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Cat. No.: ARXOISK-100 (for 100T)

Description

Amplex Red Xanthine Oxidase Inhibitor Screening Kit, also known as the Amplex Red XO Inhibitor Screening Kit or Amplex Red XOD Inhibitor Screening Kit, is a simple, rapid, and sensitive assay based on the Amplex Red probe for high-throughput screening of xanthine oxidase inhibitors using fluorescence detection.

Xanthine oxidase (XO or XOD) is a crucial enzyme in nucleic acid metabolism within the body. It catalyzes the oxidation of hypoxanthine to xanthine and further oxidizes xanthine to uric acid, producing hydrogen peroxide and superoxide anions in the process. This enzyme plays an important role in purine degradation metabolism across various species, including humans. In humans and other primates, XO is typically present in serum, lungs, liver, and intestinal mucosa. In rodents, XO is expressed in most tissues. XO activity in the blood is generally low, but it increases in serum and lungs during influenza infection. When liver function is impaired, XO is released into the bloodstream, making the detection of XO levels a sensitive indicator for assessing severe liver damage (such as jaundice). XO may also be related to the pathogenesis of gout, as xanthine oxidase is involved in uric acid formation; therefore, xanthine oxidase inhibitors like allopurinol are used to treat gout. Additionally, research shows that inhibiting XO activity can alleviate cardiovascular diseases and chronic obstructive pulmonary disease.

Components

• XO Assay Buffer
• XO (20X)
• XO Indicator (10X)
• XO Substrate (10X)
• Febuxostat (10µM)

Principle

Under the catalysis of xanthine oxidase, xanthine reacts with oxygen to produce uric acid and H2O2. The XO Probe provided in this kit is a highly sensitive fluorescent probe for H2O2. In the presence of horseradish peroxidase (HRP), XO Probe reacts 1:1 with H2O2 to produce a strong red fluorescent substance, resofurin. Resofurin has a maximum excitation wavelength of 571 nm and a maximum emission wavelength of 585 nm, with fluorescence intensity proportional to the amount of H2O2 produced by the catalysis of xanthine oxidase. If an xanthine oxidase inhibitor is added to the reaction, the activity of xanthine oxidase will be inhibited, leading to a reduced production of H2O2 and, consequently, a decrease in fluorescence. The fluorescence intensity is inversely proportional to the inhibitory effect of the inhibitor, allowing for the detection of the inhibitor's efficacy.

Features

• This kit includes a positive control for inhibitors, making it easy to use. The kit provides XO assay buffer, xanthine oxidase (XO), XO substrate, the positive control febuxostat, and the probe (XO Probe) and horseradish peroxidase (HRP) needed for fluorescence generation. Febuxostat is a selective non-purine XO inhibitor commonly used in the clinical treatment of gout and hyperuricemia. Typically, the IC50 of febuxostat is around 2 nM to 20 nM.
• This kit offers rapid detection with high sensitivity. Compared to the xanthine oxidase inhibitor screening kit using the WST-8 method, this kit provides further improvements in detection efficiency and sensitivity, with the entire process completed in approximately 15 minutes. The amounts of XO and XO Substrate used in this kit have been optimized, allowing for the detection of inhibitors with both very low and relatively high IC50 values.
• We also provide Xanthine Oxidase Inhibitor Screening Kit with WST-8.

Storage

Store at -20ºC, with a shelf life of one year. XO Probe and HRP must be stored protected from light.

Precautions

• XO Probe is unstable in air and should be used as soon as possible after opening, while ensuring protection from light during use.
• The reaction products of XO Probe are highly unstable in the presence of reducing agents. The concentration of dithiothreitol (DTT) or β-mercaptoethanol in the final reaction system should be below 10μM.
• Ensure that the pH of the sample is between 7 and 8, or that the pH of the reaction system is within this range after adding the sample, as deviations can affect the stability and fluorescence of XO Probe.
• XO Assay Buffer and XO Probe need to be completely thawed and equilibrated to room temperature before use to avoid affecting the test results. XO and HRP should be kept on ice during use. After use, all reagents should be immediately stored according to the conditions specified in the kit.
• The solvent for the inhibitor being tested may interfere with the detection. It is recommended to prepare and dilute the inhibitor using XO Assay Buffer, Milli-Q grade water, or DMSO, and add the same volume of solvent to the control wells to exclude interference. If other solvents are used, ensure that their concentration in the final reaction system does not exceed 5%. If it does, include a solvent control group to test the impact of the solvent on enzyme activity and exclude interference.
• As the quantities of XO, HRP, XO Substrate, and other reagents are relatively small, please centrifuge and mix them appropriately before use.
• For fluorescence detection, it is recommended to use 96-Well Black Opaque Plates.
• This product is for scientific research use only by professionals. It should not be used for clinical diagnosis or treatment, for food or drug purposes, and should not be stored in a residential setting.
• For your safety and health, please wear lab coats and disposable gloves while handling.

Related: Xanthine Oxidase Inhibitor Screening Kit with WST-8