Porcine Cortisol ELISA Kit
Catalog No. CSB-E06811p
（96T）
This immunoassay kit allows for the in vitro quantitative determination of porcine
Cortisol 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 Cortisol and
antibody preparation specific for Cortisol 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 Cortisol in the samples is then
determined by comparing the O.D. of the samples to the standard
curve.
DETECTION RANGE
0.31 ng/ml-80 ng/ml. The standard curve concentrations used for the
ELISA’s were 80 ng/ml, 20 ng/ml, 5 ng/ml, 1.25 ng/ml, 0.31 ng/ml.
SPECIFICITY
This assay recognizes recombinant and natural porcine Cortisol. No
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significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of porcine Cortisol is typically less than
0.2 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
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Standard S1 S 2 S3 S4 S5
Concentration
（ng/ml）
0.31 1.25 5 20 80
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 ℃
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sunlight.
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 minutes at 1000 g.
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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 standard curve by
reducing the data using computer software capable of generating a four
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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 Cortisol 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.
Any variation in 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 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.
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
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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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