ELISPOT protocol

General ELISPOT procedure outlining plate and sample preparation, cell incubation, enzymatic detection, and analysis.

Last edited Tue 31 Jan 2023

ELISPOT is a technique related to ELISA that was developed for the detection of secreted proteins, such as cytokines and growth factors. It is also called enzyme-linked immunospot.

ELISPOT is performed using a PVDF or nitrocellulose membrane 96-well plate pre-coated with an antibody specific to the secreted protein. Cells are added to the plate, where they attach to the coated membrane. Cells are then stimulated, and the secreted protein binds to the antibody. Next, a detection antibody is added that binds specifically to the bound protein.

The resulting antibody complex can be detected either through enzymatic action to produce a colored substrate or with fluorescent tags. An advantage to using fluorescence is the ability to identify more than one secreted protein at a time.

The membrane can be analyzed by manually counting the spots or with an automated reader designed for this purpose. Each secreting cell appears as a spot of color or fluorescence, thus this is a quantitative method for evaluating protein secretion.

Stage 1 - Plate preparation and coating with the capture antibody

Materials required

Steps

Prepare PVDF membranes in the 96-well plates by incubating in 35% ethanol for 30 seconds.

Ensure the ethanol is completely removed by washing thoroughly with PBS. Any remaining ethanol can affect cell viability, as well as antibody binding.

Coat 96-well plate with capture antibody diluted in phosphate buffered saline (PBS).

Incubate overnight at 4°C.

Empty the wells, tapping them dry, and wash with PBS.

Do not use a plate washer at this stage.
ELISPOT plates are more delicate than ELISA plates and should be handled with care. When tapping dry, do so gently.

Add 100 µL per well 2% dry skim milk to block non-specific binding to the membrane.

Wash the plate 3 times in PBS and leave to dry.

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 down-regulate the signal-transducing zeta chain of CD3, suppressing T cell function).

If preparation and plating are not possible within 8 hours:

  1. 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 4oC).
  2. 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.
  3. Ship fresh samples at ambient temperature. Note that transport temperatures can be below 4oC. Commercially available containers can be used to keep samples at an ambient temperature.
  4. 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.

Be careful to minimize the mixing of the sample and the Ficoll-PaqueTM.

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.

Rapid freezing damages cells.
To check the standardization of results, keep a large sample of cells frozen down from a good donor to use as a control in a series of ELISPOT experiments.

Steps

Thaw cells quickly.

Do not vortex cells at any time.

Gently transfer cells into a 50 mL tube containing 15 mL of culture media.

Fill tube to 50 mL with culture media.

Spin down cells at 300 x g for 5 minutes.

Pour off supernatant and flick tube gently to re-suspend the pellet.

Cells are now ready to use in the ELISPOT assay.

Steps

Tissue homogenization.

Centrifuge the cells for 5 minutes at 600 x g and re-suspend the cell pellet in medium recommended for the following procedure.

Consistent results can be obtained if the splenocytes are pre-stimulated for 24-48 hours with appropriate stimuli for cytokine release before addition to the ELISPOT plate with the same supplement for further incubation of 8-16 hours to allow spot formation.

Stage 3 - Cell incubation

Steps

Use cells prepared in the previous stage.

Dilute cells to the required concentration and add the cell suspension to wells.

Culture overnight at 37°C in CO2 incubator. Do not shake the plates.

Don't stack the plates if you have more than one to prevent edge effects.
Do not move the plates while the cells are culturing. This will lead to 'snail trail' spots that will not be well defined.

Optional - Indirect ELISPOT

Positive control stimulation

Ensure you are stimulating the PBMCs with the correct stimulant for the detection of your target cytokine.

Wash away the cells and the unbound cytokine by incubating with PBS 0.1% Tween 20 for 10 min.

Do not use a plate washer at this stage.
Ensure you include Tween 20 in the wash buffer. Some cells will have started attaching after culture overnight (eg some stem cells are known to do this). Tween 20 will help wash these off the membrane.

Stage 4 - Incubation with detection antibody

Materials required

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.

The incubation time may require optimization.

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.

HRP substrates
The substrate for HRP is hydrogen peroxide. The cleavage of hydrogen peroxide is coupled to the oxidation of a hydrogen donor, which changes color during the reaction
TMB (3,3’,5,5’-tetramethylbenzidine)
Add TMB solution to each well, incubate for 15–30 min, add an equivalent volume of stopping solution (2 M H2SO4), and measure the optical density at 450 nm
OPD (o-phenylenediamine dihydrochloride)
Keep and store the substrate in the dark, as it is light-sensitive. Add the OPD solution and incubate for 10-30 min. The reaction can be stopped using sulfuric acid 2.5-3.0 M H2SO4. For stopped reactions, measure the reaction using a plate reader at 450 nm; for reactions that are not stopped, measure the reaction at 490 nm.
ABTS (2,2’-azino-di-[3-ethyl-benzothiazoline-6 sulfonic acid] diammonium salt)
ABTS is an HRP substrate. Add ABTS solution to each well and incubate for 20 min on a rocker. If color generation occurs rapidly, then reduce the concentration of the conjugated antibody in the assay. Do not dilute this solution. This highly sensitive substrate produces a green product. The end product is measured at 405 nm. Always handle carefully and wear gloves as some enzyme substrates are considered hazardous (potential carcinogens)
AP substrate
P-Nitrophenyl-phosphate (pNPP) is the most widely used substrate for most applications. Measure the yellow color of nitrophenol at 405 nm after a 15–30 min incubation at room temperature and stop the reaction by adding an equivalent volume of 0.75 M NaOH.

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.

Spots may become sharper if membranes are stored overnight at 4°C and may continue to improve for up to 2 weeks. If storing, wrap membranes in foil and keep them at 4°C.

Stage 6 - Readout and analysis

Steps

Punch the membranes out of the wells onto a sticky plastic sheet.

Measure the sheet and analyze the membrane circles.

In the analysis software, set the following parameters for measurement.

These parameters can be saved and used for subsequent experiments for standardized results.

We recommend reading each plate 3 times and averaging the results in order to minimize errors in the measurements.