Frequently asked questions ELISPOT

The enzyme-linked immunospot (ELISPOT) assay is a widely used method for monitoring immune responses. The assay is a highly sensitive method for the ex vivo quantification of cytokine or antibody secreting cells after stimulation with an appropriate stimulus in vitro (Cox et al. 2006). In fact, each cell can be detected by the assay as long as a characteristic protein is released and specific high affinity antibodies recognizing the protein are available.

The ELISPOT assay is very similar to an enzyme-linked immunosorbent assay (ELISA) and is based on the same immunochemical 'sandwich' principle. The major difference is that an ELISPOT is a combination of both an immunoassay and bioassay because living cells are cultured directly in the wells of the ELISPOT plate.
As first step, coating antibodies, specific for the protein of interest, are immobilized onto a solid phase (96-well plate). Isolated cells (for example, PBMC with activated T or B cells) are subsequently brought into the wells and incubated for a certain length of time. The released proteins are trapped by the coating antibody in the area directly surrounding the producing cells. These areas are then visualized by a combination of a second specific detector antibody, a conjugate and a precipitating substrate.
A single cell forms a colored 'footprint' (spot) on the bottom of the well representing its secretory activity. The frequency of spot forming cells can be quantified from the number of spots in the well and the cell input. The term 'spot-forming cells' or SFC, is used as a quantitative measure for the number of cytokine or antibody secreting cells in the ELISPOT assay.

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ELISPOT assays are among the most sensitive and specific methods available for monitoring T and B cell responses permitting the ex vivo identification of cells actively secreting signaling proteins or antibodies. Moreover the assay is relatively fast, highly efficient and can detect a single cell out of a million cells.
Major advantages of the ELISPOT assay are its relatively easy performance, its potential for high throughput screening and no requirement for expensive instruments. Because of the short-term in vitro culture, the measured response closely mirrors the in vivo T or B cell frequency of an individual.

The T cell ELISPOT assay has the lowest detection threshold among the assays available for detection of T cell responses. The classical T cell assays are lymphoproliferation assays (LPA) and cytotoxic T lymphocyte (CTL) assays that measure CD4+ and CD8+ T cell-mediated immune responses, respectively. Both the LPA and CTL assays have their drawbacks including the use of radioactivity, low throughput screening, decreased sensitivity in cryopreserved specimens and technical burden. A more modern test, RT-PCR analysis, is a sensitive assay to measure T cell responses. However, this assay detects mRNA instead of actually secreted protein.

The B cell ELISPOT assay is the assay of choice to identify and determine the number of antibody secreting cells (ASC) and quantitate the frequency of circulating memory B cells. The traditional method to monitor a B cell response generated after immunization or infection is to quantify specific antibody titers in serum by ELISA. However, antibody levels in serum do not provide information about the number and location of the ASC and the status of the immune response such as non-responsiveness to vaccines. Vaccination normally induces long-term or even life-long protection mediated by the presence of circulating antigen specific memory B cells that do not spontaneously produce antibodies ex vivo. Therefore, memory B cells require antigenic stimulation to differentiate into ASCs, what can be done in the B cell ELISPOT assay.

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An ELISA determines the total concentration of the secreted signaling protein or antibody, whereas an ELISPOT detects individual cytokine or antibody secreting cells answering the question 'what is the frequency of secreting cells?'. Therefore ELISPOT should be used not 'instead of ' but rather 'in addition to' ELISA.
An ELISPOT assay can 100 to 400 times more sensitive than a conventional ELISA because the secreted protein is captured directly onto the well of an ELISPOT plate before having the chance to be diluted in the culture supernatant, degraded by proteases or captured by receptors on adjacent cells.

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The ELISPOT assay can be used in many areas of research such as cancer, infectious disease, autoimmune disease, allergy and organ transplantation. The assay is particularly effective for the measurement of antigen-specific responses post-vaccination in peripheral blood cell preparations. For more information, please see ELISPOT applied in research.

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One key piece of information that can be obtained from ELISPOT assays is the frequency of antigen-specific T or B cells within a pool of different cell types. This frequency reflects the clonal size of the cells and, therefore, the magnitude of the cellular of humoral immune response.

Pictures:

Example of human IgG B cell ELISPOT results:
Used in the assay: 1x10E5 human PBMC/well
PBMC of an individual vaccinated with Tetanus toxiod more than 15 year ago.
Memory B cells were activated with IL-2 and R848 during an in vitro preincubation step.
Upper: Number of Tetanus toxoid specific IgG secreting cells
Lower: Total number of IgG secreting cells
In this assay enzymatic staining was applied resulting in red spots.

Example of human IFN-γ T cell ELISPOT results:
Used in the assay: 2x10E5 human PBMC/well
PBMC were stimulated with 0.1 µg/ml Leishmania major antigen.
Upper: Response of PBMC of a patient with cutaneous leishmaniasis
Lower: Response of PBMC of a a healthy individual
In this assay silver staining was applied resulting in black spots.

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Whole blood should be kept at room temperature (RT) until processing. Do not refrigerate. If collected elsewhere, samples should be shipped at ambient temperature to the desired laboratory. Samples should not be at RT for more than 8h after draw.

The recommended anti-coagulants are citrate and heparin. For both substances there are no reported adverse effects on cellular function in the ELISPOT assay. EDTA, on the other hand, inhibits coagulation through calcium chelation which may impair cytokine induction during antigen-specific re-stimulation and is therefore dissuaded for use as an anti-coagulant.

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Monocytes are critical as antigen-presenting cells (APCs)  for T cells and a low number of monocytes can lead to lower spot forming cells (SFCs) in the T cell ELISPOT assay. Particularly cryopreserved samples may contain a decreased number of monocytes. Replenishment of monocytes may therefore be necessary.

The presence of dead cells in PBMC preparations can impact the functioning of both the T and B cell ELISPOT assay and a cut-off of ≥ 80% viability is recommended as a threshold. In some cases, even though the number of dead cells is low, there may be a lower number of spots because of apototic activity induced in T or B cells during isolation and culture.

Pictures:
Example of human IFN-gamma ELISPOT
2x10E5 PBMC/well
Upper: monocyte depleted sample
Lower: normal sample

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ELISPOT sensitivity critically depends on high cell density. A high number of cells enhances the probability of contact between stimulating and responding cells.
For antigen-specific responses, optimal stimulation of T cells can be achieved when the total cell number approaches 5x10E5 to 10E6 cells/ml. In a 96-well ELISPOT plate, a maximum of 3x10E5 cells can be put into each well because this number of cells forms a tight monolayer on the bottom of the wells. Higher numbers of cells will lead to piling up of cells and linearity between cell input and detected spot frequency is lost. In general, 2x10E5 PBMC generate less than 100 antigen-specific SFC per well.
For polyclonal stimuli such as mitogens or PMA + ionomycin, 10-100-fold less cells should be plated (2x10E3 to 2x10E4) to obtain individual spots at a spot frequency of 50-100 SFC/well. Too many spots will lead to overlap of individual spots and high background staining.

Picture:
Example of Mouse IFN-gamma ELISPOT:
stimulus: concanavalin A

Upper: 2.5x10E4 mouse (Balb/c) splenocytes/well
Lower: 1x10E5 mouse (Balb/c) splenocytes/well
(resulting in too many spots per well)

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A 24-42h preincubation step at high cell density (>10E6 cells/well/ml) is required when full-length proteins or long peptides  are used for re-stimulation. These antigens must first be internalized, processed and presented by antigen-presenting cells (APCs) via MHC class I/II molecules before they can stimulate cytokine release by T cells. The high number of cells enhances the probability of contact between stimulating and responding cells. Omitting this step leads to a significant lower frequency of spot forming cells (SFCs). On the other hand, small (synthetic) peptides (8-12 mer) can directly be presented by APC to CD8+ cells and consequently need no preincubation step.
For more information please see our T cell ELISPOT instruction manual. To download the manual please visit 'Manuals' under 'General Information' on our website.

Example of IL-2 specific spots produced by mouse (C57bl/6) splenocytes using the Mouse IL-2 ELISPOT kit (CT435-PR):

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For the 'direct' human ELISPOT assay (no preincubation step), serum-free medium (AIM-V) has proven to be the best choice for obtaining an optimal T cell response. On the other hand, the 'indirect' assay (with preincubation step) requires medium (like RPMI-1640) supplemented with 10% FCS or human serum. AIM-V medium should not be used in the 'indirect' assay.

It should be mentioned that some batches of fetal calf serum (FCS) or human serum may non-specifically activate the cells and can therefore be the cause of background spot formation. Different batches of FCS and human serum should therefore be pre-tested before using it in the ELISPOT assay.

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Appropriate controls are important for the validation of the ELISPOT assay. Choice of controls depends on the cells if interest and type of experiment.

Positive control in the T cell ELISPOT assay
- Cell are activated with antigen-specific or polyclonal stimuli.
- High number of spots indicates that the assay works properly.

Both antigen-specific and polyclonal stimuli can be used. However, antigen-specific stimuli are preferred since cells of various species respond differently to polyclonal stimuli. For the human system, vaccine proteins (Tetanus toxoid, Hepatitis B, etc.) or a pool of synthetic peptides of common human viral epitopes are excellent positive controls. When using vaccine proteins, it should of course be known whether individuals have been vaccinated.

Negative control in the T cell ELISPOT assay
- Cells are incubated without any stimuli.
- No spots or spontaneously occurring spots are revealed.

Background control in the T cell ELISPOT assay
- Run the ELISPOT assay without adding cells to the well of the ELISPOT plate.
- No spots or reagents/cell culture media produce false spot-like structures.

Positive control in the B cell ELISPOT assay (Memory B cells only)
- Cells are incubated in wells coated with an antibody to species-specific immunoglobulin.
- High number of spots provides information whether the assay works properly and provides data to determine the ratio of antigen-specific versus total antibody secreting cells.

Background control in the B cell ELISPOT assay
- Cells are incubated in wells not coated with antigen or antibody.
- No spots or reagents/cell culture media produce false spot-like structures.

To: An overview of general cell stimuli commonly used in ELISPOT and FluoroSpot assays
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In case cells in the transparent polystrene-bottomed ELISPOT plate are contaminated with infectious agents such as enveloped viruses (e.g. HIV, SIV), the wells can be decontaminated after the preferred incubation time by first removing the cells from the well, adding 300 µl 1% Triton X-100 (in PBS) and then to led it stand for 30 minutes at room temperature. Thereafter wells can be safely washed as usual.
When PVDF membrane-bottomed ELISPOT plates are used, they can be decontaminated after the preferred cell incubation time by first removing the cells from the well, washing 5 times with PBS, adding 300 µl 4% Paraformaldehyde (in PBS) and then to led it stand for 5 minutes (max) at room temperature. Thereafter wells can be safely washed as usual.
These procedures may lead to a slightly higher background staining but has no adverse effect on spot number and size.

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Selection of optimal primary and secondary antibodies in an ELISA system does not imply that these are also the most optimal antibodies for an ELISPOT assay. The ELISPOT antibody pairs are selected by the manufacturer on basis of extensive optimalization and validation and should be used as a matched antibody pair for the ELISPOT assay.
One of the most important parameters to standardize is the total amount of antibody used for coating. A general guideline is that approximately 1 µg per well (10 µg/ml) of coating antibody result in well-defined spots. Antibody concentrations that are too low result in not only fainter spots but also fewer detectable spots. The total amount of the detection antibody can range from 0.1 to 2 µg/ml. Therefore, the most efficient antibody concentration needs to be established to concur with cost-effectiveness without compromising spot quality and ultimately spot count.

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For optimal results, the coating antibodies should be left in the wells of the ELISPOT plate for minimal 16 h at 4°C. The blocking buffer, detector antibodies and conjugate can be incubated for 1 h at 37°C, 2 h at room temperature or overnight at 4°C without any problem.
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Antibody coated plates (both polystyrene- and PVDF membrane-bottomed) can be prepared 7 days ahead. Seal the plate against evaporation and store these plates at 4ºC. Be aware that for long-term storage, sterility should be guaranteed. Moreover, it is recommended that after coating, the wells are washed with sterile PBS and blocked with Blocking solution. This blocking solution is left in the wells until use.

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Horseradish peroxidase (HRP) (only for PVDF), or gold particles (for polystyrene and PVDF membrane-bottomed plates) can be used as conjugates.
The advantage of using HRP is its fast turnover rate (spots develop fast). The HRP substrate AEC (3'-amino-9-ethylcarbazole) forms intense red colored spots, but may bleach in short period of time when stored in daylight. Therefore, plates should be protected from light and moisture during storage at room temperature. 
A highly effective way of spot staining can be achieved with gold-labeled anti-biotin antibodies using silver precipitation for spot visualization. Because of the high stability of silver, spots do not fade and ELISPOT plates can be reanalyzed after being stored for several years at room temperature.

Pictures:
Example of Human IFN-gamma ELISPOT
2x10E5 PBMC/well; stimulus: viral peptide pool
Upper: Silver staining (transparent polystyrene-bottomed well)
Lower: AEC staining (PVDF membrane-bottomed well)

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Each spot within an ELISPOT well is the 'footprint' of a single cell that has released a relatively high amount of cytokines. True spots have a dense center with a light outer ring caused by the diffusion of the cytokine from the producing cell.
The color depth or the size of spots depends on the amount of secreted cytokines. Only strong and well-defined spots should be counted; any small or faint spot is likely to be an artifact and should be ignored.
Artifactual spots can be caused by the aggregation of antibodies used in the process of coating and detection. Also incomplete removal of cells from the plate after incubation can be a source for artifactual spots. A simple measure to minimize false spot formation is to thoroughly wash the plate between all incubation steps and after completion of the coloring reaction.

Sometimes small and very dark spots appear in the background that are not generated by secreted cytokines. In the visual evaluation, these spots are differentiated from 'true' spots by their sharper edges. True spots always have a dark center with fading color intensity towards the edges. These dark spots have a homogeneous intensity and may originate from small cell membrane fragments firmly attached to the solid support carrying receptor bound cytokines.

The average size of a true spot is 5000-10,000 square microns, although this varies depending on incubation time, microtiter plates, antibody source and concentration, enzyme activity, substrates and other materials used as well as the functional state of the cytokine-secreting cells.
Manual counting of spots by light microscopy is a laborious task and strongly dissuaded in case many wells need to be analyzed. The availability of sophisticated computer-controlled ELISPOT readers from different companies offers a complete solution for precise and automatic evaluation of ELISPOT data and offers various procedures to overcome variable background intensity problems and to distinguish true spots from artifacts by image analysis.

In all cytokine ELISPOT assays, a wide spectrum of spot sizes and densities can be seen. Thus when analyzing ELISPOT results, cut-off values need to be set for the minimum spot size to be counted. The maximum spot size must likewise be defined so that clusters of cells can be identified as such. The minimum and maximum 'gates' set will critically affect the number of spots counted. For this reason, one of the main goals of ELISPOT image analysis has been to establish absolute criteria for gating.

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Certain stimuli trigger the release of chemokines which causes the T cells to move around during incubation producing a cytokine trail in the well. This is particularly true when T cells are activated with polyclonal stimuli such as mitogens and anti-CD3/CD28 antibodies. Also the movement of plates during cell incubation may cause the cells to roll generating irregular or oblong-shaped spots.

Picture:
Chemokine mediated spurring of spots

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There are two possibilities. Due to static electricity, dust particles attach to the underside and/or inside of the well of the transparent polystyrene-bottomed plates. Upon visual evaluation, these spots vary in size and are highly irregular. By cleaning the underside of the well with 70% ethanol, followed by blowing 4-5 bar compressed air against the underside and into the well, this problem can easily be solved.
The second possibility are cell wall fragments containing receptor-bound cytokines that stick to the coating antibodies in the well. These particles are very small, have sharp edges and cannot easily be removed by standard washing procedures. Remarkably, only PBMCs from certain individuals generate these 'sticky' spots. By proper gating, these small artifactual spots are ignored by standard ELISPOT readers.

Pictures:
Upper: example of dust particles
Lower: example of spots generated by cell wall fragments

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ELISPOT assays can be performed using either 96-well transparent polystyrene-bottomed plates or 96-well PVDF membrane-bottomed plates. Both solid supports bind proteins by hydrophobic interactions (Van der Waal's forces).
PVDF membranes have a higher retaining capacity for antibodies because of a larger surface area than polystyrene plates. However, this increased surface area is not required for ELISPOT analyses since there is no flow of reagents through or across the membrane but rather a diffusion of cell-secreted molecules towards coating antibodies immobilized on top of the membrane. This explains that for both membrane- and polystyrene-bottomed plates the optimal concentration of the antibody for coating is more or less similar. It should be stressed, however, that only a very few types of 96-well PVDF membrane- and polystyrene-bottomed plates have a sufficient high antibody binding capacity for use in ELISPOT analyses.

There are both sterile and non-sterile microtiter plates available for ELISPOT analyses. Since ELISPOT requires short culture times and includes the use of antibiotics in the culture medium, non-sterile plates can be safely used without contamination problems.

When using PVDF membrane-bottomed plates a prewetting step with 70% ethanol is required. The treatment with ethanol makes the membrane hydrophilic and ensures optimal binding of the coating antibody resulting in better sensitivity (increased spot number) and more accurate quantitation (more sharply defined spots) (Weiss A.J. 2012). After maximum 2 minutes of prewetting with 25 µl 70% ethanol, the membrane should be rinsed thoroughly by adding coating buffer to the well to efficiently wash out the ethanol before the coating antibody is added to the wells. Once the membrane is ethanol-treated, it must be kept wet for the entire assay procedure. Overtreatment with larger volumes of ethanol, more concentrated ethanol and longer exposure time can lead to trapping of residual liquid between the membrane and underdrain, which may result in poor assay performance. This prewetting step is not necessary for polystyrene-bottomed plates.

The U-CyTech ELISPOT kits are either suited for the use with PVDF membrane-bottomed plates (96-well plates from Millipore [cat. no. MSIP S4510] are recommended) or contain 96-well polystyrene-bottomed plates. When needed, extra polystyrene-bottomed plates can be ordered separately (cat. no. CT350). All our 2-plate format T cell ELISPOT kits do contain 96-well plates.

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For the pipetting of cells it is important to use tips with a wide opening to avoid shearing stress upon the cells that may induce apoptosis and thus inhibition of spot formation.

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A critical issue in ELISPOT analyses, is the wash procedure. Washing of ELISPOT plates is necessary to remove cells and residual biotinylated antibody and staining reagents. Stimulated cells become very sticky and their complete removal may require incubation with an enzymatic cell-detachment solution. Some protocols recommend up to 0.05% Tween-20 in the wash buffer. A drawback of using Tween-20 is that it sometimes reduces the hydrophobicity of the PVDF membrane to such an extent that the PVDF membrane leaks, completely ruining the experiment.

Washing can be performed in several ways: manually (multichannel pipette or squirt bottle) or (semi)automatically using a multichannel washer or an ELISA microplate autowasher.
Since damaging of the membrane is a serious risk when using an automatic plate washer and the fact that both sides of the membrane need to be washed, a squirt bottle is the best choice for effective washing of membrane-bottomed plates. On the other hand, an automatic plate washer is highly convenient and effective for polystyrene-bottomed plates. It should be realized however, that the use of an automatic washing device may cause problems in case the probes are placed too far or too close to the bottom of the wells generating a poor wash or well-washed concentric areas in the center of the well with clearly defined spots. Automated washers also require consistent decontamination and cleaning procedures, otherwise accumulation of debris in the probes will create high and irregular background staining and slow flow.
After completing the wash, tap the plates on a dry paper towel.

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This phenomenon is typically caused by foam formation during washing of the plates.
Particularly squirt bottles with a narrow spout produce excessive foam preventing an effective and uniform wash. The use of an automatic washing device or squirt bottle with a wide spout is usually sufficient to overcome this problem.

Picture:
Example of areas in the well lacking spots due to foam formation

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It is critical that the plate is equally heated during incubation. Stacking of plates will lead to variation in temperature of the individual wells and consequently the size and frequency of spots will vary.

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Both polystyrene-bottomed and PVDF membrane-bottomed plates should be dried without a lid upside down at room temperature. When dry, PVDF membrane-bottomed plates with enzymatically colored spots should be stored in the dark at a dry place at room temperature to prevent bleaching of spots. Storage of plates with silver-stained spots do not require special storage conditions.

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The researchers of U-CyTech have over 20 years experience with the development and performance of the ELISPOT assay. Not only for human but also for monkey, mouse and rat species. They are more than happy to provide you with additional advice and to share their experiences with you.

Please contract our customer service:
E-mail: cs @ ucytech.com

U-CyTech biosciences
Yalelaan 48
3584 CM Utrecht
The Netherlands

Phone: +31.85 073 1460

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