ELISPOT Assay

Materials

  • IFNgamma Capture antibody: monoclonal anti human IFNg, low endotoxin, no azide Pierce Biotechnology catalog no. M-700A
  • Biotinylated anti IFNg: Pierce Biotechnology catalog no. M-701B
  • HRP-streptavidin: Pierce Biotechnology catalog no. M-701B
  • Plates: Millipore cat. no. MAHA S45 10
  • Substrate: TMB blotting from Pierce Biotechnology catalog number 34018. Other peroxidase substrates, those suitable for immunohistochemical staining, can be used.

Procedure

  1. Dilute anti IFNg antibody to 10 ug/ml in sterile PBS. Add antibody to Millipore HA plate at 100 ul of antibody per well. Store overnight at 2-8°C.
  2. Remove antibody solution by flicking into a basin. Wash plate four times with sterile PBS by adding 200 ul of PBS per well and flicking.
  3. Add 200 ul per well PBS 10% FBS and incubate at 37°C for at least 30 minutes.
  4. Prepare antigens and anti CD3 (as a positive control). We have had best results with M1 peptide at a concentration of 20 ug/mL and anti-CD3 at 1 ug/mL. Other antigens should be tested to find the best concentration. Prepare 0.4 mL to test in triplicate.
  5. When antigens are ready, flick out PBS 10% FBS and add medium, anti-CD3 and antigens to plate. Add 100 ul per well.
  6. Thaw PBMC to be tested and adjust cell concentration to 2.5 x 106 per ml. Add cells to plate at 100 ul per well.
  7. Incubate 40-48 hours. Do not disturb plates during incubation or spots will be blurred.
  8. Flick out cells and wash plate four times with PBS followed with four washes with PBS-Tween. (0.05% Tween 20)
  9. Add 100 ul per well of biotinylated anti IFNg (dilute to 1 ug/mL in PBS 4% BSA).
  10. Incubate 1 hour at room temperature.
  11. Wash 4 times with PBS-Tween.
  12. Add 100 ul per well of HRP-streptavidin. Dilute HRP-streptavidin 1:10,000 in PBS-Tween.
  13. Incubate 30 minutes at room temperature.
  14. Wash 4 times with PBS-Tween.
  15. Add 100 ul per well of TMB substrate.
  16. Incubate 5 minutes at room temperature. Stop reaction by rinsing plate with tap water.
  17. Remove plastic backing from plate and allow to dry.