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Peroxidase Detection

Peroxidase is a small molecule (MW ~40 KD) that can usually be conjugated to an antibody. Due to its small size, it rarely causes steric hindrance problem with antibody/antigen complex formation. Peroxidase is inexpensive compared to other labeling enzymes. The major disadvantage associated with peroxidase is its low tolerance to many preservatives, such as sodium azide, that inactivates peroxidase activity even at low concentration. HRP conjugates are extensively used as secondary detection reagents in ELISAs, immuno-histochemical techniques as well as Northern, Southern and Western blot analyses.

Amplite® Colorimetric Peroxidase Assay Kit (Cat# 11551) uses Amplite® Blue, our ultrasensitive chromogenic HRP substrate. Amplite® Blue is a chromogenic peroxidase substrate that is much more sensitive to both H2O2 and peroxidase than other chromogenic peroxidase substrates such as TMB, ABTS, OPD and K-Blue. Amplite® Blue generates a highly absorptive material that has maximum absorption at 664 nm. This near infrared absorption minimizes the background absorption often caused by the autoabsorption of biological samples that rarely absorb light beyond 600 nm. The signal can be easily read by an absorbance microplate reader at 664 ± 5 nm.


HRP dose responses were measured with Amplite Colorimetric Peroxidase Assay Kit (Cat# 11551) in a 96-well white wall/clear bottom plate. As low as 3 mU/mL peroxidase was detected.


Amplite® Fluorimetric Peroxidase Assay Kits (Cat# 11552 & 11553) are quick (10 min) HRP assays in a one-step, homogeneous, no wash assay system. They can be used for ELISAs, characterizing kinetics of enzyme reaction and high throughput screening of oxidase inhibitors, etc. The kits provide an optimized 'mix and read' assay protocol that is compatible with HTS liquid handling instruments. Kit 11552 uses fluorogenic Amplite® Red HRP substrate to quantify peroxidase in solutions. It can be used for ELISAs, characterizing kinetics of enzyme reaction, and high throughput screenings. The kit provides an optimized 'mix and read' assay protocol that is compatible with HTS liquid handling instruments. As low as 10 µU/mL HRP was detected. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format. Its signal can be easily read by either a fluorescence microplate reader at Ex/Em = ~540/590 nm or an absorbance microplate reader at ~576 nm.


HRP dose responses were measured with Amplite® Fluorimetric Peroxidase Assay Kit (Cat# 11552) in a 384-well black plate using a Gemini fluorescence microplate reader (Molecular Devices). As low as 10 µU/mL peroxidase was detected with 30 minutes incubation (n=3).


Kit 11553 uses Amplite® IR, our near infrared fluorogenic HRP substrate. Amplite® IR generates a substance that has maximum absorption of 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. Its signal can be easily read by either a fluorescence microplate reader at Ex/Em = 600 to 650/650 to 690 nm (maximum Ex/Em = 640/680 nm) or an absorbance micropalte reader at 647 ± 5 nm. As low as 1 mU/mL HRP was detected with Kit 11553.


HRP dose responses were measured with Amplite® Fluorimetric Peroxidase Assay Kit (Cat# 11553) in a solid black 384-well plate using a Gemini fluorescence microplate reader. As low as 1 mU/mL peroxidase was detected with 30 minutes incubation (n=3).


Myeloperoxidase (MPO), most abundantly present in neutrophils and monocytes, is a green hemoprotein having peroxidase activity. It catalyzes the reaction of hydrogen peroxide and halide ions to form cytotoxic acids and other intermediates; and plays an important role in the oxygen-dependent killing of tumor cells and microorganisms. MPO deficiency is a hereditary deficiency of the enzyme, which predisposes to immune deficiency. There are considerable interests in the development of therapeutic MPO inhibitors.

Amplite® Myeloperoxidase Assay Kit (Cat# 11301) provides a quick and sensitive method for the measurement of myeloperoxidase in solution and in cell lysates. The kit uses our Amplite® Red substrate which enables a dual recordable mode. The signal can be easily read by either a fluorescence microplate reader at Ex/Em = 540/590 nm or an absorbance microplate reader at ~576 nm. With Amplite® Myeloperoxidase Assay Kit, as low as 0.1 mU/mL myeloperoxidase was detected in a 100 µL reaction volume.


Myeloperoxidase dose responses were measured with Amplite® Fluorimetric Myeloperoxidase Assay Kit (Cat# 11301) in a 96-well black solid plate using a Gemini fluorescence microplate reader (Molecular Devices). As low as 0.1 mU/mL myeloperoxidase was detected with 60 minutes incubation (n=3).



The chemical structure of Amplite® ADHP (Cat# 11000).
Amplite® ADHP (Cat# 11000) is chemically the same as Amplex® Red. It is a sensitive fluorogenic peroxidase substrate that has much lower background than the materials from other commercial vendors. 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 ADHP is minimal. So far ADHP has been known as the most sensitive and stable fluorogenic probe for detecting HRP and H2O2. ADHP has been widely used to detect HRP in many immunoassays. On the other hand, ADHP can also be used to detect trace amount of H2O2. The ADHP-based H2O2 detection is at least one order of magnitude more sensitive than the commonly used scopoletin assay for H2O2. Because H2O2 is produced in many enzymatic redox reactions, ADHP can be used in coupled enzymatic reactions to detect the activity of many oxidases and/or related enzymes/substrates or cofactors, such as glucose, acetylcholine and cholesterol, L-glutamate, amino acids, etc.

Horseradish peroxidase (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 microplates or test tubes. ABTS solution allows HRP reaction done in a single addition. The assay solution changes the color to light green upon its reaction with HRP or HRP conjugates in the presence of hydrogen peroxide.

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. Use of a stop solution enhances sensitivity 2-4 fold and the resulting yellow solution can be read at 450 nm. ReadiUse™ TMB Substrate Solution provides a convenient and ultrasensitive quantitative substrate system.


HRP does responses were measured with ReadiUse™ TMB Substrate Solution (Cat# 11003) in a clear 96-well plate. As low as 3 µU/well peroxidase was detected with 10 minutes incubation.


Amplite® Red (Cat# 11011) is a sensitive fluorogenic peroxidase substrate generating a highly red fluorescent product that has maximum absorption at 571 nm and maximum emission at 585 nm. Unlike other HRP substrates, such as dihydrofluoresceins and dihydrorhodamines, Amplite® Red has minimum air-oxidation. Amplite® Red is one of the most sensitive and stable fluorogenic probes for detecting HRP and H2O2. Amplite® Red has been widely used to detect HRP in many immunoassays. On the other hand, Amplite® Red can also be used to detect trace amount of H2O2. The Amplite® Red-based H2O2 detection is at least one order of magnitude more sensitive than the commonly used scopoletin assays for H2O2. Because H2O2 is produced in many enzymatic redox reactions, Amplite® Red can be used in coupled enzymatic reactions to detect the activity of many oxidases and/or related enzymes/substrates or cofactors, such as glucose, acetylcholine and cholesterol, L-glutamate, amino acids, etc


HRP dose responses were measured with Amplite® Red (Cat# 11011). As low as 10 µU/mL peroxidase was detected with 30 minutes incubation (n=3).

 

Table 1. Peroxidase Detection Assay Kits and Probes

Cat No.
Product Name
Ex (nm)
Em (nm)
Unit Size
Price
11000Amplite® ADHP [10-Acetyl-3,7-dihydroxyphenoxazine] *CAS#: 119171-73-2*57158525 mg$217
11551Amplite® Colorimetric Peroxidase (HRP) Assay Kit *Blue Color*664 500 Tests$217
11552Amplite® Fluorimetric Peroxidase (HRP) Assay Kit *Red Fluorescence*575590500 Tests$217
11553Amplite® Fluorimetric Peroxidase (HRP) Assay Kit *Near Infrared Fluorescence*647670500 Tests$327
11301Amplite® Fluorimetric Myeloperoxidase Assay Kit *Red Fluorescence*571585200 Tests$217
11011Amplite® Red5715851000 Assays$106
11001ReadiUse™ ABTS Substrate Solution *Optimized for ELISA Assays with HRP Conjugates*420 1 L$194
11003ReadiUse™ TMB Substrate Solution *Optimized for ELISA Assays with HRP Conjugates*650 1 L$547
11010Signal Guard™ HRP conjugate stabilizer 50 mL$106