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An ELISpot assay (enzyme-linked immunospot assay) counts the individual cells in a sample that secrete a given protein, such as a cytokine. Each spot on the membrane marks one secreting cell, so the result is a frequency of responding cells, not a concentration of protein. That difference is why ELISpot, ELISA and flow cytometry can rank the same donors differently.
Definition and scope
Mabtech’s documentation describes ELISpot as an immunoassay used to quantify analyte-secreting cells, and as an extremely sensitive sandwich assay that works down to the single-cell level. The most commonly used cells, at least from human samples, are peripheral blood mononuclear cells (PBMCs); other cell types work if they are viable.
This page covers the concept and the consumable choices the assay forces. It does not give a bench protocol; follow your assay kit’s instructions for use for that.
How the assay works in practice
The assay is built on a 96-well plate whose wells have a PVDF membrane at the bottom. According to Mabtech, the PVDF provides a larger membrane surface, which increases the amount of capture antibody that can be bound. Capture antibodies coat the membrane and catch the secreted protein right after it leaves the cell, during stimulation. That immediate capture near the secreting cell is the source of the assay’s sensitivity.
After the stimulation period, a detection antibody, an enzyme system and a substrate are used to reveal where protein was captured. Mabtech lists biotinylated detection antibodies for two-step formats and enzyme-conjugated ones for one-step formats, with alkaline phosphatase (substrate BCIP/NBT-plus) or horseradish peroxidase (substrate TMB) as the enzyme options. The substrate must precipitate on the membrane so that it forms a visible spot; Mabtech says not to use ELISA substrates for ELISpot. The dried plate is then counted, usually with an automated reader. For judging a positive response, Mabtech describes the DFR statistical method rather than simple subtraction of negative-control spots.
Each experiment includes positive, negative and background controls alongside the stimulated cells.
ELISpot vs ELISA vs flow cytometry
The three methods are often run on the same cell samples, but they answer different questions. A multi-donor study of IFN-γ in human PBMCs (Hagen et al., Cells 2015) ran ELISPOT side by side with ELISA and flow cytometry. The study used five donors and a single mitogen stimulation, and all authors are employed by a company that sells ELISpot, ELISA and flow cytometry products, so the donor-level figures should not be generalised.
| Method | What it reports in the Hagen et al. comparison | What the study found |
|---|---|---|
| ELISpot | Number of spot-forming cells (SFCs) secreting IFN-γ | Donor ranking generally matched flow cytometry; donors with similar flow counts showed 2-3-fold differences in SFCs |
| ELISA | Cytokine amount in the sample | ELISA values did not correlate with either ELISPOT or flow cytometry; donors with the same ELISA IFN-γ showed six-fold differences in ELISPOT |
| Flow cytometry | Cells expressing IFN-γ | Generally matched ELISPOT ranking of donors |
The study’s conclusion was that the three assays are complementary and are best run together.
What to check when specifying plates and reagents
The assay constrains several consumable choices. Each item below comes from Mabtech’s documentation, except where noted.
- Plate membrane: PVDF membrane in each of the 96 wells.
- Plate format: MSIP plates have a plastic underdrain and one cut corner, in clear (MSIPS45) or white (MSIPS4W); MAIPSWU (ELIIP) plates are white, with a detachable tray and two cut corners.
- Substrate: a precipitating substrate, not an ELISA substrate.
- Enzyme system: alkaline phosphatase (BCIP/NBT-plus) or horseradish peroxidase (TMB), matched to the detection format.
- Controls: positive, negative and background wells.
- Readout: an automated reader and an analysis rule such as DFR, agreed before the run.
- Sample: viable cells; PBMCs are the most commonly used.
Common misunderstandings
More spots means more cytokine
Not necessarily. Hagen et al. state that a higher number of SFCs in ELISPOT does not guarantee that those cells secrete larger amounts of cytokine. ELISpot counts cells, not protein.
An ELISA result can stand in for a spot count
The same study found no correlation between ELISA values and ELISPOT or flow cytometry results in the donors tested, so the two numbers should not be treated as interchangeable.
ELISA reagents can be reused in an ELISpot plate
The membrane format and the precipitating substrate differ. Mabtech says not to use ELISA substrates for ELISpot.
FAQ
What does ELISpot stand for?
Enzyme-linked immunospot. Each spot marks one protein-secreting cell.
What does an ELISpot assay measure?
The number of analyte-secreting cells in a sample, down to the single-cell level.
Is ELISpot the same as ELISA?
No. Both are sandwich-type assays, but ELISpot counts secreting cells on a membrane and needs a precipitating substrate, while ELISA reports the amount of protein in a sample.
Which cells are used?
PBMCs are the most commonly used cells, at least from human samples; other cell types can be used if they are viable.
Related CASRAI pages
For background see what immunology covers and the ImmPort immunology data repository. Related methods: ELISA antibodies, how a flow cytometer works and PBMC preparation for flow cytometry.
Sources
- Mabtech, “ELISpot step by step” (manufacturer documentation): https://mabtech.com/knowledge-center/assay-principles/elispot-assay-principle/elispot-step-step (checked 2026-10-29).
- Hagen J, et al. “Comparative Multi-Donor Study of IFNg Secretion and Expression by Human PBMCs Using ELISPOT Side-by-Side with ELISA and Flow Cytometry Assays.” Cells 2015;4(1):84-95: https://pmc.ncbi.nlm.nih.gov/articles/PMC4381211 (checked 2026-10-29).
