EZQuant–ELN Knowledge Base

Direct ELISA	Table of Contents

Plate coating:

• Add 50μl of antigen diluted in Carbonate-bicarbonate buffer to 96-well ELISA plates.
• Incubate for 2 hr at 37°C, or overnight at 4°C.
• Aspirate the antigen solution from the wells.
• Wash with 100μl of TBS and aspirate from the wells.

Blocking:

• Add 200μl of TBS+3% BSA to each well and incubate the plate for 2 hr
  at room temperature.
• Wash with 100μl of TBS+0.05% Tween 20 and aspirate from the wells.

Antibodies and detection: 

• Add 50μl of primary antibody diluted in TBS+0.05% Tween 20.
• Incubate plate for 2 hr at 37°C or overnight at 4°C.
• Wash 3 x 100μl of TBS+0.05% Tween 20 and aspirate from the wells.
• Add 50μl of enzyme-conjugated secondary antibody, diluted in TBS+0.05% Tween 20 and incubate the plate for 2 hr at 37°C.
• Wash 5 x 100μl of TBS+0.05% Tween 20 and aspirate from the wells.
• Add 200μl of substrate to each well and watch for color development.
• Read the OD values in a spectrophotometer at the appropriate wavelength.