GENERAL GUIDELINES FOR ELISA


NOTE:
To achieve the best results, optimization of many conditions must be done.  Assay conditions such as antibody and antigen working concentrations, incubation temperatures, and incubation times must be evaluated.  For optimal results, each laboratory should determine the best working conditions for their specific assay. 

A. ELISA - INDIRECT:

  1. Coat antigen in wells of a microtiter plate (100ul/well) diluted in 0.02M sodium phosphate buffer pH 7.4, PBS pH 7.4, or 0.05M Carbonate buffer pH 9.6.  Cover the plate with plastic film to prevent evaporation.  Incubate at room temperature for 4 hours or at 4ºC overnight.
  2. Remove coating solution from the wells.  Wash the wells 2X with PBS-0.05% Tween20. Tap the plates on absorbent paper to remove residual wash solution.
  3. Block the wells with 1% BSA-PBS (300ul/well).  Incubate at room temperature for 2 hours.
  4. Remove the blocking solution.  Do not wash the plates.
  5. Add antibody diluted in 1% BSA-PBS to the plate (100ul/well).   Incubate at room temperature for 1 hour.
  6. Wash 5X with PBS-0.05% Tween20.  Tap the plates on absorbent paper to remove residual wash solution.
  7. Add HRP-labeled secondary antibody diluted in 1% BSA-PBS.  Incubate at room temperature for 1 hour.
  8. Wash 5X with PBS-0.05% Tween20.  Tap the plates on absorbent paper to remove residual wash solution.
  9. Add TMB substrate solution (100ul/well).   Incubate until color is developed.
  10. Add 50ul of stop solution (0.4M H2SO4).  Tap the side of the plate gently to mix.
  11. Read the absorbance at OD450nm.
B. ELISA - SANDWICH:
  1. Coat capture antibody in wells of a microtiter plate (100ul/well) diluted in 0.02M sodium phosphate buffer pH 7.4 or PBS pH 7.4.  Cover the plate with plastic film to prevent evaporation.  Incubate at room temperature for 4 hours or at 4ºC overnight.
  2. Remove coating solution from the wells.  Wash the wells 2X with PBS-0.05% Tween20.  Tap the plates on absorbent paper to remove residual wash solution.
  3. Block the wells with 1% BSA-PBS (300ul/well).  Incubate at room temperature for 2 hours.
  4. Remove the blocking solution.  Do not wash the plates.
  5. Add antigen diluted in 1% BSA-PBS to the plate (100ul/well).  Incubate at room temperature for 1 hour.
  6. Wash 5X with PBS-0.05% Tween20.  Tap the plates on absorbent paper to remove residual wash solution.
  7. Add HRP-labeled detection antibody diluted in 1% BSA-PBS.  Incubate at room temperature for 1 hour.
  8. Wash 5X with PBS-0.05% Tween20.  Tap the plates on absorbent paper to remove residual wash solution.
  9. Add TMB substrate solution (100ul/well).  Incubate until color is developed.
  10. Add 50ul of stop solution (0.4M H2SO4).  Tap the side of the plate gently to mix.
  11. Read the absorbance at OD450nm.