The following Elispot protocol is a general recommendation, and applies best for testing of PBMC, Lymph node or spleen cells for reactivity against peptides and peptide pools.

Day 1
Coat plate:

  1. 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.
  2. Coat plate with 100µl capture antibody in PBS (1µg total antibody per well works well in most instances).
  3. Incubate overnight at 4oC.

Day 2
Prepare plate:

    1. Decant antibody solution.
    2. Wash plate once with 100µl PBS.
    3. 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.)
      Prepare antigen(s):
    4. Dilute stock solution of peptide(s) to 2x final concentration with T-cell medium.
    5. Dilute stock solutions of control peptide pools (e.g., CEF, CEFT, CMVpp65) to 2x final concentration with T-cell medium.
    6. Dilute mitogen (e.g., PHA or ConA) to 2x final concentration with T-cell medium.
    7. 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.)
    8. Decant blocking medium.
    9. Add 50µl of antigen or negative control buffer to appropriate wells.

Prepare effector cells:

  1. 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.)
  2. Wash effector cells in T-cell medium, count and resuspend at a final concentration of 4-6×106 cells/ml in T-cell medium.
  3. Gently plate out effector cells (recommendation: 2-3×105 cells/well) in 50µl T-cell medium/well.
  4. Incubate for 16-20 hours (IFN-ɣ assay) in 37oC, 5% CO2.

Day 3

  1. Discard cells.
  2. Wash plate rigorously 6 x with PBS/0.05% Tween 20.
  3. 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.
  4. Incubate for at least 2 hours at 37oC.
  5. Wash plate 3 x with PBS.
  6. Add 100µl of Avidin-Enzyme-Complex/well.
  7. Incubate for at least 1 hour at room temperature.
  8. Discard Avidin-Enzyme-Complex, wash 3x with PBS.
  9. Add 100µl substrate per well following the manufacturer’s instructions. (Note: most substrate require an incubation time of 4 – 10 minutes.)
  10. Stop spot development under tap water.
  11. Remove underdrain and all excess liquid from wells.
  12. Dry back of wells thoroughly with a paper towel.
  13. Let plate dry overnight in darkness before reading or shipment to ZellNet for evaluation. (Note: spots are stable for many weeks and often months.)
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