Hydrogen Peroxide Assay

Catalog Number DE3700

For the quantitative determination of hydrogen peroxide (H2O2)

concentrations in cell culture supernates, serum, plasma, urine,
and other biological fluids.

This package insert must be read in its entirety before using this product.

FOR RESEARCH USE ONLY.

NOT FOR USE IN DIAGNOSTIC PROCEDURES.
 TABLE OF CONTENTS
Contents Page
INTRODUCTION 2
PRINCIPLE OF THE ASSAY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
TECHNICAL HINTS 2
REAGENTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
STORAGE 3
OTHER SUPPLIES REQUIRED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
PRECAUTIONS 3
SAMPLE COLLECTION AND STORAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
SAMPLE PREPARATION 4
REAGENT PREPARATION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
ASSAY PROCEDURE 5
CALCULATION OF RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
TYPICAL DATA 5
PRECISION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
RECOVERY 6
LINEARITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
SENSITIVITY 7
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

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INTRODUCTION
Hydrogen peroxide (H2O2) is a reactive oxygen metabolic by-product that serves as a key
regulator for a number of oxidative stress-related states (1, 2). Functioning through NFk-B and
other factors, hydroperoxide-mediated pathways have been linked to asthma, inflammatory
arthritis, atherosclerosis, diabetic vasculopathy, osteoporosis, a number of neurodegenrative
diseases and Down's Syndrome (3 - 11). A recent report indicates that antibodies have the
capacity to convert molecular oxygen into hydrogen peroxide to contribute to the normal
recognition and destruction processes of the immune system (12, 13). Measurement of this
reactive species may help to determine how oxidative stress modulates varied intracellular
pathways.

PRINCIPLE OF THE ASSAY

This colorimetric H2O2 assay is a complete kit for the determination of hydrogen peroxide in cell
culture supernates, serum, plasma, urine, and other biological fluids. This kit is designed to
measure low concentrations of H2O2 in biological matrices. It uses a color reagent that contains
xylenol orange dye in an acidic solution with sorbitol and ammonium iron sulfate that reacts to
produce a purple color in proportion to the concentration of H2O2 in the sample being tested.
The exact mechanism of the color reaction is not known, but probably involves coordinated iron
reacting with H2O2 and the dye molecule.

TECHNICAL HINTS
· FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
· The kit should not be used beyond the expiration date on the kit label.
· Do not mix or substitute reagents with those from other sources or lots.
· When mixing or reconstituting protein solutions, always avoid foaming.
· Pre-rinse the pipette tips when pipetting standards.
· To avoid cross-contamination, change pipette tips between additions of each standard
level, between sample additions, and between reagent additions. Also, use separate
reservoirs for each reagent.
· Pipette standards and samples to the bottom of the wells.
· Add all other reagents to the side of the wells to avoid contamination.

REAGENTS
Half-Area Microplate (Part R80-0915) - Ready to use microplate.
Hydrogen Peroxide Color Reagent (Part R80-0968) - 11 mL of colorimetric substrate solution in dilute
acid.
Hydrogen Peroxide Standard (Part R80-0941) - 0.5 mL of H2O2 (100,000 ng/mL) in deionized water
with preservatives.
Plate Covers - 2 adhesive strips.

2
STORAGE
Unopened Kit Store the unopened kit at £ -20° C. Do not use past kit expiration date.
Store at £ -20° C in a manual defrost freezer.* Avoid
Color Reagent
repeated freeze-thaw cycles.
Opened
Standard Store at 2 - 8° C.*
Reagents
Unused wells should be covered tightly with a plate cover
Microplate
and may be stored at 2 - 8° C.* Do not reuse wells.

*Provided this is within the expiration date of the kit.

OTHER SUPPLIES REQUIRED

· Microplate reader capable of measuring absorbance at 550 nm (540 nm and 570 nm).
· Pipettes and pipette tips.
· Multi-channel pipette, squirt bottle, or manifold dispenser.
· 50 mM phosphate, pH 6.0 (for use as a Sample Diluent).

PRECAUTIONS
The Hydrogen Peroxide Standard is light-sensitive and should be protected from direct light for
prolonged periods of time.
Care should be taken when handling and disposing of the contents of the plate.
This kit has been tested with a variety of samples; however, it is possible that high levels of
interfering substances may cause variation in assay results.

SAMPLE COLLECTION AND STORAGE

Cell Culture Supernates - Remove particulates by centrifugation. Assay immediately or
aliquot and store at £ -20° C. Avoid repeated freeze-thaw cycles.
Serum - Use a serum separator tube (SST). Allow samples to clot for 30 minutes. Centrifuge
for 15 minutes at approximately 1000 x g. Assay immediately or aliquot and store the serum at
£ -20° C. Avoid repeated freeze-thaw cycles.
Plasma - Collect plasma using EDTA, heparin, or citrate as an anticoagulant. Centrifuge for
15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store
the plasma at £ -20° C. Avoid repeated freeze-thaw cycles.
Urine - Aseptically collect the first urine of the day (mid-stream), voided directly into a sterile
container. Centrifuge to remove particulate matter. Assay immediately or aliquot and store the
urine at £ -20° C. Avoid repeated freeze-thaw cycles.
Other Biological Fluids - The recovery and linearity characteristics of this assay using other
biological fluids has not been evaluated.

3
SAMPLE PREPARATION
Serum, plasma, and urine samples require a 64-fold dilution. A suggested 64-fold dilution is
5 mL of sample + 315 mL of Sample Diluent.

REAGENT PREPARATION
The Hydrogen Peroxide Color Reagent must be kept at 2 - 8° C during use. Bring all
other reagents to room temperature before use.

Hydrogen Peroxide Standard* - Pipette 966 mL of Sample Diluent into the 3400 ng/mL tube.
Pipette 500 mL of Sample Diluent into the remaining tubes. When pipetting standards, it is
important to pre-rinse the pipette tips. Use the 100,000 ng/mL standard stock to produce a
dilution series (below). Mix each tube thoroughly by vortexing and change pipette tips between
each transfer. The 3400 ng/mL standard serves as the high standard and the Sample Diluent
serves as the zero standard (0 ng/mL).
*When running cell culture supernate samples, use cell culture media (CCM) to produce
the standard dilution series and as the zero standard. Changes in binding have been
associated with running the standards and samples in media.

500 m L 500 m L 500 m L 500 m L 500 m L

34 mL
std.

100,000 3400 1700 850 425 212 106

ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL

4
ASSAY PROCEDURE
The Hydrogen Peroxide Color Reagent must be kept at 2 - 8° C during use. Bring all other
reagents and samples to room temperature before use. It is recommended that all samples and
standards be assayed in duplicate.
1. Prepare all reagents, working standards, and samples as directed in the previous sections.
2. Determine the number of wells to be used. Cover any unused wells tightly with a plate sealer.
DO NOT REUSE WELLS.
3. Add 50 mL of Sample Diluent or cell culture media (CCM) into duplicate Blank (zero standard)
wells.
4. Add 50 mL of each Standard into duplicate wells.
5. Add 50 mL of sample* into duplicate wells.
6. Add 100 mL of Hydrogen Peroxide Color Reagent to all the wells.
7. Mix well by tapping the side of the plate gently for 10 seconds.
8. Incubate for 30 minutes at room temperature.
9. Determine the optical density (OD) of each well using a microplate reader set to 550 nm.

*Serum, plasma, and urine samples require dilution. See Sample Preparation section.

CALCULATION OF RESULTS
Average the duplicate readings for each standard and sample and subtract the average zero standard
optical density.
Create a standard curve by reducing the data using computer software capable of generating a linear
curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each
standard on a y-axis against the concentration on the x-axis and draw the best fit curve through the
points on the graph.
If samples have been diluted, the concentration read from the standard curve must be multiplied by the
dilution factor.

TYPICAL DATA
This standard curve is provided for demonstration only. A standard curve should be generated for each set
of samples assayed.

ng/mL O.D. Average Corrected

0.306
0 0.306 0.306 ___
0.337
106 0.346 0.341 0.035
0.421
212 0.421 0.421 0.115
0.584
425 0.586 0.585 0.279
0.894
850 0.904 0.899 0.593
1.257
1700 1.266 1.262 0.956
1.405
3400 1.419 1.412 1.106

5
PRECISION
Intra-assay Precision (Precision within an assay)
Three samples of known concentration were tested sixteen times on one plate to assess
intra-assay precision.
Inter-assay Precision (Precision between assays)
Three samples of known concentration were tested in eight separate assays to assess
inter-assay precision.

Intra-assay Precision Inter-assay Precision

Sample 1 2 3 1 2 3
n 16 16 16 8 8 8
Mean
314 772 1606 326 768 1733
(pg/mL)
Standard
31 23 64 7.2 13 98
deviation
CV (%) 9.9 3.0 4.0 2.2 1.7 5.6

RECOVERY
The recovery of H2O2 spiked into samples in various matrices was evaluated.
Sample Type Average % Recovery Range
Cell Culture Media (n=4) 106 92.5 - 116%
Human Serum* (n=1) 95 ___

Equine Heparin Plasma* (n=4) 94 88.7 - 99.2%

Human Urine* (n=1) 91 ___

*Samples were diluted prior to assay as directed in Sample Preparation.

6
LINEARITY
To assess the linearity of the assay, Sample Diluent spiked with H 2O2 was assayed using
serial 2-fold dilutions.
Observed Expected % Observed
Dilution (ng/mL) (ng/mL) Expected
Neat 2880 ___ ___
1:2 1340 1440 93
1:4 598 720 83
1:8 296 360 82
1:16 163 180 90
1:32 70 90 78

SENSITIVITY
The sensitivity of the H2O2 assay is typically less than 51.3 ng/mL.
Sensitivity was determined by subtracting two standard deviations to the mean absorbance
value of sixteen zero standard replicates and calculating the corresponding concentration.

REFERENCES
1. Davies, K.J. (1999) IUBMB Life 48(1):41.
2. Zhang, J. et al. (2001) Antioxid. Redox Signal 3(3):493.
3. Li, N. and M. Karin (1999) FASEB 13:1137.
4. Kim, D.K. et al. (2001) J. Cell Sci. 114:4329.
5. Emelyanov, A. et al. (2001) Chest 120:1136.
6. Uesuge, M. et al. (2000) Immunol. 165:6532.
7. Okuda, M. et al. (2001) Artherioscler. Thromb. Vasc. Biol. 21(9):1483.
8. Peiro, C. et al. (2001) Br. J. Pharmacol. 133(7):967.
9. Mody, N. et al. (2001) Free Radic. Biol. Med. 31(4):509.
10. Halliwell, B. (2001) Drugs Aging 18(9):685.
11. Sanji, E. et al. (2001) Biochem. Biophys. Res. Commun. 287(4):1003.
12. Wentworth Jr., P. et al. (2001) Science 293:1806.
13. Wentworth, A.D. et al. (2000) Proc. Natl. Acad. Sci. USA 97(20):10930.

Manufactured for R&D Systems by Assay Designs, Inc.

11.03 751037.0 11/03