-h co-culture were chosen for these experiments.
due to low sfc numbers , ie- <dig> stimulation results showed increased variability compared to pp <dig> and did not allow a reliable linearity calculation .
elispot assay results following pp <dig> stimulation showed a good linear correlation for both donor pairs, with r <dig> values of  <dig>   and  <dig>   in the linear regression analysis .
precision and repeatability of the optimized assay were evaluated by calculating the intra-assay, inter-assay, inter-operator and inter-site variability.
in each case, pbmc from three cmv-seropositive healthy donors were tested in quadruplicate, and variability was assessed by calculating the coefficient of variation , defined as the ratio of the standard deviation to the mean.
cv for elispot values <  <dig> sfc/ <dig>  pbmc were not calculated .
cv intra-assay reached 14% for ie- <dig> stimulation and 6% for pp <dig> stimulation .
cv inter-assay did not exceed 17% for ie- <dig> and 22% for pp <dig> .
cv inter-operator was below 13% and 18% for ie- <dig> and pp <dig> respectively .
finally, inter-site variation, which is essential for assay validation for diagnostics purposes, was evaluated.
whole blood samples were simultaneously collected from three cmv-seropositive healthy donors and shipped  to four different laboratories in germany.
pbmc were freshly isolated and elispot assays performed according to the optimized protocol by a total of  <dig> operators.
cv inter-site reached a maximum of 39% for ie- <dig> and of 28% for pp <dig> .
evaluation of at least four independent measurements has been recommended to achieve statistically significant elispot results .
to address the influence of intra-replicate variations on the assay outcome, we compared elispot results of quintuplicate and quadruplicate measurements of each control and antigen stimulations.
no significant variation of test results was found .
therefore, quadruplicate measurements of unstimulated and t-activated® ie-1- and pp65-stimulated conditions allow assay reliability, as well as practicability in combination with the use of 8-well strips.
staphylococcal enterotoxin b  is a powerful superantigen, and phytohemagglutinin  a potent mitogen, both inducing massive ifn-γ secretion by t cells  <cit> .
thus seb and pha are suitable positive controls for cell viability, successful stimulation of cytokine secretion and overall t cell functionality.
this is particularly important for result interpretation when low t cell frequency is expected, for instance in recipients of allogeneic stem cell transplantation.
in the optimized elispot assay, stimulations of test samples with either seb or pha are performed in duplicate.
in addition, an effector cell-independent operator control was established to validate proper assay performance.
this operator control is based on the detection of recombinant ifn-γ upon direct incubation with the immobilized anti-human-ifn-γ mab  capture antibody.
this control, also performed in duplicate, should yield a homogeneous dark staining of the pvdf membrane.
technical assay validation: definition of a positivity cut-off
to ease result interpretation of the ifn-γ elispot assay and to maximize specificity , a technical cut-off was defined.
ifn-γ elispot assays were performed according to the optimized protocol on pbmc from  <dig> healthy donors, of which  <dig> were cmv igg-seropositive .table  <dig> technical validation cohort of healthy donors
median and range of sfc values from unstimulated pbmc of cmv-seronegative and cmv-seropositive individuals were comparable .
spot counts in ie-1- and pp65-stimulated pbmc of cmv-seronegative subjects were low .
in cmv-seropositive individuals, sfc levels in ie-1- and pp65-stimulated pbmc reached  <dig> and  <dig> spot counts/ <dig>  pbmc .
for the determination of a technical cut-off, spot counts in the unstimulated control as well as in t-activated® pp65- and ie-1-stimulated conditions of cmv-seronegative and cmv-seropositive individuals were taken into consideration.
positivity threshold was determined using z-statistics  on log10-transformed geometric mean values.
values =  <dig> were replaced by values near detection limit, which was assumed to be  <dig> .
standard deviation  of elispot measurements for the unstimulated control, ie- <dig> stimulation and pp <dig> stimulation was respectively  <dig> ,  <dig>  and  <dig> .
considering a sd of  <dig>  and assuming that  <dig> replicates are measured for each negative control and test samples, a criterion that the ratio of geometric means of stimulated to unstimulated values is at least  <dig>  was calculated.
on the other hand, precision profiles were generated from both ie-1- and pp65-specific test results, whereby a coefficient of variation  no higher than 40% was used as a limit of acceptance of assay validity to determine the respective limit of quantitation .
precision profiles for ie-1- and pp65-specific elispot results from the  <dig> healthy donors yielded loq values of  <dig>  and  <dig>  respectively .
of note, a similar analysis performed on a cohort of  <dig> hemodialysis patients provided comparable sd values within unstimulated and antigen-stimulated conditions  and yielded loq values of  <dig>   and  <dig>    <cit> .
based on these analyses, a cut-off of  <dig> sfc/ <dig>  pbmc was chosen to define positive test results.fig.
<dig> assay validation in immunocompetent donors.
pbmc isolated from whole blood of  <dig> healthy donors  were assayed using the optimized ifn-γ elispot.
a positivity cut-off of  <dig> sfc/ <dig>  pbmc  was defined .
considering a test result as positive when the geometric mean for at least one of the ie- <dig> or pp <dig> stimulated approach is ≥  <dig> sfc/ <dig>  pbmc and when the ratio of geometric means of stimulated to unstimulated conditions is ≥  <dig> , positive agreement  and negative agreement  of the optimized ifn-γ elispot test results with cmv serology within this collective of healthy donors was 97% and 85% respectively
altogether, using t-activated® pp <dig> and ie- <dig> antigens and the optimized ifn-γ elispot assay, test results are considered positive if the geometric mean of the spots resulting from pp <dig> or ie <dig> stimulations are ≥  <dig> sfc/ <dig>  pbmc and if the ratio of the geometric means of stimulated to non-stimulated conditions is ≥  <dig> .
according to these definitions, the collective of  <dig> healthy donors  revealed a sensitivity  of 97% and a specificity  of 85% .
functional assay validation: t-activated antigens stimulate a broad spectrum of clinically relevant cmv-reactive effector cells
urea-formulated recombinant  proteins are processed by both the exogenous  and endogenous  antigen processing and presentation pathways  <cit> .
stimulation of pbmc by t-activated® proteins thus reproduces more closely a natural infection, potentially resulting in the activation of a broad spectrum of clinically relevant cmv-reactive cells , which might contribute to the high sensitivity of the ifn-γ elispot assay.
to further characterize the cells targeted by t-activated® ie- <dig> and pp <dig> antigens and to investigate the possible inter-individual variability in the effector cells response, intracellular ifn-γ staining and flow cytometry analyses were performed in parallel to ifn-γ elispot assays.
freshly isolated pbmc from six cmv-seropositive healthy donors were stimulated with t-activated® ie- <dig> and pp <dig> proteins for 19 h and ifn-γ-producing cells enumerated according to the optimized elispot assay.
the same pbmc preparations  were stimulated for 6 h with the same batch of t-activated® ie- <dig> and pp <dig> proteins in the presence of co-stimulatory anti-cd <dig> and anti-cd49d antibodies.
surface markers  and intracellular ifn-γ staining were analysed by flow cytometry, as described in the methods section.
ifn-γ+ subpopulations of cd3+cd4+ , cd3+cd8+ , cd3+cd56+  and cd3−cd56+  lymphocytes were enumerated following the gating strategy illustrated in additional file  <dig>  all six donors differed in their capacity to elicit an ie-1- and/or pp65-dependent response in the ifn-γ elispot assay.
the intensity of the response was also heterogeneous among the six donors, with values ranging from  <dig> to  <dig>  sfc  and from  <dig> to  <dig> sfc  per  <dig>  pbmc .
similarly, individual donors differed in the frequency and ratio of the various cell subpopulations investigated by flow cytometry .
interestingly, healthy cmv-seropositive individuals with lower spot counts  also showed lower frequencies of ifn-γ+ lymphocytes by flow cytometry , highlighting the ability of the elispot assay to distinguish low from high responders.
activation of each lymphocyte subpopulation investigated was detected in some but not all donors.
for instance, weak to strong cd4+ t cell activation was detected in  <dig> out of  <dig> donors  in response to either ie- <dig>  pp <dig>  or both.
similarly,  <dig> out of  <dig> donors  showed cd8+ t cell activation in one or both stimulation conditions.
cd3-cd56+  were apparent in one donor  in response to both antigens.
cd3+cd56+  cells were weakly activated in  <dig> out of  <dig> individuals  in response to both ie- <dig> and pp <dig> .
remarkably, a strong pp65-specific cd4+ activation in d <dig> correlated with a strong pp65-specific response in elispot, and a strong pp65-specific  and ie-1-specific  cd8+ activation was associated with a high spot count in the corresponding elispot , suggesting that these cmv-reactive effector cells significantly contribute to the detected elispot signals.
altogether, these data demonstrate the ability of t-activated® ie- <dig> and pp <dig> proteins to stimulate a broad range of cmv-specific effector cells.
these experiments also revealed the high heterogeneity of responses among healthy cmv-seropositive individuals.
notably, the observation that some individuals respond to either ie- <dig> or pp <dig> emphasizes the importance of assessing the response to both antigens.
monitoring both ie-1- and pp65-specific responses in the optimized ifn-γ elispot assay might improve the overall sensitivity of the test.fig.
<dig> t-activated® pp <dig> and ie- <dig> cmv antigens stimulate a broad range of cmv-reactive effector cells.
comparative analysis of ifn-γ secreting cells by elispot  and flow cytometry  following stimulation of pbmc with t-activated® pp <dig> and ie- <dig> antigens.
pbmc  from six cmv-seropositive healthy donors  years;  <dig> male and  <dig> female) were stimulated with t-activated® pp <dig> and ie- <dig> antigens according to the optimized ifn-γ elispot  and to the protocol of intracellular and surface marker staining and flow cytometry described in the methods section .
bar graphs in  depict ifn-γ-dependent sfc per  <dig>  pbmc, as before.
bar graphs in  represent the number of ifn-γ-expressing cd3+cd4+ , cd3+cd8+ , cd3−cd56+  and cd3+cd56+  cells per  <dig>  lymphocytes.
note that the y-axis scales in  were adjusted for a better resolution of the respective data.
individual age and gender of donors were as follows: d <dig>  37-year-old male; d <dig>  55-year-old female; d <dig>  46-year-old female; d <dig>  41-year-old female; d <dig>  24-year-old female; d <dig>  22-year-old male
discussion
we describe here the development and technical validation of an optimized and standardized ifn-γ elispot protocol for the monitoring of cmv-reactive effector cells of cell-mediated immunity .
the particularity of the assay is the use of t-activated® proteins  <cit>  as stimulant of cmv-reactive effector cells.
we now demonstrate that urea-formulated ie- <dig> and pp <dig> proteins are capable of activating not only cmv-reactive cd4+  and cd8+  lymphocytes, but also innate lymphocytes , likely by bystander activation as well as by activation of cmv-specific memory nk cells  <cit> .
this finding is important given the acknowledged role played by these effector cells in the protection against cmv reactivation in vivo .
inter-individual variability in the response of lymphocyte subpopulations was high among cmv-seropositive healthy donors, therefore further emphasizing the importance of an antigen formulation capable of activating a broad spectrum of cmv-reactive effector cells.
in this, the optimized ifn-γ elispot assays outperforms existing elispot- and elisa-based cmv-specific cmi monitoring tools.
in addition, the characteristics and high sensitivity of our assay predict a performance independent of the hla-type of the donor.
the second particularity of the described assay is the combined use of two t-activated® proteins, ie- <dig> and pp <dig>  as antigens.
we show here that both antigens can elicit the activation of distinct cmv-specific effector cells and that while some cmv-seropositive healthy individuals are reactive to both antigens, some are only reactive to one , as previously reported  <cit> .
therefore, consideration of both test results is expected to improve the sensitivity of the assay, notably in cohort studies.
this is particularly relevant for the monitoring of cmv-specific cmi in immunocompromized patients.
accordingly, we recently demonstrated the benefit of measuring the response to both t-activated® ie- <dig> and pp <dig> antigens in terms of assay sensitivity, in clinical studies assessing cmv-specific cmi during pregnancy  <cit> , in hemodialysis patients  <cit>  and in renal transplant recipients .
the assessment of the response to both ie- <dig> and pp <dig> antigens is further justified by the respective relevance and differential contribution of ie-1- and pp65-reactive effector cells to the protection against cmv reactivation and related clinical complications, both in healthy cmv-seropositive individuals and in transplant recipients .
the differential contribution of ie-1- and pp65-specific effector cells likely reflect the dynamic of the cell-mediated immune response .
mechanisms of immune evasion inhibiting mhc-i-dependent ie- <dig> antigen processing and presentation might also explain the lower frequency of ie-1-specific ifn-γ-producing cells detected in our elispot assay  <cit> .
in addition, reduced processing and presentation efficiency due to protein stability, size and nuclear localization might play a role in the reduced reactivity to ie- <dig>  as opposed to pp <dig>  <cit> .
this proposition is supported by the observation that higher concentrations of ie- <dig> were required in our elispot assay, in comparison to pp <dig>  to trigger a significant response.
another particularity of our ifn-γ elispot assay was its ability to elicit positive test results in cmv-seronegative individuals, notably following stimulation with ie- <dig>  in the cohort of  <dig> healthy donors, out of the  <dig> cmv-seronegative individuals  <dig> showed ie-1-specific values ≥  <dig> sfc/ <dig>  pbmc , corresponding to a negative agreement with cmv igg-serology of 85%.
similar results were obtained in a cohort of  <dig> hemodialysis patients  <cit> .
numerous studies reported discordant results between cellular and humoral immunity against cmv .
given the high sensitivity of our elispot assay and its ability to detect a broad range of cmv-reactive cells, it is possible that cellular reactivity in cmv-seronegative individuals reflects a previous exposure to cmv that failed to mount a humoral immune response, rather than a false-positive elispot result.
further experiments will be necessary to address this possibility.
nonetheless, this observation raises the question of the accuracy of cmv serology to identify immunocompromised patients at increased risk of cmv reactivation  <cit> .
the optimized and standardized ifn-γ elispot assay exhibited robust performance in terms of assay variability, precision and linearity.
intra-assay, inter-assay and inter-operator coefficients of variations  did not exceed 22%, which is below the range of recommended precision  for cell-based assays  <cit> .
as predicted, inter-site variability involving different operators was higher, but remained acceptable with a cv of no more than 39%.
this is an important criterion for the reliable longitudinal monitoring of cmv-specific immune responses in patients.
linearity of the assay is also a critical parameter, in particular for the monitoring of immunocompromized patients with reduced and/or functionally impaired t cells, such as children or recipients of allogeneic stem cell transplantation.
good linearity and high signal-to-noise ratio were obtained for both ie- <dig>  and pp <dig>  in the range of  <dig>  to  <dig>  pbmc per well.
two multi-center clinical studies are currently being conducted, in renal transplant recipients under immunosuppressive therapy and in allogeneic stem cell transplantation patients, to assess the sensitivity of the optimized cmv-specific ifn-γ elispot assay in this clinical context and to correlate results of the assay to clinical outcome.
CONCLUSIONS
altogether, this optimized, standardized and user-friendly cmv-specific ifn-γ elispot assay meet all the conditions of a sensitive and reliable diagnostic test for the monitoring of the functionality of cmv-specific cmi.
beside its validation in healthy individuals , its suitability to assess cmv-specific cell-mediated immunity in a cohort of hemodialysis patients representative of patients prior to transplantation has been demonstrated  <cit> .
further validation of the assay in immunocompromised patients, such as transplant recipients is necessary to determine its suitability in assisting clinicians to evaluate the risk of cmv reactivation and disease, and possibly individualize the therapeutic management of patients.
additional files
additional file 1: assay variability.
intra-assay , inter-assay , inter-operator  and inter-site  variability was assessed as described in the respective legends.
additional file 2: positivity cut-off definition.
results obtained using the optimized ifn-γ elispot assay on pbmc isolated from whole blood of  <dig> healthy donors  were used to generate precision profiles for each ie- <dig> and pp <dig> stimulation.
limits of quantitation  for ie- <dig> and pp <dig> were  <dig>  and  <dig>  sfc/ <dig>  pbmc respectively.
loq calculated on a cohort of  <dig> hemodialysis patients  <cit>  were in a similar range .
therefore, a positivity cut-off of  <dig> sfc/ <dig>  pbmc was chosen for the standardized assay.
in addition, intra-assay standard deviation  within both cohorts of healthy donors  and hemodialysis patients  for stimulated and unstimulated measurements was the basis for the calculation of a criterion that the ratio of geometric means of stimulated to unstimulated values is at least  <dig> .
finally, considering a test result as positive when geometric mean for at least one of the ie- <dig> or pp <dig> stimulated approach is ≥  <dig> sfc/ <dig>  pbmc, positive agreement  and negative agreement  of the optimized ifn-γ elispot test results with cmv serology within the collective of  <dig> healthy donors was 97% and 85% respectively .
additional file 3: gating strategy of flow cytometry analyses following surface marker  and intracellular ifn-γ staining.
pbmc  from six cmv-seropositive healthy donors were stimulated with t-activated® pp <dig> and ie- <dig> antigens for 6 h, as indicated in the legend to fig.
<dig>   cells were first gated on single events in a forward scatter-area  x fsc-height  dot plot .
based on fsc-a x side scatter-area  properties, a gate was next set around lymphocytes .
by plotting cd <dig> against cd <dig>  cd <dig> single positive cells , cd3/cd <dig> double positive cells  and cd <dig> single positive cells  were separated.
cd <dig> single positive cells  were further subdivided into cd3/cd <dig> double positive  and cd3/cd <dig> double positive cells .
representative dot plots  of stained pbmc of donor d <dig>  in each plot, percentage value in gated area is the mean frequency  of ifn-γ-expressing cd3+cd4+ , cd3+cd8+ , cd3−cd56+  and cd3+cd56+  cells.
abbreviations
cmicell-mediated immunity
cmvcytomegalovirus
ctlcytotoxic t lymphocyte
cvcoefficient of variation
elisaenzyme-linked immunosorbent assay
elispotenzyme-linked immunospot
hlahuman leukocyte antigen
icsintracellular cytokine staining
ie-1immediate early- <dig> protein
ifn-γinterferon gamma
loqlimit of quantitation
nknatural killer cell
nktnatural killer t cell
pbmcperipheral blood mononuclear cell
pp6565 kda lower matrix phosphoprotein
rsdrelative standard deviation
sdstandard deviation
sfcspot-forming cell
tht helper cell

 human cytomegalovirus is endemic in all human populations, with a seroprevalence ranging from to 100% depending on age, gender and location. in healthy individuals, cmv replication is efficiently controlled by cell-mediated immunity. in immunocompromized individuals however, the reduced frequency and functionality of cmv-reactive effector cells is associated with severe clinical complications due to uncontrolled virus replication. quantitative assessment of functional cmv-reactive effector cells in immunocompromized individuals might help to identify patients at increased risk for cmv-mediated clinical complications and to adjust antiviral and immunosuppressive therapy. reliable monitoring of cmv-specific cmi in immunocompromized individuals, such as solid-organ or allogeneic stem cell transplant recipients, requires a specific, standardized but also highly sensitive assay capable of detecting low numbers of cmv-reactive cells. such sensitivity might be achieved by using highly immunogenic stimulants and via the reactivation of a broad spectrum of physiological effector cells involved in the protection against cmv replication in vivo, notably cd4+, cd8+, but also natural killer and natural killer t cells. current diagnostic methods to detect and monitor cmv-specific cmi are mostly based on the restimulation of cd4+ and/or cd8+ effector cells with pools of overlapping peptides, cocktails of preselected immunodominant cmv-peptides or lysates of cmv-infected cells and the subsequent measurement of induced cytokine production or cell proliferation, by flow cytometry, enzyme-linked immunosorbent assay or enzyme-linked immunospot assay. a different approach consists in the direct staining with cmv-peptide-loaded multimers and enumeration of cmv-specific cd8+ t cells by flow cytometry. however, this method is lacking a functional readout and is restricted to certain hla types, impeding its use in routine diagnostics. elisa-based assays, such as quantiferon®-cmv are advantageous in that they require low blood volumes and are easy to perform. however, they are restricted to the detection of ifn-γ-producing cd8+ t cells and do not allow single-cell-level measurement. due to analyte dilution, elisa-based assays usually result in reduced sensitivity and often yield indeterminate results, especially in immunocompromized patients. intracellular cytokine staining and subsequent flow cytometric analysis is usually more sensitive than elisa and allows the assessment of both the functionality and the phenotype of cmv-reactivated cytokine-producing cells. however, this method is difficult to standardize and only detects the intracellular analyte, not the biologically active cytokine secreted over the stimulation period. elispot-based assays identify and enumerate biologically active, cytokine-secreting cells from isolated peripheral blood mononuclear cells, at the single-cell level both qualitatively and quantitatively. elispot represents the most sensitive read-out system and thus is most appropriate for the detection of low-level responses. in particular, elispot is more sensitive than ics for the detection of antigen-specific cells, such as in vitro-reactivated memory t cells, which produce only low amounts of cytokines. elispot was recently successfully employed over elisa in the detection of congenital cmv infection. conventional cmv-specific elispot assays are based on the detection of ifn-γ-producing cd4+ and/or cd8+ t cells, depending on the antigens used for pbmc stimulation. the careful selection and formulation of antigens is crucial to ensure specificity, sensitivity and thus a diagnostic value to the assay. we have previously shown that urea-formulated recombinant proteins are processed by the exogenous and endogenous antigen processing machinery, resulting in the presentation of naturally generated peptides by mhc class ii and class i molecules. stimulation of pbmc by t-activated® proteins thus mimics more closely a natural infection, resulting not only in the specific activation of memory t and nk cells, but also possibly in the bystander activation of nk and nkt-like cells present in the pbmc population. we describe here the establishment, optimization and standardization of a highly sensitive elispot assay that takes advantage of both the ifn-γ elispot readout and the immunodominant and highly immunogenic t-activated® ie- and pp cmv proteins as stimulants. we also compared the performance of the optimized assay to that of intracellular staining and flow cytometry in healthy cmv-seropositive donors, and demonstrated the ability of t-activated® ie- and pp antigens to activate a broad spectrum of cmv-reactive cells, including cd3+cd4+, cd3+cd8+, cd3−cd56+ and cd3+cd56+ cells. therefore, the combined use of t-activated® ie- and pp cmv proteins with our optimized ifn-γ elispot defines a highly sensitive assay meeting all conditions for a reliable and standardized immune monitoring diagnostic tool. methods proteins, elispot plate precoating and detection conjugate urea-formulated t-activated® proteins were prepared as previously described. the t-activated® immunodominant region of cmv pp was provided by mikrogen. full length ie- was kindly provided by christina paulus and michael nevels. t-activation® of ie- was performed by lophius biosciences. staphylococcal enterotoxin b and phytohemagglutinin were used as positive controls for pbmc stimulation. 96-well elispot plates and 8-well elispot strips each pre-coated with anti-human-ifn-γ mab 1-d1k as well as the detection conjugate mab-ap coupled with alkaline phosphatase were obtained from microcoat biotechnologie gmbh. blood collection and pbmc preparation blood samples were collected in lithium heparin tubes from healthy individuals with known cmv serostatus by venipuncture and stored for up to 8 hours at room temperature until further processing. isolation of peripheral blood mononuclear cells was performed using standard ficoll-paque density centrifugation as specified by the manufacturer. pbmc were finally suspended in serum-free aim-v® medium and counted either manually in a neubauer’s chamber or using the hem-o-test cell-counting device. automated cell counting was performed in the whole blood venous mode of the calibrated analyzer. elispot assay ifn-γ elispot assays were performed as previously described unless specified otherwise in the results section. briefly, × freshly isolated pbmc were plated in four replicates into 96-well elispot plates or 8-well elispot strips precoated with anti-human-ifn-γ mab 1-d1k and stimulated for 19 h at 37 °c under 5% co with either 3 μg/ml t-activated® pp antigen or 15 μg/ml t-activated® ie- protein. as unstimulated control, cells were incubated for 19 h in cell culture medium. after cell removal, plates were developed for 2 h at room temperature in the presence of u/ml ifn-γ-specific alkaline phosphatase-coupled mab 7-b6- spot detection was performed following incubation for 6 min in the dark with a 1-step nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate substrate. ifn-γ-specific spot-forming cells were counted using a bioreader® eα. of note, comparable results were obtained on two other readers. intracellular cytokine staining intracellular cytokine staining was performed as previously described, with the following modifications. pbmc from six healthy cmv-seropositive donors were isolated and four replicates each were stimulated for 6 h at 37 °c and 5% co with 3 μg/ml t-activated® pp antigen or with 15 μg/ml t-activated® ie- protein in the presence of co-stimulatory anti-cd and anti-cd49d monoclonal antibodies. the unstimulated pbmc control cells were incubated with co-stimulatory anti-cd and anti-cd49d molecules only. stimulation with seb and anti-cd28 + anti-cd49d served as positive control. after the first two hours of incubation, 1 μg/ml brefeldin a was added to prevent cytokine secretion from activated cells. surface markers were stained for 30 min at 4 °c using the following conjugated antibodies: anti-cd apc-cy anti-cd brilliant violet anti-cd fitc, anti-cd percp. cells were fixed and permeabilized for 30 min at 4 °c in bd cytofix/cytoperm. intracellular staining of ifn-γ was performed using anti-ifn-γ apc in bd perm/wash buffer for 45 min at 4 °c. samples were analyzed on a bd facscanto™ ii flow cytometer. live-gating of cells was performed during acquisition. mean number of event acquisition was. results were reported as percentage of the gated population producing ifn-γ and as the number of ifn-γ+ cells/ lymphocytes. serology the cmv serological testing of blood donors was performed using the fully automated cmv immunoglobulin m and igg tests on the architect instrument or the bep® iii system by the diagnostics department of the institute for medical microbiology and hygiene. cmv igg-serology was used as primary reference measurement procedure. statistics statistics applied to assay development was performed using sigmaplot version and graphpad prism. at the start of this study, a positive elispot test result was defined by a statistically significant difference between quadruplicate sfc values of non-stimulated and specifically stimulated approaches of at least one of the two antigens used, calculated using the mann–whitney u-test. two-sided exact p values are reported. p values <  were considered statistically significant. statistical analyses to assess variations between two or more settings were performed by generating arithmetic means for all replicates of each given setting. those means were grouped to generate an overall arithmetic mean and standard deviation. the coefficient of variation in % was calculated in microsoft excel. the curve fitting for sigmoidal curves of the titration experiment was performed using the four parameter logistic function of graphpad prism. for the assessment of the elispot assay sensitivity in seropositive healthy blood donor collectives, the non-parametric mann–whitney u-test was performed to determine if a significant difference exists between the groups of negative control and stimulation quadruplicates. multiple-group comparisons were performed using a non-parametric one-way anova with dunn’s multiple comparison post test. sfc values were depicted using graphpad prism either as tukey box plots showing median values, interquartile ranges, lower and upper whiskers and outliers, or as histograms. for assay validation of optimized elispot, positivity cut-off was calculated on ifn-γ elispot results obtained from a collective of healthy donors using sas software and vfp version.fig. determination of optimal concentrations of t-activated® pp and ie- for the stimulation of pbmc. pbmc were isolated from one cmv-seropositive healthy donor blood sample and stimulated for 19 h with increasing concentrations of t-activated® pp or ie-. ifn-γ elispot results of four replicates are expressed as mean sfc/ pbmc. error bars represent standard deviations. the y-axis scale was adjusted to pp65- and ie-1- specific values to optimize data resolution. curve fitting was made using the four parameter logistic function of graphpad prism  ifn-γ elispot assay optimization following pbmc stimulation with t-activated® ie- and pp cmv antigens freshly isolated pbmc were used for the elispot assay. to prevent loss in t cell functionality, for instance due to activated granulocytes, heparinized blood samples were processed with no further additives within 8 h. a total number of × pbmc per well was chosen for the development of the elispot assay protocol as this cell count is below confluency and can usually be obtained from samples of less than 15 ml whole blood. the cmv immediate-early protein ie- and the late tegument protein pp represent well-characterized immunodominant t cell antigens. full-length ie- and a amino-acid c-terminal fragment of pp were produced and formulated in the presence of urea to increase their stimulatory capacity for different types of cmv-reactive effector cells of cell-mediated immunity. optimal t-activated® antigen concentration was first determined by performing dose–response experiments. freshly isolated pbmc of one healthy cmv-seropositive donor were stimulated with  fg/ml to  μg/ml t-activated® pp or with to  μg/ml t-activated® ie- and the number of ifn-γ secreting cells was determined by ifn-γ elispot. t-activated® pp revealed a much stronger capacity to stimulate ifn-γ secreting effector cells than t-activated® ie- reaching a plateau of responsiveness between and  ng/ml pp vs. approximately  μg/ml for ie-. accordingly, t-activated® antigen concentrations of 3 μg/ml pp and 15 μg/ml ie- were selected for further pbmc stimulations and elispot assays. assay sensitivity and specificity were determined by stimulating pbmc isolated from each cmv-seropositive and cmv-seronegative healthy donors with the defined pp and ie-1 t-activated® antigen concentrations. the number of reactive effector cells was quantified by ifn-γ elispot. significant stimulation was defined using a mann–whitney u-test as a statistically significant difference between sfc values of non-stimulated and cmv antigen-stimulated conditions. t-activated® pp and ie- induced a significant activation of responsive effector cells in out of and out of pbmc preparations from individual cmv-seropositive donors, respectively. in this collective, t-activated® pp showed an overall greater capacity to activate responsive cells with a median of sfc/ pbmc, compared to t-activated® ie- with a median of sfc/ pbmc. nevertheless, substantial response of up to sfc/ pbmc was detected in response to t-activated® ie- in individual samples of cmv-seropositive donors. all pbmc samples from different cmv-seronegative donors showed negative test results after stimulation with pp, while out of pbmc samples from cmv-seronegative individuals were negative after stimulation with ie-. spot count within cmv-seronegative ie-1-stimulated pbmc was higher than in cmv-seronegative pp65-stimulated pbmc but did not exceed sfc/ pbmc.fig. determination of assay sensitivity and specificity. pbmc from each cmv-seropositive and cmv-negative healthy blood donors were left unstimulated or were stimulated with t-activated® pp or ie- and ifn-γ elispot assays were conducted as before. mean sfc values per pbmc of replicates are shown as box plots. median values are indicated in brackets. y-axis scales were adjusted in each graph for better resolution of sfc counts. median age and range of cmv-seronegative and cmv-seropositive subjects was and years. gender distribution in cmv-seronegative and cmv-seropositive groups was comparable. differences between unstimulated and stimulated conditions were tested using the non-parametric two-sided mann–whitney u test. p-values <  were considered statistically significant ifn-γ has been shown to be secreted continuously during antigen stimulation. thus, signal intensity in ifn-γ elispot is dependent on the duration of stimulation. the duration of antigen stimulation in ifn-γ elispot reported in the literature usually ranges from to 24 h. to address the influence of the incubation time on the test results, ifn-γ elispot were performed on pbmc from independent cmv-seropositive healthy donors following stimulation with t-activated® pp and ie- antigens for and 21 h. no statistically significant differences in sfc numbers were detected between the conditions, demonstrating signal stability in this time range. thus, an incubation time of 19 h was chosen for the optimized elispot assay.fig. effect of duration of antigen stimulation on ifn-γ elispot test results. sfc counts in ifn-γ elispot following stimulation of pbmc samples from three cmv-seropositive healthy donors with t-activated® ie- or pp for and 21 h. unstimulated pbmc were used as a negative control. differences between stimulation durations were tested using the non-parametric two-sided one-way anova kruskal-wallis test. p-values <  were considered statistically significant numerous protocol variables can affect elispot test results. for instance, the medium used for primary cell culture often includes serum which contains various batch-dependent non-characterized bioactive molecules in different concentrations. in order to define standardized cell culture conditions, the impact on the assay performance of different serum-containing media and of serum-free media was investigated. serum-free media yielded the best effector cell responses, comparable to that of rpmi supplemented with 5% fcs. in addition, aim-v® exhibited lowest background signals in unstimulated conditions, thus maximizing signal-to-noise ratio. consequently, the elispot protocol was further established using aim-v® serum-free medium.fig. evaluation of different cell culture media on the performance of ifn-γ elispot. a boxplot diagram showing elispot results upon stimulation of pbmc of cmv-seropositive healthy blood donors years; 40% male and 60% female) with t-activated® pp in various cell culture media. median values are indicated in brackets. differences between medium conditions among stimulated conditions were tested using the non-parametric two-sided one-way anova kruskal-wallis test. b elispot results of the non-stimulated cells incubated in aim-v or in ultraculture serum-free media are shown separately with an expanded y-axis scale. differences between both conditions were tested using the non-parametric two-sided mwu test. p-values <  were considered statistically significant. serum-containing media were composed of rpmi supplemented with 5% serum: fcs, nta, nts or human ab elispot assays can be performed with various membrane materials, including nitrocellulose, mixed cellulose ester and polyvinylidene fluoride. we compared ifn-γ elispot results from pbmc samples of six healthy individuals employing the most commonly used mce plates, pvdf plates and pvdf strips from millipore. pvdf membranes require an activation step with ethanol prior to binding of the capture antibody. in addition, more stringent washing steps prior to spot development were needed using pvdf-based plates compared to mce plates, to avoid undesirable background staining of membranes. nevertheless, the resolution of the detected spots in terms of sharpness and homogeneity was improved on pvdf membranes, resulting in higher spot counts compared to mce membranes. since pvdf 8-well strips were more performant and because their use might allow reduced costs, in particular when single patient samples are tested in clinical routine, the pvdf 8-well-strip format was chosen for the optimized assay development.fig. comparison of ifn-γ elispot performance using different microtiter plates. elispot were performed using pbmc from three cmv-seropositive healthy donors on various microtiter plate materials. age of donors was, and. sfc values per pbmc are shown as box plots for non-stimulated pbmc, and for t-activated® ie-1- and pp65-stimulated pbmc. median values are indicated in brackets. differences between microtiter plate materials were tested using the non-parametric two-sided one-way anova kruskal-wallis test optimal coating of microtiter plates with capture antibody is crucial for a robust assay performance. the density of ifn-γ capture antibody bound to the pvdf membrane should not be limiting and, in particular, should allow the reliable detection of high spot counts. pvdf strips were coated with increasing concentrations of anti-ifn-γ antibody. pbmc from five cmv-seropositive donors were seeded into the coated wells and either left unstimulated or stimulated with t-activated® ie- or pp in the range of to  μg/ml, increasing antibody concentrations had no effect on background staining in unstimulated pbmc. similarly, ie-1-specific low-to-moderate sfc levels were comparable in all coating-antibody conditions. the same was true for pp65-specific responses up to ~ sfc/ pbmc. in contrast, in individual donors with higher spot counts, detection of pp65-reactive cells increased in a dose-dependent manner with the concentration of ifn-γ capture antibody used for coating, especially between and 5 μg/ml antibody. at antibody concentrations of 5 μg/ml and beyond, spot counts remained stable. based on these results, a concentration of 5 μg/ml ifn-γ capture antibody was chosen for coating in the optimized elispot protocol.fig. titration of the ifn-γ capture antibody. pbmc from five cmv-seropositive healthy donors years; male and female) were seeded on pvdf microtiter strips coated with increasing concentrations of ifn-γ capture antibody. cells were left unstimulated or were stimulated with t-activated® ie- or pp cmv antigens, as before. sfc mean values per pbmc are shown as box plots for the collective of donors and as histograms for pp65-stimulated pbmc of individuals donors. differences between coating conditions were tested using the non-parametric two-sided one-way anova kruskal-wallis test elispot assays rely on the detection of captured cytokine by cytokine-specific antibodies, which can be either directly coupled to a reporter enzyme, like alkaline phosphatase, or a combination of a biotinylated secondary antibody and a streptavidin-conjugated reporter enzyme. a one-step assay development saves handling time and prevents operator errors. ap-conjugated mab was used as detection conjugate for the standardized elispot protocol. different incubation parameters were tested. spot counts were comparable in all conditions. however, spot morphology and thus proper detection was best upon incubation with ap-conjugate for 2 h at rt, compared to other conditions. increase in the concentration of the detection conjugate from to u/ml resulted in slightly elevated sfc median values for pp and the maximum spot counts exceeded those generated by the two-step assay development. therefore, a one-step assay development for 2 h at rt with u/ml detection conjugate was chosen. finally, standardization of the sfc staining reaction was addressed to complete assay optimization. duration of incubation with the ap substrate affects spot size and/or background level, and is thus critical for reliable spot enumeration. a one-step chromogenic alkaline phosphatase substrate nbt/bcip was used as staining solution, and incubation times ranging from to 13 min in the dark were evaluated. sfc counts were comparable in all conditions. however, shorter incubation times resulted in smaller spot diameter, while longer incubation times yielded enhanced background levels. therefore, a staining duration of six minutes in the dark was defined for the optimized elispot protocol. linearity and precision of the optimized cmv elispot assay the optimized elispot protocol was used to determine the working range of pbmc that ensure assay linearity. pbmc from one healthy cmv-seropositive donor were seeded at a density ranging from × to × pbmc per well. for cell numbers between × and × pbmc per well, elispot counts were directly proportional to the number of pbmc seeded, following stimulation with either ie- or pp. because of the usually lower spot count resulting from ie- stimulation, the use of × pbmc per well was chosen for the standardized elispot assay, to ensure sufficient sfc values.fig. ifn-γ elispot assay linearity. a working range of pbmc per elispot assay. increasing number of pbmc from one cmv-seropositive healthy donor were seeded per well and elispot assays were performed as described, following stimulation with t-activated® ie- or pp mean sfc values and standard deviation obtained for - pbmc per well are depicted. scale of the y-axes was adjusted for pp and ie- for a better data resolution. b linearity between the number of cmv-reactive pbmc and enumerated sfc. the indicated numbers of pbmc from one cmv-seropositive healthy donor were mixed with pbmc from one cmv-seronegative donor and stimulated with t-activated® pp antigen according to the optimized protocol. mean sfc values and standard deviation of quadruplicate measurements are shown for two donor pairs. in both panels, regression lines and corresponding coefficient of determination r were generated using the regression line tool of graphpad prism to further verify the linearity between the number of cmv-reactive effector cells and enumerated sfc, increasing numbers of pbmc from one cmv-seropositive healthy donor were seeded and the total number of pbmc was adjusted to x per well using pbmc from one cmv-seronegative donor. two donor pairs that showed no allo-reactivity in a 19-h co-culture were chosen for these experiments. due to low sfc numbers, ie- stimulation results showed increased variability compared to pp and did not allow a reliable linearity calculation. elispot assay results following pp stimulation showed a good linear correlation for both donor pairs, with r values of and in the linear regression analysis. precision and repeatability of the optimized assay were evaluated by calculating the intra-assay, inter-assay, inter-operator and inter-site variability. in each case, pbmc from three cmv-seropositive healthy donors were tested in quadruplicate, and variability was assessed by calculating the coefficient of variation, defined as the ratio of the standard deviation to the mean. cv for elispot values <  sfc/ pbmc were not calculated. cv intra-assay reached 14% for ie- stimulation and 6% for pp stimulation. cv inter-assay did not exceed 17% for ie- and 22% for pp. cv inter-operator was below 13% and 18% for ie- and pp respectively. finally, inter-site variation, which is essential for assay validation for diagnostics purposes, was evaluated. whole blood samples were simultaneously collected from three cmv-seropositive healthy donors and shipped to four different laboratories in germany. pbmc were freshly isolated and elispot assays performed according to the optimized protocol by a total of operators. cv inter-site reached a maximum of 39% for ie- and of 28% for pp. evaluation of at least four independent measurements has been recommended to achieve statistically significant elispot results. to address the influence of intra-replicate variations on the assay outcome, we compared elispot results of quintuplicate and quadruplicate measurements of each control and antigen stimulations. no significant variation of test results was found. therefore, quadruplicate measurements of unstimulated and t-activated® ie-1- and pp65-stimulated conditions allow assay reliability, as well as practicability in combination with the use of 8-well strips. staphylococcal enterotoxin b is a powerful superantigen, and phytohemagglutinin a potent mitogen, both inducing massive ifn-γ secretion by t cells. thus seb and pha are suitable positive controls for cell viability, successful stimulation of cytokine secretion and overall t cell functionality. this is particularly important for result interpretation when low t cell frequency is expected, for instance in recipients of allogeneic stem cell transplantation. in the optimized elispot assay, stimulations of test samples with either seb or pha are performed in duplicate. in addition, an effector cell-independent operator control was established to validate proper assay performance. this operator control is based on the detection of recombinant ifn-γ upon direct incubation with the immobilized anti-human-ifn-γ mab capture antibody. this control, also performed in duplicate, should yield a homogeneous dark staining of the pvdf membrane. technical assay validation: definition of a positivity cut-off to ease result interpretation of the ifn-γ elispot assay and to maximize specificity, a technical cut-off was defined. ifn-γ elispot assays were performed according to the optimized protocol on pbmc from healthy donors, of which were cmv igg-seropositive.table technical validation cohort of healthy donors median and range of sfc values from unstimulated pbmc of cmv-seronegative and cmv-seropositive individuals were comparable. spot counts in ie-1- and pp65-stimulated pbmc of cmv-seronegative subjects were low. in cmv-seropositive individuals, sfc levels in ie-1- and pp65-stimulated pbmc reached and spot counts/ pbmc. for the determination of a technical cut-off, spot counts in the unstimulated control as well as in t-activated® pp65- and ie-1-stimulated conditions of cmv-seronegative and cmv-seropositive individuals were taken into consideration. positivity threshold was determined using z-statistics on log10-transformed geometric mean values. values =  were replaced by values near detection limit, which was assumed to be. standard deviation of elispot measurements for the unstimulated control, ie- stimulation and pp stimulation was respectively, and. considering a sd of and assuming that replicates are measured for each negative control and test samples, a criterion that the ratio of geometric means of stimulated to unstimulated values is at least was calculated. on the other hand, precision profiles were generated from both ie-1- and pp65-specific test results, whereby a coefficient of variation no higher than 40% was used as a limit of acceptance of assay validity to determine the respective limit of quantitation. precision profiles for ie-1- and pp65-specific elispot results from the healthy donors yielded loq values of and respectively. of note, a similar analysis performed on a cohort of hemodialysis patients provided comparable sd values within unstimulated and antigen-stimulated conditions and yielded loq values of and. based on these analyses, a cut-off of sfc/ pbmc was chosen to define positive test results.fig. assay validation in immunocompetent donors. pbmc isolated from whole blood of healthy donors were assayed using the optimized ifn-γ elispot. a positivity cut-off of sfc/ pbmc was defined. considering a test result as positive when the geometric mean for at least one of the ie- or pp stimulated approach is ≥  sfc/ pbmc and when the ratio of geometric means of stimulated to unstimulated conditions is ≥ , positive agreement and negative agreement of the optimized ifn-γ elispot test results with cmv serology within this collective of healthy donors was 97% and 85% respectively altogether, using t-activated® pp and ie- antigens and the optimized ifn-γ elispot assay, test results are considered positive if the geometric mean of the spots resulting from pp or ie stimulations are ≥  sfc/ pbmc and if the ratio of the geometric means of stimulated to non-stimulated conditions is ≥ . according to these definitions, the collective of healthy donors revealed a sensitivity of 97% and a specificity of 85%. functional assay validation: t-activated antigens stimulate a broad spectrum of clinically relevant cmv-reactive effector cells urea-formulated recombinant proteins are processed by both the exogenous and endogenous antigen processing and presentation pathways. stimulation of pbmc by t-activated® proteins thus reproduces more closely a natural infection, potentially resulting in the activation of a broad spectrum of clinically relevant cmv-reactive cells, which might contribute to the high sensitivity of the ifn-γ elispot assay. to further characterize the cells targeted by t-activated® ie- and pp antigens and to investigate the possible inter-individual variability in the effector cells response, intracellular ifn-γ staining and flow cytometry analyses were performed in parallel to ifn-γ elispot assays. freshly isolated pbmc from six cmv-seropositive healthy donors were stimulated with t-activated® ie- and pp proteins for 19 h and ifn-γ-producing cells enumerated according to the optimized elispot assay. the same pbmc preparations were stimulated for 6 h with the same batch of t-activated® ie- and pp proteins in the presence of co-stimulatory anti-cd and anti-cd49d antibodies. surface markers and intracellular ifn-γ staining were analysed by flow cytometry, as described in the methods section. ifn-γ+ subpopulations of cd3+cd4+, cd3+cd8+, cd3+cd56+ and cd3−cd56+ lymphocytes were enumerated following the gating strategy illustrated in additional file all six donors differed in their capacity to elicit an ie-1- and/or pp65-dependent response in the ifn-γ elispot assay. the intensity of the response was also heterogeneous among the six donors, with values ranging from to sfc and from to sfc per pbmc. similarly, individual donors differed in the frequency and ratio of the various cell subpopulations investigated by flow cytometry. interestingly, healthy cmv-seropositive individuals with lower spot counts also showed lower frequencies of ifn-γ+ lymphocytes by flow cytometry, highlighting the ability of the elispot assay to distinguish low from high responders. activation of each lymphocyte subpopulation investigated was detected in some but not all donors. for instance, weak to strong cd4+ t cell activation was detected in out of donors in response to either ie- pp or both. similarly, out of donors showed cd8+ t cell activation in one or both stimulation conditions. cd3-cd56+ were apparent in one donor in response to both antigens. cd3+cd56+ cells were weakly activated in out of individuals in response to both ie- and pp. remarkably, a strong pp65-specific cd4+ activation in d correlated with a strong pp65-specific response in elispot, and a strong pp65-specific and ie-1-specific cd8+ activation was associated with a high spot count in the corresponding elispot, suggesting that these cmv-reactive effector cells significantly contribute to the detected elispot signals. altogether, these data demonstrate the ability of t-activated® ie- and pp proteins to stimulate a broad range of cmv-specific effector cells. these experiments also revealed the high heterogeneity of responses among healthy cmv-seropositive individuals. notably, the observation that some individuals respond to either ie- or pp emphasizes the importance of assessing the response to both antigens. monitoring both ie-1- and pp65-specific responses in the optimized ifn-γ elispot assay might improve the overall sensitivity of the test.fig. t-activated® pp and ie- cmv antigens stimulate a broad range of cmv-reactive effector cells. comparative analysis of ifn-γ secreting cells by elispot and flow cytometry following stimulation of pbmc with t-activated® pp and ie- antigens. pbmc from six cmv-seropositive healthy donors years; male and female) were stimulated with t-activated® pp and ie- antigens according to the optimized ifn-γ elispot and to the protocol of intracellular and surface marker staining and flow cytometry described in the methods section. bar graphs in depict ifn-γ-dependent sfc per pbmc, as before. bar graphs in represent the number of ifn-γ-expressing cd3+cd4+, cd3+cd8+, cd3−cd56+ and cd3+cd56+ cells per lymphocytes. note that the y-axis scales in were adjusted for a better resolution of the respective data. individual age and gender of donors were as follows: d 37-year-old male; d 55-year-old female; d 46-year-old female; d 41-year-old female; d 24-year-old female; d 22-year-old male discussion we describe here the development and technical validation of an optimized and standardized ifn-γ elispot protocol for the monitoring of cmv-reactive effector cells of cell-mediated immunity. the particularity of the assay is the use of t-activated® proteins as stimulant of cmv-reactive effector cells. we now demonstrate that urea-formulated ie- and pp proteins are capable of activating not only cmv-reactive cd4+ and cd8+ lymphocytes, but also innate lymphocytes, likely by bystander activation as well as by activation of cmv-specific memory nk cells. this finding is important given the acknowledged role played by these effector cells in the protection against cmv reactivation in vivo. inter-individual variability in the response of lymphocyte subpopulations was high among cmv-seropositive healthy donors, therefore further emphasizing the importance of an antigen formulation capable of activating a broad spectrum of cmv-reactive effector cells. in this, the optimized ifn-γ elispot assays outperforms existing elispot- and elisa-based cmv-specific cmi monitoring tools. in addition, the characteristics and high sensitivity of our assay predict a performance independent of the hla-type of the donor. the second particularity of the described assay is the combined use of two t-activated® proteins, ie- and pp as antigens. we show here that both antigens can elicit the activation of distinct cmv-specific effector cells and that while some cmv-seropositive healthy individuals are reactive to both antigens, some are only reactive to one, as previously reported. therefore, consideration of both test results is expected to improve the sensitivity of the assay, notably in cohort studies. this is particularly relevant for the monitoring of cmv-specific cmi in immunocompromized patients. accordingly, we recently demonstrated the benefit of measuring the response to both t-activated® ie- and pp antigens in terms of assay sensitivity, in clinical studies assessing cmv-specific cmi during pregnancy, in hemodialysis patients and in renal transplant recipients. the assessment of the response to both ie- and pp antigens is further justified by the respective relevance and differential contribution of ie-1- and pp65-reactive effector cells to the protection against cmv reactivation and related clinical complications, both in healthy cmv-seropositive individuals and in transplant recipients. the differential contribution of ie-1- and pp65-specific effector cells likely reflect the dynamic of the cell-mediated immune response. mechanisms of immune evasion inhibiting mhc-i-dependent ie- antigen processing and presentation might also explain the lower frequency of ie-1-specific ifn-γ-producing cells detected in our elispot assay. in addition, reduced processing and presentation efficiency due to protein stability, size and nuclear localization might play a role in the reduced reactivity to ie- as opposed to pp. this proposition is supported by the observation that higher concentrations of ie- were required in our elispot assay, in comparison to pp to trigger a significant response. another particularity of our ifn-γ elispot assay was its ability to elicit positive test results in cmv-seronegative individuals, notably following stimulation with ie- in the cohort of healthy donors, out of the cmv-seronegative individuals showed ie-1-specific values ≥  sfc/ pbmc, corresponding to a negative agreement with cmv igg-serology of 85%. similar results were obtained in a cohort of hemodialysis patients. numerous studies reported discordant results between cellular and humoral immunity against cmv. given the high sensitivity of our elispot assay and its ability to detect a broad range of cmv-reactive cells, it is possible that cellular reactivity in cmv-seronegative individuals reflects a previous exposure to cmv that failed to mount a humoral immune response, rather than a false-positive elispot result. further experiments will be necessary to address this possibility. nonetheless, this observation raises the question of the accuracy of cmv serology to identify immunocompromised patients at increased risk of cmv reactivation. the optimized and standardized ifn-γ elispot assay exhibited robust performance in terms of assay variability, precision and linearity. intra-assay, inter-assay and inter-operator coefficients of variations did not exceed 22%, which is below the range of recommended precision for cell-based assays. as predicted, inter-site variability involving different operators was higher, but remained acceptable with a cv of no more than 39%. this is an important criterion for the reliable longitudinal monitoring of cmv-specific immune responses in patients. linearity of the assay is also a critical parameter, in particular for the monitoring of immunocompromized patients with reduced and/or functionally impaired t cells, such as children or recipients of allogeneic stem cell transplantation. good linearity and high signal-to-noise ratio were obtained for both ie- and pp in the range of to pbmc per well. two multi-center clinical studies are currently being conducted, in renal transplant recipients under immunosuppressive therapy and in allogeneic stem cell transplantation patients, to assess the sensitivity of the optimized cmv-specific ifn-γ elispot assay in this clinical context and to correlate results of the assay to clinical outcome.  altogether, this optimized, standardized and user-friendly cmv-specific ifn-γ elispot assay meet all the conditions of a sensitive and reliable diagnostic test for the monitoring of the functionality of cmv-specific cmi. beside its validation in healthy individuals, its suitability to assess cmv-specific cell-mediated immunity in a cohort of hemodialysis patients representative of patients prior to transplantation has been demonstrated. further validation of the assay in immunocompromised patients, such as transplant recipients is necessary to determine its suitability in assisting clinicians to evaluate the risk of cmv reactivation and disease, and possibly individualize the therapeutic management of patients. additional files additional file 1: assay variability. intra-assay, inter-assay, inter-operator and inter-site variability was assessed as described in the respective legends. additional file 2: positivity cut-off definition. results obtained using the optimized ifn-γ elispot assay on pbmc isolated from whole blood of healthy donors were used to generate precision profiles for each ie- and pp stimulation. limits of quantitation for ie- and pp were and sfc/ pbmc respectively. loq calculated on a cohort of hemodialysis patients were in a similar range. therefore, a positivity cut-off of sfc/ pbmc was chosen for the standardized assay. in addition, intra-assay standard deviation within both cohorts of healthy donors and hemodialysis patients for stimulated and unstimulated measurements was the basis for the calculation of a criterion that the ratio of geometric means of stimulated to unstimulated values is at least. finally, considering a test result as positive when geometric mean for at least one of the ie- or pp stimulated approach is ≥  sfc/ pbmc, positive agreement and negative agreement of the optimized ifn-γ elispot test results with cmv serology within the collective of healthy donors was 97% and 85% respectively. additional file 3: gating strategy of flow cytometry analyses following surface marker and intracellular ifn-γ staining. pbmc from six cmv-seropositive healthy donors were stimulated with t-activated® pp and ie- antigens for 6 h, as indicated in the legend to fig.  cells were first gated on single events in a forward scatter-area x fsc-height dot plot. based on fsc-a x side scatter-area properties, a gate was next set around lymphocytes. by plotting cd against cd cd single positive cells, cd3/cd double positive cells and cd single positive cells were separated. cd single positive cells were further subdivided into cd3/cd double positive and cd3/cd double positive cells. representative dot plots of stained pbmc of donor d in each plot, percentage value in gated area is the mean frequency of ifn-γ-expressing cd3+cd4+, cd3+cd8+, cd3−cd56+ and cd3+cd56+ cells. abbreviations cmicell-mediated immunity cmvcytomegalovirus ctlcytotoxic t lymphocyte cvcoefficient of variation elisaenzyme-linked immunosorbent assay elispotenzyme-linked immunospot hlahuman leukocyte antigen icsintracellular cytokine staining ie-1immediate early- protein ifn-γinterferon gamma loqlimit of quantitation nknatural killer cell nktnatural killer t cell pbmcperipheral blood mononuclear cell pp6565 kda lower matrix phosphoprotein rsdrelative standard deviation sdstandard deviation sfcspot-forming cell tht helper cell 
remarkably, stimulation of peripheral blood mononuclear cells with t-activated® ie- and pp proteins resulted in the activation of a broad range of cmv-reactive effector cells, including cd3+cd4+, cd3+cd8+, cd3−cd56+ and cd3+cd56+ cells. objective of this work was the optimization and technical validation of an ifn-γ elispot assay for a standardized, sensitive and reliable quantification of cmv-reactive effector cells. accordingly, the optimized ifn-γ elispot assay revealed very high sensitivity in a cohort of healthy donors, of which were cmv igg-seropositive. the combined use of t-activated® ie- and pp proteins for the stimulation of pbmc with the optimized ifn-γ elispot assay represents a highly standardized, valuable tool to monitor the functionality of cmv-specific cmi with great sensitivity and reliability. close monitoring of cmv-specific cmi is therefore clinically relevant and might allow a reliable prognosis of cmv disease as well as assist personalized therapeutic decisions. all basic assay parameters and reagents were tested and optimized to establish a user-friendly protocol and maximize the signal-to-noise ratio of the elispot assay. functional impairment of cmi in immunocompromized individuals however can lead to uncontrolled cmv replication and severe clinical complications. optimized and standardized elispot revealed low intra-assay, inter-assay and inter-operator variability and cv inter-site was lower than 40%. in healthy individuals, cytomegalovirus infection is efficiently controlled by cmv-specific cell-mediated immunity.
 in healthy individuals, cytomegalovirus infection is efficiently controlled by cmv-specific cell-mediated immunity. functional impairment of cmi in immunocompromized individuals however can lead to uncontrolled cmv replication and severe clinical complications. close monitoring of cmv-specific cmi is therefore clinically relevant and might allow a reliable prognosis of cmv disease as well as assist personalized therapeutic decisions. methods objective of this work was the optimization and technical validation of an ifn-γ elispot assay for a standardized, sensitive and reliable quantification of cmv-reactive effector cells. t-activated® immunodominant cmv ie- and pp proteins were used as stimulants. all basic assay parameters and reagents were tested and optimized to establish a user-friendly protocol and maximize the signal-to-noise ratio of the elispot assay.  optimized and standardized elispot revealed low intra-assay, inter-assay and inter-operator variability and cv inter-site was lower than 40%. good assay linearity was obtained between × and × pbmc per well upon stimulation with t-activated® ie- and pp antigens. remarkably, stimulation of peripheral blood mononuclear cells with t-activated® ie- and pp proteins resulted in the activation of a broad range of cmv-reactive effector cells, including cd3+cd4+, cd3+cd8+, cd3−cd56+ and cd3+cd56+ cells. accordingly, the optimized ifn-γ elispot assay revealed very high sensitivity in a cohort of healthy donors, of which were cmv igg-seropositive.  the combined use of t-activated® ie- and pp proteins for the stimulation of pbmc with the optimized ifn-γ elispot assay represents a highly standardized, valuable tool to monitor the functionality of cmv-specific cmi with great sensitivity and reliability. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords cytomegaloviruscmvie-1pp65cell-mediated immunityelispotcd4+cd8+t helper cytotoxic t lymphocyte natural killer nkt-likehttp://dx.doi.org/ /501100002745bayerische forschungsstiftungaz 924-10az 1070- wagner ralf deml ludwig issue-copyright-statement© the author 2017 