Competitive ELISA Protocol and Animation

Competitive ELISA is a technique used to estimate antibodies present in a specimen, such as serum.

• The principle of the test is that two specific antibodies, one conjugated with enzyme and the other present in test serum (if serum is positive for antibodies), are used.
• Competition occurs between the two antibodies for the same antigen.
• The appearance of color indicates a negative test (absence of antibodies), while the absence of color indicates a positive test (presence of antibodies).
• The central event of competitive ELISA is a competitive binding process executed by the original antigen (sample antigen) and add-in antigen.
• The procedures of competitive ELISA are different in some respects compared with other forms of ELISA.

Steps/Process of Competitive ELISA

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

Competitive ELISA Results

• In this test, microtiter wells are coated with antigen.
• The sera to be tested are added to these wells and incubated at 37°C and then washed.
• If antibodies are present in the test serum, antigen–antibody reaction occurs.
• The antigen– antibody reaction is detected by adding enzyme-labeled-specific antibodies.
• In a positive test, no antigen is left for these antibodies to act.
• Hence, the antibodies remain free and are washed away during the process of washing.
• When substrate is added, no enzyme is available to act on it.
• Therefore, positive result is indicated by absence of color reaction.
• In a negative test, in which no antibodies are present in the serum, antigen in the coated wells is available to combine with enzyme-conjugated antibodies and the enzyme acts on the substrate to produce color.

Advantages of Competitive ELISA

1. High specificity, since two antibodies are used and the antigen/analyte is specifically captured and detected
2. Suitable for complex samples, since the antigen does not require purification prior to measurement
3. Flexibility and sensitivity, since both direct and indirect detection methods can be used

Protocol of Competitive ELISA

For most applications, a polyvinylchloride (PVC) microtiter plate is best.

1. Add 50 μL of diluted primary antibody (capture) to each microtiter well. Allow to incubate 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 the 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 the plate overnight at 4°C to allow complete binding.
• Add primary capture antibody (as above).

2. Wash the wells twice with PBS. The antibody solution washes can be removed by flicking the plate over a suitable container.
3. The remaining sites for protein binding on the microtiter plate must be saturated by incubating with blocking buffer. Fill the wells to the top with 3% BSA/PBS with 0.02% sodium azide. Incubate for 2 hrs. to overnight in a humid atmosphere at room temperature.
4. Wash wells twice with PBS.
5. Add 50 μL of the standards or sample solution to the wells. All dilutions should be done in the 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.

6. Add 50 μL of the antigen-conjugate solution to the wells (the antigen solution should be titrated). All dilutions should be done in the blocking buffer (3% BSA/PBS with 0.05% Tween-20). Incubate for at least 2 hrs. at room temperature in a humid atmosphere.
7. Wash the plate four times with PBS.
8. Add substrate. After suggested incubation time has elapsed, optical densities at target wavelengths can be measured on an ELISA reader.

Competitive ELISA Animation