ELISA protocol

Add 100 ul of antigen into the wells of a EIA plate. As a rule of thumb, each sample should be plated out in triplicate. Furthermore, a good experimental design should include various antigen and antibody dilution, as well as controls. Incubate overnight at 40C.

  • Wash 1x with TBS (Tris buffered saline)
  • Block for 1 hour with 5% BSA in TBS. (100 ul). Place the plates on a rocker platform (room temperature).
  • Wash 3x with TBS.
  • Add primary antibody that has been diluted in TBS-Tween (0.1%). The antibody should be diluted so that 100 ul is added into each well. Incubate for 2 hours at room temperature. Note: for low affinity polyclonal antibodies, it may be necessary to let the reaction to proceed overnight. If so, incubate the reaction at 4oC.
  • Wash 3x with TBS
  • Add secondary antibodythat has been diluted in TBS-Tween.The antibody should be diluted so that 100 ul is added into each well.Incubate for 2 hours at room temperature.
  • Wash 3x in TBS.
  • Add developer. Read the absorbance of the reactions after the recommended period of time has elapsed. You may have to stop colour development after some time. These considerations depend on the enzyme/substrate system that you are using.
Sham Nair 2014