The major steps involved in competitive ELISA includes:
- Primary antibody (unlabeled) is incubated with sample antigen.
- Antibody-antigen complexes are then added to well plates which are pre-coated with same antigen.
- 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.”)
- The secondary antibody that is specific to primary antibody and conjugated with an enzyme is added.
- A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
For most applications, a polyvinylchloride (PVC) microtiter plate is most suitable /best. The common procedure for Competitive ELISA is outlined as follows:
- 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:
- 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).
- Incubate plate overnight at 4°C to allow complete binding.
- Add primary capture antibody (as above).
- Wells are washed twice with PBS. The antibody solution washes can be removed by flicking the plate over a suitable container.
- 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.
- Wash wells twice with PBS.
- 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.
- 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.
- Plate was washed with PBS for four to five times.
- 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