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Chicken Corticosterone（CORT）
ELISA kit
（96 tests）
This immunoassay kit allows for the in vitro rapid detection of chicken CORT
concentrations in plasma, serum 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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INTRODUCTION
Corticosterone is a steroid hormone secreted by the outer layer,
or cortex, of the adrenal gland . Classed as a glucocorticoid,
corticosterone helps regulate the conversion of amino acids into
carbohydrates and glycogen by the liver, and helps stimulate
glycogen formation in the tissues. Corticosterone is similar in
structure, although somewhat less potent, than the other
glucocorticoids cortisol and cortisone . It is produced in response
to stimulation by the pituitary substance adrenocorticotropic
hormone （ACTH）. In some species, but not in humans,
corticosterone is the predominant glucocorticoid secreted by the
adrenal. It is a precursor in the synthesis of aldosterone , another
adrenal cortical steroid.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with
goat-anti-rabbit antibody. Standards or samples are added to the
appropriate microtiter plate wells with an antibody specific for
CORT and Horseradish Peroxidase （HRP） conjugated CORT,
and incubated. A competitive inhibition reaction is launched
between CORT （Standards or samples） and Horseradish
Peroxidase （HRP） conjugated CORT with the antibody specific
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for CORT. 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 CORT in the samples is then determined by
comparing the O.D. of the samples to the standard curve.
DETECTION RANGE
0.04ng/ml-10ng/ml. The standard curve concentrations used for
the ELISA’s were 10ng/ml, 2.5ng/ml, 0.62ng/ml, 0.16ng/ml
0.04ng/ml.
SPECIFICITY
This assay recognizes CORT. No significant cross-reactivity or
interference was observed.
SENSITIVITY
The minimum detectable dose of chicken CORT is typically less
than 0.025 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 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 S2 S3 S4 S5
Concentration
（ng/ml）
0.04 0.16 0.62 2.5 10
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.
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.
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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 to room temperature before use.
2. Wash Buffer If crystals have formed in the concentrate,
warm up 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.
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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 x g. Remove serum and assay immediay
or aliquot and store samples at -20°C. Centrifuge t he 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 x g within
30 minutes of collection. Assay immediay or aliquot and
store samples at -20° C. Centrifuge the sample agai n 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 without any solution. Add 50μl of Standard or
Sample per well.
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2. Add 50μl of HRP-Conjugate and 50μl of Antibody to each
well. Not to Blank well!
3. Cover with the adhesive strip. Incubate for 1 hour at 37° C.
4. Aspirate each well and wash, repeating the process three
times for a total of three washes. Wash by filling each well
with Wash Buffer （200μl） using a squirt bottle, multi-channel
pipette, manifold dispenser or autowasher. 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.
5. Add 50μl of Substrate A and 50μl of Substrate B to each
well. Incubate for 15 minutes at 37°C. Keeping the plate away
from drafts and other temperature fluctuations in the dark.
6. 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.
7. Determine the optical density of each well within 30 minutes,
using a microplate reader set to 450 nm.
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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 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 CORT 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.
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If samples generate values higher than the highest standard,
dilute the samples with the appropriate Diluent and repeat the
assay.
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.
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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