Substrates for Oxidases, Including Amplex Red Kits - Section 10.5

Amplex Red Reagent: Stable Substrate for Peroxidase Detection

In the presence of horseradish peroxidase (HRP), the Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine, A12222, A22177; ) reacts with H₂O₂ with a 1:1 stoichiometry to produce highly fluorescent resorufin (R363, Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1, Figure 10.59). The Amplex Red reagent has greater stability, yields less background and produces a red-fluorescent product 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. Using the Amplex Red reagent in conjunction with HRP, we have found that release of hydrogen peroxide to the medium by as few as 2000 phorbol ester–stimulated neutrophils can be detected in a fluorescence microplate reader.

The Amplex Red reagent has been used to detect the release of H₂O₂ from activated human leukocytes, to measure the activity of monoamine oxidase in cow brain tissue, to demonstrate the extracellular production of H₂O₂ produced by UV light stimulation of human keratinocytes and to measure L-glutamate in food samples. Using the Amplex Red reagent, researchers have discovered that antibodies can convert molecular oxygen to H₂O₂, which may be important in understanding a new chemical arm of the immune system, as well as the evolution of antibodies and the role they may play in human diseases. The sensitivity of the Amplex Red reagent in detecting the activity of D-amino acid oxidase has been reported to be 5- to 25-times better than that of the QuantaBlu fluorogenic peroxidase substrate, with a lower limit for detection of D-alanine of 2 picomoles. The Amplex Red reaction can be used to routinely detect as little as 10 picomoles of H₂O₂ in a 100 µL volume (50 nM, Figure 10.60), at least a 10-fold greater sensitivity than that attained with the commonly used scopoletin assay for H₂O₂. In the scopoletin assay, HRP catalyzes conversion of the fluorescent scopoletin to a nonfluorescent product. Unlike scopoletin, the Amplex Red reagent is a fluorogenic substrate with very low background fluorescence. Consequently, assays using Amplex Red as the substrate result in an increase in fluorescence, not a decrease — an inherently superior method for enzymatic assays. Other advantages of the Amplex Red reaction over scopoletin-based H₂O₂ assays include high chemical stability of the Amplex Red reagent and its fluorescent product, resorufin, and the long-wavelength spectra of resorufin. Because resorufin has excitation/emission maxima () of ~570/585 nm (versus 360/460 nm for scopoletin), there is much less interference from autofluorescence in most biological samples. The Amplex Red reagent can be purchased separately 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 10.59 Principle of coupled enzymatic assays using our 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 our Amplex Red Glucose/Glucose Oxidase Assay Kit (A22189).

Figure 10.60 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.

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

Our Amplex UltraRed reagent (A36006) improves upon the performance of the Amplex Red reagent, offering brighter fluorescence and enhanced sensitivity on a per-mole basis in horseradish peroxidase or horseradish peroxidase-coupled enzyme assays (Figure 10.62). Fluorescence of the oxidized Amplex UltraRed reagent is also less sensitive to pH (Figure 10.63), and the substrate and its oxidation product exhibit greater stability that the Amplex Red reagent in the presence of hydrogen peroxide (H₂O₂) or thiols such as dithiothreitol (DTT). Like the Amplex Red reagent, the 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) (). Because the reaction product has long-wavelength spectra, there is little interference from the blue or green autofluorescence found in most biological samples.

The Amplex UltraRed reagent can provide increased sensitivity in peroxidase-based enzyme-linked immunosorbent assays (ELISAs). With a high extinction coefficient and good quantum efficiency, the fluorescence-based Amplex UltraRed reagent is more sensitive than standard colorimetric reagents and provides a broader measurement range for ELISAs. In contrast to commonly used ELISA reagents such as ABTS and TMB, the Amplex UltraRed reagent is exceptionally resistant to autooxidation, making it a superior alternative for peroxidase detection (Advantages of the Amplex UltraRed reagent over chromogenic reagents - Table 10.5). Like the Amplex Red reagent, the versatile Amplex UltraRed reagent can be detected using either fluorescence- or absorption-based instrumentation. The Amplex UltraRed reagent, which should be suitable for any of the applications described for the Amplex Red reagent (Substrates for Oxidases, Including Amplex Red Kits - Section 10.5), is available as a set of five vials, each containing 1 mg of the substrate (A36006).

Figure 10.62 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 10.63 Comparison of pH-dependent fluorescence 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.

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, acetylcholine and cholesterol (Figure 10.65). Advantages of Amplex Red reagent–and Amplex UltraRed reagent–based assays include the following:

• The 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.
• The 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.
• Detection can be by either absorption or fluorescence.
• The 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(R) Red Hydrogen Peroxide/Peroxidase Assay Kit)
• Amplex Red Catalase Assay Kit (A22180, Amplex(R) Red Catalase Assay Kit)
• Amplex Red Monoamine Oxidase Assay Kit (A12214, Amplex(R) Red Monoamine Oxidase Assay Kit)
• Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit (A12221, Amplex(R) Red Glutamic Acid/Glutamate Oxidase Assay Kit)
• Amplex Red Glucose/Glucose Oxidase Assay Kit (A22189, Amplex(R) Red Glucose/Glucose Oxidase Assay Kit)
• Amplex Red Galactose/Galactose Oxidase Assay Kit (A22179, Amplex(R) Red Galactose/Galactose Oxidase Kit)
• Amplex Red Neuraminidase (Sialidase) Assay Kit (A22178, Amplex(R) Red Neuraminidase (Sialidase) Assay Kit)
• Amplex Red Cholesterol Assay Kit (A12216, Amplex(R) Red Cholesterol Assay Kit)
• Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217, Amplex(R) Red Acetylcholine/AChE Assay Kit)
• Amplex Red Phosphatidylcholine-Specific Phospholipase C Assay Kit (A12218, Probes for Lipid Metabolism and Signaling - Section 17.4, Amplex(R) Red Phosphatidylcholine-Specific Phospholipase C Assay Kit)
• Amplex Red Phospholipase D Assay Kit (A12219, Probes for Lipid Metabolism and Signaling - Section 17.4, Amplex(R) Red Phospholipase D Assay Kit)
• Amplex Red Sphingomyelinase Assay Kit (A12220, Sphingolipids, Steroids, Lipopolysaccharides and Related Probes - Section 13.3, Amplex(R) Red Sphingomyelinase Assay Kit)
• Amplex Red Uric Acid/Uricase Assay Kit (A22181, Amplex(R) Red Uric Acid/Uricase Assay Kit)
• Amplex Red Xanthine/Xanthine Oxidase Assay Kit (A22182, Amplex(R) Red Xanthine/Xanthine Oxidase Assay Kit)
• Amplex Red ELISA Kits #1 and #2 (A22170, A22171; Amplex(R) Red ELISA Kit #1, with Goat Anti–Mouse IgG, Horseradish Peroxidase Conjugate, Amplex(R) Red ELISA Kit #2, with Protein G, Horseradish Peroxidase Conjugate)

The Amplex UltraRed assay kits include:

• Amplex ELISA Development Kits for Mouse IgG and for Rabbit IgG (A33851, A33852; Amplex(R) ELISA Development Kit for Mouse IgG with Amplex(R) UltraRed Reagent, Amplex(R) ELISA Development Kit for Rabbit IgG with Amplex(R) UltraRed Reagent)
• EnzChek Myeloperoxidase (MPO) Activity Assay Kit (E33856, EnzChek(R) Myeloperoxidase (MPO) Activity Assay Kit)
• Zen Myeloperoxidase (MPO) ELISA Kit (Z33857, Zen Myeloperoxidase (MPO) ELISA Kit)
• EnzChek Ultra Phytase Assay Kit (E33701, EnzChek(R) 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. The Amplex Red and Amplex UltraRed kits and reagents are sold for noncommercial use and for high-throughput screening applications only.

Figure 10.63 Comparison of pH-dependent fluorescence 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 Hydrogen Peroxide/Peroxidase Assay Kit

The Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (A22188) provides a simple, sensitive, one-step assay for detecting H₂O₂ or the activity of horseradish peroxidase either by measuring fluorescence with a fluorescence-based microplate reader or a fluorometer (Figure 10.66) 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 and is also useful for detecting H₂O₂ that is produced as a product of enzyme-coupled reactions. The Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxide (HRP)
• Hydrogen peroxide (H₂O₂) for use as a positive control
• Concentrated reaction buffer
• Detailed protocols (Amplex(R) 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 10.66 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.

Amplex Red ELISA Kits

Molecular Probes' Amplex Red ELISA Kits offer an extremely sensitive fluorometric or colorimetric detection method for horseradish peroxidase (HRP)–amplified enzyme-linked immunosorbent assays (ELISAs). The Amplex Red ELISA Kit #1 (A22170) contains an HRP goat anti–mouse IgG antibody conjugate, which can be used for the ELISA detection of any mouse IgG antibody. The Amplex Red ELISA Kit #2 (A22171) contains the versatile protein G conjugate of HRP, which can be used for the ELISA detection of IgGs from most commonly used species, including human, mouse, rabbit, goat, sheep, bovine and horse. The Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine, ) provided in these kits is a highly sensitive and stable probe for the detection of HRP activity. In the presence of HRP, the Amplex Red reagent reacts with hydrogen peroxide with a 1:1 stoichiometry to form the fluorescent product resorufin (R363, Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1; absorption/emission maxima ~571/ 585 nm, Figure 10.59). Because resorufin also has strong absorption, the assay can be performed either fluorometrically or spectrophotometrically. The Amplex Red ELISA Kit #1 with the HRP–goat anti–mouse IgG antibody conjugate has detection limits of as little as 10 pg/microplate well of a mouse IgG by fluorometry or 50 pg/microplate well by colorimetry (Figure 7.50). The Amplex Red ELISA Kit #2 with HRP–protein G has detection limits of as little as 1 ng/microplate well of a mouse IgG by fluorometry or 3 ng/microplate well by colorimetry (Figure 7.51) in 96-well plates.

Figure 7.50 Detection of a mouse monoclonal antibody using the Amplex Red ELISA Kit #1, with the horseradish peroxidase conjugate of goat anti–mouse IgG antibody (A22170). The wells of a microplate were first coated with an excess of a fluorescein conjugate of bovine serum albumin (BSA) and then blocked with PBS–BSA. The indicated amounts of anti-fluorescein/Oregon Green mouse monoclonal 4-4-20 antibody (A6421) were then applied in 100 µL volumes and incubated for one hour. The wells were washed and then assayed using the reagents and protocol provided in this kit. The reactions were incubated for 50 minutes and then measured both A) for fluorescence (excitation/emission of 530 ± 12.5 nm/590 ± 10 nm) and B) for absorbance (576 ± 5 nm). The data points represent the average of three reactions. For the fluorescence plot, a background of 280 (arbitrary units) has been subtracted from each reading; for the absorption plot, a background of 0.040 has been subtracted from each reading.

Figure 7.51 Detection of a mouse monoclonal antibody using the Amplex Red ELISA Kit #2 (A22171). The assay can detect as little as 1 ng of a monoclonal antibody in the well of a microplate by fluorometry (panel A) or 3 ng by colorimetry (panel B). Conditions of the assay are essentially as described in the caption of Figure 7.50, except that a horseradish peroxidase conjugate of protein G was used instead of the horseradish peroxidase conjugate of goat anti–mouse IgG antibody.

Each Amplex Red ELISA Kit contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Concentrated reaction buffer
• Hydrogen peroxide (H₂O₂)
• Horseradish peroxidase (HRP) conjugate of goat anti–mouse IgG antibody (in Kit #1, A22170) or of protein G (in Kit #2, A22171)
• Detailed ELISA protocols (Amplex(R) Red ELISA Kit #1, with Goat Anti–Mouse IgG, Horseradish Peroxidase Conjugate, Amplex(R) Red ELISA Kit #2, with Protein G, Horseradish Peroxidase Conjugate)

Each kit provides sufficient reagents for approximately 1000 ELISA assays using either a fluorescence- or absorption-based microplate reader and a reaction volume of 100 µL per assay. Our HRP conjugates of the goat anti–mouse IgG antibody (G21040), goat anti–rabbit IgG antibody (G21234) and protein G (P21041) are available separately. HRP conjugates of additional antibodies that can be used with the Amplex Red reagent (A12222, A22177; Substrates for Oxidases, Including Amplex Red Kits - Section 10.5) are listed in Alkaline phosphatase and horseradish peroxidase enzyme conjugates and Zenon Labeling Kits - Table 7.4.

Figure 10.59 Principle of coupled enzymatic assays using our 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 our Amplex Red Glucose/Glucose Oxidase Assay Kit (A22189).

Amplex ELISA Development Kits for Rabbit and Mouse IgG

The Amplex ELISA Development Kits for Mouse IgG (A33851) and for Rabbit IgG (A33852) provide a comprehensive set of components for creating a fluorescence-based ELISA using a mouse or rabbit primary antibody, respectively. This assay is based on the Amplex UltraRed reagent, a fluorogenic substrate for horseradish peroxidase (HRP) that 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) (). Because the Amplex UltraRed peroxidation product has long-wavelength spectra, 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. In a sandwich ELISA format using C-reactive protein, Molecular Probes scientists are routinely able to detect 75 pg of antigen using goat anti–mouse IgG antibody and 1 pg using goat anti–rabbit IgG antibody); these detection limits are 25-fold lower than those obtained from the same sandwich ELISA format using the common colorimetric reagent TMB. Each Amplex ELISA Development Kit contains:

• Amplex UltraRed reagent
• Dimethylsulfoxide (DMSO)
• Concentrated phosphate-buffered saline (PBS), pH 7.2
• Horseradish peroxidase conjugate of goat anti–mouse IgG antibody (in A33851) or goat anti—rabbit IgG antibody (in A33852)
• Amplex stop reagent
• Hydrogen peroxide (H₂O₂)
• 0.1 M sodium bicarbonate buffer, pH ~9.3
• Bovine serum albumin (BSA)
• Tween 20
• Nunc-Immuno MaxiSorp U96 plate
• Detailed protocols (Amplex(R) ELISA Development Kit for Mouse IgG with Amplex(R) UltraRed Reagent, Amplex(R) ELISA Development Kit for Rabbit IgG with Amplex(R) UltraRed Reagent)

Sufficient reagents are provided in each kit for 500 microplate assays in a 96-well fluorescence microplate format (100 µL per assay).

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, Molecular Probes has 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 (Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2) 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:

• 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)

Each kit provides sufficient materials to stain approximately 200 slides.

Myeloperoxidase (MPO, EC 1.11.1.7) is a lysosomal hemeoprotein 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, the major strong oxidant with powerful antimicrobial activity and broad-spectrum reactivity with biomolecules. MPO has been considered as an important marker for inflammatory and 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 redox and enzyme intermediate compound MPO-I, which oxidizes chloride (Cl^–) to HOCl; these reactions make up the chlorination cycle. 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 (AH) 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. 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 with excitation/ emission maxima of ~568/581 nm (). 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). 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. Each 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)
• Detailed protocols (EnzChek(R) 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).

Zen Myeloperoxidase (MPO) ELISA Kit

The Zen Myeloperoxidase (MPO) ELISA Kit (Z33857) provides a comprehensive set of components for accurate and sensitive quantitation of MPO in a variety of biological samples, including human serum. This assay is based on the Amplex UltraRed reagent, a fluorogenic substrate for horseradish peroxidase (HRP) that reacts with H₂O₂ in a 1:1 stoichiometric ratio to produce the Amplex UltraRed product, a brightly fluorescent and strongly absorbing product with excitation/ emission maxima of ~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. Each 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 (primary capture antibody)
• Rabbit anti-MPO antibody (secondary capture antibody)
• Zen microplates
• 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 microplate-based formulation provides speed and convenience yet is highly sensitive, and it can be used to detect MPO over a broad dynamic range (0.2 to 100 ng/mL) at room temperature.

The Amplex Red Catalase Assay Kit (A22180) provides an ultrasensitive, yet simple, 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 10.67). 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.

Resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively (). Because the absorption is strong, the assay can be performed either fluorometrically or spectrophotometrically. 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(R) 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 10.67 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). Molecular Probes has found that the 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 10.59). 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 10.25) 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 10.68). We have even shown that the Amplex Red reagent can detect glucose liberated from native dextrans by dextranase and from carboxymethylcellulose. 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(R) Red Glucose/Glucose Oxidase Assay Kit)

The 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 10.25 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 10.68 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 (Amplex(R) Red Galactose/Galactose Oxidase Kit) 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 10.14). 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(R) 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 10.15, Figure 10.16). Because of the high absorption of resorufin, the absorptimetric 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 sialoglyconjugate. This product is described in detail in Detecting Glycosidases - Section 10.2.

Figure 10.14 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 10.15 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 10.16 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 13.38) 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 10.65). 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(R) 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 13.35 Measurement of sphingomyelinase activity using the Amplex Red Sphingomyelinase Assay Kit (A12220). Each reaction contained 50 µM Amplex Red reagent, 1 U/mL horseradish peroxidase (HRP), 0.1 U/mL choline oxidase, 4 U/mL of alkaline phosphatase, 0.25 mM sphingomyelin and the indicated amount of Staphylococcus aureus sphingomyelinase in 1X reaction buffer. Reactions were incubated at 37°C for one hour. Fluorescence was measured with a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm.

Figure 10.65 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 10.69).

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. Each 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(R) Red Monoamine Oxidase Assay Kit)

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

Figure 10.69 Detection of plasma amine oxidase (an 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 16.34). 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 40 µU/mL (Figure 16.35). The Amplex Red reagent has been used to quantitate the activity of glutamate-producing enzymes in a high-throughput assay for drug discovery. Each 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(R) 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.

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 potential uses for this kit include screening for AChE inhibitors and measuring the release of ACh from synaptosomes. The Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit can also be used for the ultrasensitive and specific assay of free choline, an "essential nutrient," in foods.

In the assay, AChE activity is monitored indirectly using the Amplex Red reagent (Figure 10.65). 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 16.27). 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 16.28). 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(R) 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.

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 the 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.

In healthy individuals, xanthine oxidase is present in appreciable amounts only in the liver and jejunum. However, in various liver disorders the enzyme is released into circulation. Therefore, determination of serum xanthine oxidase level 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 absorption 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 18.4). This kit can also be used to detect as little as 200 nM hypoxanthine or xanthine (Figure 18.5), and, when coupled to the purine nucleotide phosphorylase enzyme, to detect inorganic phosphate.

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

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• Hydrogen peroxide (H₂O₂)
• Concentrated reaction buffer
• Xanthine oxidase from buttermilk
• Hypoxanthine
• Xanthine
• Detailed protocols (Amplex(R) 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.

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(R) 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.