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Amplite® Fluorimetric Xanthine Oxidase Assay Kit *Red Fluorescence*

Xanthine oxidase (XO) is an enzyme that catalyzes the oxidation of hypoxanthine to xanthine and can further catalyze the oxidation of xanthine to uric acid. It plays an important role in the catabolism of purines. Xanthine oxidase is normally found in liver and jejunum. During severe liver damage, xanthine oxidase is released into blood, so a blood assay for XO is a way to determine if liver damage has happened. Xanthinuria is a rare genetic disorder where the lack of xanthine oxidase leads to high concentration of xanthine in blood and can cause health problems such as renal failure. The Amplite® Fluorimetric Xanthine Oxidase Assay Kit provides a quick and ultrasensitive method for the measurement of xanthine oxidase activities. It can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. In the assay, xanthine oxidase catalyzes the oxidation of purine bases, hypoxanthine or xanthine to uric acid and superoxide , which spontaneously degrades to hydrogen peroxide (H2O2). The kit uses our Amplite® Red substrate which enables a dual recordable mode. The fluorescent signal can be easily read by either a fluorescence microplate reader or an absorbance microplate reader. With the Amplite® Xanthine Oxidase Assay Kit, we have detected as little as 0.15 mU/mL xanthine oxidase in a 100 µL reaction volume.

Example protocol

AT A GLANCE

Protocol summary

  1. XO standards or test samples (50 µL)
  2. Add XO working solution (50 µL)
  3. Incubate at room temperature for 15 - 30 minutes
  4. Read fluorescence intensity at Ex/Em = 540/590 nm (cut off 570 nm)

Important notes
Thaw all the kit components to room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Amplite™ Red stock solution (250X):
Add 40 µL of DMSO (Component F) into the vial of Amplite™ Red substrate (Component A). The stock solution should be used promptly. Note: The Amplite™ Red substrate is unstable in the presence of thiols such as dithiothreitol (DTT) and 2-mercaptoethanol. The final concentration of DTT or 2-mercaptoethanol in the reaction should be no higher than 10 µM. The Amplite™ Red substrate is also unstable at high pH (>8.5). Therefore, the reaction should be performed at pH = 7 – 8. The provided assay buffer, pH = 7.4, is recommended.

2. HRP stock solution (500X):
Add 100 µL of Assay Buffer (Component B) into the vial of Horseradish Peroxidase (Component C).

3. Xanthine Oxidase (XO) standard solution (1 U/mL)
Add 200 µL of Assay Buffer (Component B) into the vial of Xanthine Oxidase Standard (Component E).

PREPARATION OF STANDARD SOLUTION

XO standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11304

Add 10 µL of 1 U/mL XO stanndard solution into 990 µL of Assay Buffer (Component B) to make 10 mU/mL XO standard solution (XO7). Perform 1:3 serial dilutions to get remaining serially diluted XO standards (XO6-XO1).

PREPARATION OF WORKING SOLUTION

Add 20 μL of Amplite™ Red stock solution (250X), 10 μL of HRP stock solution (500X), and 50 μL of Xanthine (100X, Component D) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.08 mL Xanthine Oxidase (XO) working solution. Protect from light.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of XO standards and test samples in a solid black 96-well microplate.

BLBLTSTS
XO1XO1......
XO2XO2......
XO3XO3  
XO4XO4  
XO5XO5  
XO6XO6  
XO7XO7  

Table 2. Reagent composition for each well.

WellVolumeReagent
XO1 - XO750 µLserial dilution (0.01 to 10 mU/mL)
BL50 µLAssay Buffer (Component B)
TS50 µLsample
  1. Prepare XO standards (XO), blank controls (BL), and test samples (TS) into a solid black 96-well microplate according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 50 µL of XO working solution into each well of the XO standards, blank control, and test samples to make the total XO assay volume of 100 µL/well. For a 384-well plate, add 25 µL of XO working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction for 15 to 30 minutes at room temperature, protected from light.

  4. Monitor the fluorescence intensity with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm), cut off = 570 nm.

Spectrum

Citations

View all 5 citations: Citation Explorer
Evaluation of anti-aging and antioxidant properties of a new rose variety, Ever-rose
Authors: Han, Se Jik and Belousova, Polina and Kwon, Sangwoo and Jang, Jihui and Lee, Jun Bae and Kim, Hyunjae and You, Gayeon and Song, Jihyeon and Mok, Hyejung and Ha, Ho Su and others,
Journal: Chemical and Biological Technologies in Agriculture (2024): 1--17
Protective effects of Cyclocarya paliurus on hyperuricemia and urate-induced inflammation
Authors: Zhu, Li-Hua and Xu, Ying-Yin and Zhu, Li-ping and Zheng, Xian and Jiang, Cui-Hua and Liu, Jian-Jing and Zhang, Jian and Yin, Zhi-Qi
Journal: Journal of Functional Foods (2022): 105130
Studies on Status of Oxidative Stress related Molecules and Enzymes in Obese with and without Diabetes in the Northern region of India
Authors: Singh, Sukhpal and Mahajan, Amita and Kaur, Jaspreet
Journal: Research Journal of Pharmacy and Technology (2020): 801--809
Xanthine oxidoreductase regulates macrophage IL1$\beta$ secretion upon NLRP3 inflammasome activation
Authors: Ives, Annette and Nomura, Johji and Martinon, Fabio and Roger, Thierry and LeRoy, Didier and Miner, Jeffrey N and Simon, Gregoire and Busso, Nathalie and So, Alexander
Journal: Nature communications (2015): 1--11
Xanthine oxidoreductase regulates macrophage IL1β secretion upon NLRP3 inflammasome activation
Authors: Ives, Annette and Nomura, Johji and Martinon, Fabio and Roger, Thierry and LeRoy, Didier and Miner, Jeffrey N and Simon, Gregoire and Busso, Nathalie and So, Alex and er, undefined
Journal: Nature communications (2015)

References

View all 52 references: Citation Explorer
Phenotyping of N-acetyltransferase type 2 and xanthine oxidase with caffeine: when should urine samples be collected
Authors: Jetter A, Kinzig M, Rodamer M, Tomalik-Scharte D, Sorgel F, Fuhr U.
Journal: Eur J Clin Pharmacol (2009): 411
Measurement of xanthine oxidase inhibition activity of phenolics and flavonoids with a modified cupric reducing antioxidant capacity (CUPRAC) method
Authors: Ozyurek M, Bektasoglu B, Guclu K, Apak R.
Journal: Anal Chim Acta (2009): 42
In vitro xanthine oxidase inhibitory activity of the fractions of Erythrina stricta Roxb
Authors: Umamaheswari M, Asokkumar K, Sivashanmugam AT, Remyaraju A, Subhadradevi V, Ravi TK.
Journal: J Ethnopharmacol (2009): 646
Increased xanthine oxidase in the skin of preeclamptic women
Authors: Bainbridge SA, Deng JS, Roberts JM.
Journal: Reprod Sci (2009): 468
Immuno-spin trapping of a post-translational carboxypeptidase B1 radical formed by a dual role of xanthine oxidase and endothelial nitric oxide synthase in acute septic mice
Authors: Chatterjee S, Ehrenshaft M, Bhattacharjee S, Deterding LJ, Bonini MG, Corbett J, Kadiiska MB, Tomer KB, Mason RP.
Journal: Free Radic Biol Med (2009): 454
Page updated on December 11, 2024

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Spectral properties

Excitation (nm)

571

Emission (nm)

584

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

Platform

Fluorescence microplate reader

Excitation540nm
Emission590nm
Cutoff570nm
Recommended plateSolid black

Components

Xanthine oxidase dose response was measured with Amplite® Fluorimetric Xanthine Oxidase Assay Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices).
Xanthine oxidase dose response was measured with Amplite® Fluorimetric Xanthine Oxidase Assay Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices).
Xanthine oxidase dose response was measured with Amplite® Fluorimetric Xanthine Oxidase Assay Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices).