The following Elispot protocol is a general recommendation, and applies best for testing of PBMC, Lymph node or spleen cells for reactivity against peptide and peptides pools.
- Prewet PVDF plate (e.g., MSIPS4510; Millipore) with 15µl 70% Ethanol until entire well turns dark, decant Ethanol and wash plate 3x with 150µl PBS.
- Coat plate with 100µl capture antibody in PBS (1µg total antibody per well works well in most instances).
- Incubate overnight at 4oC.
- Decant antibody solution.
- Wash plate once with 100µl PBS.
- Block with 150µl of T-cell medium (10% serum) for at least 2 hours at 37oC. (In case of serum-free medium: use PBS + 1% BSA, Fraction V.)
- Dilute stock solution of peptide(s) to 2x final concentration with T-cell medium.
- Dilute stock solutions of control peptide pools (e.g., CEF, CEFT, CMVpp65) to 2x final concentration with T-cell medium.
- Dilute mitogen (e.g., PHA or ConA) to 2x final concentration with T-cell medium.
- Prepare negative control (e.g., T-cell medium + DMSO at concentration equal to peptide conditions)
Note: (Avoid repeated thawing and freezing cycles of peptides and mitogens to avoid degradation and loss of activity.)
- Decant blocking medium.
- Add 50µl of antigen or negative control buffer to appropriate wells.
Prepare effector cells:
- Purify immune cells of interest or thaw frozen cells, e.g., PBMC. (Note: Consider overnight resting when working with frozen PBMC, also see Elispot Harmonization Guidelines and this publication.)
- Wash effector cells in T-cell medium, count and resuspend at a final concentration of 4-6×106 cells/ml in T-cell medium.
- Gently plate out effector cells (recommendation: 2-3×105 cells/well) in 50µl T-cell medium/well.
- Incubate for 16-20 hours (IFN-ɣ assay) in 37oC, 5% CO2.
- Discard cells.
- Wash plate rigorously 6 x with PBS/0.05% Tween 20.
- Add 100µl/well biotinylated detection antibody in PBS/0.5% BSA after filtering the antibody diluted in buffer with low-protein-binding filter, 0.22µm pore size.
- Incubate for at least 2 hours at 37oC.
- Wash plate 3 x with PBS.
- Add 100µl of Avidin-Enzyme-Complex/well.
- Incubate for at least 1 hour at room temperature.
- Discard Avidin-Enzyme-Complex, wash 3x with PBS.
- Add 100µl substrate per well following the manufacturer’s instructions. (Note: most substrate require an incubation time of 4 – 10 minutes.)
- Stop spot development under tap water.
- Remove underdrain and all excess liquid from wells.
- Dry back of wells thoroughly with a paper towel.
- Let plate dry overnight in darkness before reading or shipment to ZellNet for evaluation. (Note: spots are stable for many weeks and often months.)