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Cat. No.: ARXOAK-100 (for 100T)

Cat. No.: ARXOAK-500 (for 500T)

Description

Amplex Red Xanthine Oxidase Assay Kit, also known as the Amplex Red XO Assay Kit or Amplex Red XOD Assay Kit, is a reagent kit designed for rapid and highly sensitive detection of xanthine oxidase activity in biological fluids such as serum, plasma, and urine, as well as in cell culture supernatants and tissue or cell samples. The assay uses the Amplex Red probe, which can detect xanthine oxidase activity via fluorescence or absorbance. Typically, 0.1-1 µl of serum or plasma is sufficient for fluorescence-based detection with this kit.

Xanthine oxidase (XO or XOD) is an important enzyme in nucleic acid metabolism within the body. It catalyzes the oxidation of hypoxanthine to xanthine and further oxidizes xanthine to uric acid, while generating hydrogen peroxide and superoxide anions through a series of oxidation reactions. This enzyme plays a significant role in purine catabolism 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 blood is usually low, but during influenza A infection, XO activity in serum and lungs increases. When liver function is impaired, XO is released into the bloodstream in large amounts, making blood XO levels a sensitive indicator for assessing severe liver damage (e.g., jaundice).

In this kit, Amplex Red is a highly sensitive fluorescent probe for H2O2. In the presence of horseradish peroxidase (HRP), Amplex Red reacts 1:1 with H2O2 to produce a strong red fluorescent product, resofurin. Resofurin has a maximum excitation wavelength of 571 nm and a maximum emission wavelength of 585 nm, with strong visible light absorption at the excitation wavelength. Therefore, this kit can be used for detection by both absorbance and fluorescence methods.

Components

• Buffer A for Metabolic Assay
• XO Assay Buffer
• Amplex Red
• XO (1U/ml)
• XO Developer
• XO Substrate

Principle

Under the catalysis of xanthine oxidase (XO), xanthine and oxygen undergo an oxidation reaction to produce uric acid and H2O2. The enzyme activity of xanthine oxidase is then calculated by measuring the fluorescence intensity or absorbance of the reaction product resofurin, which is formed from the reaction between H2O2 and Amplex Red. The fluorescence intensity and absorbance of resofurin are proportional to the activity of xanthine oxidase in the sample.

Features

• This kit offers high sensitivity, a wide linear range, and requires a small sample volume. With a sample volume of 20 µl, absorbance detection can measure xanthine oxidase activity as low as 2 μU, with a good linear relationship within the 0.1-2 mU/ml (2-40 μU) activity range. Fluorescence detection can measure xanthine oxidase activity as low as 0.2 μU, with a good linear relationship within the 0.01-1 mU/ml (0.2-20 μU) activity range. Fluorescence detection is approximately 10 times more sensitive than absorbance detection, allowing for the use of smaller sample volumes. The kit includes XO (1 U/ml) for preparing xanthine oxidase standard solutions, which can be used to generate a standard curve and calculate the xanthine oxidase activity in samples.

• This kit offers flexible detection methods and fast detection speed. Compared to the xanthine oxidase activity assay kit using the WST-8 method, this kit provides higher sensitivity and more flexible detection options. The kit supports both fluorescence and absorbance detection, with the entire process completed in approximately 40 minutes.

• This kit has a wide range of applications. It can be used for detecting biological fluids such as serum, plasma, and urine from mice, rats, humans, as well as cell culture supernatants, and tissue or cell samples. It is suitable for both small sample sizes and high-throughput screening automation systems.

• The assay buffer provided in this kit has broad utility. Cells or tissue samples lysed with Buffer A for Metabolic Assay can also be used for detection in other metabolic assay kits that use Buffer A for Metabolic Assay, demonstrating strong versatility. Additionally, it can be used for protein concentration assays, SDS-PAGE, or Western blotting of some easily soluble proteins.

Storage

Store at -20ºC for one year. Amplex Red and XO Developer should be stored protected from light.

Precautions

• Amplex Red is not very stable in air; it should be used as soon as possible after opening, and it should be kept protected from light during use.
• The reaction product of Amplex Red is very unstable in the presence of reducing agents. Therefore, the concentration of dithiothreitol (DTT), β-mercaptoethanol, or similar reducing agents in the final reaction system should be less than 10 µM.
• Ensure that the pH of the reaction system is between 7 and 8, as this affects the stability and fluorescence of Amplex Red.
• Amplex Red and XO Assay Buffer should be completely thawed and equilibrated to room temperature before use, as this will otherwise affect the test results. Other solutions should be kept on ice during use.
• Serum and other samples should not be stored at 4ºC for more than 2 weeks, as this may affect the accuracy of the test results. Serum samples are typically best stored at -20ºC, with -80ºC being preferable.
• To minimize errors caused by background from the dilution buffer, the dilution of samples and XO (1U/ml) should be based on the type of sample. For samples lysed with Buffer A for Metabolic Assay, use Buffer A for Metabolic Assay for dilution. For other samples like blood, use XO Assay Buffer for dilution.
• This product is intended for scientific research use by professionals only and is not for clinical diagnosis or treatment, food, or drugs, nor should it be stored in a regular household.
• For your safety and health, please wear a lab coat and disposable gloves while handling.