Disclaimer

This procedure should be used as a guideline only. Please keep in mind that Creative Biolabs cannot guarantee specific results for ELISPOT assays.

ELISPOT is a technique related to ELISA for the detection of secreted proteins, such as cytokines and growth factors. It is also known as an enzyme-linked immunospot. It is a method for the quantitative assessment of protein secretion. Creative Biolabs offers specialized antibody development services for ELISPOT assays.

How ELISPOT Works

ELISPOT is performed using PVDF or nitrocellulose membrane 96-well plates. These plates are pre-encapsulated with antibodies specific for the secreted protein. Cells are added to the plates and attached to the encapsulated membranes. The cells are stimulated to secrete the proteins, which bind to the antibody. Next, a detection antibody that binds specifically to the bound protein is added.

The etection of the resulting antibody complexes is performed by the action of enzymes that produce colored substrates or by using fluorescent labels. One advantage of using fluorescence is the ability to recognize multiple secreted proteins simultaneously.

Membranes can be analyzed by manually counting spots or by using an automated reader designed for this purpose. Each secretory cell is shown as a colored or fluorescent spot, so this is a quantitative method for assessing protein secretion.

Procedure

1. PVDF membranes in 96-well plates are prepared by incubating them in 35% ethanol for 30 seconds. (It is important to ensure that the ethanol is completely removed by thorough washing with PBS. Any remaining ethanol will affect cell viability as well as antibody binding.)
2. Coat a 96-well plate with capture antibody diluted in phosphate-buffered saline (PBS). Incubate overnight at 4°C. (Approximately 0.5–1 µg of antibody should be used per well for well-defined spots. The capture concentration of the kit is optimized for optimal performance and is typically 100 µl per well.)
3. Empty the wells and tap them dry and wash them with PBS. (ELISPOT plates are more delicate than ELISA plates and should be handled carefully and gently. Do not use a plate washer at this stage.)
4. Add 100 µl of 2% dry skim milk to each well to block non-specific binding to the membrane. The plates are incubated at room temperature for 2 hours.
5. Wash the plates 3 times with PBS and leave them to dry. If necessary, the plates can be stored at this stage. Store at 4°C for no more than 2 weeks in a sealed plastic bag with desiccant.
6. Peripheral blood mononuclear cells (PBMCs) are prepared from fresh blood. The cells are counted using a viability dye, such as trypan blue. They should have more than 95% viability. Dilute the cells to the desired concentration and add the cell suspension to the wells. If optimizing the cell count assay, use a 1 : 2 dilution series. Do not shake the plate.
   The number of cells per well should be optimized. For example, if a low percentage of cells are expected to secrete the target cytokine, use more cells. Please refer to the specific target kit protocol for recommendations on assay control, and the number of cells per well. Typically, the number of cells per well should be between 2x10⁵ and 4x10⁵ PBMC cells.
   If possible, use serum-free medium, as serum contains many proteins that may affect the results. Alternatively, several batches of serum can be tested to find the serum with the best reaction-to-noise ratio. This batch of sera can then be stored and used for subsequent experiments.
7. Incubate overnight in a CO₂ incubator at 37°C. Do not shake the plates. (Do not move the plate during cell culture. This will result in "snail tracks" that are not well defined. If you have multiple plates, do not stack them to prevent edge effects.)
8. During the overnight incubation, the cells will secrete cytokines that bind to primary antibodies. (If the cells take time to respond to the stimulus, see the indirect method below.)
9. Incubate with PBS 0.1% Tween 20 for 10 minutes to wash off cells and unbound cytokines. (Make sure you include Tween 20 in the wash buffer. Some cells will begin to attach after being incubated overnight, as some stem cells do, for example. Tween 20 will help wash these cells away from the membrane. Do not use a plate washer at this stage.)
10. Dilute the conjugated detection antibody in PBS 1% BSA. Add to wells and incubate for 1–2 hours at room temperature (incubation time may need to be optimized.) Wash the plate 3 times with PBS 0.1% Tween 20 to remove non-specific antibody binding.
11. Add the conjugated detection antibody. Dilute the antibody in PBS 1% BSA. Incubate for 1–2 hours at room temperature (incubation time may need to be optimized.) Wash the plate 3 times with PBS 0.1% Tween 20 to remove non-specific antibody binding.
12. Add the enzyme substrate solution to each well. (For enzymatic assay protocols, the substrate should be removed from the bottom of the plate prior to adding the substrate/chromogenic agent in order to thoroughly clean the membrane. For example, after incubation with the streptavidin alkaline phosphatase conjugate, remove the substrate and wash both sides of the membrane under running distilled water. This helps prevent high background, as some reagents can leak through the membrane into the bottom tray of the plate.)
13. After replacing the base solution and adding the substrate, carefully monitor the spot formation. Once the development rate slows down, gently wash the plate with PBS 0.1% Tween 20 to stop the reaction. Remove the substrate from the plate and wash both sides of the membrane with distilled water to stop spot formation.
14. Wipe the plate dry and allow the film to dry at room temperature. (If the film is stored overnight at 4°C, spots may become sharper and may continue to improve for up to 2 weeks. If stored, membranes may be wrapped in foil and kept at 4°C.)
15. Punch the membranes through the hole onto an adhesive plastic sheet. This step will depend on the requirements of the readers.
16. Measure the sheet and analyze the circles of the membrane.

Data analysis

In the analysis software, set the following measurement parameters:

1. Size/spot diameter
2. Intensity/saturation
3. Roundness/shape
4. Spot development/slope

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

We recommend reading each plate 3 times and averaging the results to minimize measurement errors.

Indirect ELISPOT

If the cells take some time to respond to the stimulus, they may need to be pretreated with the stimulus in a separate 96-well culture dish prior to transfer to the ELISPOT plate.

Positive control stimulation

Experiments using ELISPOT for cytokine detection will require the use of positive controls. In these positive control wells, the cells should be stimulated with an agent known to induce expression of the cytokine being assayed. This can be used to compare with a negative control—no treatment or stimulation of a different secreted protein.

Make sure you stimulate PBMCs with the correct stimuli to detect your target cytokines. Typical stimuli include:

1. LPS to stimulate IL1β and IL6 secretion
2. PMA and ionomycin stimulate IL2 and IL4 secretion
3. PHA, 10 µg/ml for IFN gamma
4. Anti-CD3/CD28 antibodies for IFN gamma, IL4, IL10, and Granzyme B

EPISPOT sample preparation

Most ELISPOT experiments are performed with isolated PBMC (peripheral blood mononuclear cells). Both freshly prepared and cryopreserved cells can be used for this experiment. However, it is recommended that frozen cells be allowed to rest for at least one hour after thawing to allow for debridement of cellular debris prior to addition to the plate.

PBMCs should be prepared and plated within 8 hours of blood sample collection to preserve cellular function. If blood samples are left longer than this time, granulocytes (neutrophils) mixed with PBMCs will be activated. When PBMCs are separated from granulocytes, this may alter their buoyancy profile, and therefore granulocytes may contaminate the PBMC layer. Activated granulocytes may also start to activate some PBMCs (they can downregulate the CD3 signaling zeta chain and inhibit T cell function.)

If preparation and plating cannot be performed within 8 hours:

1. Dilute the blood sample immediately. This helps reduce granulocyte contamination. For example, dilute 1 : 1 in RPMI or PBS. Keep it at room temperature.
2. Remove granulocytes by cross-linking red blood cells and granulocytes, then separate from PBMCs. (Disadvantage: some PBMC may be lost.)
3. Transport fresh samples at ambient temperature. Note that transport temperatures may be below 4°C.
4. The freezing of samples should be optimized. Use serum-free media whenever possible (serum contains mitogens and inhibitors that may affect results.)
5. Density gradient separation is performed on the whole blood sample.

Preparing mouse spleen samples

1. Tissue homogenization: Gently pick the tissue with sterile stainless steel and disperse it into 30 mL of the recommended medium. Further disperse clumps by gently pipetting up and down several times. Remove the remaining cell clumps and debris.
2. Centrifuge the cells at 2,000 RPM for 5 minutes and resuspend the cell pellet in the medium recommended by the following procedure.

Note: Consistent results are obtained if splenocytes are pre-stimulated with the appropriate cytokine release stimulant for 24–48 hours and further incubated for 8–16 hours to allow for spot formation prior to the addition of splenocytes to ELISPOT plates.

Freezing cells for storage

1. Make up 20% DMSO in the cell culture medium. keep it on ice.
2. Label frozen vials.
3. Resuspend cells in ice-cold medium at 40 x 10⁶ cells/ml. Keep it on ice.
4. Dispense 0.5 mL of cell suspension into cryovials. Gently add 20% DMSO to the cell suspension at a ratio of 1 : 1. The final suspension will be 20 x 10⁶ cells/mL.
5. Place the cryovial immediately into the freezing container.

Note: Rapid freezing can damage the cells. Keeping a large number of frozen samples of cells from a good donor as a control in a series of ELISPOT experiments can be a useful tool for checking the standardization of results.

Thawing frozen cells for the ELISPOT assay

1. Thaw cells quickly in a 37°C water bath or in a warm water beaker. Do not vortex the cells at any time.
2. Gently transfer the cells to a 50 mL tube containing 15 mL of medium (0.5–5.0 mL of cells per 50 mL tube.)
3. Fill the 50 mL tubes with the medium. Gently invert the tubes to mix.
4. Spin the cells at 1,200 RPM for 5 minutes.
5. Pour out the supernatant and gently shake the tube to resuspend the cell pellet. Count in the appropriate medium and adjust the cells to the desired density.
6. Cells are now ready for ELISPOT assay.

ELISPOT Troubleshooting

High Background

No Spots or Very Few Spots

Blank Areas

Blank Center

False Positives (Checked by Running a Media Negative Control)

Confluent Spots

Poorly Defined Spots

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