APPLIED BIOANALYTICAL LABS
providing tools for oxidative stress research
Oxidative Stress Assay Kits
Total Antioxidant Capacity                 Inhibition assay of chemiluminescence caused by peroxyl radicals
Catalase Activity                                UV rate assay based hydrogen peroxide decomposition
Catalase Activity                                Colorimetric assay based on HRP-catalyzed oxidation of undecomposed H2O2
Superoxide Dismutase Activity           Colorimetric rate assay of SOD inhibition of hematoxylin oxidation
Myeloperoxidase Activity                        Colorimetric chlorination activity assay by trapping HOCl with taurine
Superoxide/Total Oxidant Species     Chemiluminescent cellular oxidative burst test with patented enhancer
Product Name:  Total Antioxidant capacity Determination Kit (for 30 test tubes assays or 100 microplate assays)
Catalog number                                    Unit Price
TAC-Peroxyl                                                                     $525

Benefits:
  • Simple method applicable to plasma, semen plasma, CSF, tissue homogenates, urine, tea, fruits, wines,
    juices, extracts.
  • Using a biologically relevant free radical generator.
  • Extensively tested and validated.

Introduction:
Reactive oxygen free radicals (ROS) have been implicated in more than 100 human diseases and in aging process.
Tissue damage caused by free radicals is also well documented in trauma, toxin shocks, ischemia/reperfusion.  
ROS are generated endogenously by aerobic respiration, inflammation and lipid peroxidation, to name a few.  
Exogenously generated ROS pose un-precedent challenge to human kind because of deteriorating environment,
tobacco smoking, ionization radiation, UV-light exposure, organic solvents, anesthetics, pesticides and
medications. Organisms have developed a powerful antioxidant defense system to minimize or prevent
deleterious effects from ROS exposure.  Enzymes such as superoxide dismutase, glutathione peroxidase and
catalase aid in the decomposition of  harmful radical species. Small free radical-scavenging molecules such as
ascorbic acid, glutathione, uric acid, vitamin-E and CoQ-10 act as free radical scavengers. Macromolecules work to
chelate metals and adsorb free radicals.   The overall antioxidant status is also related to other factors such as
disease, life-style (exercise and diet) and an organism’s stress load in general. Numerous methods have been
described in literature to evaluate total antioxidant capacity (TAC) of samples.  These methods can be categorized
into scavenging assays against 1) superoxide anion radical, 2) hydrogen peroxide, 3) hypochlorous acid, 4)
hydroxyl radical, 5) peroxyl radicals, 6) peroxylnitrite.  There are also methods using less biologically relevant
systems such as those measuring capacity to reduce ferric ion and cuperic ion, and those measuring scavenging
ability toward 2,2-diphenyl-1picryhydrazyl (DPPH) radical and towards N,N-dimethyl-p-phenyleneamine (DMPD)
radical.   These methods tend to give varied results  because one particular antioxidant compound has differed
ability to scavenge a given free radical, and in a complex biological samples yields different contributions to the
total antioxidant capacity in the different assay platforms.  Therefore, it is important to describe TAC data in terms
of a specific system used.

Assay Principle:
This assay is the most popular method for the analysis of a wide range of biological samples such as serum
(plasma), CSF, semen plasma, tissue homogenates and urine. It can also been used in the analysis of tea, wine,
fruits and botanical extracts. The platform of this kit is an artificial system where biologically relevant peroxyl free
radicals are generated by thermal decomposition of 2,2’-azobis(2-amidinopropane) (ABAP).   The ABAP
decomposition products are a pair of C-centered free radicals R. and a nitrogen molecule. The R. free radicals
further react with oxygen molecules to form peroxyl radicals ROO., which are similar to those found in vivo during
lipid peroxidation.  These peroxyl radicals react with an indicator molecule, luminol (LH2), to generate a luminol
radical (LH.) that results in emission of blue lights centered at ~425 nm.  When antioxidants  are present, such a
light production is inhibited until the antioxidants are exhausted. The time of inhibition or the induction time to
light production is proportional to the total concentration of antioxidants.  The antioxidant concentration is
determined by comparing induction time to that of a water-soluble Vitamin E (tocopherol) analog, Trolox.  Typical
data of this assay is shown in the following figure:
Product Name:        Catalase Activity Determination Kit (30 cuvette assays or 100 microplate assays)
Catalog number                                    Unit Price
CAT-240                                                                           $360

Benefits:
  • Determine H2O2 decomposition rate (catalase activity) directly by measuring UV absorbance change
    at 240 nm.
  • Convenience, determine catalase activity in minutes in RBC lysate & tissue homogenates.
  • Eliminate O2 bubble problem encountered in other similar commercial kits.
  • Proprietary formulation increases stability of H2O2 and catalase for more accurate results.


Introduction:
Catalase is an antioxidant enzyme ubiquitously present in aerobic cells. It catalyses the decomposition of
hydrogen peroxide (H2O2) to water and oxygen. Hydrogen peroxide is formed in cells by controlled pathways.
H2O2 elicits a broad spectrum of cellular response ranging from mitogenic growth stimulation to apoptosis to
necrosis at different concentration levels.  Locally intense amount of hydrogen peroxide is produced by
inflammatory cells to kill pathogens.  Hydrogen peroxide at high concentration is deleterious to cells and its
accumulation causes oxidation of cellular targets such as DNA, proteins, and lipids leading to mutagenesis and
cell death. Removal of the H2O2 from the cell by catalase provides protection against oxidative damage to the
cell. The role of catalase in oxidative stress related diseases has been widely known. Catalase activity varies
greatly between tissues. The activity is highest in the liver, kidney and erythrocyte, and lowest in connective
tissues. In eukaryotic cells the enzyme is concentrated in the subcellular organelles called peroxisomes.
The enzyme consists of 4 subunits of the same size, each of which contains a heme active site to accelerate
decomposition of hydrogen peroxide. Catalase exhibits an unusual kinetic behaviour, i.e., it is not possible to
saturate the enzyme with substrate H2O2 up to 5 M concentration but there is a rapid inactivation of the enzyme
above 0.1 M H2O2.  Therefore, its activity assay is typically carried out at 10 – 50 mM H2O2.  Because
substantially lower concentration than saturated substrate is used, the enzyme activity is dependent on precise
concentration of H2O2.  The most common definition of one catalase unit is the amount of catalase decomposing
1.0 micromoles of hydrogen peroxide per minute at pH 7.0 at 25oC, with initial H2O2 concentration of 10.3 mM.

Assay Principle:
Our method is essentially that described by Beers and Sizer (1952) in which the decomposition of peroxide is
followed spectrophotometrically at 240 nm, with modifications to increase robustness and convenience. The
reaction scheme is shown below. Instead of having to calibrate precise H2O2 concentration to 10.3 mM in a
tedious process, our assay uses a certified catalase standard with known activity unit. Because catalase
concentration in sample is obtained by comparing to catalase standards, calibration of precise H2O2
concentration is not necessary in our assay.  Similarly, experiments can be carried out at room temperature and
under conditions that are more accurate and convenient (also eliminating erratic oxygen gas bubble interference
with measurement in other similar commercial kits). Modifications are also made in our formulations to overcome
problems associated with instability of diluted hydrogen peroxide and diluted enzyme standards at the room
temperature.   
Product Name:        Catalase Activity Determination Kit (30 cuvette assays or 100 microplate assays)
Catalog number                                    Unit Price
CAT-650                                                                          inquire

Benefits:
  • Undecomposed H2O2 is determined by HRP-catalyzed oxidation of a substrate to generate a
    chromophor.  Unlike UV wavelength rate assay in CAT-240, this end-point assay at a visbible
    wavelength is compatible with all kinds of spectrophotometers and microplate readers.
  • Suitable for high through-put assays for RBC lysates and tissue homogenates.
  • Proprietary formulation increases stability of H2O2 and catalase for more accurate results.


Introduction:
Catalase is an antioxidant enzyme ubiquitously present in aerobic cells. It catalyses the decomposition of
hydrogen peroxide (H2O2) to water and oxygen. Hydrogen peroxide is formed in cells by controlled pathways.
H2O2 elicits a broad spectrum of cellular response ranging from mitogenic growth stimulation to apoptosis to
necrosis at different concentration levels.  Locally intense amount of hydrogen peroxide is produced by
inflammatory cells to kill pathogens.  Hydrogen peroxide at high concentration is deleterious to cells and its
accumulation causes oxidation of cellular targets such as DNA, proteins, and lipids leading to mutagenesis and
cell death. Removal of the H2O2 from the cell by catalase provides protection against oxidative damage to the
cell. The role of catalase in oxidative stress related diseases has been widely known. Catalase activity varies
greatly between tissues. The activity is highest in the liver, kidney and erythrocyte, and lowest in connective
tissues. In eukaryotic cells the enzyme is concentrated in the subcellular organelles called peroxisomes.
The enzyme consists of 4 subunits of the same size, each of which contains a heme active site to accelerate
decomposition of hydrogen peroxide. Catalase exhibits an unusual kinetic behaviour, i.e., it is not possible to
saturate the enzyme with substrate H2O2 up to 5 M concentration but there is a rapid inactivation of the enzyme
above 0.1 M H2O2.  Therefore, its activity assay is typically carried out at 10 – 50 mM H2O2.  Because
substantially lower concentration than saturated substrate is used, the enzyme activity is dependent on precise
concentration of H2O2.  The most common definition of one catalase unit is the amount of catalase decomposing
1.0 micromoles of hydrogen peroxide per minute at pH 7.0 at 25oC, with initial H2O2 concentration of 10.3 mM.

Assay Principle:
In our kit, an assay cocktail containing H2O2 is incubated with catalase sample for exactly 2 minutes. The
catalase reaction is quenched by adding an inhibitor.  The remaining H2O2 is measured by Horseradish
Peroxidase (HRP) catalyzed oxidation of TMB (3,5,3’,5’-tetramethylbenzidine) to generate a blue colored cation
free radical with a peak absorbance at 653 nm. Catalase concentration in sample is determined by comparing
absorbance at 653 nm to that of a certified catalase standard, eliminating a need to calibrate precise H2O2
concentration.  Experiments can be carried out at room temperature under conditions that are more accurate and
convenient. Modifications are also made in our formulations to overcome problems associated with instability of
diluted hydrogen peroxide and diluted enzyme standards at the room temperature.
Product Name:        Superoxide Dismutase (SOD) Activity Determination Kit (30 cuvette assays or 100
microplate assays)
Catalog number                                    Unit Price
SOD-560                                                                           $360

Benefits:
  • Simple and convenient rate assay to determine SOD activity in standard "cytochrome c" unit in
    biological samples.
  • Determines total SOD activity, or CuZnSOD or MnSOD activity separately (1 - 2 mM cyanide can be used
    to inhibit CuZnSOD activitywithout affecting hematoxylin auto-oxidation rate, cyanide not included in the kit).

Introduction:
The discovery of enzyme superoxide dismutase (SOD) by McCord and Fridovich in 1969 started a new era of
research on the role of free radicals in biology and medicine. Now it has been found that SOD is ubiquitous in
every aerobic organism from microbes to human. In animal cells, there are two kinds of SODs, a cellular SOD
containing a CuZn active site and a mitochondria SOD containing a Mn active site.  An extracellular CuZn-SOD (EC-
SOD) is also found in mammalian extracellular fluids such as plasma, lymph, synovial fluid, cerebrospinal fluid and
seminal plasma.  The EC-SOD is probably bound to heparan sulfate proteoglycans on cell surfaces, in basal
membranes and in connective tissue matrix.  Prokaryotic SODs are more diverse in active site composition
consisting of CuZn, or Mn, or Fe, or Ni metal centers.

SOD decomposes superoxide anion into hydrogen peroxide and oxygen at close to highest reaction rate
possible.  Superoxide radical is involved in diverse physiological and pathophysiological processes.  It is produced
in respiratory and cytochome P450 electron transport chain reactions as a by-product.  An intense amount is also
produced during oxidative burst by activated neutrophils and macrophages.  A very interesting chemistry involves
interaction between superoxide with nitric oxide (NO) that is a vasodilator and a cellular signal molecule.  
Superoxide can react with NO at very fast rate to form peroxynitrite – a very powerful oxidant that cause
damages to DNA, protein and other biological molecules.  Removal of superoxide provides a defense against
these damages.  The relationship between SOD and various human diseases has been well documented in
literature.  One SOD activity unit is defined as the amount of SOD that inhibits the rate of cytochrome c reduction
by half at pH 7.8 at 25oC under specific conditions. The “standard” method of SOD activity assay is the inhibition
of cytochrome c reduction coupled with superoxide generation by xanthine oxidase. Adjusting amounts of
cytochrome c, xanthine oxidase and samples are tedious.

Assay Principle:
The method used in our kit as essentially that described by Martin J. P., Jr etc, with modifications to increase
robustness and reliability.  Briefly, auto-oxidation of hematoxylin (with increasing absorbance at 560 nm) is
inhibited by SOD at the assay pH, The percentage of inhibition is linearly proportional to the amount of SOD
present within a specific range.  The SOD activity in a samples is determined by measuring ratio of auto-oxidation
rates of hematoxylin in the presence and absence of the sample.  Additional advantage of our assay is that the
rate is not affected by cyanide and other reagents used to distinguish CuSOD and MnSOD activity, total SOD
activity can be determined, or CuSOD and MnSOD activities can be determined separately using the same kit.  
Typical data of rate assay assay are shown below.
Product Name:        Myeloperoxidase Chlorination Activity Determination Kit (30 cuvette assays)
Catalog number                                    Unit Price
MPO-412                                                                         $495

Benefits:
  • Simple and convenient assay to determine myeloperoxidase chlorination in tissue homogenates, intact
    and extracted neutrophils.
  • Highly specific.  Our kit determines chlorination activity while other myelperoxidase kits measure
    peroxidative activity that is interfered by the peroxidases coexisted in biological samples.

Introduction:
Myeloperoxiase (MPO), the most abundant protein in neutrophils (also found in monocytes), is the focus of
inflammatory pathologies. Most recent work has indicated that it is an excellent biomarker for human
cardiovascular risk.  Its ability to catalyze reaction between chloride and hydrogen peroxide (H2O2) to form
hypochlorous acid is unique among mammalian enzymes and is considered to be the dominant function of MPO in
vivo. Hypochlorous acid is a powerful antimicrobial agent, and extremely reactive with biological molecules
causing much of the damage mediated by neutrophils in inflammatory diseases.

MPO also exhibits peroxidase activity that catalyzes oxidation of a number of substrates by (H2O2).  This activity
has been widely used to assess the amount of MPO.  However, its specificity is very poor for unpurified biological
samples because of presence of other peroxidases.  Peroxidases, however, generally do not produce
hypochlorous acid. The only exception is eosinophil peroxidase that produces hypochlorous acid at pH below 5.  
The chlorination activity of MPO has a pH optinum of near neutral pH, therefore assay conditions can be set so
that only MPO activity is specifically measured.

Assay Principle:
Our kit provides a simple and easy colorimetric assay for the study of MPO activity in various biological and
purified samples.  Our method has been described by Weiss and coworkers (1982).  Briefly, hypochlorous acid
(HOCl) is formed from MPO catalyzed reaction between chloride and hydrogen peroxide.  HOCl is rapidly trapped
by
beta-amino acid taurine to form a stable oxidant taurine chloramine.  Taurine prevents accumulation of
hypochlorous acid that could deactivate MPO and does not react with MPO enzyme intermediate to interfere MPO
catalysis.  After incubation for specific time, the MPO catalyzed reaction is stopped by adding catalase to eliminate
hydrogen peroxide.  Taurine chloramine thus formed is then allowed to react with 5-thio-2-nitrobenzoic acid
(TNB).  TNB has a chromophore that has maximal absorbance at 412 nm while its reaction product with taurin
chloramine, 5-5’-dithiobis(2-nitrobenzoic acid) or DTNB is colorless. By following decrease of absorbance at 412
nm, MPO activity is measured. One unit is the amount of MPO that can produce 1.0 nmoles of taurine chloramine
(hypochlorous acid) at pH 6.5 and 25oC during 30 minutes in the presence of 100 mM chloride and 100 microM of
hydrogen peroxide.
Product Name:       Chemiluminescent Assay Kit for Superoxide / Total Oxidant Species
                               (For 30 test tube assays or 100 microplate assays)
Catalog number                                    Unit Price
Superoxide/TOS-Luminol                                            $470

Benefits:
  • Simple and convenient assay to determine Superoxide or total oxidant species in whole blood or
    isolate cells (e.g., neutrophils).
  • Ultimate sensitivity with the help of a patented and non-toxic enhancer, minimal amount of sample
    required.

Introduction:
Superoxide and other reactive oxygen species play very important role in diseases and pathology related to
oxidative stress.  Its extremely low steady-state concentration in vivo requires sensitive methods of
measurement.  A number of techniques have been described in the literature including ferricytochrome c
reduction, nitroblue tetrazolium reduction, aconitase activity inhibition, nitrone spin trapping, electrochemical
detection, and chemiluminescent assays with luminol and lucigenin.

Assay Principle:
Luminol reacts with superoxide to produce a luminophore with an emission peak at ~425 nm.  The luminescence
intensity is proportional to the amount of superoxide in the sample.  Most recently, the luminol assay is criticized
as a specific quantitative method to determine superoxide production in cells.  In addition to its sensitivity to a
number of reactive oxygen species (hypochlorite, peroxynitrite and hydroxyl radical from H2O2+metal/heme-
proteins), the first intermediate in the reaction can react with oxygen to generate superoxide resulting in higher
apparent superoxide production rate than actual cellular production rate.  However, this assay does provide a
valid gross assessment of  reactive oxygen species where their production is known.  Indeed, luminol assay is
one of the most important methods in the analysis of metabolic activities of phagocytes with combined
advantages of simplicity and sensitivity.                

TOS-Luminol kit utilizes a luminescence enhancer (sodium orthovanadate) which further increases sensitivity to
superoxide.  A number of compounds such as iodophenol are known to increase the intensity of luminescent
emission from luminol and are widely used in immunological protein and DNA detection kits.  However, their
toxicity limits the usage only in cell-free systems. Orthovanadate is not toxic to cells and does not induce
chemiluminescence by itself.  The enhanced luminescence (~50x) allows fewer cells to be taken for testing.  This
feature may be particularly important for monitoring treatment of patients with hereditary chronic granulomatous
disease (CGD) that lacks NADPH oxidase and therefore ability to generate superoxide.  Similarly, it is desirable to
remove as few cells as possible from a cell culture that is under frequent monitoring for superoxide or total
oxidant production.  Orthovanadate also enhances detection of superoxide in cell-free systems.

The mechanism of orthovanadate action is not completely understood, it is believed that orthovanadate may act
as an electron transporter or may lower activation energy for luminol activation. Therefore, its sensitivity to
different type of oxidants (or specificity) may not be the same as that in the system without the enhancer. For
monocytic cells, this enhanced chemiluminescent signal after stimulation is inhibited by superoxide dismutase
(SOD), but not significantly by catalase (H2O2 scanvenger) and not by azide - myeloperoxidase inhibitor. The
signal is also linearly proportional to the number of cells up to approxiamtely 10(6) cell. This indicates that
superoxide is mainly responsible for chemiluminescent signal in these cells, but one should be very careful in
extending these results to different cell types/stimulants where a different oxidant may be more dominant source
for chemiluminescence.