Horseradish Peroxidase (HRP)
Definition
The most common enzyme label in immunoassays. When the substrate TMB (3,3'-5,5'-tetramethylbenzidine) is added, HRP converts it to a blue product that turns yellow on acidification and is read at 450 nm.
- Role
- Enzyme label in immunoassays
- Common substrate
- TMB
- Colour change
- Blue product, turns yellow on acidification
- Read wavelength
- 450 nm
Common questions
Why is the reaction stopped with acid before reading the plate?+
Acidification converts the blue TMB oxidation product to a more stable yellow product and halts further enzyme activity, giving a fixed endpoint reading rather than a signal that keeps changing while the plate is being read.
Why is HRP favoured over alternative enzyme labels like alkaline phosphatase?+
HRP has a fast turnover rate, is relatively inexpensive and stable, and works with a range of chromogenic and chemiluminescent substrates, making it a practical default choice for many ELISA-based forensic serology and toxicology assays.
Related terms
- Cross-Reactivity Matrix
- A table documenting the assay's optical density response to blood from multiple non-target species, expressed as a percentage of the human blood...
- Limit of Detection (LOD)
- The lowest concentration of analyte that produces a signal reliably distinguishable from the instrument noise, conventionally three times the standard deviation of...
- Optical Density (OD)
- The absorbance reading from a microplate reader that measures how much light is absorbed by the coloured product. Higher OD means more...
- Sandwich ELISA
- An ELISA format in which a capture antibody immobilised on the plate binds one epitope of the target antigen, and a separate...
- Standard Curve
- A series of known antigen concentrations run on the same ELISA plate, used to convert the OD reading for an unknown sample...