The ELISpot Assay

29/9/22, 14:16 The ELISpot Assay Enables Functional Analysis of Cellular Immunology
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The ELISpot Assay Enables Functional Analysis of Cellular Immunology
The ELISpot Assay Enables Functional Analysis of

Cellular Immunology
ABSTRACT
Under optimal conditions, the enzyme-linked immunosorbent spot (ELISpot ) assay enables
visualization of multiple secretory products from a single responding cell. Thus, the ELISpot provides
both qualitative (type of immune protein) and quantitative (number of responding cells) information.
By virtue of this assay’s unsurpassed sensitivity, frequency analysis of rare cell populations (e.g.,
antigen-specific responses) which were not possible before are now relatively easy. Recent
improvements to the design of multiwell microplates, including use of membranes with reduced
background fluorescence, have bolstered the widespread application of Elispot assays. When assay
sensitivity, ease of use, and cost are all taken into consideration, the Elispot platform is likely the
superior choice for the development of multifunctional T cell assays for the research, therapeutic,
and diagnostic communities.
THE IMMUNE RESPONSE AND THE ELISPOT – NECESSITY IS THE MOTHER

OF INNOVATION
Precise regulation of effector function is critical to mounting a potent, yet specific immune response.
T lymphocytes provide the framework for this process, exquisitely orchestrating the body’s defense
against infections and cancer. This is accomplished through highly selective engagement and
activation of antigen-specific effector cell lineages. Depending on the strength and nature of the
stimuli, a wide range of effector functions may be elicited, including cytolytic activity, secretion of
multiple cytokines and other bio-active molecules, proliferation, and selective homing to sites of
infection. As these T lymphocytes and their responses represent true correlates of clinical outcome,
the ultimate goal in immune diagnostics has been to reliably identify that small fraction of
responders, qualify their mode(s) of action, and accurately quantify the degree of response. While no
one assay can measure all relevant parameters simultaneously, the ELISpot offers multi-dimensional,
quantitative assessment of effector function(s) at the single cell level with superior sensitivity and
resolution.
Developed in 1983, 1,2 the ELISpot assay represents the convergence of plate-based enzyme-linked
immunosorbent assays (ELISAs) with membrane-based Western blotting technologies, permitting
detection of secreted analytes at the single cell level. Membranes offer vastly improved binding
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characteristics over standard polystyrene surfaces. While a number of options exist, the majority of
Elispots are currently performed on polyvinylidene fluoride (PVDF) membrane plates. Binding of
capture antibody (Ab) is governed by hydrophobic interactions between amino acids such as
phenylalanine or leucine and PVDF; this association is much stronger than the electrostatic
3,4
interactions at nitrocellulose surfaces. Stronger binding interactions translate to greater Ab
4
density on the membrane’s surface, resulting in better-defined spots. Because the readout for an
ELISpot is "spots/well", the PVDF membrane’s white color provides the ideal backdrop for spot
detection and analysis. The microplate format further offers greater throughput and is amenable to
automation; more samples, more stimuli, or greater numbers of different cytokines can be assayed
simultaneously in neighboring wells.
1,2
Originally conceived for the enumeration of B cells secreting antigen-specific antibodies , the
ELISpot has been adapted for many tasks, the most prominent being the quantification of antigen-
specific cytokine responses (Figure 1). In the standard assay, cytokine-specific Abs are immobilized
on membrane-bottomed 96-well plates. Next, cells (commonly, total peripheral blood mononuclear
cells (PBMCs) or purified subsets) are seeded in the presence or absence of stimulating agents. Over
time, activated cells begin to secrete cytokines, which bind to the capture Ab in the immediate
vicinity of the expressing cell. Cells are then washed away and spot detection is accomplished
through substrate deposition following either a one-step (enzyme-conjugated, cytokine-specific Ab)
or two-step (biotinylated Ab/streptavidin-enzyme) antibody binding process. Once the signals are
developed, spot numbers can be tallied manually or through use of image-based spot readers with
accompanying analysis software. The frequency and total number of responder cells is determined by
comparing the number of spots between stimulated and untreated/control wells.
This review will highlight the unique performance characteristics, workflow attributes, and cost
benefits which, when considered together, clearly identify the ELISpot assay as an excellent platform
for elucidating the complexities underlying immune responses. In addition, we outline recent
advances related to this technology and how these improvements provide greater benefit to the
research community, whether focused on mechanistic studies, diagnostics, or therapeutic design.
Lastly, the assay protocol will be discussed in detail with an emphasis on standard best practices and
troubleshooting guides.
THE UNMATCHED POWER OF THE ELISPOT PLATFORM FOR T-CELL

FUNCTIONAL ANALYSIS
The complexity of any given immune response is underscored by the multitude of parameters that
may need to be assessed to gain clarity on the physiological mechanisms underlying the process.
While many assay formats exist, those most commonly used in the study of ex vivo T cell effector
function include flow cytometry, ELISpot, ELISA, multiplex bead arrays, and quantitative PCR. While
all have specific strengths and limitations, Elispot assays present clear advantages, which will be
highlighted in the following section.
The ELISpot , like flow cytometry-based intracellular cytokine staining (ICS), directly determine the
p , y y y g( ), y
frequency of antigen (Ag)-specific T cells, a core competency for immune diagnostics. Such resolving
power is unattainable with supernatant-based assays, such as ELISAs or multiplex bead arrays, where
measurements are based on bulk cytokine production by all cells in a given sample well. In acute HIV
subjects, the frequency of cells producing IFNγ in response to common recall antigens (e.g., TT or
5
PPD) was comparable to healthy donors; however, spot size is dramatically reduced . This result
suggests that HIV-specific T-cell function, and not cell number, was impaired. Similarly, T cells
recently activated in vivo may show increased per cell cytokine production when compared to
6,7
"e;older"e; memory T cells . The ability to distinguish between long-term memory and recently
activated subsets has implications for T cell diagnostics of autoimmune disorders and chronic
infections. Results from bulk assays are also confounded by the contribution of background signal(s)
from the innate immune system. Dilution of the Ag-specific response results in overall signal
flattening; this issue is most relevant for detecting the presence of rare populations, such as
circulating tumor cells (CTC) in PBMC or disseminated tumor cells in bone marrow, both early
8
markers of metastasis .
Figure 1. The ELISpot Assay Workflow. (1) Coat membrane with capture antibody. Add immune cells and stimulate.
(2) Responding cells produce cytokines. The cytokine of interest binds to the capture Ab beneath the cell. (3) Wash
to remove cells. Add a second cytokine-specific biotinylated Ab, which binds to the cytokine-Ab complex. (4) Add
streptavidin-enzyme conjugate. (5) Add enzyme substrate and develop. Within a well, each responding cell will
result in the development of one spot.
For the T cell repertoire to be capable of recognizing a potentially infinite number of infective agents
while simultaneously distinguishing them from self, the total naive pool contains ≥ 10 12 unique T-cell
receptor (TCR) specificities. Consequently, in the absence of infection, the frequency of circulating
memory cells with specificity to any one antigen is quite low, typically in the range of 1:10,000 -
1,000,000 9,10 . Detection of such rare events can present a significant challenge to flow-based
platforms, where the lower limit of sensitivity is reported to be 0.02% 11 . Relative to ELISpots, the
sensitivity threshold for cytokine measurements in culture supernatants is further diminished by
analyte dilution in the surrounding milieu, absorption by bystander cells, and enzymatic degradation.
By contrast, ELISpot assays demonstrate a detection threshold of less than 25 IFNγ-producing T cells
per million PBMC (0.0025%) 12,13 ; this equates to a near 10-fold increase in detection sensitivity. The
ELISpot assay’s high sensitivity is also important for allergy research, where identifying the very low
frequency Th2 cytokine-producing cells is critical for both disease monitoring and development of
immune therapies 14 Specifically both flow cytometry and ELISA platforms demonstrate insufficient
immune therapies . Specifically, both flow cytometry and ELISA platforms demonstrate insufficient
detection of IL-4, the predominant indicator of a Th2-driven response 15 .
The ELISpot is one of the few techniques permitting quantitative single cell analysis of biological
function (e.g., cytokine release). With intracellular cytokine staining (ICS), where cytokine detection
occurs prior to release, there is the potential for misleading results due to post-translational
modulation before or during the secretory process 16 . The duration of an ICS assay is limited by the
toxicity of protein transport inhibitors such as Brefeldin A or Momensin. For quantitative RT-PCR,
detection is even further removed from actual function, since the target being measured is mRNA.
ELISpot assays are also independent of secretion kinetics, a significant fact given the unsynchronized
nature of the responding T cells pool. For ICS, all cells are killed via fixation at a pre-determined time.
Cytolytic response mediators, such as granzyme B and perforin, are stored in granules then released
17-19
upon proper stimuli . Due to this unique regulatory mechanism, ICS will falsely identify all
effector memory cells (~20% of total T cells) as perforin-positive. Perhaps of greater significance is
the Lysispot assay, a modified ELISpot capable of enumerating Ag-specific cytotoxic CD8+ T cell
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effector function through direct target cell lysis . Until the development of the Lysispot assay, since
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most cytotoxicity assays are performed on bulk cultures , IFNγ ELISpots were commonly used as
22-24
correlates of CD8+ cellular immunity .Use of the Lysispot in the study of HIV revealed that not
25
all IFNγ producing cells were capable of killing . This finding also highlights the need for greater
multiplicity of detection in single cell immunoassays.
T cells occur in a wide range of effector classes, and expression of one or more of these can vary
greatly depending on the type of pathogen and the subject’s immune status. Cumulative findings in
the area of tuberculosis (TB) diagnostics suggest that differences in the cytokine signature may
provide a clearer distinction between asymptomatic latent and active forms of the infection. The
rapid identification of active cases is most critical as these individuals pose the greatest health risk to
26-30
the community . While bead-based quantitation in supernatants offers multiparameter analysis,
it suffers from limitations precluding acceptance as a diagnostic platform for TB and other diseases.
By contrast, ELISpots are amenable to multiplex analyses carried out simultaneously (single well) or
in parallel. Well-established dual-color ELISpots, using both enzymatic and fluorescent approaches,
are currently used in many research settings. Fluorescent ELISpots, or FluoroSpots, offer significant
advantages over colorimetric formats, particularly in the areas of multiplexing and automated spot
detection. Moreover, as spot development is not enzymatic, signal intensity is directly proportional to
the amount of analyte within the spot and therefore far more quantitative.
Increasing the multiplexing capacity beyond two colors requires membrane surfaces with minimal
fluorescent background signal. Due to their highly porous nature, membrane surfaces are very rough.
For this reason, they scatter light and exhibit high fluorescence background. While PVDF membrane
®
(Immobilon -P membrane) is purported to be a better surface than nitrocellulose for FluoroSpots,
®
the Immobilon -FL PVDF membrane variant was designed specifically for fluorescence detection in
Western blotting applications and exhibits background fluorescence signal that is nearly 1/100 that
®
of standard PVDF Data showing the use of Immobilon -FL membrane in two-color IFNγ/IL-2
of standard PVDF. Data showing the use of Immobilon FL membrane in two color IFNγ/IL 2
FluoroSpots is presented in Figure 2 (images) and Figure 5 (page 6, spot counts). Beyond multiplexing,
FluoroSpots permit distinction of two simultaneously measured functional outputs. Multiplexing also
serves to reduce required sample size. The ever-expanding availability of discrete fluorochromes,
when combined with multi-fluorescent imaging instrumentation and fully automated sample
acquisition and data analysis, provides the framework for unsurpassed polyfunctional analysis of Ag-
specific T cell responses via ELISpots.
Figure 2. Representative images from two-color IFNγ/IL-2 FluoroSpot assays performed on the Multiscreen®HTS
plates fitted with FluoroSpot-optimized Immobilon®-FL PVDF membrane. CEF pool refers to pool of peptides
covering epitopes of Cytomegalovirus, Epstein-Barr virus and flu virus.
Beyond the membrane and plate material, plate color can also greatly impact the success of the
FluoroSpot. The data presented in Figure 3 highlights the differences in image quality between
different Multiscreen ® HTS plate formats. IFNγ/IL-2 FluoroSpots were performed on PBMC cells
following culture (250K/well) in the presence of CEF peptides. From a strictly visual perspective, spot
clarity was roughly equivalent on the clear and black formats (Figure 3A). By contrast, white plates
showed high background signal, making spot detection difficult, particularly in the Green channel
(IFNγ-FITC). The high background occurred even after a significant reduction in exposure time
(roughly 1/5). High background was most likely due to increased reflectance as compared to black or
clear frames where light is either absorbed by or passes through the surrounding plate material
clear frames where light is either absorbed by or passes through the surrounding plate material,
respectively. A comparison of spot counts demonstrated a significant reduction in "spots counted" on
white plates when compared to either black or clear formats (Figure 3B-C). Once again, the
discrepancy was more significant for IFNγ spots, where almost 80% reduction in total spots was
observed. The background issue may be eliminated if wells were punched out and analyzed
separately; however, this may not be practical if large experiments are to be performed. Given their
similarities in performance, the clear plate format offers the more practical option, as it also
facilitates visually monitoring reagent addition. It should be noted that all analyses were performed
using the iSpot™ system (AID). Due to differences in performance characteristics, other fluorescent
plate readers, may not demonstrate the same plate preference.
Figure 3. Shown are representative well images for dual color IFNγ/IL-2 FluoroSpot performed on total PBMC in
three different Multiscreen®HTS plate formats (Clear, Black, and White) using Mabtech’s FluoroSpot kits. For CEF-
stimulated wells, displayed are images for each individual cytokine as well as the overlay image. For unstimulated
wells, only the IFNγ single color data are shown. (B) The graph presents summation data for each plate format
across three culture conditions. Each bar is segregated into three parts – IFNγ, IL-2, and dual responder spots. All
bars represent the average of 3 replicates. (C) The two bar graphs show comparative spot data for each cytokine
measured. All bars represent the average of 3 replicates.
Unlike flow cytometry, where instrument priming can result in sample loss, every cell in an ELISpot is
measured. The ELISpot also, on average, requires one-tenth as many cells per test, which provides a
crucial advantage under conditions where samples are precious (remote settings) and/or limiting
(pediatric or immunosuppressed test subjects). One long-standing problem with the 96-well
microplates has been the waste of unused wells in small-scale assays such as that occurring in
diagnostic analysis of a single patient sample. We offer 8-well strips (Product No. M8IPS4510)
designed for the diagnostic community; this format is particularly attractive to resource-limited
countries where diseases such as TB and HIV are most devastating (Figure 4A). 28 Constructed in a
transparent format, the strips are suitable for FluoroSpots, as well as standard enzymatic options,
and perform comparably to the standard 96-well plate (Figure 4B, 5). The 8-well strips are currently
part of Oxford Immunotech’s T-SPOT.TB Test, an FDA-approved IFNγ ELISpot test designed
specifically for diagnosis of tuberculosis infection.
Figure 4. (A) Membrane-bottom, 8-well strip plate designed specifically for diagnostic ELISpot. This format is part
of T-SPOT.TB (Oxford Immunotech), a commercially available IFNγ ELISpot kit designed specifically as a diagnostic
for tuberculosis infection. (B) Representative images (single and overlay) from two-color FluoroSpot performed in
8-well strips. IFNγ/IL-2 FluoroSpots were performed on healthy untreated PBMCs left untreated and following
stimulation with CEF peptides. All assays were performed using FluoroSpot kits (Mabtech).
Figure 5. 8-well strips perform similarly to the standard 96-well MultiScreen® plates. The bar graph presents
summation data for each format across three culture conditions. Each bar is segregated into three parts – IFNγ, IL-
2, and dual responder spots. All bars represent the average of 3 replicates. Working with far fewer cells per assay
also means that multiple replicates can be performed, thereby increasing statistical power. Such discriminatory
capacity is not possible with bulk assays. Although ELISpot and flow cytometry assays have similar protocol steps,
ELISpot data acquisition/analysis is far easier to perform and less time-consuming than flow cytometry. In fact, data
from a 96-well ELISpot plate can be acquired and analyzed by an image-based platform as rapidly as it takes a
skilled flow cytometer operator to analyze one sample containing 300,000 cells. For certain cytokines, the
signal:noise ratios for ICS are low and often non-bimodally distributed, making gating decisions arbitrary and
difficult. While flow cytometry struggles with a lack of user-independent gating algorithms for sub-population
analysis, and therefore suffers from subjectivity and lab-to- lab variation, automated platforms promote the
standardization of ELISpot data analysis and greater reproducibility across sites31. This combination of features
also makes ELISpot the ideal choice for high-throughput testing applications, which could be applied in large-scale
subject profiling. For example, IFNγ ELISpots are commonly used as a correlate of vaccine efficacy to identify
potential candidates for HIV and other diseases.32-33
Assay miniaturization can simultaneously reduce cell requirements while increasing throughput. The
data presented in Figure 6 is part of ongoing studies performed at Cellular Technology Limited (CTL)
34
to validate the application of ELISpots to a 384-well format. In this example, IFNγ ELISpots were
performed on PBMCs following stimulation with CEF-7 peptide. Plates were imaged and analyzed
®
using CTL’s ImmunoSpot S6 Micro Analyzer. For the range of seeding densities tested, the assay
demonstrated a strong linear relationship (R2=0.9866) between spot-forming units (SFU) and cell
number (Figure 6B). Lastly, modifications to microplate design have increased compatibility with
existing robotics systems, thereby also improving potential throughput. These plate adaptations
include stricter dimensional specifications and rigid side walls. Plates are now fully compatible with
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standard fluidics platforms, plate washers, and devices for imaging and image analysis.
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Figure 6. (A) A representative image from an IFNγ ELISpot performed in 384-well plates on four seeding densities
of PBMC (n=96 per level). Well F12 has been magnified to demonstrate spot clarity. (B) In this graph, IFNγ spot
forming units (SFU) were plotted against seeding density. Each data point represents the average of 96 replicates
and error bars represent standard deviation. The assay shows a strong linear response for range of cell quantities
tested.
ELISPOT OPTIMIZATION
While ELISpot assays permit frequency determination for very rare events, data interpretation can
become ambiguous when (1) spot numbers in antigen-containing wells are low, (2) spot counts in
negative control wells are elevated, and particularly when both occur simultaneously. Thus, the
primary task, even before statistics are employed, must be the optimization of basic assay
parameters and reagents to maximize the signal-to- noise ratio. While the use of highly specific
ELISpot–validated Ab pairs is key to assay success, proper consideration and execution of a number
of other steps are required to ensure optimal performance. This section will provide an overview of
the standard T cell ELISpot assay with particular emphasis placed on the experimental rationale
behind underlying pivotal steps and suggestions for troubleshooting erroneous or ambiguous results.
INITIAL THOUGHTS
Choice of Plate (Membrane) – PVDF membrane plates (Catalogue Nos. MSIPS4W10, MSIPS4510,
MAIPSWU10, MAIPS4510) are recommended, over a mixed cellulose ester format (Catalogue No.
MSHAS4510), due to slightly improved binding of capture Ab and superior performance in spot
detection, particularly for fluorescent applications. The one drawback of PVDF plates is the extreme
hydrophobicity of the material, 33 a property that may necessitate pre-wetting with alcohol prior to
addition of the coating Ab. The potential pitfalls of this step are outlined in a later section. Since the
mixed cellulose membrane is hydrophilic, ELISpots can be performed without pre-wetting, 36,37
Negative/Positive Controls – Relevant controls are crucial to measuring Ag-specific responses via
ELISpot. Negative controls routinely consist of cells cultured without stimuli, whereas polyclonal T-
cell activators are commonly used as positive controls to confirm both cell and assay functionality.
Positive controls include anti-CD3/CD28 Abs, phytohemagglutinin (PHA) and concanavalin A
(ConA). These activators induce secretion of many common cytokines including IFNγ, IL-2 (Th1), IL-4,
IL-5, IL-10 and IL-13 (Th2). Another common control is the commercially available CEF
(Cytomegalovirus, Epstein-Barr virus, Influenza virus) peptide pools. These consist of multiple
epitopes from each of the three viruses, to which most healthy individuals (~ 90%) possess CD8-
responding T-cells. 38
Plate organization - Edge effects – The plate is an array of 8 rows with 12 wells in each. Wells at the
periphery of the plate (columns 1 and 12, Rows A and H) are in greater direct contact with
surrounding environment and thus may differ from interior wells. Specifically, medium evaporation
from peripheral wells in prolonged cultures may impact overall assay performance. Where possible,
the use of "media only" wells around the periphery of the true sample wells can minimize this effect.
THE QUESTION OF PRE-WETTING

Comparative testing has previously demonstrated that proper ethanol pre-treatment of PVDF-based
MultiScreen ® HTS plates (15 µL of freshly prepared 35% v/v ethanol followed immediately by water
washes) can lead to increased spot number (better sensitivity) and more sharply defined spots (for
more accurate quantitation) (Figure 7) 35 . That said, pre-wetting is not universally applicable to all
ELISpots ; its requirement is dependent on the inherent hydrophobicity of the capture Ab; therefore,
the pre-wetting protocol should be optimized prior to application 35,36 . Overtreatment with larger
volumes of alcohol, longer exposure time, or more concentrated alcohol can lead to trapping of
residual liquid between the membrane and underdrain, which may result in poor assay performance
or, more critically, well leakage. Leakage associated with alcohol pre-wetting is not a concern when
using ELISpot plates lacking an underdrain (Catalogue No. MAIPSWU10); however, this format may
suffer from potential media evaporation during extended culturing as well as sterility issues
surrounding the exposed base membrane. Another alternative is to use microplates made with
hydrophilic membrane such as mixed cellulose ester. It is also important to note that once plates are
ethanol-treated, they must be kept wet for the entire assay.
Figure 7. (A) Representative well images for mouse IFN gamma ELISpot performed on PVDF membrane-bottom
plates with and without ethanol pre-wetting. (B) A schematic showing the architecture of an individual well in a
MultiScreen®HTS plate. Recent design changes to increase the spacing between the base of the membrane and top
of the underdrain have reduced the incidence of leaking.
COATING WITH CAPTURE ANTIBODY

In traditional ELISAs, binding to the surface occurs via passive adsorption and requires alkaline
conditions (0.2 M sodium carbonate/bicarbonate pH>9) to maximize the electrostatic component of
the protein:polystyrene interaction. By contrast, coating of PVDF membranes for ELISpot is
mediated solely by hydrophobic forces. For this reason, a phosphate-buffered saline (PBS) buffer (pH
7.4) is commonly used. Membranes also offer significantly greater surface area (300X) for binding
than do polystyrene plates 39 . To ensure performance while maximizing cost efficiency, it is critical to
standardize the amount of capture Ab used per well. For optimal performance, we recommend initial
titration of both the coating and detection Abs in tandem. Typically, a good starting point for ELISpot
coating is 0.5-1 µg Ab per well (5-10 µ/mLin 100 µL); this is 5-10X greater input than for ELISAs.
Lower input can result in more diffuse spot morphology as well as reduced spot number. Both
parameters need to be considered when validating new assay protocols, particularly when
determining quantitative expression (spot size) or low frequency events, respectively. Mabtech offers
a wide range of fully validated ELISpot Ab pairs (coating and detection) for the assay of human
samples as well as other species (For details, see www.mabtech.com).
BLOCKING STEP
Following incubation with capture Ab, plates should be washed extensively, then blocked and
equilibrated for 2 hours at 37 °C with the same culture medium (200 µL/well) that will be used during
cell stimulation (minus activator). Once in blocking medium, sealed PVDF plates can be stored
overnight at 4 °C. Longer storage can result in protein precipitation and reduced spot resolution. It is
important to note that when plates are removed from 4 °C and allowed to reach room temperature,
the sealing tape must also be removed to prevent leakage due to gas expansion.
CELLS - PLATING AND STIMULATION

Fresh vs. frozen - PBMCs or enriched T-cell subsets constitute the bulk of ELISpot assays. Once
40
purified from blood, PBMC can be cryopreserved and thawed without loss of functional activity .
Given the often precious nature of disease samples, there are multiple benefits to cryopreservation:
(1) data can be independently reproduced, (2) multiple analytes can be assessed, (3) patient samples
can be stockpiled and assayed simultaneously, thus minimizing the potential inter-assay variability.
For optimal recovery of viable cells, follow these recommendations: (A) during freezing, have cells
and freezing medium at room temperature prior to mixing and (B) during thawing, to minimize
osmotic lysis during washing of thawed cells, transfer cells to a 15 mL conical tube on ice and slowly
add cold medium.
The benefit of Benzonase ® nuclease - An additional challenge with using frozen PBMCs is cell
clumping during the thawing process. Clumping is often caused by the presence of free DNA and cell
debris; it appears to be related to both the donor source and blood handling. In particular, clumping
occurs more frequently when blood has been stored overnight prior to PBMC isolation. Spot count
results for overnight-stored blood showed a dramatic decrease when compared with responses for
the corresponding PBMCs isolated from fresh blood. 36 The greatest decreases in signal were
detected for samples in which the highest degree of cell clumping was observed. To improve assay
performance, we recommend addition of Benzonase ® nuclease (Product No. 71205), which degrades
all forms of DNA and RNA, to the assay medium for the first two wash steps during the thawing
procedure. The results from overnight blood PBMCs processed with Benzonase ® nuclease more
closely approximate the results obtained with cells isolated from fresh blood. Moreover, Benzonase ®
nuclease addition resulted in no changes to cell viability or changes in the expression of certain
surface markers, including CD 4 , CD 8 , CD 38 , or CD 62 L. 36
Cell counting and the value of percent viability – Once Ab steps have been standardized, differences
in quantified cell yield and integrity of each sample presents the greatest source of assay variability.
While total cell counts are important, a more critical factor to consider when setting up the culturing
component is cell viability. Determining the percentage of dead and apoptotic cells is not only
important for culture setup, it also provides quantitative information on the overall quality of the
sample. The latter component is particularly useful when assessing success/failure of the freeze-
thaw process. Manual counting methods, such as Trypan Blue exclusion using a hemacytometer, lack
accuracy due to user subjectivity. Further, these methods do not provide a measurement of the
apoptotic fraction. Automated cell counting via flow cytometry using fluorescent dyes, such as the
Muse™ cell analyzer and ViaCount ® reagent, demonstrated superior precision to manual methods
for the enumeration of viable cells. 41
Cells per well and replicates – On average, T-cell ELISpot counts show linearity for PBMCs in the
range of 100,000-800,000 cells 5,9-13 . Where possible, cells should be serially diluted and plated in
2
triplicate. Unfortunately, given the restrictions of well size in 96-well plates (0.3 cm ), seeding more
than 400,000 cells per well may result in overcrowding and cell stacking. The consequence here is
creation of diffuse spots due to indirect contact of the cells with the Ab-coated membrane. To best
monitor instances where the frequency of Ag-specific responders is low, and higher cell loads are
required, either perform assays in larger wells or perform replicate wells at maximal cell density. By
using replicate wells, spot counts from all the wells can be summed to derive the response frequency
(SFU/total cells seeded).
During incubation, we do not recommend plate-stacking, as this can lead to variations in temperature
between the plates and potentially differences in spot size and/or number. Also, it is important that
plates are subject to minimal agitation, because movement can lead to localized cytokine diffusion
and loss of spot sharpness.
DETECTION - CHROMOGENIC VS. FLUORESCENT OPTIONS

Following stimulation, cells are removed, wells extensively washed, and a second analyte-specific Ab
is applied. At this and all subsequent steps, washing is critical for the complete removal of cells,
nonspecifically bound Ab, and detection reagent. Incomplete removal of unbound reagents will lead
to an overall increase in background signal. Potential challenges surrounding washing and spot
development will be addressed in the troubleshooting section (page 10).
ELISpot assays may be performed either with antibodies directly conjugated to the detection motif
(enzyme or fluorochrome) or as a two-step process involving a biotin/streptavidin-conjugated Ab
pair. While the two-step process offers greater intensity due to signal amplification, and therefore
may be preferable in cases where cytokine production per cell is low (allergy/Th2 responses), this
protocol also suffers from a greater potential for background staining due to nonspecific interaction
with the coating Ab. With enzymes, such as horseradish peroxidase (HRP), a precipitating substrate
(TMB or AEC) is used for spot detection. Due to HRP’s high turnover rate, spot development is fast
(≤5 minutes). By contrast, spot development using alkaline phosphatase-conjugated Abs is far slower
but with apprecibly lower background. For chromogenic assays performed on MultiScreen ® HTS
plates (those with underdrains), it is recommended that the underdrain be removed before substrate
addition; failure to do so can result in high background staining. Once removed, plates should be
propped up to minimize membrane contact. To enhance spot visualization, plates should be dried
without a lid, upside down, at room temperature for several hours. For long-term storage, plates
should be kept in a dark, dry place at room temperature to prevent bleaching of spots.
As previously discussed, the use of fluorescent conjugates offers significant advantages over
colorimetric schemes especially for dual cytokine applications or where greater quantitative
assessments of individual spots is desired. While FITC- and Cy3-conjugated Abs are commonly used,
the choice of fluorescent probe is limited only by the availability of conjugates and detection
platforms.
SPOT COUNTING AND ANALYSIS – WHAT IS A REAL SPOT?

Each spot represents the ‘cytokine signature’ of a single cell. Due to diffusion properties, a true spot
p p y g g p p , p
has a densely colored center which fades toward the edges; the size and/or color intensity of the
spots is determined by the amount of cytokine released. That said, due to differences in analyte
measured, incubation time, antibody concentration, enzyme activity, substrates and other materials
used as well as the functional state of the cytokine-secreting cells, spot size and density can vary
greatly. Artifactual spots may appear and can be caused by the aggregation of antibodies or the
incomplete removal of cells and cellular debris. Morphologically, these spots can be differentiated
from ‘true’ spots by their homogeneity in color intensity and sharper (non-rounded) edges.
From the above description, manual spot counting by light microscopy would be classified as a highly
subjective process, fraught with a great degree of inter-user variability. Further, when considering
the sheer number of wells that may need to quantified in a standard vaccine trials, the task of ELISpot
data analysis becomes a far too laborious task for human eyes. The availability of sophisticated
ELISpot readers offers a complete solution for precise evaluation of spot data. These instruments
include features to overcome problems with variable background intensity and the ability to
distinguish true single cell spots from artifacts. The latter capability relies upon the use of minimum
and maximum threshold values for spot size and intensity, permitting the exclusion of weak
bystander responses and clusters containing multiple cells, respectively. Beyond speed, spot analysis
software offers process standardization, a critical component when studies are performed across
sites, such as is the case for diagnostic testing and vaccine trials. Moreover, ELISpot readers and
analysis software open the door for more precise measurements of spots, permitting the
quantitation of secretion of multiple cytokines on a per-cell basis.
A troubleshooting guide for interpreting and correcting ambiguous Elispot result
Issue Solution or Explanation
1. High levels of background staining
Were cells washed prior to the incubation Washing prevents the carryover of secreted cytokines
step? in the preincubation medium.
®
Antibody filtration using Steriflip (Catalogue No.
®
SE1M003M00) or Millex filters (Catalogue No.
Was the secondary/detection antibody
SLHV033RS) reduces background staining or false
filtered prior to use?
positive spots that may arise due to protein
aggregates.
As plate washers are less vigorous than manual

Was the recommended number of wash
methods, we recommend 1.5X the standard number
steps performed throughout the assay?
of washes if a plate washer is used.
p
A troubleshooting guide for interpreting and correcting ambiguous Elispot result
Were sterile technique and reagents Culture contaminants may result in nonspecific
employed during assay execution? background staining.
Wet/damp membranes can display a dark blue

background color. Drying overnight at 4 °C may
Was the membrane dried completely prior to
increase contrast between background and spots.
analysis?
Drying at temperatures greater than 37 °C may cause
membrane cracking.
Due to diffusion, background staining or diffuse spots

Was the plate moved/knocked during
can arise if plates are moved during the cell incubation
incubation?
step.
Was the percentage of live/dead cells A high number of dead cells may result in high
estimated prior to incubation? background staining and/or lack of spots.
Was the cell seeding density optimized prior We recommend prior optimization of input cell
to the commencement of the assay? number and stimuli concentration.
Was the secondary/detection antibody

Excess biotinylated secondary/detection antibody or
concentration, enzyme conjugate (HRP-
enzyme conjugate is likely to contribute to
Streptavidin or AP-Streptavidin) and enzyme
background. A reduction in the concentration of
substrate optimized prior to the
reagents or reaction time will reduce background.
commencement of the assay?
Some PBS formulations may benefit from filtering

Was PBS filtered prior to use?
with a 0.2 µM filter prior to use.
2. No spots/blank wells
Was a membrane pre-

Inadequate pre-wetting may result in an absence of signal, non-
wetting step performed?
staining areas or poorly defined spots due to poor capture Ab
Did the membrane turn
binding. Make sure that the 35% EtOH is prepared immediately
gray/translucent after pre-
before use and is a true 35% solution (not 35% of 95% EtOH).
i ?
be o e use a d s a t ue 35% so ut o ( ot 35% o 5% tO ).
wetting?
Have you chosen the Ensure that the capture and detection Abs react with different
correct antibody pairs? antigenic epitopes.
Was the cell seeding

density optimized prior to An absence of spots may indicate that the frequency of
the commencement of the responder cells is very low.
study?
Have you stimulated your

For T cell responses, we recommend using a polyclonal activator
cytokine/protein of
such as PHA for a positive control.
interest appropriately?
Did the culture medium

If so, a high percentage of cells may have undergone
turn yellow during
apoptotic/necrotic cell death.
stimulation?
Were cells resuspended

into a single cell
Clumping may lead to the underestimation of spot-forming cells
suspension prior to
and inconsistent results.
addition to the Elispot
plate?
Were your cells stored Cell viability should be assessed prior to culture set-up and
appropriately prior to stimulation. We recommend the guava easyCyte™ benchtop
®
stimulation? flow cytometry system and ViaCount reagent
Was PBST (PBS + 0.5%

Tween ® 20) used for the Detergents, such as Tween ® -20, can inhibit enzyme reactions.
final wash before spot Use “PBS only” for final wash steps.
development?
3. Fuzzy/poorly
defined/conflue nt
spots
Was a pre- Pre-wetting is not universally applicable to all Elispots; its requirement is
wetting step dependent on the inherent hydrophobicity of the capture Ab; therefore,
f d? th tti t l h ld b ti i d i t li ti
performed? the pre-wetting protocol should be optimized prior to application.
Was
primary/capture
A common cause of large, diffuse spots is insufficient capture antibody. It
antibody
is good practice to determine optimal Ab concentrations before use.
concentration
Using validated kits will ensure that all reagent concentrations are
optimized prior
optimal.
to starting the
assay?
Were plates
stacked during Stacking plates may affect the even distribution of heat across plates or
the incubation individual wells.
step?
Were the
developing
The HRP and AP enzymatic reaction(s) perform optimally at room
reagents allowed
temperature. Poorly defined spots may be the result of
to come to room
underdevelopment due to addition of cold substrate.
temperature
prior to use?
Was incubation The longer cells are incubated, the more cytokine/protein they will
time optimized secrete, resulting in larger spots that start to merge and become
prior to indistinguishable. Incubation time can vary (18-48 hours) according to
commencement cell type and cytokine/protein of interest. The amount of stimulant may
of the assay? also require optimization.
Was the plate

allowed to dry Drying overnight at 4 °C may help increase the contrast between
completely background and spots.
before reading?
ACKNOWLEDGEMENTS
We would like to thank Tomas Ernemar (Mabtech) for providing all the FluoroSpot data as well as
manuscript review. The 384-well data was kindly provided by Jodie Hansen (CTL). We would also like
to thank Sylvia Janetzki (ZellNet Consulting) for manuscript review.
Referencia del producto Descripción SDS Precios
™
Brightmax Sealing Films
Z732117 Expandir
for luminescence and microscopy, non-sterile
Referencia del producto Descripción ® SDS Precios

ExtrAvidin –Alkaline Phosphatase
E2636 Expandir
buffered aqueous solution
Monoclonal Anti-β-Galactosidase−Biotin
Conjugate antibody produced in mouse
B0271 Expandir
clone GAL-13, purified immunoglobulin,
IL-2 ELISPOT Antibody Pair, Mouse

ELI-002-M Expandir
from rat, clone JES6-1A12, clone JES6-5H4

ELI-004-M Expandir
from rat, clone 1B11, clone BVD6-24G2

ELI-006-M Expandir
from rat, clone TRFK5, clone TRFK4

ELI-008-M Expandir
from rat, clone MP5-20F3, clone MP5-32C11
Anti-Mouse IgG (Fc specific)–Alkaline

Phosphatase antibody produced in goat
A1418 Expandir
affinity isolated antibody, buffered aqueous
solution
Anti-Mouse IgG (Fc specific)–Biotin antibody
produced in goat
B9904 Expandir
affinity isolated antibody, buffered aqueous
solution
MultiScreen-BV Filter Plate, 1.2 µm, clear,
non-sterile
MABVN1250 Receptor binding assays, compound Expandir
generation sample prep, resin/bead based
assays, sample prep
MultiScreen-BV Filter Plate, 1.2 µm, opaque,
non-sterile
MABVN0B50 Expandir
Receptor binding assays, resin/bead based
assays
MultiScreen Carrier Rack for 4 mL vials
This carrier rack for 4 ml vial is used for
MACR08124-M Expandir
Radionucleotide counting by transfer of
individual filters to tubes.
MultiScreen Carrier Rack for 12 mm x 75 mm
tubes
MACR81275-M This carrier rack for 12mm x 75mm tubes is Expandir
Referencia del producto used for Radionucleotide counting by transfer

Descripción SDS Precios
of individual filters to tubes.
MultiScreen Centrifuge Alignment Frame,
blue, aqueous applications
MACF09604 This Centrifuge Alignment Frame is used to Expandir
align MultiScreen plate to 96-well receiver
plate for centrifugation of aqueous media.
MultiScreen Column Loader

MACL09625 Expandir
25µL, Black, Non-Sterile

MACL09645 Expandir

MACL09680 Expandir

MACL09600 Expandir
MultiScreen Deep, 96 Well Solvinert

Opaque, Non-Sterile, 0.45 µm pore size,
MDRLN0410 Expandir
Hydrophilic Polytetrafluoroethylene (PTFE),
10 plates, volume 1.9mL
Clear, Non-Sterile, 0.45 µm pore size,
MDRPNP410 Expandir
Hydrophobic Polytetrafluoroethylene (PTFE),
10 plates, volume 1.9mL with prefilter
Mostrar más
Materials
Referencia del producto Descripción SDS Precios
™
Brightmax Sealing Films
Z732117 Expandir
for luminescence and microscopy, non-sterile
®
ExtrAvidin –Alkaline Phosphatase
E2636 Expandir
Monoclonal Anti-β-Galactosidase−Biotin Conjugate
antibody produced in mouse
B0271 Expandir
clone GAL-13, purified immunoglobulin, buffered
aqueous solution
ELI-002-M Expandir
from rat, clone JES6-1A12, clone JES6-5H4
ELI-004-M Expandir
from rat, clone 1B11, clone BVD6-24G2
IL 5 ELISPOT Antibody Pair, Mouse
ELI-006-M
Referencia del producto Descripción SDS Expandir
Precios
from rat, clone TRFK5, clone TRFK4
ELI-008-M Expandir
from rat, clone MP5-20F3, clone MP5-32C11
Anti-Mouse IgG (Fc specific)–Alkaline Phosphatase
A1418 antibody produced in goat Expandir
affinity isolated antibody, buffered aqueous solution
Anti-Mouse IgG (Fc specific)–Biotin antibody
B9904 produced in goat Expandir
affinity isolated antibody, buffered aqueous solution
MultiScreen-BV Filter Plate, 1.2 µm, clear, non-

sterile
MABVN1250 Expandir
Receptor binding assays, compound generation sample
prep, resin/bead based assays, sample prep
MultiScreen-BV Filter Plate, 1.2 µm, opaque, non-
MABVN0B50 sterile Expandir
Receptor binding assays, resin/bead based assays
MultiScreen Carrier Rack for 4 mL vials
This carrier rack for 4 ml vial is used for
Radionucleotide counting by transfer of individual
filters to tubes.
MultiScreen Carrier Rack for 12 mm x 75 mm tubes
This carrier rack for 12mm x 75mm tubes is used for
Radionucleotide counting by transfer of individual
filters to tubes.
MultiScreen Centrifuge Alignment Frame, blue,
aqueous applications
MACF09604 This Centrifuge Alignment Frame is used to align Expandir
MultiScreen plate to 96-well receiver plate for
centrifugation of aqueous media.
MACL09625 Expandir
MACL09645 Expandir
MACL09680 Expandir
MACL09600 Expandir
Opaque, Non-Sterile, 0.45 µm pore size, Hydrophilic
MDRLN0410 Expandir
Polytetrafluoroethylene (PTFE), 10 plates, volume
1.9mL
Referencia del producto Clear, Non-Sterile, 0.45 µm pore size, Hydrophobic

Descripción SDS Precios
MDRPNP410 Expandir
Polytetrafluoroethylene (PTFE), 10 plates, volume
1.9mL with prefilter
Mostrar más
References
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6/0022-1759(83)90308-3
2. Sedgwick J, Holt P. 1983. A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting cells.
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3. Starita-Geribaldi M, Sudaka P. 1990. Fast electrotransfer of human serum proteins in their native state from polyacrylamide thin
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799(91)90064-o
5. Helms T, Boehm BO, Asaad RJ, Trezza RP, Lehmann PV, Tary-Lehmann M. 2000. Direct Visualization of Cytokine-Producing Recall
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4049/jimmunol.164.7.3723
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2)00328-9
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9(97)00184-1
13. Streeck H, Frahm N, Walker BD. 2009. The role of IFN-? Elispot assay in HIV vaccine research. Nat Protoc. 4(4):461-469. http://dx.
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325-7_1
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10.1089/aid.2007.0125
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cytotoxic T cells by the Lysispot assay: independent regulation of cytokine secretion and short-term killing. Nat Med. 9(2):231-236.
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21. Brunner K, Mauel J, Cerottini J, Chapuis B. 1968. Quantitative assay of the lytic action of immune lymphoid cells of 51Cr-labelled
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Marker for Circulating Cytotoxic T Lymphocytes That Recognize an HLA A2-Restricted Epitope of Human Cytomegalovirus
Phosphoprotein pp65. Clin. Diagn. Lab. Immunol.. 8(3):628-631. http://dx.doi.org/10.1128/cdli.8.3.628-631.2001
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24. Betts MR. 2006. HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells. Blood. 107(12):4781-
4789. http://dx.doi.org/10.1182/blood-2005-12-4818
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Measured Directly Ex Vivo by the Lysispot Assay Can Be Higher or Lower Than the Frequencies of IFN-?-Secreting Cells: Anti-HIV
Cytotoxicity Is Not Generally Impaired Relative to Other Chronic Virus Responses. J Immunol. 176(4):2662-2668. http://dx.doi.or
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26. Casey R, Blumenkrantz D, Millington K, Montamat-Sicotte D, Kon OM, Wickremasinghe M, Bremang S, Magtoto M, Sridhar S,
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Tuberculosis. PLoS ONE. 5(12):e15619. http://dx.doi.org/10.1371/journal.pone.0015619
27. Sester U, Fousse M, Dirks J, Mack U, Prasse A, Singh M, Lalvani A, Sester M. Whole-Blood Flow-Cytometric Analysis of Antigen-
Specific CD4 T-Cell Cytokine Profiles Distinguishes Active Tuberculosis from Non-Active States. PLoS ONE. 6(3):e17813. http://d
x.doi.org/10.1371/journal.pone.0017813
28. Hirsch CS, Hussain R, Toossi Z, Dawood G, Shahid F, Ellner JJ. 1996. Cross-modulation by transforming growth factor beta in
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Academy of Sciences. 93(8):3193-3198. http://dx.doi.org/10.1073/pnas.93.8.3193
29. Harris J, De Haro SA, Master SS, Keane J, Roberts EA, Delgado M, Deretic V. 2007. T Helper 2 Cytokines Inhibit Autophagic
Control of Intracellular Mycobacterium tuberculosis. Immunity. 27(3):505-517. http://dx.doi.org/10.1016/j.immuni.2007.07.022
30. Tincati C, Cappione III AJ, Snyder-Cappione JE. Distinguishing Latent from Active Mycobacterium tuberculosis Infection Using
Elispot Assays: Looking Beyond Interferon-gamma. Cells. 1(2):89-99. http://dx.doi.org/10.3390/cells1020089
31. Boaz MJ, Hayes P, Tarragona T, Seamons L, Cooper A, Birungi J, Kitandwe P, Semaganda A, Kaleebu P, Stevens G, et al. 2009.
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35. Weiss AJ. 2012. Overview of Membranes and Membrane Plates Used in Research and Diagnostic ELISPOT Assays.243-256. http://
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016/s0022-1759(03)00226-6
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An Introduction to Antibodies: Antibody-Antigen Interaction
Antibody Standard Validation
An Introduction to Antibodies: Antigens, Epitopes and Antibodies
Protein Conformation Array Multiplexing
Conferma™ Sandwich ELISAs
ELISA Troubleshooting Guide
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Multiscreen® Filter Plates
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29/9/22, 14:16 The ELISpot Assay Enables Functional Analysis of Cellular Immunology

Introduzca el nombre de producto, el número de lote, etc.

The ELISpot Assay Enables Functional Analysis of Cellular Immunology

The ELISpot Assay Enables Functional Analysis of

THE IMMUNE RESPONSE AND THE ELISPOT – NECESSITY IS THE MOTHER

THE UNMATCHED POWER OF THE ELISPOT PLATFORM FOR T-CELL

THE QUESTION OF PRE-WETTING

COATING WITH CAPTURE ANTIBODY

CELLS - PLATING AND STIMULATION

DETECTION - CHROMOGENIC VS. FLUORESCENT OPTIONS

SPOT COUNTING AND ANALYSIS – WHAT IS A REAL SPOT?

A troubleshooting guide for interpreting and correcting ambiguous Elispot result

Issue Solution or Explanation

1. High levels of background staining

As plate washers are less vigorous than manual

Wet/damp membranes can display a dark blue

Due to diffusion, background staining or diffuse spots

Was the secondary/detection antibody

Some PBS formulations may benefit from filtering

Was a membrane pre-

Was the cell seeding

Have you stimulated your

Did the culture medium

Were cells resuspended

Was PBST (PBS + 0.5%

Was the plate

Referencia del producto Descripción SDS Precios

Referencia del producto Descripción ® SDS Precios

IL-2 ELISPOT Antibody Pair, Mouse

IL-4 ELISPOT Antibody Pair, Mouse

IL-5 ELISPOT Antibody Pair, Mouse

IL-6 ELISPOT Antibody Pair, Mouse

Anti-Mouse IgG (Fc specific)–Alkaline

Referencia del producto used for Radionucleotide counting by transfer

MultiScreen Column Loader

MultiScreen Column Loader

MultiScreen Column Loader

MultiScreen Column Loader

MultiScreen Deep, 96 Well Solvinert

MultiScreen-BV Filter Plate, 1.2 µm, clear, non-

Referencia del producto Clear, Non-Sterile, 0.45 µm pore size, Hydrophobic

Antibody Standard Validation

An Introduction to Antibodies: Antigens, Epitopes and Antibodies

Protein Conformation Array Multiplexing

Conferma™ Sandwich ELISAs

ELISA Troubleshooting Guide

RELATED PRODUCT CATEGORIES

Sealing Films, Foils & Tapes

Classical Media & Buffers

¿Le ha resultado útil el contenido de esta página?

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