Competitive ELISA (Protocol)

Competitive ELISA is enzyme linked immune assay involving two antibodies (one present in serum and another conjugated with enzyme) competing for binding the antigen to form antigen antibody complex.



The major steps involved in competitive ELISA includes:

  1. Primary antibody (unlabeled) is incubated with sample antigen.
  2. Antibody-antigen complexes are then added to well plates which are pre-coated with same antigen.
  3. Unbound antibody is removed by washing plate. (The more antigen in sample, less antibody will be able to bind to antigen in well, hence “competition.”)
  4. The secondary antibody that is specific to primary antibody and conjugated with an enzyme is added.
  5. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.


Competitive ELISA Protocol



For most applications, a polyvinylchloride (PVC) microtiter plate is most suitable /best. The common procedure for Competitive ELISA is outlined as follows:


  1. 50 μL of diluted primary antibody (capture) is added to each microtiter well & incubated for 4 hrs. at room temperature or 4°C overnight.

Note: If a purified capture antibody is not available, plate should first be coated with a purified secondary antibody, directed against the host of the capture antibody according to the following procedure:

  1. Bind unlabeled secondary antibody to the bottom of each well by adding approximately 50 μL of antibody solution to each well (20 μg/mL in Phosphate buffered saline (PBS).
  2. Incubate plate overnight at 4°C to allow complete binding.
  3. Add primary capture antibody (as above).


  1. Wells are washed twice with PBS. The antibody solution washes can be removed by flicking the plate over a suitable container.
  2. The remaining sites for protein binding on the microtiter plate must be saturated by incubating with blocking buffer. Wells are filled to the top with 3% BSA/PBS with 0.02% sodium azide and incubated for 2 hrs. or overnight in a humid atmosphere at room temperature.


Competitive ELISA Protocol


  1. Wash wells twice with PBS.
  2. 50 μL of standards or sample solution are added to wells. All dilutions should be done in blocking buffer (3% BSA/PBS with 0.05% Tween-20)

Note: Sodium azide is an inhibitor of horseradish peroxidase. Do not include sodium azide in buffers or wash solutions, if an HRP-labeled conjugate will be used for detection.


  1. 50 μL of the antigen-conjugate solution is added to wells (antigen solution should be titrated). All dilutions should be done in blocking buffer (3% BSA/PBS with 0.05% Tween-20) & incubated for at least 2 hrs. at room temperature in a humid atmosphere.
  2. Plate was washed with PBS for four to five times.
  3. Substrate is added. After suggested incubation time has elapsed, optical densities at target wavelengths can be measured on an ELISA reader.


Download Link: Compettitve ELISA (Protocol).pdf

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