ELISpot Protocol

Detailed procedure for ELISpot. View our ELISpot Troubleshooting to solve your problem.

Reagents

• PBS
• Coating buffer: 8.4 g NaHCO₃, 3.56 g Na₂CO₃, add ddH₂O up to 1.0 L, pH to 9.5.
• Blocking buffer
• Wash buffer: PBS containing 0.1% Tween 20
• Antibody dilution buffer: PBS containing 1% BSA
• Substrate solution

Do not use sodium azide in any buffers or solutions as sodium azide inactivates the horseradish-peroxidase (HRP) enzyme.

General ELISpot Procedure

• Prepare the Plate
1. Prepare the polyvinylidene difluoride (PVDF) membrane 96-well ELISpot plates by soaking them in 35% ethanol for 30 seconds.
2. Wash thoroughly with PBS to remove any residual ethanol.

Note: Any remaining ethanol can negatively affect cell viability, as well as antibody binding.

• Coat the Plate
1. Coat 96-well plate with appropriate diluted capture antibody. Incubate overnight at 4°C.

Note: Approximately 0.5-1 µg per well of antibody should be used for well-defined spots.

2. Empty the wells, tapping them dry. Wash the plate 3 times in PBS.

Note: a) ELISpot plates are more delicate than ELISA plates and thus should be handled carefully. b) Do not use an automatic plate washer, since this could compromise the integrity of the PVDF membrane.

• Block the Plate
1. Block plates by adding 100 µl 2% milk per well. Incubate 2 h at RT. Wash the plate 3 times in PBS.

Note: The plates can be stored at this stage. Store at 4°C for not more than 2 weeks.

• Set-Up Tissue Culture and Add Antigen or Mitogen
1. Prepare peripheral blood mononuclear cells (PBMCs) from fresh blood. 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.

Note: The number of cells per well should be optimized. If optimizing the assay for cell number, use a 1:2 dilution series. Do not shake plates.

2. 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.

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

• Add Detection Antibody
1. Wash away the cells and the unbound cytokine by incubating with PBS containing 0.1% Tween 20 for 10 min.
2. Dilute the conjugated detection antibody in PBS containing 1% BSA. Add to wells and incubate for 1-2 h at RT. Wash plate 3 times with PBS containing 0.1% Tween 20 to remove non-specific detection antibody binding.
• Add Substrate
1. Add the enzyme substrate solution to each well. Carefully monitor spot formation.
2. Stop the reaction by gently washing the plate with PBS containing 0.1% Tween 20 once development appears to slow. Take the base off the plates. Wash both sides of the membrane with distilled water to stop the spot formation.
3. Dry the plates and allow the membranes to dry at RT.
4. Measure the sheet and analyze the membrane circles.

Typical Stimulates

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

Indirect ELISpot

If the cells take some time to respond to stimulation, they may require pretreatment with the stimulant in a separate 96-well culture dish before transferring to the ELISpot plate.

Control Setting

• Positive control wells: The cells should be stimulated with an agent known to induce expression of the cytokine being detected.
• Negative control wells: No treatment or stimulation of a different secreted protein.

Precautions

• PBMC, or other blood samples should be treated as potentially infectious biohazard. Handle everything that comes in contact with specimen as potential biohazard, level II.
• Wear disposable gloves and personal protection throughout the entire procedure and thoroughly wash hands after handling the test reagents.
• Wipe any spills immediately with a laboratory-approved disinfectant, such as 10% bleach followed by 70% ethanol.
• All test specimens and materials used in the procedure must be disposed of as biohazardous waste.