ELISpot measures the frequency of individual cells secreting a target analyte, most commonly cytokines such as IFN-γ, IL-2 or TNF-α. Capture antibody is immobilised on a membrane-bottom plate, cells are stimulated in the wells, secreted analyte is captured locally, and spots are developed after the cells are removed. Reliable ELISpot depends on healthy cells, validated capture/detection antibody pairs, controlled stimulation, careful washing and objective spot-counting criteria.
Enzyme-linked immunospot (ELISpot) is a highly sensitive cell-based immunoassay used to enumerate cells that secrete a defined protein during a controlled incubation period. Unlike a conventional ELISA, which reports the accumulated concentration of analyte in bulk solution, ELISpot preserves the spatial footprint of secretion around individual cells. Each developed spot therefore represents a local secretion event rather than simply total soluble protein concentration.
When is ELISpot useful?
- Measuring antigen-specific T-cell or B-cell responses
- Quantifying cytokine-secreting cells after peptide, protein or mitogen stimulation
- Immune monitoring in vaccine, infection, cancer and immunotherapy research
- Detecting low-frequency responder populations that may be difficult to resolve by bulk cytokine assays
- Comparing response frequency across treatment, genotype or stimulation conditions
Bio-Techne notes that optimized ELISpot assays can detect very low-frequency cytokine-secreting cells, while Abcam describes the assay as a single-cell secretion readout suitable for PBMCs and other immune cell populations.
ELISpot vs ELISA vs intracellular cytokine flow cytometry
| Method | Primary readout | Strength | Limitation |
|---|---|---|---|
| ELISpot | Frequency of secreting cells | Very sensitive for rare responder populations | Limited phenotyping unless combined with additional methods |
| ELISA | Bulk analyte concentration | Straightforward quantitative soluble-protein measurement | Does not identify how many cells produced the analyte |
| Intracellular cytokine flow cytometry | Cytokine-positive cells plus phenotype markers | Multiparameter cell phenotyping | Requires fixation/permeabilisation and measures retained intracellular cytokine rather than secreted spots |
Equipment
- 96-well ELISpot plate with PVDF or nitrocellulose membrane
- Multichannel pipette and sterile tips
- Cell counter or haemocytometer
- 37°C cell-culture incubator with appropriate CO₂ control
- Plate shaker where required
- ELISpot plate reader or validated imaging system
- Inverted microscope for cell-quality checks
Reagents
- Capture antibody specific to the secreted target
- Biotinylated or otherwise labelled detection antibody recognising a non-competing epitope
- Streptavidin-enzyme conjugate for biotin-based detection
- Compatible chromogenic or fluorescent substrate
- Blocking buffer
- Wash buffer
- Cell-culture medium and supplements appropriate to the cells
- Experimental antigen/stimulus
- Positive-control stimulant where appropriate
Experimental design before starting
Choose the biological readout
Define the cytokine or secreted protein that answers the research question. IFN-γ is common in T-cell studies, but the correct target depends on the cell population and biology being studied.
Plan the controls
- Unstimulated/vehicle control: establishes spontaneous secretion background.
- Positive stimulation control: confirms cell competence and assay function.
- Medium-only blank: monitors membrane and detection background.
- Cell-free antibody/detection control: useful when troubleshooting substrate background.
- Replicate wells: strongly recommended because biological responses can be heterogeneous.
Optimise cell number
Too few cells reduce sensitivity; too many cells can create overlapping or confluent spots that cannot be counted accurately. Run a cell-number titration for a new cell type, stimulus or expected response magnitude.
Detailed ELISpot protocol
1. Prepare the membrane plate
Follow the plate manufacturer's instructions. PVDF plates may require brief ethanol wetting before PBS washing. Abcam's general protocol uses 35% ethanol for approximately 30 seconds, followed by thorough PBS washing; residual ethanol must be removed because it can affect both cell viability and antibody binding.
2. Coat with capture antibody
- Dilute the capture antibody in PBS or the recommended coating buffer.
- Add the validated volume per well, commonly around 100 µL.
- Incubate overnight at 4°C or according to the antibody-pair/kit instructions.
- Wash carefully without damaging the membrane.
Abcam gives approximately 0.5–1 µg capture antibody per well as a general starting point for development assays, but optimized kits may use different concentrations. Use product-specific recommendations where available.
3. Block the plate
- Add blocking buffer to cover the membrane completely.
- Incubate for approximately 1–2 hours at room temperature or as validated.
- Remove blocking solution immediately before adding cells.
4. Prepare cells
- Use freshly prepared, viable cells wherever possible.
- Count cells accurately and assess viability.
- Resuspend in complete culture medium at the required concentration.
- Keep stimulation and handling times consistent across samples.
5. Add cells and stimulus
- Add cells to each well at the optimized density.
- Add antigen, peptide, mitogen, drug or vehicle according to the experiment.
- Include positive- and negative-control wells on the same plate.
- Avoid disturbing the plate once cells have settled because local secretion must remain spatially confined for discrete spot formation.
6. Incubate for secretion
Incubation time depends on the target and stimulation model. Overnight or approximately 12–24 hour incubations are common for many cytokine ELISpot assays. Longer incubations can increase background or spot spreading, while short incubations may under-sample slower responses.
7. Remove cells and wash
- Discard cells and culture medium.
- Wash the membrane thoroughly using the recommended buffer.
- Use sufficient washes to remove cells and soluble analyte without physically damaging the membrane.
8. Add detection antibody
- Dilute the detection antibody in the recommended assay buffer.
- Add to each well and incubate for the validated time, often 1–2 hours at room temperature.
- Wash thoroughly.
9. Add enzyme conjugate
For biotinylated detection systems, add streptavidin-HRP or streptavidin-alkaline phosphatase at the validated dilution, incubate, then wash extensively.
10. Develop spots
- Add the compatible substrate.
- Monitor spot formation and stop development before high background or spot merging occurs.
- Wash with water or the manufacturer's stop procedure.
- Allow the plate to dry completely before automated counting if required by the reader.
11. Count and analyse
Use an ELISpot reader with consistent sensitivity, spot-size and intensity thresholds across comparable groups. Report spot-forming cells or spot-forming units per defined number of input cells, after applying the pre-specified background subtraction rule.
How should ELISpot data be interpreted?
- Compare stimulated wells with matched unstimulated controls.
- Inspect the plate images, not only the exported count.
- High background in negative-control wells reduces confidence even if the stimulated condition is higher.
- Confluent wells are not reliably countable and should be repeated at a lower cell density.
- Do not treat every machine-detected object as a biological spot; reader settings should be validated against representative wells.
Optimisation priorities
| Variable | If too low | If too high |
|---|---|---|
| Cell number | Missed rare responses | Overlapping/confluent spots |
| Capture antibody | Poor local capture | Potential non-specific background/cost |
| Stimulation | Weak biological response | Toxicity, non-specific activation |
| Incubation time | Insufficient secretion | Diffuse spots and increased background |
| Detection reagent | Weak spots | High background |
| Substrate time | Faint spots | Merged/dark background |
Limitations
- ELISpot measures secretion frequency, not detailed multiparameter phenotype.
- Spot size/intensity can vary with secretion kinetics and should not automatically be interpreted as per-cell secretion quantity without validation.
- Cell viability and sample handling materially affect results.
- Different readers/software settings can change spot counts.
- Cross-reactivity in capture/detection antibody pairs can create false signal.
Troubleshooting
| Problem | Likely cause | What to try |
|---|---|---|
| Few/no spots in positive control | Poor cell viability, inactive stimulus, weak antibody pair | Check viability, verify stimulant and antibody reagents, confirm incubation conditions. |
| High background in unstimulated wells | Spontaneous activation, inadequate washing, overdevelopment | Reduce handling stress, increase wash quality, shorten substrate development. |
| Spots too large/confluent | Too many cells, long incubation or strong stimulation | Reduce cell number and/or incubation time. |
| Very faint spots | Low secretion, weak capture/detection pair or short development | Titrate antibodies, verify stimulus and extend substrate development cautiously. |
| High well-to-well variability | Inconsistent cell counting/pipetting or uneven stimulation | Use calibrated multichannel pipettes, mix cell suspension gently and consistently, use replicates. |
Frequently asked questions
Does one ELISpot spot always equal one cell?
It is best understood as one local secretion event from a secreting cell under the assay conditions. Overlapping cells, migrating cells and spot merging can complicate one-to-one interpretation, so counting settings and cell density must be validated.
How many cells should I plate?
There is no universal number. Start with a titration based on expected response frequency and choose the range that gives discrete, countable spots in stimulated wells while keeping background manageable.
Can ELISpot replace flow cytometry?
No. ELISpot is exceptionally sensitive for secretion frequency, while flow cytometry can identify phenotype, intracellular markers and multiparameter cell states. They answer related but different questions.
Related ABMIUM methods
Explore reagents for ELISpot
Need a capture/detection antibody pair, recombinant cytokine control or help checking species reactivity? Use Find your MATCH or contact ABMIUM with the target, species, cell type and intended readout. We can search across the ABMIUM catalogue rather than limiting the shortlist to one manufacturer.