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Human Nitric Oxide（NO）
ELISA Kit
（96T）
? This immunoassay kit allows for the in vitro quantitative determination of human
NO 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 an
antibody specific to NO. Standards or samples are then added to the
appropriate microtiter plate wells with a biotin-conjugated polyclonal
antibody preparation specific for NO and Avidin conjugated to Horseradish
Peroxidase （HRP） is added to each microplate well and incubated. Then a
TMB （3,3',5,5' tetramethyl-benzidine） substrate solution is added to each
well. Only those wells that contain NO, biotin-conjugated antibody and
enzyme-conjugated Avidin will exhibit a change in color. 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 NO in the samples is
then determined by comparing the O.D. of the samples to the standard
curve.
DETECTION RANGE
0.32 ng/ml -20 ng/ml. The standard curve concentrations used for the
ELISA’s were 20 ng/ml, 10 ng/ml, 5 ng/ml, 2.5 ng/ml, 1.25 ng/ml, 0.62 ng/ml,
0.32 ng/ml.
SPECIFICITY
This assay recognizes human NO. No significant cross-reactivity or
interference was observed.
SENSITIVITY
The minimum detectable dose of human NO is typically less than 0.08
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.
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MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standard 2
Sample Diluent 2 x 20 ml
Biotin-antibody Diluent 1 x 10 ml
HRP-avidin Diluent 1 x 10 ml
Biotin-antibody 1 x 120μl
HRP-avidin 1 x 120μl
Wash Buffer
1 x 20 ml
（25×concentrate）
TMB Substrate 1 x 10 ml
Stop Solution 1 x 10 ml
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.
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REAGENT PREPARATION
Bring all reagents to room temperature before use.
1. Wash Buffer If crystals have formed in the concentrate, warm up to
room temperature and mix gently until the crystals have compley
dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or
distilled water to prepare 500 ml of Wash Buffer.
2. Standard Centrifuge the standard vial at 6000-10000rpm for 30s.
Reconstitute the Standard with 1.0 ml of Sample Diluent. This
reconstitution produces a stock solution of 20ng/ml. Allow the standard
to sit for a minimum of 15 minutes with gentle agitation prior to making
serial dilutions. The undiluted standard serves as the high standard
（20ng/ml）. The Sample Diluent serves as the zero standard （0 ng/ml）.
Prepare fresh for each assay. Use within 4 hours and discard after use.
3. Biotin-antibody Centrifuge the vial before opening. Dilute to the
working concentration using Biotin-antibody Diluent（1:100）,
respectively.
4. HRP-avidin Centrifuge the vial before opening. Dilute to the working
concentration using HRP-avidin Diluent（1:100）, respectively.
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.
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? 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 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. Recommend to dilute the serum or plasma samples with Sample
Diluent（1:200） before test. The suggested 200-fold dilution can be
achieved by adding 5μl sample to 95μl of Sample Diluent. Complete the
200-fold dilution by adding 25μl of this solution to 225μl of Sample
Diluent. The recommended dilution factor is for reference only. The
optimal dilution factor should be determined by users according to their
particular experiments.
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2. Add 100μl of Standard, Blank, or Sample per well. Cover with the
adhesive strip. Incubate for 2 hours at 37°C.
3. Remove the liquid of each well, don’t wash.
4. Add 100μl of Biotin-antibody working solution to each well. Incubate
for 1 hour at 37°C. Biotin-antibody working solution may appear
cloudy. Warm up to room temperature and mix gently until solution
appears uniform.
5. Aspirate each well and wash, repeating the process three times for a
total of three washes. Wash: Fill each well with Wash Buffer （200μl） and
let it stand for 2 minutes, then remove the liquid by flicking the plate
over a sink. The remaining drops are removed by patting the plate on a
paper towel. Complete removal of liquid at each step is essential to
good performance.
6. Add 100μl of HRP-avidin working solution to each well. Cover the
microtiter plate with a new adhesive strip. Incubate for 1 hour at 37°C.
7. Repeat the aspiration and wash five times as step 5.
8. Add 90μl of TMB Substrate to each well. Incubate for 10-30 minutes at
37°C. Keeping the plate away from drafts and other temperature
fluctuations in the dark.
9. Add 50μl of Stop Solution to each well when the first four wells
containing the highest concentration of standards develop obvious blue
color. If color change does not appear uniform, gently tap the plate to
ensure thorough mixing.
10. Determine the optical density of each well within 30 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.
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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 x-axis
against the concentration on the y-axis and draw a best fit curve through the
points on the graph. The data may be linearized by plotting the log of the
NO 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.
? It is important that the Standard Diluent selected for the standard curve
be consistent with the samples being assayed.
? If samples generate values higher than the highest standard, dilute the
samples with the appropriate Standard Diluent and repeat the assay.
? Any variation in Standard Diluent, 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 Immunoassay, the possibility of
interference cannot be excluded.
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TECHNICAL HINTS
? Centrifuge vials before opening to collect contents.
? 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.
? 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 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.