Substrates for Oxidases, Including Amplex Red Kits—Section 10.5

Page Contents

• Peroxidases
• Myeloperoxidase
  
• Catalase
  
• Glucose and Glucose Oxidase
• Galactose and Galactose Oxidase
  
• Cholesterol and Cholesterol Oxidase
• Monoamine Oxidase
  
• Glutamic Acid and Glutamate Oxidase
  
• Acetylcholine, Acetylcholinesterase and Choline Oxidase
  
• Xanthine and Xanthine Oxidase
  
• Uric Acid and Uricase
  
• Data Table
• Ordering Information

Oxidases, the most useful of which is undoubtedly horseradish peroxidase (HRP), are important enzymes that are used in a wide variety of bioassays. Peroxidase activity is also present in many cells. Reagents for quantitating peroxidase and the activity of a variety of other oxidases are described in this section; reagents for detecting the activity of cellular peroxidases and the oxygen radicals produced by these peroxidases are described in Probes for Following Receptor Binding and Phagocytosis—Section 16.1 and Generating and Detecting Reactive Oxygen Species—Section 18.2. Antibody and streptavidin conjugates of horseradish peroxidase are listed in the product list for this section and described in Secondary Immunoreagents—Section 7.2 and Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6. Tyramide signal amplification (TSA) technology, described in TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2 makes extensive use of peroxidase-conjugated reagents and fluorescent dye– or hapten-labeled tyramides to deposit a detectable product at the site of enzymatic activity.

We have used our extremely versatile Amplex Red reagent—the most stable and sensitive fluorogenic substrate known for horseradish peroxidase—to develop a variety of novel fluorogenic and chromogenic assays for enzymes that produce hydrogen peroxide. Furthermore, these coupled assays permit the ultrasensitive quantitation of a diverse assortment of analytes, including glucose, galactose, cholesterol, glutamic acid, xanthine (or hypoxanthine), uric acid, choline and acetylcholine, as well as hydrogen peroxide. The P_iPer Phosphate and P_iPer Pyrophosphate Assay Kits (P22061, P22062), described in Detecting Enzymes That Metabolize Phosphates and Polyphosphates—Section 10.3, also utilize the Amplex Red reagent for the continuous assays of enzymes that produce either inorganic phosphate or pyrophosphate.

Amplex Red Reagent: Stable Substrate for Peroxidase Detection

In the presence of horseradish peroxidase (HRP), Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine, A12222, A22177) reacts with H₂O₂ in a 1:1 stoichiometry to produce highly fluorescent resorufin (R363, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1, Figure 10.5.1). Amplex Red reagent has greater stability, yields less background and produces a red-fluorescent product (excitation/emission maxima ~571/585 nm) that is more readily detected than the similar reduced methylene blue derivatives commonly used for colorimetric determination of lipid peroxides in plasma, sera, cell extracts and a variety of membrane systems.

Amplex Red reagent has been used to detect the release of H₂O₂ from activated human leukocytes, to measure the activity of monoamine oxidase in bovine brain tissue, to demonstrate the extracellular production of H₂O₂ produced by UV light stimulation of human keratinocytes and for microplate assays of H₂O₂ and lipid hydroperoxide generation by isolated mitochondria. Amplex Red reagent is available in a single 5 mg vial (A12222) or packaged as a set of 10 vials, each containing 10 mg of the substrate, for high-throughput screening applications (A22177).

Figure 10.5.1 Principle of coupled enzymatic assays using Amplex Red reagent. Oxidation of glucose by glucose oxidase results in generation of H₂O₂, which is coupled to conversion of the Amplex Red reagent to fluorescent resorufin by HRP. The detection scheme shown here is used in the Amplex Red Glucose/Glucose Oxidase Assay Kit (A22189).

Amplex UltraRed Reagent: Brighter and More Sensitive than the Amplex Red Reagent

Amplex UltraRed reagent (A36006) improves upon the performance of Amplex Red reagent, offering brighter fluorescence and enhanced sensitivity on a per-mole basis in horseradish peroxidase or horseradish peroxidase–coupled enzyme assays (Advantages of the Amplex UltraRed reagent over chromogenic reagents—Table 10.5, Figure 10.5.2). Fluorescence of oxidized Amplex UltraRed reagent is also less sensitive to pH (Figure 10.5.3), and the substrate and its oxidation product exhibit greater stability than Amplex Red reagent in the presence of H₂O₂ or thiols such as dithiothreitol (DTT). Like Amplex Red reagent, nonfluorescent Amplex UltraRed reagent reacts with H₂O₂ in a 1:1 stoichiometric ratio to produce a brightly fluorescent and strongly absorbing reaction product (excitation/emission maxima ~568/581 nm) (). Although the primary applications of the Amplex UltraRed reagent are enzyme-linked immunosorbent assays (ELISAs) and HRP-coupled solution assays, it is also frequently used (in combination with HRP) to detect H₂O₂ production by isolated mitochondria and cell cultures.

Figure 10.5.2 Detection of H2O2 using Amplex UltraRed reagent (red square) or Amplex Red reagent (blue triangle). Reactions containing 50 µM Amplex UltraRed or Amplex Red reagent, 1 U/mL HRP and the indicated amount of H2O2 in 50 mM sodium phosphate buffer, pH 7.4, were incubated for 30 minutes at room temperature. The inset shows the sensitivity and linearity of the Amplex UltraRed assay at low levels of H2O2.

Figure 10.5.3 Comparison of pH-dependent fluorescence of the products derived from oxidation of Amplex UltraRed reagent (solid blue circles) and Amplex Red reagent (open blue squares). Fluorescence intensities were measured using excitation/emission of ~570/585 nm.

Amplex Red/UltraRed Stop Reagent

Amplex Red and Amplex UltraRed Assay Kits provide sensitive biomolecular assays based on hydrogen peroxide–generating enzyme systems linked to peroxidase–mediated oxidation of the fluorogenic Amplex Red or Amplex UltraRed reagent. Typically, detection reactions are performed in microplate wells and are initiated by adding the fluorogenic Amplex or Amplex UltraRed reagent, resulting in a continuous fluorescence increase that proceeds for 30 minutes or more. Ultimately, unknown analyte concentrations are determined by referencing fluorescence intensities measured at a certain time point during the reaction to parallel measurements at the same time point on standard samples of known concentration. Clearly, it is quite critical to ensure that the timing of the standard and unknown sample measurements is the same. The Amplex Red/UltraRed stop reagent (A33855) provides convenience and control by allowing the fluorescence signal-generating reaction to be terminated at a user-determined time point. After addition of the stop reagent, the fluorescence signal remains stable for at least 3 hours. The Amplex Red/UltraRed stop reagent is compatible with all Amplex Red and Amplex UltraRed Assay Kits and will terminate reactions containing up to 0.1 units/mL HRP and 5 µM H₂O₂.

Coupled Enzymatic Reactions with the Amplex Red and Amplex UltraRed Reagents

Because H₂O₂ is produced in many different enzymatic reactions, the Amplex Red and Amplex UltraRed reagents can be used in coupled enzymatic reactions to detect the activity of many different enzymes such as NADPH oxidase and lysyl oxidase, or, when the substrate concentration is limited, to assay solutions for metabolically active constituents such as glucose, pyruvate, acetylcholine and cholesterol. Advantages of Amplex Red reagent–and Amplex UltraRed reagent–based assays include the following:

• Amplex Red and Amplex UltraRed reagents are fluorogenic substrates with extremely low background color or fluorescence.
• Stock solutions of the Amplex Red and Amplex UltraRed reagents are chemically stable.
• Fluorescent peroxidase reaction products are also stable.
• The long-wavelength spectra of the peroxidase reaction products result in little interference from blue or green autofluorescence in biological samples.
• Peroxidase reaction products can be detected by either fluorescence- or absorption-based methods.
• In most cases, Amplex Red and Amplex UltraRed assays can detect either unlabeled natural biomolecules, including amino acids, sugars or lipids, or the activity of enzymes that metabolize these substrates.

The Amplex Red reagent is also utilized as the detection reagent in our many Amplex Red assay kits. Substituting the Amplex UltraRed reagent in these kits should offer even greater sensitivity. The Amplex Red assay kits include:

• Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (A22188, Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit)
• Amplex Red Catalase Assay Kit (A22180, Amplex Red Catalase Assay Kit)
• Amplex Red Monoamine Oxidase Assay Kit (A12214, Amplex Red Monoamine Oxidase Assay Kit)
• Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit (A12221, Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit)
• Amplex Red Glucose/Glucose Oxidase Assay Kit (A22189, Amplex Red Glucose/Glucose Oxidase Assay Kit)
• Amplex Red Galactose/Galactose Oxidase Assay Kit (A22179, Amplex Red Galactose/Galactose Oxidase Kit)
• Amplex Red Neuraminidase (Sialidase) Assay Kit (A22178, Amplex Red Neuraminidase (Sialidase) Assay Kit)
• Amplex Red Cholesterol Assay Kit (A12216, Amplex Red Cholesterol Assay Kit)
• Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217, Amplex Red Acetylcholine/AChE Assay Kit)
• Amplex Red Phosphatidylcholine-Specific Phospholipase C Assay Kit (A12218, Probes for Lipid Metabolism and Signaling—Section 17.4)
• Amplex Red Phospholipase D Assay Kit (A12219, Probes for Lipid Metabolism and Signaling—Section 17.4)
• Amplex Red Sphingomyelinase Assay Kit (A12220, Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3)
• Amplex Red Uric Acid/Uricase Assay Kit (A22181, Amplex Red Uric Acid/Uricase Assay Kit)
• Amplex Red Xanthine/Xanthine Oxidase Assay Kit (A22182, Amplex Red Xanthine/Xanthine Oxidase Assay Kit)

The Amplex UltraRed assay kits include:

• Amplex ELISA Development Kits for Mouse IgG and for Rabbit IgG (A33851, A33852; TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2)
• EnzChek Myeloperoxidase (MPO) Activity Assay Kit (E33856, EnzChek Myeloperoxidase (MPO) Activity Assay Kit)
• Zen Myeloperoxidase (MPO) ELISA Kit (Z33857, Zen Myeloperoxidase (MPO) ELISA Kit)
• EnzChek Ultra Phytase Assay Kit (E33701, EnzChek Ultra Phytase Assay Kit)

Most of these Amplex Red and Amplex UltraRed kits are further discussed in this section; however, some are only presented in the sections listed above.

Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit

The Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (A22188) provides a simple, sensitive, one-step assay for detecting H₂O₂ (Figure 10.5.4) or the activity of horseradish peroxidase either by measuring fluorescence with a fluorescence-based microplate reader or a fluorometer (Figure 10.5.5) or by measuring absorption with an absorption-based microplate reader or a spectrophotometer. The Amplex Red peroxidase substrate can detect the presence of active peroxidases and the release of H₂O₂ from biological samples, including cells and cell extracts.

The Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxide (HRP)
• H₂O₂ for use as a positive control
• Concentrated reaction buffer
• Detailed protocols (Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit)

Each kit provides sufficient reagents for approximately 500 assays using a fluorescence- or absorption-based microplate reader and a reaction volume of 100 µL per assay.

Figure 10.5.4 Detection of H2O2 using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (A22188). Reactions containing 50 µM Amplex Red reagent, 1 U/mL HRP and the indicated amount of H2O2 in 50 mM sodium phosphate buffer, pH 7.4, were incubated for 30 minutes at room temperature. Fluorescence was measured with a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 580 ± 25 nm. Background fluorescence (24 units), determined for a no-H2O2 control reaction, was subtracted from each value. The inset shows the sensitivity and linearity of the assay at low levels of H2O2.

Figure 10.5.5 Detection of HRP using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (A22188). Reactions containing 50 µM Amplex Red reagent, 1 mM H2O2 and the indicated amount of HRP in 50 mM sodium phosphate buffer, pH 7.4, were incubated for 30 minutes at room temperature. Fluorescence was measured with a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. Background fluorescence (3 units), determined for a no-HRP control reaction, was subtracted from each value. The inset shows the sensitivity of the assay at very low levels of HRP.

Other Substrates for Peroxidase Assays

Although HRP is an important enzyme for both histochemistry and ELISAs, fluorogenic peroxidase substrates have not been extensively used for its detection. Fluorogenic peroxidase substrates such as the dihydrofluoresceins (also known as fluorescins) (D399, C400, C13293), dihydrocalcein AM (D23805), dihydrorhodamines (D632, D633, D23806; Probes for Nitric Oxide Research—Section 18.3) and dihydroethidium (hydroethidine; D1168, D11347, D23107; Generating and Detecting Reactive Oxygen Species—Section 18.2) are converted to highly fluorescent products in the presence of the enzyme and hydrogen peroxide. Because these substrates are insufficiently stable for routine use in ELISA assays, we have converted the dihydrofluoresceins to diacetates. When used in intracellular applications, the acetates are cleaved by endogenous esterases, releasing the intact substrate. However, when used for in vitro assays, an esterase or a mild base must first be added to cleave the acetates, releasing the substrate. The dihydrofluoresceins have been used to measure peroxidase activity and to detect hydroperoxide formation.

In addition to being a reagent for derivatization of aldehydes and ketones (Reagents for Modifying Aldehydes and Ketones—Section 3.3) and detection of nitric oxide (Probes for Nitric Oxide Research—Section 18.3), NBD methylhydrazine (N-methyl-4-hydrazino-7-nitrobenzofurazan, M20490) has been reported to be useful as a fluorogenic peroxidase substrate, with a sensitivity limit for detection of H₂O₂ of about 75 nM.

Luminol and MCLA: Chemiluminescent Peroxidase Substrates

Nonisotopic immunoassays utilizing peroxidase conjugates and the chemiluminescent horseradish peroxidase substrate luminol (L8455) have provided a rapid and sensitive method for quantitating a wide variety of analytes, including cholesterol, digoxin and acetylcholine. Addition of trace amounts of luciferin (L2911, L2912, L2916; Substrates for Microsomal Dealkylases, Acetyltransferases, Luciferases and Other Enzymes—Section 10.6) has been shown to considerably enhance the sensitivity in the assay of thyroxine, digoxin, α-fetoprotein and other analytes. A method that employs luminol has been developed for the quantitation of very limiting samples of human DNA from single hairs, saliva, small blood stains and paraffin-embedded and fixed tissue sections. Using a biotinylated oligodeoxynucleotide probe to membrane-immobilized DNA, horseradish peroxidase streptavidin and luminol, researchers have detected 150 pg of human DNA.

MCLA (M23800) is principally utilized as a superoxide-sensitive chemiluminescent probe (Generating and Detecting Reactive Oxygen Species—Section 18.2). MCLA has also been utilized for the determination of both horseradish peroxidase and myeloperoxidase.

DAB Histochemistry Kits

The use of horseradish peroxidase (HRP) for enzyme-amplified immunodetection—commonly referred to as immunoperoxidase labeling—is a well-established standard histochemical technique. The most widely used HRP substrate for these applications is diaminobenzidine (DAB), which generates a brown-colored polymeric oxidation product localized at HRP-labeled sites. The DAB reaction product can be visualized directly by bright-field light microscopy or, following osmication, by electron microscopy. We offer DAB Histochemistry Kits for detecting mouse IgG primary antibodies (D22185) and biotinylated antibodies and tracers (D22187). Each kit contains sufficient materials to stain approximately 200 slides, including:

• Diaminobenzidine (DAB)
• HRP-labeled goat anti–mouse IgG antibody (in Kit D22185) or streptavidin (in Kit D22187) conjugate
• Hydrogen peroxide (H₂O₂) reaction additive
• Blocking reagent
• Staining buffer
• Detailed staining protocols (Diaminobenzidine Histochemistry Kits)

Myeloperoxidase (MPO, EC 1.11.1.7) is a lysosomal hemoprotein located in the azurophilic granules of polymorphonuclear (PMN) leukocytes and monocytes. It is a dimeric protein composed of two 59 kD and two 13.5 kD subunits. MPO is a unique peroxidase that catalyzes the conversion of hydrogen peroxide (H₂O₂) and chloride to hypochlorous acid, a strong oxidant with powerful antimicrobial activity and broad-spectrum reactivity with biomolecules. MPO is considered an important marker for inflammatory diseases, autoimmune diseases and cancer. MPO is also experimentally and clinically important for distinguishing myeloid from lymphoid leukemia and, due to its role in the pathology of atherogenesis, has been advocated as a prognostic marker of cardiovascular disease.

The ferric, or native, MPO reacts with hydrogen peroxide (H₂O₂) to form the active intermediate MPO-I, which oxidizes chloride (Cl^–) to HOCl; these reactions make up the chlorination cycle (Figure 10.5.6). MPO also oxidizes a variety of substrates, including phenols and anilines, via the classic peroxidation cycle. The relative concentrations of chloride and the reducing substrate determine whether MPO uses hydrogen peroxide for chlorination or peroxidation. Assays based on measurement of chlorination activity are more specific for MPO than those based on peroxidase substrates such as tetramethylbenzidine (TMB).

EnzChek Myeloperoxidase (MPO) Activity Assay Kit

The EnzChek Myeloperoxidase (MPO) Activity Assay Kit (E33856) provides assays for rapid and sensitive determination of both chlorination and peroxidation activities of MPO in solution and in cell lysates (Figure 10.5.6). For detection of chlorination, the kit provides nonfluorescent 3'-(p-aminophenyl) fluorescein (APF), which is selectively cleaved by hypochlorite (^–OCl) to yield fluorescein. Peroxidation is detected using nonfluorescent Amplex UltraRed reagent, which is oxidized by the H₂O₂-generated redox intermediates MPO-I and MPO-II to form a fluorescent product. The EnzChek Myeloperoxidase Activity Assay Kit can be used to continuously detect these activities at room temperature over a broad dynamic range (1.5 to 200 ng/mL) (Figure 10.5.7). The speed (30 minutes), sensitivity, and mix-and-read convenience make this kit ideal for measuring MPO activities and for high-throughput screening for MPO-specific inhibitors.

The EnzChek Myeloperoxidase (MPO) Activity Assay Kit contains:

• 3'-(p-aminophenyl) fluorescein (APF)
• Amplex UltraRed reagent
• Human myeloperoxidase (MPO) standard
• Chlorination inhibitor
• Peroxidation inhibitor
• Hydrogen peroxide (H₂O₂)
• Phosphate-buffered saline (PBS)
• Dimethylsulfoxide (DMSO)
• Detailed protocols (EnzChek Myeloperoxidase (MPO) Activity Assay Kit)

Sufficient reagents are provided to perform 200 assays for chlorination and 200 assays for peroxidation activity in a 96-well fluorescence microplate format (100 µL per assay).

Figure 10.5.6 Schematic diagram for detection of chlorination and peroxidation activity of MPO using the EnzChek Myeloperoxidase (MPO) Activity Assay Kit (E33856). AH₂ represents the nonfluorescent Amplex UltraRed substrate, and A represents its fluorescent oxidation product.

Figure 10.5.7 Typical standard curves for detection of MPO using the APF-based chlorination assay (panel A) and Amplex UltraRed–based peroxidation assay (panel B) provided in the EnzChek Myeloperoxidase (MPO) Activity Assay Kit (E33856). Reactions were incubated at room temperature for 30 minutes. Values on the x-axes are concentrations of MPO in the standards prior to adding the detection reagent. Fluorescence was measured with a fluorescence microplate reader using fluorescence excitation and emission at 485 and 530 nm, respectively, for the APF assay, or excitation and emission at 530 and 590 nm, respectively, for the Amplex UltraRed assay. The background fluorescence measured for each zero-MPO control reaction was subtracted from each fluorescence measurement before plotting.

Zen Myeloperoxidase (MPO) ELISA Kit

The Zen Myeloperoxidase (MPO) ELISA Kit (Z33857) provides a comprehensive set of components for accurate and sensitive quantitation of human MPO in a variety of biological samples, including human serum. This sandwich immunoassay utilizes the Amplex UltraRed reagent, a fluorogenic substrate for horseradish peroxidase (HRP) that reacts with H₂O₂ in a 1:1 stoichiometric ratio to produce the brightly fluorescent and strongly absorbing Amplex UltraRed oxidation product (excitation/ emission maxima ~568/581 nm). Because the Amplex UltraRed product has long-wavelength emission, there is little interference from the blue or green autofluorescence found in most biological samples. With a high extinction coefficient, good quantum efficiency, and resistance to autooxidation, the fluorescence-based Amplex UltraRed reagent delivers better sensitivity and a broader assay range than colorimetric reagents.

The Zen Myeloperoxidase (MPO) ELISA Kit contains:

• Amplex UltraRed reagent
• Dimethylsulfoxide (DMSO)
• Concentrated phosphate-buffered saline (PBS)
• Horseradish peroxidase (HRP)–labeled goat anti–rabbit IgG antibody
• Amplex stop reagent
• Hydrogen peroxide (H₂O₂)
• MPO standard
• Bovine serum albumin (BSA)
• Tween 20
• Mouse anti-MPO antibody (capture antibody)
• Rabbit anti-MPO antibody (detection antibody)
• Zen microplates for oriented capture antibody coating
• Detailed protocols (Zen Myeloperoxidase (MPO) ELISA Kit)

Sufficient reagents are provided for 200 assays in a microplate format, using a 100 µL per well reaction volume. The Zen Myeloperoxidase (MPO) ELISA Kit can be used to detect from 0.2 to 100 ng/mL MPO at room temperature (Figure 10.5.8).

Figure 10.5.8 Typical standard curve for detection of MPO using the Zen Myeloperoxidase (MPO) ELISA Kit (Z33857). The sandwich ELISA was carried out as described in the protocol using a mouse anti-MPO primary capture antibody, MPO standards ranging from 0.2 ng/mL to 100 ng/mL, and a rabbit anti-MPO detection antibody.

The Amplex Red Catalase Assay Kit (A22180) provides a sensitive subtractive assay for measuring catalase activity. Catalase is a heme-containing redox protein found in nearly all animal and plant cells, as well as in aerobic microorganisms. In eukaryotic cells, it is concentrated in the peroxisomes. Catalase is an important enzyme because H₂O₂ is a powerful oxidizing agent that is potentially damaging to cells. By preventing excessive buildup of H₂O₂, catalase allows important cellular processes that produce H₂O₂ as a by-product to take place safely.

In the assay, catalase first reacts with H₂O₂ to produce water and oxygen (O₂). Next, the Amplex Red reagent reacts with a 1:1 stoichiometry with any unreacted H₂O₂ in the presence of horseradish peroxidase to produce the highly fluorescent oxidation product, resorufin. Therefore, as catalase activity increases, the signal from resorufin decreases (Figure 10.5.9). The results are typically plotted by subtracting the observed fluorescence from that of a no-catalase control. Using this kit, it is possible to detect catalase activity in a purified system at levels as low as 50 mU/mL.

The Amplex Red Catalase Assay Kit contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• Hydrogen peroxide (H₂O₂)
• Concentrated reaction buffer
• Catalase
• Detailed protocols (Amplex Red Catalase Assay Kit)

Each kit provides sufficient reagents for approximately 400 assays using either a fluorescence- or absorption-based microplate reader and a reaction volume of 100 µL per assay.

Figure 10.5.9 Subtractive detection of catalase using the Amplex Red Catalase Assay Kit (A22180). Reactions contained the indicated amount of catalase and 20 µM H2O2 in 1X reaction buffer and was incubated for 30 minutes. The final reaction containing 50 µM Amplex Red reagent and 0.2 U/mL HRP and was incubated at 37°C. After 30 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. The change in fluorescence is reported as the observed fluorescence intensity subtracted from that of a no-catalase control.

Glucose oxidase is widely used for glucose determination and, when conjugated to antibodies, for use in enzyme immunoassays (EIAs). Amplex Red reagent can be utilized for the ultrasensitive detection of both glucose and glucose oxidase. In this enzyme-coupled assay, glucose oxidase reacts with glucose to form gluconolactone and H₂O₂. The H₂O₂ is then detected using the Amplex Red reagent peroxidase substrate (Figure 10.5.1). The Amplex Red Glucose/Glucose Oxidase Assay Kit (A22189) can be used to detect glucose levels as low as 3 µM or 0.5 µg/mL (Figure 10.5.10) and is at least 10-fold more sensitive than assays using o-dianisidine as the peroxidase substrate. This kit can also be used to detect glucose oxidase levels as low as 0.05 mU/mL (Figure 10.5.11). We have even shown that the Amplex Red reagent can detect glucose liberated from native dextrans by dextranase and from carboxymethylcellulose by cellulase.

The Amplex Red Glucose/Glucose Oxidase Assay Kit contains:

• Amplex Red reagent
• DMSO
• Horseradish peroxidase (HRP)
• Hydrogen peroxide (H₂O₂) for use as a positive control
• Concentrated reaction buffer
• Glucose oxidase
• D-glucose
• Detailed protocols (Amplex Red Glucose/Glucose Oxidase Assay Kit)

Each kit provides sufficient reagents for approximately 500 assays using either a fluorescence- or absorption-based microplate reader and a reaction volume of 100 µL per assay.

Figure 10.5.10 Detection of glucose using the Amplex Red Glucose/Glucose Oxidase Assay Kit (A22189). Reactions containing 50 µM Amplex Red reagent, 0.1 U/mL HRP, 1 U/mL glucose oxidase and the indicated amount of glucose in 50 mM sodium phosphate buffer, pH 7.4, were incubated for one hour at room temperature. Fluorescence was then measured with a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. Background fluorescence (arbitrary units), determined for a no-glucose control reaction, has been subtracted from each value. The inset shows the sensitivity and linearity of the assay at low levels of glucose (0–15 µM).

Figure 10.5.11 Detection of glucose oxidase using the Amplex Red Glucose/Glucose Oxidase Assay Kit (A22189). Reactions containing 50 µM Amplex Red reagent, 1 U/mL HRP, 50 mM glucose and the indicated amount of glucose oxidase in 50 mM sodium phosphate buffer, pH 7.4, were incubated for 30 minutes at room temperature. Fluorescence was measured with a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. Background fluorescence (19 units) determined for a no–glucose oxidase control reaction was subtracted from each value. The inset shows the assay's sensitivity at low levels of glucose oxidase (0–0.2 mU/mL).

The Amplex Red Galactose/Galactose Oxidase Assay Kit (A22179) provides the reagents and a general protocol for the assay of terminal galactosylated proteins, galactose-producing enzymes and for the assay of galactose oxidase. Unlike glucose oxidase, galactose oxidase can produce H₂O₂ from either free galactose or from polysaccharides—including glycoproteins in solution or on cell surfaces—in which galactose is the terminal residue, producing an aldehyde moiety on the 6-position of the galactose (Figure 10.5.12). We have used our Amplex Red galactose oxidase assay for the quantitative assay of mucin-type glycoproteins by using a method similar to one described by Kinosita and collaborators.

The Amplex Red Galactose/Galactose Oxidase Assay Kit (A22179) contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• Hydrogen peroxide (H₂O₂)
• Concentrated reaction buffer
• Galactose oxidase from Dactylium dendroides
• D-Galactose
• Detailed protocols (Amplex Red Galactose/Galactose Oxidase Kit)

Sufficient reagents are provided for approximately 400 assays using either a fluorescence- or absorption-based microplate reader and a reaction volume of 100 µL per assay. The Amplex Red galactose/galactose oxidase assay accurately measures as low as 4 µM galactose and 2 mU/mL galactose oxidase activity (Figure 10.5.13, Figure 10.5.14). Because of the high absorption of resorufin, the spectrophotometric assay has only slightly lower sensitivity than the fluorometric assay. The Amplex Red Neuraminidase (Sialidase) Assay Kit (A22178) utilizes this galactose oxidase–coupled chemistry for continuous assay of neuraminidase-catalyzed hydrolysis of fetuin, a sialoglycoprotein. This product is described in detail in Detecting Glycosidases—Section 10.2.

Figure 10.5.12 Detection scheme utilized in the Amplex Red Galactose/Galactose Oxidase Assay Kit (A22179). Oxidation of the terminal galactose residue of a glycoprotein, glycolipid or polysaccharide results in the generation of H₂O₂, which, in the presence of horseradish peroxidase (HRP), reacts stoichiometrically with the Amplex Red reagent to generate the red-fluorescent oxidation product, resorufin.

Figure 10.5.13 Detection of galactose using the Amplex Red Galactose/Galactose Oxidase Assay Kit (A22179). Each reaction contained 50 µM Amplex Red reagent, 0.1 U/mL HRP, 2 U/mL of galactose oxidase and the indicated amount of galactose in 1X reaction buffer. Reactions were incubated at 37°C. After 30 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. A background fluorescence of 93 units was subtracted from each data point.

Figure 10.5.14 Detection of galactose oxidase activity using the Amplex Red Galactose/Galactose Oxidase Assay Kit (A22179). Each reaction contained 50 µM Amplex Red reagent, 0.1 U/mL HRP, 100 µM galactose and the indicated amount of galactose oxidase in 1X reaction buffer. Reactions were incubated at 37°C. After 20 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 530 ± 12.5 nm with fluorescence detection at 590 ± 17.5 nm.

The Amplex Red Cholesterol Assay Kit (A12216) provides an exceptionally sensitive assay for both cholesterol and cholesteryl esters in complex mixtures that is suitable for use with either fluorescence microplate readers or fluorometers. The assay provided in this kit can detect as little as 5 ng/mL (5 × 10^-4 mg/dL) cholesterol (Figure 10.5.15) and can accurately measure the cholesterol or cholesteryl ester content in the equivalent of 0.01 µL of human serum. The assay uses an enzyme-coupled reaction scheme in which cholesteryl esters are hydrolyzed by cholesterol esterase into cholesterol, which is then oxidized by cholesterol oxidase to yield H₂O₂ and the corresponding ketone steroidal product (Figure 10.5.16). The H₂O₂ is then detected using the Amplex Red reagent in combination with HRP. The Amplex Red cholesterol assay is continuous and requires no separation or wash steps. These characteristics make the assay particularly well suited for the rapid and direct analysis of cholesterol in blood and food samples using automated instruments. By performing two separate measurements in the presence and absence of cholesterol esterase, this assay is also useful for determining the fraction of cholesterol that is in the form of cholesteryl esters within a sample. In addition, by adding an excess of cholesterol to the reaction, this assay can be used to sensitively detect the activity of cholesterol oxidase.

The Amplex Red Cholesterol Assay Kit contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• Hydrogen peroxide (H₂O₂) for use as a positive control
• Concentrated reaction buffer
• Cholesterol oxidase from Streptomyces
• Cholesterol esterase from Pseudomonas
• Cholesterol for preparation of a standard curve
• Detailed protocols (Amplex Red Cholesterol Assay Kit)

Each kit provides sufficient reagents for approximately 500 assays using a fluorescence microplate reader and a reaction volume of 100 µL per assay.

Figure 10.5.15 Detection of cholesterol using the Amplex Red Cholesterol Assay Kit (A12216). Each reaction contained 150 µM Amplex Red reagent, 1 U/mL HRP, 1 U/mL cholesterol oxidase, 0.1 µM cholesterol esterase and the indicated amount of the cholesterol in reaction buffer. Reactions were incubated at 37°C for 30 minutes. Fluorescence was measured with a fluorescence microplate reader using excitation at 560 ± 10 nm and fluorescence detection at 590 ± 10 nm. Background fluorescence (340 arbitrary units), determined for the no-cholesterol control reaction, has been subtracted from each value.

Figure 10.5.16 Enzyme-coupled Amplex Red assays. Enzyme reactions that produce H₂O₂ can be made into Amplex Red assays. The Amplex Red Cholesterol Assay Kit (A12216) uses cholesterol oxidase to produce H₂O₂, which is then detected by the Amplex Red reagent in the presence of horseradish peroxidase (HRP). Similarly, the Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217) uses choline oxidase to produce H₂O₂.

Monoamine oxidase, which inactivates several primary, secondary and tertiary amines via oxidative transamination, serves to regulate tissue levels of amine neurotransmitters and dietary amines. The Amplex Red Monoamine Oxidase Assay Kit (A12214) provides a simple fluorometric method for the continuous measurement of amine oxidase activity in tissue homogenates or purified preparations. We have found that the assay is able to sensitively detect both monoamine oxidase (MAO) activity and semicarbazide-sensitive amine oxidase (SSAO) activity and is useful for performing both end-point and continuous measurements of amine oxidase activity. The assay is able to detect both MAO-A and MAO-B from cow brain tissue using as little as 200 µg of total protein per sample and has been used to measure plasma amine oxidase (an SSAO) activity levels as low as 1.2 × 10^-5 U/mL using a commercially available enzyme (Figure 10.5.17).

To facilitate discrimination of MAO-A and MAO-B activity, two MAO substrates and two MAO inhibitors are included in the kit. p-Tyramine is a substrate for both MAO-A and MAO-B, whereas benzylamine is a substrate for MAO-B. Both p-tyramine and benzylamine are also substrates for SSAO enzymes. Clorgyline is a specific inhibitor of MAO-A activity, and pargyline is a specific inhibitor of MAO-B activity. The potential applications of this kit include the measurement of amine oxidase activity in normal and diseased tissues, blood samples and other biological fluids, the screening of drugs as possible MAO inhibitors or substrates and the determination of kinetic constants for different amine oxidase substrates.

The Amplex Red Monoamine Oxidase Assay Kit contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• Hydrogen peroxide (H₂O₂) for use as a positive control
• Concentrated reaction buffer
• Benzylamine, a substrate for MAO-B and SSAO enzymes
• p-Tyramine, a substrate for MAO-A, MAO-B and SSAO enzymes
• Clorgyline, a specific inhibitor of MAO-A activity
• Pargyline, a specific inhibitor of MAO-B activity
• Resorufin, for use as a reference standard
• Detailed protocols (Amplex Red Monoamine Oxidase Assay Kit)

Each kit provides sufficient reagents for approximately 500 assays using a reaction volume of 200 µL per assay.

Figure 10.5.17 Detection of semicarbazide-sensitive amine oxidase (SSAO) activity using the Amplex Red Monoamine Oxidase Assay Kit (A12214) and benzylamine as the substrate. Each reaction contained 1 mM benzylamine, 1 U/mL HRP, 200 µM Amplex Red reagent and the indicated amount of SSAO in 50 mM potassium phosphate, pH 7.4. Reactions were incubated at room temperature for 15 minutes. Fluorescence was measured with a fluorescence microplate reader using excitation at 560 ± 10 nm and fluorescence detection at 590 ± 10 nm.

The Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit (A12221) provides an ultrasensitive method for continuously detecting glutamic acid or for monitoring glutamate oxidase activity in a fluorescence microplate reader or a fluorometer. In this assay, L-glutamic acid is oxidized by glutamate oxidase to produce α-ketoglutarate, NH₃ and H₂O₂. L-Alanine and L-glutamate–pyruvate transaminase are also included in the reaction. Thus, the L-glutamic acid is regenerated by transamination of α-ketoglutarate, resulting in multiple cycles of the initial reaction and a significant amplification of the H₂O₂ produced. Hydrogen peroxide reacts with the Amplex Red reagent in a 1:1 stoichiometry in a reaction catalyzed by horseradish peroxidase (HRP) to generate the highly fluorescent product resorufin (R363, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1). Because resorufin has absorption/emission maxima of ~571/ 585 nm, there is little interference from autofluorescence in most biological samples.

If the concentration of L-glutamic acid is limiting in this assay, then the fluorescence increase is proportional to the initial L-glutamic acid concentration. The Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit allows detection of as little as 10 nM L-glutamic acid in purified systems using a 30-minute reaction time (Figure 10.5.18). If the reaction is modified to include an excess of L-glutamic acid, then this kit can be used to continuously monitor glutamate oxidase activity. For example, purified L-glutamate oxidase from Streptomyces can be detected at levels as low as 0.04 mU/mL (Figure 10.5.19). The Amplex Red reagent has been used to quantitate the activity of glutamate-producing enzymes in a high-throughput assay for drug discovery.

The Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• Hydrogen peroxide (H₂O₂)
• Concentrated reaction buffer
• L-Glutamate oxidase from Streptomyces sp.
• L-Glutamate–pyruvate transaminase from pig heart
• L-Glutamic acid
• L-Alanine
• Detailed protocols (Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit)

Each kit provides sufficient reagents for approximately 200 assays using a fluorescence microplate reader and a reaction volume of 100 µL per assay.

Figure 10.5.18 Detection of L-glutamic acid using the Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit (A12221). Each reaction contained 50 µM Amplex Red reagent, 0.125 U/mL HRP, 0.04 U/mL L-glutamate oxidase, 0.25 U/mL L-glutamate–pyruvate transaminase, 100 µM L-alanine and the indicated amount of L-glutamic acid in 1X reaction buffer. Reactions were incubated at 37°C. After 30 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm.

Figure 10.5.19 Detection of L-glutamate oxidase using the Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit (A12221). Each reaction contained 50 µM Amplex Red reagent, 0.125 U/mL HRP, 0.25 U/mL L-glutamate–pyruvate transaminase, 20 µM L-glutamic acid, 100 µM L-alanine and the indicated amount of Streptomyces L-glutamate oxidase in 1X reaction buffer. Reactions were incubated at 37°C. After 60 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. The inset represents data from a separate experiment for lower L-glutamate oxidase concentrations and incubation time of 60 minutes (0–1.25 mU/mL).

Acetylcholine, the neurotransmitter released from the nerve terminal at neuromuscular junctions, binds to the acetylcholine receptor and opens its transmitter-gated ion channel (Probes for Ion Channels and Carriers—Section 16.3). The action of acetylcholine (ACh) is regulated by acetylcholinesterase (AChE), which hydrolyzes ACh to choline and acetate. The Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217) provides an ultrasensitive method for continuously monitoring AChE activity or for detecting ACh in a fluorescence microplate reader or a fluorometer. Other uses for this kit include screening for AChE inhibitors and measuring the release of ACh from synaptosomes.

In the assay, AChE activity is monitored indirectly using the Amplex Red reagent (Figure 10.5.16). First, AChE converts the acetylcholine substrate to choline. Choline is in turn oxidized by choline oxidase to betaine and H₂O₂, the latter of which, in the presence of HRP, reacts with the Amplex Red reagent to generate the red-fluorescent product resorufin. Experiments with purified AChE from electric eel indicate that the Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit can detect AChE levels as low as 0.002 U/mL using a reaction time of one hour (Figure 10.5.20). We have been able to detect acetylcholinesterase activity from a tissue sample with total protein content as low as 200 ng/mL or 20 ng/well in a microplate assay. By providing an excess of AChE in the assay, the kit can also be used to detect acetylcholine levels as low as 0.3 µM, with a range of detection from 0.3 µM to ~100 µM acetylcholine (Figure 10.5.21).

The Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• Hydrogen peroxide (H₂O₂) for use as a positive control
• Concentrated reaction buffer
• Choline oxidase from Alcaligenes sp.
• Acetylcholine (ACh)
• Acetylcholinesterase (AChE) from electric eel
• Detailed protocols (Amplex Red Acetylcholine/AChE Assay Kit)

Each kit provides sufficient reagents for approximately 500 assays using a fluorescence microplate reader and a reaction volume of 200 µL per assay.

Figure 10.5.20 Detection of electric eel acetylcholinesterase activity using the Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217). Each reaction contained 50 µM acetylcholine, 200 µM Amplex Red reagent, 1 U/mL HRP, 0.1 U/mL choline oxidase and the indicated amount of acetylcholinesterase in 1X reaction buffer. Reactions were incubated at room temperature. After 15 and 60 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 560 ± 10 nm and fluorescence detection at 590 ± 10 nm. The inset shows the sensitivity of the 15 min () and 60 min () assays at low levels of acetylcholinesterase activity (0–13 mU/mL).

Figure 10.5.21 Detection of acetylcholine using the Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217). Each reaction contained 200 µM Amplex Red reagent, 1 U/mL HRP, 0.1 U/mL choline oxidase, 0.5 U/mL acetylcholinesterase and the indicated amount of acetylcholine in 1X reaction buffer. Reactions were incubated at room temperature. After 15 and 60 minutes, fluorescence was measured with a fluorescence microplate reader using excitation at 560 ± 10 nm and fluorescence detection at 590 ± 10 nm. The inset shows the sensitivity of the 15 min () and 60 min () assays at low levels of acetylcholine (0–3 µM).

Xanthine oxidase (E.C. 1.2.3.2) plays a key role in the production of free radicals, including superoxide, in the body. The Amplex Red Xanthine/Xanthine Oxidase Assay Kit (A22182) provides an ultrasensitive method for detecting xanthine or hypoxanthine or for monitoring xanthine oxidase activity. In the assay, xanthine oxidase catalyzes the oxidation of purine nucleotides, hypoxanthine or xanthine, to uric acid and superoxide. In the reaction mixture, the superoxide spontaneously degrades to H₂O₂, which in the presence of HRP reacts stoichiometrically with Amplex Red reagent to generate the red-fluorescent oxidation product, resorufin. Resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively, and because the extinction coefficient is high (54,000 cm^-1M^-1), the assay can be performed either fluorometrically or spectrophotometrically.

The Amplex Red Xanthine/Xanthine Oxidase Assay Kit (A22182) contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• H₂O₂
• Concentrated reaction buffer
• Xanthine oxidase from buttermilk
• Hypoxanthine
• Xanthine
• Detailed protocols (Amplex Red Xanthine/Xanthine Oxidase Assay Kit)

Each kit provides sufficient reagents for approximately 400 assays using either a fluorescence- or absorption-based microplate reader and a reaction volume of 100 µL per assay.

In healthy individuals, xanthine oxidase is present in appreciable amounts only in the liver and jejunum. In various liver disorders, however, the enzyme is released into circulation. Therefore, determination of serum xanthine oxidase levels serves as a sensitive indicator of acute liver damage such as jaundice. The Amplex Red xanthine/xanthine oxidase assay has been used as a marker of recovery from exercise stress. Previously, researchers have utilized chemiluminescence or absorbance to monitor xanthine oxidase activity. The Amplex Red Xanthine/Xanthine Oxidase Assay Kit permits the detection of xanthine oxidase in a purified system at levels as low as 0.1 mU/mL by fluorescence (Figure 10.5.22). This kit can also be used to detect as little as 200 nM hypoxanthine or xanthine (Figure 10.5.23), and, when coupled to the purine nucleotide phosphorylase enzyme, to detect inorganic phosphate.

Figure 10.5.22 Detection of xanthine oxidase using the Amplex Red Xanthine/Xanthine Oxidase Assay Kit (A22182). Each reaction contained 50 µM Amplex Red reagent, 0.2 U/mL horseradish peroxidase, 0.1 mM hypoxanthine and the indicated amount of xanthine oxidase in 1X reaction buffer. After 30 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 530 ± 12.5 nm and detection at 590 ± 17.5 nm. A background of 65 fluorescence units was subtracted from each data point. The inset shows the assay’s sensitivity and linearity at low hypoxanthine concentrations.

Figure 10.5.23 Detection of hypoxanthine using the Amplex Red Xanthine/Xanthine Oxidase Assay Kit (A22182). Each reaction contained 50 µM Amplex Red reagent, 0.2 U/mL horseradish peroxidase, 20 mU/mL xanthine oxidase and the indicated amount of hypoxanthine in 1X reaction buffer. Reactions were incubated at 37°C. After 30 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 530 ± 12.5 nm and detection at 590 ± 17.5 nm. A background of 54 fluorescence units was subtracted from each data point. The inset shows the assay’s sensitivity and linearity at low enzyme concentrations.

Serum uric acid is the end product of purine metabolism in the body tissues and is cleared through the kidneys by glomerular filtration. Most animals can metabolize uric acid to more readily excreted products, but humans lack the necessary enzyme, urate oxidase (uricase), as a result of the presence of two "nonsense mutations" in the human gene for uricase. Increased uric acid levels may result from leukemia, polycythemia, ingestion of foods high in nucleoproteins (e.g., liver and kidney) or impaired renal function. Gout results from the deposit of uric acid in body joints.

The Amplex Red Uric Acid/Uricase Assay Kit (A22181) provides an ultrasensitive method for detecting uric acid or for monitoring uricase activity. In the assay, uricase catalyzes the conversion of uric acid to allantoin, H₂O₂ and carbon dioxide. In the presence of HRP, the H₂O₂ reacts stoichiometrically with Amplex Red reagent to generate the red-fluorescent oxidation product, resorufin. Resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively, and because the extinction coefficient is high (54,000 cm^-1M^-1), the assay can be performed either fluorometrically or spectrophotometrically. Previous literature reports colorimetric detection limits at 3.6 µM, whereas the Amplex Red Uric Acid/Uricase Assay Kit can be used to detect as little as 100 nM uric acid in a purified system. A biosensor containing an encapsulated uricase–peroxidase system and the Amplex Red reagent exhibits a high specificity for uric acid in the presence of interfering species and a linear response from 20 nM to 1 µM uric acid. The Amplex Red Uric Acid/Uricase Assay Kit can also be used to detect as little as 0.2 mU/mL uricase in a purified system.

The Amplex Red Uric Acid/Uricase Assay Kit (A22181) contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• Hydrogen peroxide (H₂O₂)
• Concentrated reaction buffer
• Uricase
• Uric acid
• Detailed protocols (Amplex Red Uric Acid/Uricase Assay Kit)

Each kit provides sufficient reagents for approximately 400 assays using either a fluorescence- or absorption-based microplate reader and a reaction volume of 100 µL per assay.