When Does The Hook Effect Start?


The hook effect is a phenomenon that occurs in medical testing when the concentration of a particular substance is so high that it overwhelms the detection system. This can cause false negative results, which can be dangerous for patients. In this article, we will explore when the hook effect starts and how to recognize and prevent it.

What is the Hook Effect?

The hook effect, also known as the prozone effect, occurs when an antigen-antibody reaction is inhibited due to high antigen concentration. In other words, the antigen is present in such high quantities that it binds to all the available antibodies, leaving none for detection.

The hook effect is most commonly seen in immunoassays, which are used to detect the presence of specific proteins, hormones, or other molecules in a sample. If the concentration of the target molecule is too high, it can saturate the detection system, leading to false negative results.

When Does the Hook Effect Start?

The hook effect typically starts at very high concentrations of the target molecule. The exact concentration at which it occurs will depend on the specific immunoassay being used. However, it is generally considered to be a problem when the concentration exceeds the upper limit of the assay’s linear range.

For example, if an assay has a linear range of 0-100 ng/mL for a particular protein, the hook effect may start to occur when the concentration exceeds 100 ng/mL. At this point, the test may show a false negative result, even though the target molecule is present in the sample.

Recognizing the Hook Effect

The hook effect can be difficult to recognize, as it can produce false negative results that may be mistaken for a true negative. However, there are some signs that may indicate the presence of the hook effect:

  • Unexpectedly low or negative results, especially when the clinical symptoms suggest the presence of the target molecule
  • Inconsistent results, such as negative results on one day and positive results on another
  • Variations in results depending on dilution or concentration of the sample

If any of these signs are present, it may be necessary to repeat the test with a higher dilution or a different assay to confirm the results.

Preventing the Hook Effect

The best way to prevent the hook effect is to ensure that the concentration of the target molecule is within the linear range of the assay. This can be achieved by diluting the sample or using a different assay with a higher upper limit. It is also important to follow the manufacturer’s instructions carefully and to perform quality control checks regularly.

In some cases, it may be necessary to use a different type of assay altogether, such as a Western blot or PCR, which are less prone to the hook effect.


The hook effect is a potential problem in medical testing that can lead to false negative results. It typically occurs at very high concentrations of the target molecule, beyond the linear range of the assay. To prevent the hook effect, it is important to follow the manufacturer’s instructions carefully, perform quality control checks regularly, and consider using a different assay or diluting the sample if necessary.