Interferon-Gamma ELISA Protocol

1. Coat plate with 50ul of a 2.0ug/ml of monoclonal rat anti-murine interferon gamma antibody in 50mM sodium bicarobonate buffer (pH 9.4)
2. Incubate plates 24 hours at 4^oC
3. Aspirate wells
4. Wash five times with PBST
5. Add 200ul of 5% milk in PBS (Blotto blcok)
6. Incubate 60 minutes at room temperature
7. Wash five times with PBST
8. Make dilutions from 2000 - 0 pg/ml of mouse interferon-gamma in 4% FBS in PBS
9. Add samples to be tested. If necessary, dilute samples as in step 8
10. Incubate 1 hour at room temperature
11. Add 50ul of 0.1ug/ml biotinylated anti-mouse interferon-gamma antibody in 4% BSA in PBS
12. Incubate 1 hour at room temperature
13. Wash five times with PBST
14. Add 100ul of streptavidin labele horseradish peroxidase in 4% BSA in PBS
15. Incubate 30 minutes at room temperature
16. Wash five times with PBST
17. Add 100ul of TMB substrate with H₂O₂ to each well
18. Incubate 20 minutes at room temperature in the dark
19. Add 50ul stop solution (1M H₂SO₄)
20. Read absorbance at 450nm
21. Read absorbance at 570nm (value at 570nm is subtracted from 450nm value to control for optical abnormalities in the ELISA plates)

Note: Antibody concentrations here may not hold for all sources. These concentrations should be titrated for each source.