In enzyme-linked immunosorbent assay, what problem can arise if the amount of antigen exceeds the amount of antibody?

In an enzyme-linked immunosorbent assay (ELISA), when the amount of antigen present exceeds the amount of antibody, a phenomenon known as the "hook effect" can occur. This hook effect leads to an underestimation of the antigen concentration in the sample.

When the concentration of antigen is very high, it can saturate all available binding sites on the antibodies. As a result, fewer antigen-antibody complexes form, leading to a lower signal in the assay compared to what would be expected for that concentration of antigen. This is counterintuitive, as one might expect a higher amount of antigen to correlate with a higher signal, but the competition for binding sites prevents optimal complex formation.

This situation contrasts with the prozone effect, which refers specifically to high concentrations of antibodies leading to a similar underestimation due to insufficient antigen binding. Thus, in the context of high antigen concentrations overwhelming available antibodies, the hook effect accurately describes this issue in ELISA testing.