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Human8-Hydroxy-desoxyguano
sine（8-OhdG） ELISA Kit
（96T）
? This immunoassay kit allows for the in vitro quantitative determination of human
8-OhdG concentrations in urine, serum, plasma and other biological fluids.
? Expiration date six months from the date of manufacture
? FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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PRINCIPLE OF THE ASSAY
This assay employs the competitive inhibition enzyme
immunoassay technique. An antibody specific to 8-OHdG has
been pre-coated onto a microplate. Standards or samples are
added to the appropriate microtiter plate wells with
HRP-conjugated 8-OHdG and incubated. A competitive inhibition
reaction is launched between 8-OHdG （Standards or samples）
and HRP-conjugated 8-OHdG with the pre-coated antibody
specific for 8-OHdG. The more amount of 8-OHdG in samples,
the less antibody bound by HRP-conjugated 8-OHdG. Then the
substrate solutions are added to the wells, respectively. And the
color develops in opposite to the amount of 8-OHdG in the
sample. The color development is stopped and the intensity of
the color is measured.
DETECTION RANGE
2 ng/ml-800 ng/ml. The standard curve concentrations used for
the ELISA’s were 800 ng/ml, 200 ng/ml, 40 ng/ml, 8 ng/ml, 2
ng/ml.
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SPECIFICITY
This assay recognizes recombinant and natural human 8-OHdG.
No significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of human 8-OHdG is typically less
than 0.8 ng/ml.
The sensitivity of this assay, or Lower Limit of Detection （LLD）
was defined as the lowest protein concentration that could be
differentiated from zero.
MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standard 5 x 0.5ml
HRP-conjugate 1 x 6 ml
Wash Buffer
1 x 15 ml
（20×concentrate）
Substrate A 1 x 7 ml
Substrate B 1 x 7 ml
Stop Solution 1 x 7 ml
Standard S1 S2 S3 S4 S5
Concentration
（ng/ml）
2 8 40 200 800
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STORAGE
1. Unopened test kits should be stored at 2-8?C upon receipt
and the microtiter plate should be kept in a sealed bag with
desiccants to minimize exposure to damp air. The test kit may
be used throughout the expiration date of the kit. Refer to the
package label for the expiration date.
2. Opened test kits will remain stable until the expiring date
shown, provided it is stored as prescribed above.
3. A microtiter plate reader with a bandwidth of 10 nm or less
and an optical density range of 0-3 OD or greater at 450nm
wavelength is acceptable for use in absorbance
measurement.
REAGENT PREPARATION
1. Bring all reagents and plate to room temperature for at least
30 minutes before use. Unused wells need store at 2-8℃and
avoid sunlight.
2. Wash Buffer If crystals have formed in the concentrate,
warm up to room temperature and mix gently until the
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crystals have compley dissolved. Dilute 15 ml of Wash
Buffer Concentrate into deionized or distilled water to
prepare 300 ml of Wash Buffer.
Precaution: The Stop Solution provided with this kit is an acid solution. Wear
eye, hand, face, and clothing protection when using this material.
OTHER SUPPLIES REQUIRED
? Microplate reader capable of measuring absorbance at 450
nm, with the correction wavelength set at 540 nm or 570 nm.
? Pipettes and pipette tips.
? Deionized or distilled water.
? Squirt bottle, manifold dispenser, or automated microplate
washer.
SAMPLE COLLECTION AND STORAGE
? Serum Use a serum separator tube （SST） and allow
samples to clot for 30 minutes before centrifugation for 15
minutes at 1000 g. Remove serum and assay immediay or
aliquot and store samples at -20° C. Avoid repeated
freeze-thaw cycles.
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? Plasma Collect plasma using citrate, EDTA, or heparin as
an anticoagulant. Centrifuge for 15 minutes at 1000 g within
30 minutes of collection. Assay immediay or aliquot and
store samples at -20° C. Avoid repeated freeze-thaw cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay.
ASSAY PROCEDURE
Bring all reagents and samples to room temperature before use. It is
recommended that all samples, standards, and controls be assayed in duplicate.
1. Set a Blank well without any solution. Add 50μl of Standard or
Sample per well. Standard need test in duplicate.
2. Add 50μl of HRP-conjugate to each well （not to Blank well）,
Mix well and then incubate for 1 hour at 37°C.
3. Fill each well with Wash Buffer （about 200μl）, stay for 10
seconds and Spinning. Repeat the process for a total of three
washes. Complete removal of liquid at each step is essential
to good performance. After the last wash, remove any
remaining Wash Buffer by aspirating or decanting. Invert the
plate and blot it against clean paper towels.
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4. Add 50μl of Substrate A and Substrate B to each well, mix
well. Incubate for 15 minutes at 37°C. Keeping the plate away
from drafts and other temperature fluctuations in the dark.
5. Add 50μl of Stop Solution to each well.
6. Determine the optical density of each well within 10 minutes,
using a microplate reader set to 450 nm.
CALCULATION OF RESULTS
Average the duplicate readings for each standard, control, 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 four parameter logistic （4-PL）
curve-fit. As an alternative, construct a standard curve by plotting
the mean absorbance for each standard on the y-axis against
the concentration on the x-axis and draw a best fit curve through
the points on the graph. The data may be linearized by plotting
the log of the 8-OHdG concentrations versus the log of the O.D.
and the best fit line can be determined by regression analysis.
This procedure will produce an adequate but less precise fit of
the data. If samples have been diluted, the concentration read
from the standard curve must be multiplied by the dilution factor.
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LIMITATIONS OF THE PROCEDURE
? The kit should not be used beyond the expiration date on the
kit label.
? Do not mix or substitute reagents with those from other lots or
sources.
? If samples generate values higher than the highest standard,
dilute the samples and repeat the assay.
? Any variation in operator, pipetting technique, washing
technique, incubation time or temperature, and kit age can
cause variation in binding.
TECHNICAL HINTS
? When mixing or reconstituting protein solutions, always avoid
foaming.
? 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.
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? When using an automated plate washer, adding a 30 second
soak period following the addition of wash buffer, and/or
rotating the plate 180 degrees between wash steps may
improve assay precision.
? To ensure accurate results, proper adhesion of plate sealers
during incubation steps is necessary.
? Substrate Solution should remain colorless until added to the
plate. Keep Substrate Solution protected from light. Substrate
Solution should change from colorless to gradations of blue.
? Stop Solution should be added to the plate in the same order
as the Substrate Solution. The color developed in the wells
will turn from blue to yellow upon addition of the Stop Solution.
Wells that are green in color indicate that the Stop Solution
has not mixed thoroughly with the Substrate Solution.
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