Frequently Asked Questions ELISPOT

The enzyme-linked immunospot (ELISPOT) assay is a highly sensitive technique monitoring cellular and humoral immune responses. The assay is used to measure immune responses by detecting and quantifying individual cells that secrete cytokines or antibodies.
The ELISPOT assay is based on the same antibody "sandwich" principle as an ELISA, but with one important difference: living cells are cultured directly in the ELISPOT plate.
During the assay, antibodies specific to the target protein are coated onto the membrane on the bottom of a 96-well plate. Immune cells, such as peripheral blood mononuclear cells (PBMCs) or activated B cells, are then added and stimulated to produce cytokines or antibodies. The secreted proteins are immediately captured by the coated antibodies in the area surrounding each producing cell.
After the cells are removed, captured proteins are detected using a secondary antibody (detection antibody), and visualized by a conjugate and precipitating substrate. Each secreting cell leaves a visible colored spot, or “footprint,” on the membrane. These spots can be counted to determine the frequency of protein-secreting cells within the sample.
The result is reported as spot-forming cells (SFCs), providing a quantitative measure of antigen-specific or activated immune cells.

ELISPOT assays are among the most sensitive and specific methods available for monitoring T cell and B cell immune responses.
They enable the ex vivo identification of individual cells actively secreting cytokines, other signaling proteins, or antibodies.
The assay is relatively rapid, highly efficient, and capable of detecting frequencies as low as 1 responding cell in 100,000.
Key advantages of the ELISPOT assay include its straightforward workflow and suitability for high-throughput screening.
Because the assay requires only short-term in vitro culture, the measured response closely reflects the in vivo frequency and functionality of T or B cells within an individual.

Among currently available methods for evaluating T cell responses, the T cell ELISPOT assay offers one of the lowest detection thresholds.
Traditional assays such as the Mixed Lymphocyte Reaction (MLR) and Cytotoxic T Lymphocyte (CTL) assays assess CD4+ and CD8+ T cell-mediated immune responses. However, these methods have several limitations, including the use of radioactive materials, lower throughput, reduced sensitivity when using cryopreserved samples, and greater technical complexity. Real-time PCR (RT-PCR) represents a more modern and highly sensitive approach for assessing T cell responses. However, RT-PCR measures mRNA expression rather than the secretion of functional proteins, which may not always accurately reflect cellular activity.
 

Example of human IL-5 T cell ELISPOT (2.5x10⁴ PBMC stimulated with PMA/iono (48 h))

The B cell ELISPOT assay is considered the method of choice for identifying and quantifying antibody-secreting cells (ASCs) and determining the frequency of circulating memory B cells.
Traditionally, B cell responses following vaccination or infection have been assessed by measuring antigen-specific antibody titers in serum using ELISA. While informative, serum antibody levels do not reveal the number of ASCs, the magnitude of the memory B cell response, or the immune status of non-responsive individuals.
Vaccination often induces long-term or lifelong protection through the generation of antigen-specific memory B cells. These memory B cells do not spontaneously secrete antibodies ex vivo and therefore cannot be directly detected by conventional antibody measurements.
Following appropriate antigenic stimulation, however, memory B cells can differentiate into ASCs, enabling their quantification using the B cell ELISPOT assay.

Example of human IgG B cell ELISPOT (5x10³ PBMC stimulated with R848 + IL-2 (5 days))

An ELISA measures the total concentration of a secreted cytokine, signaling protein, or antibody present in a sample. In contrast, an ELISPOT assay detects and quantifies individual cytokine- or antibody-secreting cells, answering the question: ‘What is the frequency of secreting cells within the population?’
For this reason, the ELISPOT assays should not be viewed as a replacement for ELISA. Rather, the two techniques provide complementary information and are often most valuable when used together. While ELISA quantifies the overall amount of a secreted molecule, ELISPOT identifies the number of cells responsible for producing it.
A key advantage of the ELISPOT assay is its exceptional sensitivity. ELISPOT can be approximately 100 to 400 times more sensitive than a conventional ELISA. This increased sensitivity is achieved because the secreted protein is captured immediately around the producing cell on the membrane surface of the ELISPOT plate. As a result, the analyte is detected before it can become diluted in the culture supernatant, degraded by proteolytic enzymes, or bound by receptors on neighboring cells.
Consequently, ELISPOT is particularly well suited for detecting rare antigen-specific immune responses and low-frequency cytokine- or antibody-secreting cells that may be difficult or impossible to identify using conventional ELISA methods.

The T cell ELISPOT assay is widely used across numerous fields of immunological research, including cancer immunology, infectious diseases, autoimmune disorders, allergies, and organ transplantation. Its high sensitivity enables the detection and quantification of rare antigen-specific immune cells, making it a valuable tool for both basic and translational research.

The B cell ELISPOT assay is particularly well suited for assessing antigen-specific immune responses following vaccination. It allows for the detection and enumeration of antibody-secreting cells (ASCs) and memory B cells in peripheral blood cell preparations, providing detailed insights into vaccine-induced immunity. As a result, ELISPOT assays are frequently used to evaluate vaccine efficacy, monitor immune responses over time, and support the development of new immunotherapies and vaccines.

The primary information obtained from an ELISPOT assay is the frequency of antigen-specific T cells or B cells within a heterogeneous cell population. By detecting individual cells that secrete cytokines, growth factors, or antibodies, the assay enables highly sensitive quantification of immune responses at the single-cell level.
The measured frequency of these antigen-specific cells reflects the magnitude and quality of the cellular or humoral immune response. As a result, ELISPOT assays provide valuable insights into immune status, vaccine efficacy, infection-induced immunity, autoimmune responses, and the effectiveness of immunotherapies.
In addition to quantifying immune cell frequencies, ELISPOT assays can be used to assess the functional activity of immune cells by measuring the secretion of specific cytokines or antibodies following antigen stimulation.

In principle, any cell type capable of secreting proteins can be analyzed at the single-cell level using an ELISPOT assay. However, the technique has been most widely applied to the detection of cytokine secretion by antigen-activated T cells and antibody secretion by B cells derived from peripheral blood mononuclear cells (PBMCs) or spleen cell preparations.
Both fresh and cryopreserved PBMCs can be used successfully for ELISPOT analysis. 

However, for T cell ELISPOT assays the use of fresh rodent spleen cells is strongly recommended, as cryopreservation of rodent spleen cells can be technically challenging and may negatively affect cell viability and function. The same consideration applies to B cell ELISPOT assays, although the impact is generally less pronounced. Overall, cryopreservation can reduce spleen cell viability and functional responsiveness, potentially affecting assay sensitivity and the accurate detection of antigen-specific immune responses.

Adherent cells can also be analyzed using ELISPOT technology. When working with adherent cells, it is important to include a cell lysis step before the detection phase to ensure complete removal of cells from the ELISPOT plate. Following the incubation period, remove the majority of the cells from the ELISPOT plate and add ice-cold distilled water to each well. Incubate the plate on melting ice for 5 to 10 minutes. Subsequently, discard the solution and wash the wells five times with wash buffer before proceeding with the detection step.

Not necessarily. Some animal models are naturally skewed toward either a Th1-type or Th2-type immune response. In addition, certain animals may exhibit only a Th1 or Th2 response after in vivo stimulation with an appropriate antigen or when affected by a disease that promotes a Th1-like or Th2-like immune profile.
Therefore, before performing a T cell ELISPOT assay, verify that your animal model is capable of producing the cytokine of interest under your experimental conditions. Selecting an appropriate cytokine target requires consideration of the species, strain, immune status, and any prior antigen exposure of the animals.

Yes. Whole blood should be maintained at room temperature (RT; 20-26 °C) until processing and must not be refrigerated. If samples are collected at a remote location, they should be shipped to the receiving laboratory at ambient temperature. Blood samples should not remain at RT for more than 8 hours after collection.
For the T cell ELISPOT assay, blood samples may be gently mixed on a tube roller until peripheral blood mononuclear cells (PBMCs) are isolated.
The recommended anticoagulants are heparin and citrate, as neither has been reported to adversely affect cellular function in the ELISPOT assay. In contrast, EDTA inhibits coagulation through calcium chelation, which can impair cytokine induction during antigen-specific re-stimulation. Therefore, EDTA is not recommended as an anticoagulant for blood samples intended for use in the T cell ELISPOT assay.

Peripheral blood mononuclear cells (PBMCs) are typically isolated by density gradient centrifugation using reagents such as Ficoll. Because red blood cells can interfere with ELISPOT assay performance, the use of whole blood is generally not recommended.
Monocytes play a critical role as antigen-presenting cells (APCs) for T cells. Insufficient monocyte numbers can result in reduced spot-forming cell counts in T cell ELISPOT assays. This is particularly relevant for cryopreserved samples, which may contain fewer viable monocytes than freshly isolated samples. In such cases, replenishment of monocytes may be required to achieve optimal assay performance.
Cell viability is also an important factor. The presence of dead cells in PBMC preparations can negatively affect the performance of T cell ELISPOT and B cell ELISPOT assays. A viability threshold of ≥80% is generally recommended. In some cases, spot numbers may be reduced even when the proportion of dead cells is low, due to the presence of apoptotic cells generated during cell isolation or culture. These cells can impair immune cell function and consequently affect assay results.

The sensitivity of an ELISPOT assay is highly dependent on cell density. In T cell ELISPOT assays, a sufficient number of cells is required to promote interactions between antigen-presenting cells (APCs) and responding T cells. If too few cells are added, cell-to-cell contact may be inadequate, resulting in suboptimal responses. Conversely, excessive cell numbers can cause overlapping spots and increased background staining, making accurate spot enumeration difficult. For reliable spot counting, an optimal range is typically 50 to 250 spots per well.

For antigen-specific responses in a 96-well ELISPOT plate, a maximum of 3×10⁵ cells per well is generally recommended. At this density, the cells form a tight monolayer on the membrane surface. Higher cell numbers can lead to cell layering, causing a loss of linearity between the number of cells added and the frequency of spots detected. In most applications, 2×10⁵ cells per well produces fewer than 100 antigen-specific spot-forming cells (SFCs) per well.
For polyclonal stimulation in T cell ELISPOT assays, such as stimulation with mitogens or PMA/ionomycin, and for the detection of total immunoglobulin-secreting cells in B cell ELISPOT assays, substantially fewer cells should be used. Typically, 2×10² to 1×10⁵ cells per well are sufficient to achieve a spot frequency of approximately 50 to 100 spot-forming cells per well.

Several studies have shown that antigen-specific T cell responses in ELISPOT assay can be improved when cryopreserved PBMC are rested overnight before use. Cells are typically incubated at 37°C, 5% CO₂, and high humidity in culture medium supplemented with 10% FCS. This resting period helps remove apoptotic cells and can improve the accuracy of viable cell counts and functional T cell measurements.
However, overnight resting does not consistently enhance assay performance in every situation. The benefit depends on factors such as the laboratory's cell handling procedures, the characteristics of the PBMC cohort, and the antigens being tested. Therefore, the impact of PBMC resting should be evaluated and optimized within each laboratory's specific ELISPOT or protocol.

Quiescent memory B cells do not produce detectable levels of antibodies under normal conditions. Therefore, a preincubation step of 2 to 5 days is required to activate these cells before performing a B cell ELISPOT assay. During this period, cells should be cultured at a high density (>10⁶ cells/ml) in the presence of appropriate polyclonal stimulants, such as R848 and IL-2, to induce differentiation into antibody-secreting cells.

The choice of culture medium is important for achieving low background and strong antigen-specific responses in ELISPOT assays.
The recommended medium is RPMI-1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10% fetal calf serum (FCS). However, some FCS batches can non-specifically activate cells, leading to unwanted background spot formation. For this reason, FCS lots should be pre-tested to ensure both low background reactivity and high antigen-specific responses before use.

For T cell ELISPOT assays, human, non-human primate, or rodent serum may be used as an alternative to FCS, provided it has also been selected for low background and strong antigen-specific responses. Alternatively, a serum-free medium such as AIM-V, supplemented with penicillin and streptomycin, can also be a suitable option.

For B cell ELISPOT assays, human, non-human primate, or rodent serum should not be used as a growth supplement, as serum antibodies may interfere with spot formation.

Appropriate controls are essential for validating ELISPOT assay performance and ensuring reliable results. The choice of controls depends on the cell type, species, and experimental design.

T cell ELISPOT controls

• Positive control: Cells are stimulated with known antigen-specific or polyclonal activators. A strong spot response confirms that the assay and cells are functioning properly. Antigen-specific stimuli are generally preferred because responses to polyclonal stimulants can vary between species.
• Negative control: Cells are cultured without stimulation. This control measures spontaneous cytokine secretion and helps identify background activity.
• Background control: Assay wells contain all reagents but no cells. No spots should be detected, confirming that reagents and media do not generate false-positive signals.

B cell ELISPOT controls

• Positive control (memory B cell assays): Cells are incubated in wells coated with species-specific anti-immunoglobulin antibodies. A high spot count confirms assay performance and enables determination of the proportion of antigen-specific antibody-secreting cells.
• Background control: Cells are incubated in wells lacking antigen or capture antibody. No spots should be present, confirming the absence of non-specific spot formation.

In ELISPOT assays, each spot represents the footprint of a single cell that has secreted cytokines or antibodies. True spots typically have a dark, dense center with a gradually fading outer ring caused by protein diffusion.
Spot size and intensity generally reflect the amount of protein secreted. Only clear, well-defined spots should be counted, as small, faint, or irregular spots are often artifacts.
Artifacts may result from antibody aggregates, residual cells, DNA precipitates, tissue debris, or dust. These can usually be distinguished from true spots because they have sharper edges and uniform intensity rather than a dark center with fading borders.
To reduce artifacts, plates should be washed thoroughly between steps and kept free from dust and debris.
Because spot size and appearance can vary between assays, appropriate minimum and maximum spot-size thresholds should be applied during analysis. Automated ELISPOT readers are recommended for accurate spot detection and discrimination between true spots and artifacts.

Small, dark spot-like structures are often caused by dust particles or debris present in the wells rather than by secreted cytokines or antibodies.
These artifacts can usually be distinguished from true ELISPOT signals because they:

• Vary considerably in size
• Have highly irregular shapes
• Appear very dark or black upon visual inspection

In most cases, the problem can be easily resolved by carefully blowing compressed air (4-5 bar) into the wells to remove the particles before analysis.

Stripy, elongated, or irregularly shaped spots can occur when T cells move during the incubation period while actively secreting cytokines. Certain stimuli, particularly strong polyclonal activators such as mitogens or anti-CD3/CD28 antibodies, can induce chemokine release and promote cell migration. As the cells move, they leave a cytokine "trail," resulting in streaked or stripy spots.
In addition, moving or disturbing the ELISPOT plate during incubation can cause cells to roll across the membrane, producing oblong or irregular spot shapes. To minimize this effect, avoid handling or moving plates while the assay is in progress.

Yes. When using PVDF membrane-bottomed ELISPOT plates with samples that may contain infectious agents, such as enveloped viruses (for example HIV or SIV), the plates can be decontaminated after the cell incubation step.
A commonly used procedure is:

1. Remove the cells from the wells.
2. Wash the wells 5 times with PBS.
3. Add 300 µL of 4% paraformaldehyde (PFA) in PBS to each well.
4. Incubate for up to 5 minutes at room temperature (20-26°C).
5. Wash the wells and continue with the assay as usual.

This decontamination procedure may cause a slight increase in background staining but generally does not affect spot number or spot size in T cell ELISPOT assays.

Antibody-coated ELISPOT plates can typically be prepared up to 7 days in advance and stored at 4°C until use.
For best results:

• Seal the plate carefully to prevent evaporation.
• Maintain sterile conditions throughout storage to avoid contamination.
• After coating, wash the wells with sterile PBS and add the appropriate blocking solution.
• Leave the blocking solution in the wells during storage and remove it just before starting the assay.

ELISPOT assays are typically performed using PVDF (polyvinylidene fluoride) membrane-bottomed plates. We recommend 96-well MultiScreen®HTS Immobilon®-P (IP) plates from Millipore (e.g. Cat. No. MSIPS4510), which are optimized for ELISPOT applications.
For hydrophobic PVDF membranes, a brief pre-wetting step with 70% ethanol is required before coating. This treatment makes the membrane hydrophilic, improving antibody binding and resulting in enhanced sensitivity and better-defined spots. After pre-wetting, the membrane should be thoroughly rinsed with coating buffer to remove any residual ethanol.
Once pre-wetted, the membrane must remain wet throughout the entire assay procedure. Excessive ethanol exposure or prolonged treatment may negatively affect assay performance.
For optimal washing, it is recommended to remove the plate underdrain after detection antibody incubation and wash both sides of the membrane during each wash step.

Use wide-bore pipette tips when handling cells to minimize shear stress, which can damage cells, induce apoptosis, and reduce spot formation. Add cells and reagents gently to ensure an even distribution across the membrane. Rapid or abrupt pipetting or adding liquids after cells have settled in the wells, may result in uneven spot distribution.

The stimuli described in this overview can be used as positive controls in T cell ELISPOT and FluoroSpot assays to assess cell functionality and confirm proper assay performance.
The stimuli described for B cells can be used in B cell ELISPOT assays to activate memory B cells and promote their differentiation into antibody-secreting cells.
The concentrations listed should be considered as general guidelines. It is recommended to evaluate a range of concentrations to determine the optimal stimulation conditions for your ELISPOT assay. Up to 3×10⁵ cells can be added per well in a 96-well ELISPOT plate.
However, the number of cells should not be so high that spot counting becomes difficult. For reliable analysis, a spot count of approximately 50 to 100 spots per well is generally recommended.

Proper washing is critical for removing cells, unbound detection antibodies, and excess staining reagents while preserving membrane integrity.
For preparation of the coating antibody solution and all washing procedures, do not use PBS tablets, as tablet excipients may interfere with antibody coating and assay performance. Instead, use sterile, ready-to-use liquid PBS. We recommend PBS (Thermo Fisher Scientific, Cat. No. 10010).
For PVDF membrane-bottomed plates, manual washing with a squirt bottle fitted with a wide spout is recommended. Fill the wells completely with freshly prepared wash buffer, then remove the buffer by a firm shake-out action. Repeat the wash steps as specified in the protocol and tap the plate dry on absorbent paper after the final wash.
To improve washing efficiency, remove the plate underdrain after the detection antibody incubation step and wash both sides of the PVDF membrane during subsequent wash steps.

Stacking ELISPOT plates during incubation is not recommended.
Stacked plates may experience uneven temperature distribution, which can affect cell activity and lead to variations in spot size and spot frequency between wells and plates.

PVDF membrane-bottomed plates should be dried without the lid, upside down, and protected from light at room temperature (20 to 26°C). It is essential that the membranes are completely dry before plate evaluation, as residual moisture can affect spot visualization and counting.
After drying, plates should be stored at room temperature in a dry, dark place to prevent spot bleaching and preserve assay results.

PVDF membrane-bottomed ELISPOT plates can be evaluated using either a reflected-light microscope for manual spot counting or an automated ELISPOT reader for objective and standardized analysis.
Automated ELISPOT readers offer advantages such as faster data acquisition, improved reproducibility, and more consistent spot quantification, especially when processing large numbers of samples.

If you cannot find the answer to your question, our scientific team is happy to help. With more than 25 years of experience developing and performing ELISPOT assays for human, monkey, mouse, and rat samples, our researchers can provide expert advice and practical support.

For assistance, please contact our customer service team at cs@ucytech.com.