Competitive ELISA is a technique used for estimation of antibodies present in a specimen, such as serum. It is most complex of all the ELISA techniques. The competitive/inhibition ELISA is predominantly used to measure the concentration of an antigen or antibody in a sample by detecting interference in an expected signal output. Essentially, sample antigen or antibody competes with a reference for binding to a limited amount of labeled antibody or antigen, respectively.
Principle of the test is that two specific antibodies, one conjugated with enzyme and other present in test serum (if serum is positive for antibodies), are used. Competition occurs between two antibodies for same antigen. Appearance of color indicates a negative test (absence of antibodies), while the absence of color indicates a positive test (presence of antibodies). Higher the antigen concentration weaker the signal (i.e. color appearance).
The central event of competitive ELISA is a competitive binding process executed by original antigen (sample antigen) and add-in antigen. The procedures of competitive ELISA are different in some respects compared with other forms of ELISA (Direct, Indirect and Sandwich).
The major advantage of a competitive ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present.
In this method, a primary antibody is incubated with sample, which forms a antigen- antibody complex. The complex is then adsorbed to wells. Then, a secondary antibody is added to wells, which recognizes primary antibody only if it is not bound to the antigen. Therefore, the secondary antibody competes with antigen.
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 test serum, antigen–antibody reaction occurs. Antigen– antibody reaction i.e. Antigen-Antibody complex formation is detected by adding enzyme-labeled-specific antibodies. In a positive test, no antigen is left for these antibodies to act. Hence, antibodies remain free and are washed away during 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 serum, antigen in the coated wells is available to combine with enzyme-conjugated antibodies and the enzyme acts on the substrate to produce color.
Some competitive ELISA include enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with your sample antigen produces the signal.
- High specificity, since two antibodies are used and the antigen/analyte is specifically captured and detected.
- Sample processing is not required i.e. crude or impure sample can be used.
- Suitable for complex samples, since the antigen does not require purification prior to measurement.
- Less sensitive to sample dilution & sample matrix effects.
- Flexibility and sensitivity, since both direct and indirect detection methods can be used.
- Less variability (More consistent) between duplicate sample & assays.
Download Link: Competitive ELISA.pdf