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Rat Noradrenaline(NA)
ELISA Kit
Catalog No. CSB-E07022r
(96 T)
? This immunoassay kit allows for the in vitro quantitative determination of rat NA
concentrations in serum, plasma.
? Expiration date six months from the date of manufacture
? FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an
antibody specific to NA. Standards or samples are then added to
the appropriate microtiter plate wells with a biotin-conjugated
antibody preparation specific for NA 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 NA, 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 NA in the samples is then
determined by comparing the O.D. of the samples to the standard
curve.
DETECTION RANGE
4.69 pg/ml-300 pg/ml. The standard curve concentrations used for
the ELISA’s were , 300 pg/ml, 150 pg/ml, 75 pg/ml, 37.5 pg/ml,
18.75 pg/ml, 9.38 pg/ml, 4.69 pg/ml.
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SPECIFICITY
This assay recognizes recombinant and natural rat NA. No
significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of rat NA is typically less than 1.2
pg/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 2
Sample Diluent 1 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
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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.
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REAGENT PREPARATION
Bring all reagents to room temperature before use for 30min.
1. Wash Buffer If crystals have formed in the concentrate, warm
up to room temperature and mix gently until the crystals have
completely dissolved. Dilute 20 ml of Wash Buffer Concentrate
into deionized or distilled water to prepare 500 ml of Wash
Buffer.
2. Biotin-antibody Centrifuge the vial before opening. Dilute to
the working concentration using Biotin-antibody
Diluent(1:100), respectively. The suggested 100-fold dilution
can be achieved by adding 10 uL sample to 990uL of
Biotin-antibody Diluent for 1ml working solution.
3. HRP-avidin Centrifuge the vial before opening. Dilute to the
working concentration using HRP-avidin Diluent(1:100),
respectively. The suggested 100-fold dilution can be achieved
by adding 10 uL sample to 990uL of HRP-avidin Diluent for
1ml working solution.
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4. 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 300 pg/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 (300 pg/ml). The Sample
Diluent serves as the zero standard (0 pg/ml). Prepare fresh for
each assay. Use within 4 hours and discard after use.
300
pg/ml
150
pg/ml
75
pg/ml
37.5
pg/ml
18.75
pg/ml
9.38
pg/ml
4.69
pg/ml
Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye,
hand, face, and clothing protection when using this material.
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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 minutes at
1000 g. Remove serum and assay immediately 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 immediately 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.
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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. Add 100μl of Standard, Blank, or Sample per well. Cover with
the adhesive strip. Incubate for 2 hours at 37°C.
2. Remove the liquid of each well, don’t wash.
3. 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.
4. 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.
5. 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.
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6. Repeat the aspiration and wash five times as step 4.
7. 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.
8. 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.
9. 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.
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
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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 NA 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.
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? 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.
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.
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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.
PRECISION
Intra-assay Precision (Precision within an assay)
One sample whose concentration between the highest and the
second standard were tested twenty times on one plate to assess.
CV%<8%
Inter-assay Precision (Precision between assays)
One sample whose concentration between the highest and the
second standard were tested in thirty-five separate assays to
assess
CV%<10%
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RECOVERY
The recovery of NA spiked to three levels throughout the range of
the assay in the serum, EDTA plasma and tissue samples was
evaluated.
Sample Type Average % Recovery Range
Rat serum(n=6) 95 88 - 106%
Rat EDTA
plasma(n=6)
94 86 - 102%
*Samples were spiked then diluted prior to assay as directed in
Sample Preparation.
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PLATE LAYOUT
Use this plate layout as a record of standards and samples
assayed.
