ELISPOT protocol
General ELISPOT procedure outlining plate and sample preparation, cell incubation, enzymatic detection, and analysis.
Last edited Thu 03 July 2025
The ELISPOT (enzyme-linked immunospot) assay is a powerful immunological technique used to detect and quantify secreted proteins, such as cytokines, at the single-cell level. ELISPOT is a type of cell-based assay that relies on live, healthy cells and careful sample handling for reliable results. It is related to the enzyme-linked immunosorbent (ELISA) technique, as both utilize antibody-antigen interactions and enzyme-based detection methods. This protocol, provided by Abcam, outlines a step-by-step method for preparing PVDF membrane plates, stimulating immune cells, and visualizing secreted proteins using enzymatic or fluorescent detection. ELISPOT is widely used in immunology research to assess T cell responses, vaccine efficacy, and immune monitoring. Its high sensitivity and ability to detect rare secreting cells make it a preferred method for single-cell analysis. This guide ensures reproducibility and accuracy, offering troubleshooting tips and best practices for optimal results in your linked immunospot ELISPOT assay experiments.
Introduction to the ELISpot assay
ELISPOT is a highly sensitive assay designed to detect secreted proteins from individual cells, making it ideal for immunological studies. Developed as an extension of ELISA, ELISPOT enables researchers to visualize cytokine secretion at the single-cell level. The technique involves coating a 96-well PVDF plate with a capture antibody, adding cells, stimulating them, and detecting secreted proteins via enzyme-linked or fluorescent methods. Unlike an ELISA plate, which is a traditional plastic microplate used in ELISA assays and lacks a membrane, the ELISPOT plate contains a PVDF membrane specifically designed to capture secreted proteins at the site of secretion. Each secreting cell forms a visible spot, allowing for quantitative analysis. ELISPOT is commonly used to measure antigen-specific cytokine responses by detecting cytokine production from T cells exposed to specific antigens. This protocol provides a reliable framework for researchers working with peripheral blood mononuclear cells (PBMCs) or other immune cell types, ensuring consistent and reproducible results across various applications.
Background and principles
ELISPOT is based on the principle of capturing secreted proteins directly onto a membrane surface surrounding the secreting cell. This assay detects protein secretion from individual cells, allowing for sensitive measurement of immune responses. The assay begins with a PVDF or nitrocellulose membrane pre-coated with a specific capture antibody to the target protein, ensuring selective binding of the analyte. Upon stimulation, cells secrete proteins that bind to the capture antibody. A detection antibody is then added, followed by an enzymatic or fluorescent substrate to visualize the spots. Each spot represents a secreted analyte captured from cells producing the target protein, such as cytokines or growth factors. This enables precise quantification of cytokine-secreting cells. This method is particularly useful for detecting low-frequency immune responses and is widely used in vaccine development, cancer immunotherapy, and infectious disease research.
Comparison to other methods
Compared to ELISA, which measures bulk protein levels in solution, ELISPOT offers single-cell resolution, making it more sensitive for detecting rare secreting cells. Techniques like ELISPOT, ELISA, and flow cytometry are often performed as cell-based assays using cells prepared in tissue culture to assess immune responses in vitro. Unlike flow cytometry, which requires intracellular staining and cell permeabilization, ELISPOT preserves cell integrity and function. It also avoids the need for expensive instrumentation. While multiplexing is more limited than in Luminex assays, dual-color ELISPOT variants allow for simultaneous detection of two analytes. Overall, ELISPOT strikes a balance between sensitivity, specificity, and accessibility, making it a preferred choice for functional immune assays, especially when sample size is limited or rare cell populations are involved.
Cell culture and preparation
Proper cell culture and preparation are fundamental to achieving reliable results in the ELISPOT assay. Only viable cells should be used, as dead or damaged cells can compromise data quality and lead to false-positive results. The assay is compatible with a variety of immune cell types, including peripheral blood mononuclear cells (PBMCs), T cells, B cells, and dendritic cells, allowing for the study of diverse immune responses.
To preserve cell function, it is important to process blood samples and prepare cells within 8 hours of collection. For frozen PBMCs, allowing the cells to rest for at least 1 hour after thawing helps remove cell debris and improves overall cell viability. The choice of cell culture medium is also critical; it should support the health and activity of the specific cell populations being studied and must be free of sodium azide, which can inhibit the enzyme reactions used in the ELISPOT assay.
By ensuring that only healthy, viable cells are plated and that the cell culture conditions are optimized, researchers can accurately assess the function of antigen-specific T cells and other immune cells at the single-cell level.
Stage 1 - Plate preparation and coating with the capture antibody
Materials required
- PVDF membrane
- 96-well plates
- 35% ethanol
- 1X PBS
- Your capture antibody
- Blocking buffer such as 2% dry skim milk or 1% BSA
Steps
Prepare PVDF membranes in the 96-well plates by incubating in 35% ethanol for 30 seconds.
Coat 96-well plate with capture antibody diluted in phosphate buffered saline (PBS).
- Approximately 0.5–1 µg per well of antibody should be used for well-defined spots.
- Kits are optimized with capture concentrations for best performance (100 µL per well).
Incubate overnight at 4°C.
Empty the wells, tapping them dry, and wash with PBS.
Add 100 µL per well 2% dry skim milk to block non-specific binding to the membrane.
- Incubate the plate for 2 h at room temperature.
Wash the plate 3 times in PBS and leave to dry.
- If necessary, the plates can be stored at this stage. Store at 4°C for not more than 2 weeks in a sealed plastic pouch with desiccant.
Stage 2 - Sample preparation
Most ELISPOT experiments are done with isolated PBMCs (peripheral blood mononuclear cells). Both freshly prepared and cryopreserved cells may be used in the assay. However, it is recommended to let frozen cells rest at least 1 hour after thawing to allow the removal of cell debris before addition to the plate.
PBMCs should be prepared and plated within 8 hours of collecting the blood samples to preserve cell functionality. If the blood samples are left longer than this, the granulocytes (neutrophils) that are mixed with the PBMC can become activated. This can change their buoyancy profile when the PBMCs are separated from granulocytes using FicollTM, so the granulocytes may contaminate the PBMC layer. The activated granulocytes may also begin activating some of the PBMCs (they can downregulate the signal-transducing zeta chain of CD3, suppressing T cell function).
If preparation and plating are not possible within 8 hours:
- Dilute the blood sample immediately. This helps to minimize granulocyte contamination. For example, dilute 1:1 in RPMI or PBS. Keep at room temperature (not 4°C).
- Remove the granulocytes by cross-linking red blood cells and granulocytes, then separate them from PBMC using FicollTM. (Disadvantage: some PBMC may be lost.) Commercially available kits are available for this.
- Ship fresh samples at ambient temperature. Note that transport temperatures can be below 4°C. Commercially available containers can be used to keep samples at an ambient temperature.
- Sample freezing should be optimized. Where possible, use serum-free media (serum contains mitogens and inhibitors, which could affect the results).
Here we prepare peripheral blood mononuclear cells (PBMCs) from fresh blood by density gradient separation according to the Ficoll-PaqueTM manufacturer's protocol.
Steps
Dilute whole blood sample with an equal volume of sterile NaCl 0.9% or balanced salt solution, such as PBS or HBSS.
Layer the diluted sample over a volume of Ficoll-Paque equivalent to the initial blood volume.
Centrifuge for 20 minutes at ~700 x g at room temperature with the brake off.
Remove the PBMC from the interface between the Ficoll-Paque and the plasma layer.
Wash enriched cells with sterile NaCl 0.9% with 2 centrifugations at 5 minutes each, 600 x g, followed by one at 8 seconds at 900 x g to remove platelets.
Adjust cell density in the medium recommended for the following ELISPOT procedure.
Optional – Freezing cells for storage.
- Prepare 20% DMSO in cell culture media. Keep on ice.
- Label cryovials.
- Re-suspend cells at 40 x 106 cells/mL in the ice-cold medium. Keep on ice.
- Dispense 0.5 mL cell suspension into cryovials.
- Add gently into cell suspension 20% DMSO at a ratio of 1:1. Final suspension will be at 20 x 106 cells/ml.
- Place cryovials immediately into the freezing container.
Steps
Thaw cells quickly.
- Use a 37oC water bath or beaker of warm water.
Gently transfer cells into a 50 mL tube containing 15 mL of culture media.
- Use 0.5 to 5.0 mL of cells per 50-mL tube.
Fill tube to 50 mL with culture media.
- Gently invert the tube to mix.
Spin down cells at 300 x g for 5 minutes.
Pour off supernatant and flick tube gently to re-suspend the pellet.
- Count and adjust cells to desired density in the appropriate medium.
Cells are now ready to use in the ELISPOT assay.
Steps
Tissue homogenization.
- Gently tease the tissue through sterile stainless steel and disperse it into 30 mL of recommended medium.
- Further, disperse clumps by gently pipetting up and down several times.
- Remove remaining clumps of cells and debris.
Centrifuge the cells for 5 minutes at 600 x g and re-suspend the cell pellet in medium recommended for the following procedure.
Stage 3 - Cell incubation
Steps
Use cells prepared in the previous stage.
- Count the cells using a viability dye like trypan blue. They should be over 95% viable.
Dilute cells to the required concentration and add the cell suspension to wells.
- If optimizing the assay for cell number, use a 1:2 dilution series. Do not shake plates.
- The number of cells per well should be optimized. For example, use more cells if a low percentage of cells are expected to secrete the target cytokine. Refer to the specific target kit protocols for recommendations on assay controls and cell number per well. Typically cell numbers should usually range from between 2x105 to 4x105 PBMC cells per well.
- If possible, use serum-free media as serum contains many proteins which could affect the results. Alternatively, several batches of serum can be tested to find one with optimal response to noise ratio. This batch can then be stored and used in subsequent experiments.
Culture overnight at 37°C in CO2 incubator. Do not shake the plates.
- During the overnight incubation, the cells will secrete cytokine, which will bind to the primary antibody.
- If cells take time to respond to stimulation, please see the indirect method below.
Optional - Indirect ELISPOT
- If the cells take some time to respond to stimulation, they may require pre-treatment with the stimulant in a separate 96-well culture dish before transferring to the ELISPOT plate.
Positive control stimulation
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Experiments to detect cytokines using ELISPOT will require the use of positive control. In these positive control wells, the cells should be stimulated with an agent known to induce expression of the cytokine being detected. This can then be used to compare to the negative control (no treatment or stimulation of a different secreted protein).
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Typical stimulatants include:
- LPS to stimulate IL1β and IL6 secretion
- PMA and ionomycin stimulate IL2 and IL4 secretion
- PHA, 10 µg/mL for IFN-gamma
- Anti-CD3/CD28 antibodies for IFN-gamma, IL4, IL10, and Granzyme B
Wash away the cells and the unbound cytokine by incubating with PBS 0.1% Tween 20 for 10 min.
Stage 4 - Incubation with detection antibody
Materials required
- For our ELISPOT kits, detection antibody concentrations have been optimized for the best results.
Steps
Dilute the conjugated detection antibody in PBS 1% BSA.
Add the conjugated detection antibody to wells and incubate for 1–2 h at room temperature.
Wash the plate 3 times with PBS 0.1% Tween 20 to remove non-specific detection antibody binding.
Stage 5 - Detection
Steps
Add the enzyme substrate solution to each well.
- For enzymatic detection protocols, the base should be taken off the bottom of the plate to enable thorough washing of the membrane before adding substrate/chromogen.
- For example, after incubation with the streptavidin alkaline phosphatase conjugate, remove the base and wash both sides of the membrane under running distilled water. This helps to prevent high background as some reagents can leak through the membrane into the bottom tray of the plate.
- Add the enzyme substrate to each well and incubate with gentle agitation on a plate shaker, as directed by the manufacturer. See below for specific substrates that can be used.
After replacing the base and adding the substrate, carefully monitor spot formation.
Stop the reaction by gently washing the plate with PBS 0.1% Tween 20 once development appears to slow.
Take the base off the plates and wash both sides of the membrane with distilled water to stop the spot formation.
Dry the plates and allow the membranes to dry at room temperature.
Stage 6 - Readout and analysis
Steps
Punch the membranes out of the wells onto a sticky plastic sheet.
- This step will depend on your reader's requirements. Consult the reader manual.
Measure the sheet and analyze the membrane circles.
In the analysis software, set the following parameters for measurement.
- Size/spot diameter
- Intensity/saturation
- Circularity/shape
- Spot development/slope
These parameters can be saved and used for subsequent experiments for standardized results.
Substrate addition
The substrate addition step is pivotal in the ELISPOT assay, as it enables the visualization of cytokine-secreting cells. Selecting the appropriate substrate for the enzyme conjugate, such as AEC for horseradish peroxidase, is essential for generating clear, distinct spots. The concentration of the substrate solution should be carefully optimized to produce a strong signal from secreting cells without introducing high background noise.
Incubation time with the substrate must also be fine-tuned; too short an incubation may result in weak spots, while excessive incubation can cause overdevelopment and high background. Once optimal spot development is achieved, a stop solution, such as distilled water, should be used to halt the enzymatic reaction and preserve the integrity of the results. By paying close attention to these parameters, researchers can ensure that their ELISPOT assay accurately reflects the number and activity of cytokine-secreting cells.
Limitations
Despite its strengths, ELISPOT has several limitations. It is generally limited to detecting one or two analytes per well, making it less suitable for high-throughput multiplexing. The assay requires careful optimization of antibody concentrations and incubation times to avoid background noise or weak signals. Manual spot counting can introduce variability, although automated readers help mitigate this. Additionally, the technique is endpoint-based and does not provide kinetic data. Sample preparation, especially for PBMCs, must be handled with care to preserve cell viability and function. Finally, ELISPOT does not provide phenotypic information about the secreting cells unless combined with other methods.
Troubleshooting
Common ELISPOT issues include high background, weak signal, or inconsistent spot morphology. High background may result from insufficient blocking or non-specific antibody binding; ensure proper blocking buffers and washing steps. Weak signals often stem from low cell viability or suboptimal antibody concentrations; verify cell health and titrate antibodies. Irregular or diffuse spots may indicate overloading of cells or poor membrane quality. Always use fresh reagents and avoid using a plate washer during early steps, as ELISPOT plates are delicate. If frozen PBMCs are used, allow them to rest post-thawing to reduce debris. Consistent technique and proper controls are key to reliable results.
Conclusion
The ELISPOT assay stands out as a robust and sensitive method for detecting and quantifying cytokine-secreting cells, offering unparalleled insights into immune responses at the single-cell level. Researchers can achieve precise and reproducible results by carefully selecting high-quality materials and reagents, maintaining cell viability, and optimizing each step, from cell culture to substrate addition. The ELISPOT assay is invaluable for studying immune cells in a variety of contexts, including vaccine development, cancer immunotherapy, and basic immunology research. Attention to detail in cell preparation, reagent selection, and assay execution ensures that this technique remains a cornerstone for investigating the complex dynamics of immune cell function and the mechanisms underlying health and disease.