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Rat Asymmetrical
Dimethylarginine（ADMA）
ELISA Kit
（96T）
This immunoassay kit allows for the in vitro quantitative determination of rat
ADMA concentrations in serum, plasma.
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
The microtiter plate provided in this kit has been pre-coated with
a goat-anti-rabbit antibody. Standards or samples are then added
to the appropriate microtiter plate wells with a HRP-conjugated
ADMA and antibody preparation specific for ADMA and
incubated. Then substrate solutions are added to each well. The
enzyme-substrate reaction is terminated by the addition of a
sulphuric acid solution and the color change is measured
spectrophotometrically at a wavelength of 450 nm ± 2 nm. The
concentration of ADMA in the samples is then determined by
comparing the O.D. of the samples to the standard curve.
DETECTION RANGE
1.2 ng/ml-300 ng/ml. The standard curve concentrations used for
the ELISA’s were 300 ng/ml, 75 ng/ml, 18.75 ng/ml, 4.69 ng/ml,
1.2 ng/ml.
SPECIFICITY
This assay recognizes recombinant and natural rat ADMA. No
significant cross-reactivity or interference was observed.
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SENSITIVITY
The minimum detectable dose of rat ADMA is typically less than
0.75 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
Standards （S1-S5） 5 x 0.5 ml
HRP-conjugate 1 x 6 ml
Antibody 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 S 2 S3 S4 S5
Concentration
（ng/ml） 1.2 4.69 18.75 75 300
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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. The
test kit may be used throughout the expiration date of the kit,
provided it is stored as prescribed above. Refer to the
package label for the expiration date.
2. Opened test plate should be stored at 2-8°C in the aluminum
foil bag with desiccants to minimize exposure to damp air. The
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.
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2. Wash Buffer If crystals have formed in the concentrate,
warm to room temperature and mix gently until the 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.
An incubator which can provide stable incubation conditions
up to 37°C±0.5°C.
SAMPLE COLLECTION AND STORAGE
Serum Use a serum separator tube （SST） and allow
samples to clot for 30 minutes before centrifugation for 15
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minutes at 1000 g. Remove serum and assay immediay or
aliquot and store samples at -20°C. Centrifuge the sample
again after thawing before the assay. Avoid repeated
freeze-thaw cycles.
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. Centrifuge the sample again after
thawing before the assay. 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.
All the reagents should be added directly to the liquid level in the well. The
pipette should avoid contacting the inner wall of the well.
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）,
then 50μl Antibody to each well. Mix well and then incubate
for 1 hour at 37°C.
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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.
4. Add 50μl of Substrate A and 50μl 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. If color change does
not appear uniform, gently tap the plate to ensure thorough
mixing.
6. Determine the optical density of each well within 10 minutes,
using a microplate reader set to 450 nm.
CALCULATION OF RESULTS
Using the professional soft "Curve Exert 1.3" to make a standard curve is
recommended, which can be downloaded from our web.
Average the duplicate readings for each standard, Blank, and
sample and subtract the optical density of Blank. Create a
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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 ADMA 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.
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 with the appropriate Diluent and repeat the
assay.
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Any variation in operator, pipetting technique, washing
technique, incubation time or temperature, and kit age can
cause variation in binding.
This assay is designed to eliminate interference by soluble
receptors, binding proteins, and other factors present in
biological samples. Until all factors have been tested in the
Quantikine Immunoassay, the possibility of interference
cannot be excluded.
TECHNICAL HINTS
Centrifuge vials before opening to collect contents.
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.
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. Sealers can not be
reused.
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Substrate Solution should remain colorless or light blue until
added to the plate. Keep Substrate Solution protected from
light. Substrate Solution should change from colorless or light
blue 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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