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廈門慧嘉生物科技有限公司
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Porcine Vascuolar cell adhesion
molecule 1,VCAM-1 ELISA Kit
Catalog No. CSB-E06825p
（96 tests）
This immunoassay kit allows for the in vitro rapid detection of porcine VCAM-1
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.
2
PRINCIPLE OF THE ASSAY
This assay employs the competitive inhibition enzyme
immunoassay technique. A antibody specific to VCAM-1/CD106
has been pre-coated onto a microplate. Standards or samples
are added to the appropriate microtiter plate wells with
biotin-conjugated VCAM-1/CD106 and incubated. A competitive
inhibition reaction is launched between VCAM-1/CD106
（Standards or samples） and Biotin-conjugated VCAM-1/CD106
with the pre-coated antibody specific for VCAM-1/CD106. The
more amount of VCAM-1/CD106 in samples, the less antibody
bound by Biotin-conjugated VCAM-1/CD106. Then Avidin
conjugated to Horseradish Peroxidase （HRP） is added to each
microplate well and incubated. The substrate solutions are
added to the wells, respectively. And the color develops in
opposite to the amount of VCAM-1/CD106 in the sample. The
color development is stopped and the intensity of the color is
measured.
DETECTION RANGE
The standard curve concentrations used for the ELISA’s were
3
8000 ng/ml, 4000 ng/ml, 2000 ng/ml, 1000 ng/ml, 500 ng/ml.
SPECIFICITY
This assay recognizes VCAM-1/CD106. No significant
cross-reactivity or interference was observed.
MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standards （S1-S5） 5
HRP-avidin
Conjugate
1 x 6 ml
1 x 6 ml
Wash Buffer
1 x 15 ml
（20×concentrate）
Substrate A 1 x 6 ml
Substrate B 1 x 6 ml
Stop Solution 1 x 6 ml
Standard S1 S2 S3 S4 S5
Concentration（ng/ml） 500 1000 2000 4000 8000
STORAGE
4
1. Unopened test kits should be stored at 2-8°C upon receipt
and the microtiter plate should be kept in a sealed bag with
desiccants to minimize exposure to damp air. 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.
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.
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. 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.
3. To avoid cross-contamination, change pipette tips between
5
additions of each standard level, between sample additions,
and between reagent additions. Also, use separate reservoirs
for each reagent.
4. 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.
5. 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.
6. 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.
6
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.
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. Avoid repea ted
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. Avoid repeated freeze-thaw cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay.
7
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.
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 Conjugate to each well （not to Blank well）, Mix
well and then incubate for 1 hour at 37°C.
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 HRP-avidin to each well. Incubate for 30mins
at 37°C.
5. Repeat the aspiration and wash five times as step 4.
6. 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.
8
7. Add 50µl of Stop Solution to each well.
8. 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 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 VCAM-1/CD106 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
9
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 Calibrator 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 Calibrator 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
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