Biochemical Techniques and Instrumentation

3.2.4.3. Competitive ELISA

The competitive ELISA is used to quantify antigen using competitive method. This method is used to assay small molecules usually an antigen with one epitope such as hormones in blood.  There are competitive ELISA kits available with enzyme conjugated antigen and enzyme conjugated antibody.

In enzyme conjugated antigen assay, there is a competition between natural target antigen and enzyme conjugated antigen. The target antigen and the enzyme conjugated antigen are mixed in a microplate well. Both of them compete for the primary antibody sites. If the natural antigen is more, it displaces the ezyme conjugated antigen more leading to their reduction. The substrate is then added and colour development is recorded. If more natural antigen is bound then colour development is low. This method is routinely used to test thyrozine hormone in blood.

In the enzyme conjugated assay, there is a competition by the  enzyme-linked secondary antibody with the sample antigen which is associated with the primary antibody. The unlabeled antibody is incubated in the presence of natural target antigen. The bound antibody/antigen complexes are then added to an antigen coated microtiter well. The plate is washed to remove the unbound antibody. The more antigen in the sample, the less antibody will be able to bind to the antigen in the well. The enzyme conjugated secondary antibody, specific to the primary antibody is added. Then, a substrate is added and remaining enzymes elicit a chromogenic or fluorescent signal.

For competitive ELISA, the higher the original antigen concentration, the signal will be weaker.