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廈門慧嘉生物科技有限公司
廈門慧嘉生物科技有限公司
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閱讀：191發布時間：2013-4-19
Chicken Interleukin 17（IL-17）
ELISA Kit
Catalog No. CSB-E04607Ch
（96T）
This immunoassay kit allows for the in vitro quantitative determination of chicken
IL-17 concentrations in 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
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 IL-17
and antibody preparation specific for IL-17 and incubated. Then
substrate solution is 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 IL-17 in the
samples is then determined by comparing the O.D. of the samples
to the standard curve.
DETECTION RANGE
120 pg/ml-6000 pg/ml. The standard curve concentrations used for
the ELISA’s were 6000 pg/ml, 3000 pg/ml, 1200 pg/ml, 360 pg/ml,
120 pg/ml.
SPECIFICITY
This assay recognizes recombinant and natural chicken IL-17. No
significant cross-reactivity or interference was observed.
3
SENSITIVITY
The minimum detectable dose of chicken IL-17 is typically less than
50 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（96T）
Standards （S1-S5） 5
HRP-conjugate 1 x 6ml
Antibody 1 x 6 ml
Wash Buffer
1 x 15 ml
（20×concentrate）
Substrate A 1 x 7ml
Substrate B 1 x 7 ml
Stop Solution 1 x 7 ml
Standard S1 S2 S3 S4 S5
Concentration
（pg/ml）
120 360 1200 3000 6000
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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.
1. 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.
TECHNICAL HINTS
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. Centrifuge vials before opening to collect contents.
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3. 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.
4. 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.
5. 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.
6. To ensure accurate results, proper adhesion of plate sealers
during incubation steps is necessary. Sealers can not be
reused.
7. 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.
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8. 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.
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
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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. 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
2 hours 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 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 standard
9
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 IL-17 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.
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