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HRP (Horseradish Peroxidase) Detection 

The enzyme horseradish peroxidase (HRP), found in the roots 
of horseradish, is used extensively in biochemistry applications 
primarily for its ability to amplify a weak signal and increase 
detectability of a target molecule. 

HRP is often used in conjugates (molecules that have been joined genetically or chemically) to determine the presence of a molecular target. HRP is also commonly 
used in techniques such as ELISA and immunohistochemistry 
due to its monomeric nature and the ease with which it produces 
colored products. Peroxidase, a heme-containing oxidoreductase, 
is a commercially important enzyme which catalyzes the reductive 
cleavage of hydrogen peroxide by an electron donor.

Horseradish peroxidase is ideal in many respects for these applications because it is smaller, more stable, and less expensive 
than other popular alternatives such as alkaline phosphatase. 
It also has a high turnover rate that allows generation of strong 
signals in a relatively short time span. It should be noted that 
high concentrations of phosphate severely decrease stability of 
horseradish peroxidase. In addition to biomedical applications, 
horseradish peroxidase is one of the enzymes with important 
environmental applications. This enzyme is suitable for the removal 
of hydroxylated aromatic compounds (HACs) that are considered to 
be primary pollutants in a wide variety of industrial wastewater. 
ReadiUse™ Colorimetric HRP Substrate Solutions
HRP and HRP conjugates facilitate the ABTS oxidation in the 
presence of hydrogen peroxide, turning ABTS into its blue-green 
oxidized product. This chromogenic reaction is widely used to 
quantify HRP in ELISA assays. The oxidized ABTS product has the 
absorption maximum at 420 nm that can easily be followed with 
a spectrophotometer. ReadiUse™ ABTS Substrate Solution (Cat# 
11001) is optimized for ELISA assays that use HRP or HRP-labeled 
conjugates and hydrogen peroxide in microwell plates or test 
tubes. Our ABTS solution allows HRP reaction to be done with a 
single addition. The assay solution changes its color to light green 
Figure 1. HRP dose responses were measured with ReadiUse™ ABTS Substrate 
Solution (Cat#11001) in a 96-well clear plate using a SpectraMax® microplate reader 
(Molecular Devices).

upon its reaction with HRP or HRP conjugates. 
ReadiUse™ TMB Substrate Solution (Cat# 11003) is a premixed 
solution of TMB substrate with hydrogen peroxide. It produces a 
blue product upon interaction with HRP or HRP conjugates without 
the addition of hydrogen peroxide. The soluble blue product can 
be quantitated at 650 nm. The use of a stop solution produces 2-4 
fold increase in sensitivity and the resulting yellow solution can 
be read at 450 nm. ReadiUse™ TMB Substrate Solution provides a 
convenient and ultrasensitive quantitative substrate system.
Figure 3. HRP dose responses on a 384-well plate were measured with Amplite™ ADHP 
(Cat# 11000) and ReadiUse™ Hydrogen Peroxide Solution (Cat# 11004). 
Figure 2. HRP dose responses were measured with ReadiUse™ TMB Substrate Solution 
(Cat# 11003) in a 96-well clear plate using a SpectraMax® microplate reader (Molecular 
Devices). As low as 3 µU/well peroxidase was detected with 10 minutes incubation. 

ReadiUse™ Hydrogen Peroxide Solution

Compared with other commercial hydrogen peroxide solutions, our 
formulated ReadiUse™ Hydrogen Peroxide Solution (Cat# 11004) 
is much more stable. It is calibrated to ensure more reproducible 
peroxidase-based assays.

Amplite™ IR generates a substance that has maximum absorption 
at 647 nm with maximum emission at 670 nm. This near infrared 
absorption and fluorescence minimize the assay background 
often caused by the autoabsorption and/or autofluorescence of 
biological samples that rarely absorb light beyond 600 nm. 

Unlike  other HRP substrates, such as dihydrofluoresceins and dihydrorhodamines, the air-oxidation of Amplite™ IR is minimal. Compared 
with ADHP (also called Amplex® Red in literature), Amplite™ IR 
generates the fluorescence that is pH-independent from pH 4 to 10. 
Thus it is a superior alternative to ADHP (Amplex® Red) for detections that require low pH where ADHP (Amplex® Red) has reduced 
fluorescence. We have used Amplite™ IR to detect HRP in quite a 
few immunoassays. Amplite™ IR can also be used to detect a trace 
amount of H2O2

Fluorimetric HRP Detection Substrates
A variety of substrates are available for detecting peroxidase activity in ELISA assays. Colorimetric HRP substrates (e.g., TMB, OPD and 
ABTS) have been widely used for years. Each of these substrates 
varies greatly with its performance characteristics such as detection 
sensitivity, working range and attainable signal-to-noise ratio. 
Amplite™ Blue (Cat# 11005) is a soluble fluorogenic HRP substrate 
for the detection of peroxidase activity. Amplite™ Blue allows rapid 
HRP detection assays to be performed with greater sensitivity 
than the colorimetric HRP substrates. The fluorescent product of 
Amplite™ Blue does not photobleach. Amplite™ Blue exhibits a flat 
baseline in assays, which facilitates low-level detection sensitivity 
and allows for high signal-to-noise ratio.
Amplite™ ADHP (Cat# 11000) is chemically similar to Amplex® 
Red (Amplex® Red is the trademark of Invitrogen). It is a sensitive 
fluorogenic peroxidase substrate. Amplite™ ADHP has the highest 
quality of ADHP with much lower background than the materials 
from other commercial vendors. Amplite™ ADHP generates highly 
fluorescent resorufin that has maximum absorption at 571 nm and 
maximum emission at 585 nm. Unlike other HRP substrates, such 
as dihydrofluoresceins and dihydrorhodamines, the air-oxidation 
of Amplite™ ADHP is minimal. So far ADHP has been known as the 
most sensitive and stable fluorogenic probe for detecting HRP and 

Internal Reference: YF-PA26408