ELISPOT protocol

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

• 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 Ficoll^TM, 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 Ficoll^TM. (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-Paque^TM 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 37^oC 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

• 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).

• Typical stimulates include:

  • LPS to stimulate IL1β, IL6 secretion
  • PMA and ionomycin stimulate IL2, 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.