Amplite® Colorimetric Xanthine Oxidase Assay Kit

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® Colorimetric 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 color signal can be easily read at ~570 nm with an absorbance microplate reader. With the Amplite® Colorimetric Xanthine Oxidase Assay Kit, we have detected as little as 0.3 mU/mL xanthine oxidase in a 100 µL reaction volume.

Example protocol

AT A GLANCE

Protocol Summary

Prepare and add XO standards and/or test samples (50 µL)
Prepare and add XO Assay working solution (50 µL)
Incubate at room temperature for 30-60 minutes
Read absorbance increase at OD ratio of 570/610 nm

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

PREPARATION OF STOCK SOLUTIONS

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 HRP (Component C).
Note The unused HRP stock solution (500X) should be divided into single use aliquots and stored them at -20^oC.

3. Xanthine Oxidase (XO) stock solution

Add 200 µL of Assay Buffer (Component B) into the vial of Xanthine Oxidase Standard (Component E).
Note The unused XO stock solution should be divided into single use aliquots and stored at -20 ºC.

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/11307

Xanthine Oxidase standard

Add 10 µL of 1 U/mL XO stock solution into 990 µL of Assay Buffer (Component B) to make 10 mU/mL XO standard solution.Perform 1:3 serial dilutions to get approximately 10, 3, 1, 0.3, 0.1, 0.03, 0.01 and 0 mU/mL serially diluted XO standards.

PREPARATION OF WORKING SOLUTION

Table 1.XO Assay working solution for one clear bottom 96-well microplate (2X)

Components	Volume
Amplite&trade Red Stock Solution (250x)	20 µL
HRP Stock Solution (500X)	10 µL
Xanthine (100X)	50 µL
Assay Buffer	5 mL
Total volume	5.08 mL

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of Xanthine Oxidase standards and test samples in a clear bottom 96-well microplate. XOS = Xanthine Oxidase standard (XOS1-XOS7); BL = blank control; TS = test sample.

BL	BL	TS	TS
XOS1	XOS1	...	...
XOS2	XOS2	...	...
XOS3	XOS3
XOS4	XOS4
XOS5	XOS5
XOS6	XOS6
XOS7	XOS7

Table 2. Reagent composition for each well

Well	Volume	Reagent
XOS1 - XOS7	50 µL	Serial Dilutions (0.01 to 10 µM)
BL	50 µL	Assay Buffer (Component B)
TS	50 µL	test sample

Note The xanthine oxidase standards are for positive control only, and should not be relied on as a quantitation standard for enzyme activity.

Add XO standards and XO containing test samples into a white clear bottom microplate as described in Tables 1 and 2.
Add 50 µL of XO Assay working solution into each well of XO standard, blank control, and test samples (Table 1) to make the total XO assay volume of 100 µL/well.
Note For a 384-well plate, add 25 µL of sample and 25 µL of assay reaction mixture into each well.
Incubate the reaction for 30 to 60 minutes at room temperature, protected from light.
Monitor signal intensity with an absorbance plate reader at OD ratio of 570/610 nm.

Spectrum

Open in Advanced Spectrum Viewer

Product family

Name	Excitation (nm)	Emission (nm)
Amplite® Colorimetric Glucose Oxidase Assay Kit	571	584
Amplite® Fluorimetric Xanthine Oxidase Assay Kit Red Fluorescence	571	584

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

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