How Does the ELISA Test Work?

An ELISA test detects a specific antibody or antigen using complementary binding and an enzyme-controlled colour change. A positive result occurs when the target molecule is present and the enzyme produces a coloured product.

What does ELISA mean?

ELISA stands for enzyme-linked immunosorbent assay. It relies on the specificity of antibodies: an antibody has a binding site with a shape complementary to a particular antigen.

The exact method varies depending on whether the test is detecting an antibody or an antigen. A common method for detecting antibodies works as follows:

1. A known antigen is attached to the surface of a well in a test plate.
2. The patient's sample is added. If it contains the specific antibody, that antibody binds to the antigen.
3. The well is washed to remove any antibodies that have not bound.
4. An enzyme-linked antibody is added. This binds to the patient's antibody.
5. The well is washed again to remove unbound enzyme-linked antibodies.
6. A colourless substrate is added. The attached enzyme converts it into a coloured product.

A colour change therefore indicates that the patient's specific antibody was present. The greater the concentration of the target antibody, the more enzyme remains in the well and the stronger the colour change is likely to be. A colorimeter can measure this colour intensity.

ELISA can also detect an antigen. In this version, antibodies fixed to the well bind the target antigen from the sample. A second enzyme-linked antibody then binds to that antigen before the substrate is added.

Exam mistake to avoid

Do not say that the antibody simply “matches” the antigen. State that the antibody's binding site has a specific complementary shape to the antigen. Also explain why washing is essential: it removes unbound enzyme-linked antibodies that could otherwise produce colour and cause a false positive result.