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Human Connective Tissue
Growth Factor (CTGF)
Elisa Kit
This immunoassay kit allows for the in vitro quantitative determination of human
CTGF 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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INTRODUCTION
CTGF (connective tissue growth factor) is a cysteine-rich,
matrix-associated, heparin-binding protein. In vitro, CTGF
mirrors some of the effects of TGF beta on skin fibroblasts, such
as stimulation of extracellular matrix production, chemotaxis,
proliferation and integrin expression. CTGF can promote
endothelial cell growth, migration, adhesion and survival and is
thus implicated in endothelial cell function and angiogenesis.
CTGF gene expression had been demonstrated in human
trabecular meshwork cells, ciliary body, retinal vascular
endothelial cells, proliferative vitreoretinopathy membranes,
choroidal neovascular membranes, cataractous plaque, corneal
scars, tear fluid, and pterygia.
CTGF binds to perlecan, a proteoglycan which has been
localised in synovium, cartilage and numerous other tissues.
CTGF has been implicated in extracellular matrix remodelling in
wound healing, scleroderma and other fibrotic processes, as it is
capable of upregulating both matrix metalloproteinases (MMPs)
and their inhibitors (TIMPs). Therefore, CTGF has the potential
to activate both the synthesis and degradation of the
extracellular matrix.
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PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with
an antibody specific to CTGF. Standards or samples are then
added to the appropriate microtiter plate wells with a Horseradish
Peroxidase (HRP) -conjugated antibody preparation specific for
CTGF 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 CTGF in the samples is then
determined by comparing the O.D. of the samples to the
standard curve.
DETECTION RANGE
3.75 pg/ml-120 pg/ml. The standard curve concentrations used
for the ELISA’s were 120 pg/ml, 60 pg/ml, 26.25 pg/ml, 11.25
pg/ml, 3.75 pg/ml.
SPECIFICITY
This assay recognizes human CTGF. No significant
cross-reactivity or interference was observed.
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SENSITIVITY
The minimum detectable dose of human CTGF is typically less
than 1.87 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 5 x 0.5 ml
HRP-conjugate 1 x 6 ml
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
(pg/ml)
3.75 11.25 26.25 60 120
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.
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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.
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
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minutes at 1000 x g. Remove serum and assay immediately
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 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.
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.
2. Add 50µl of HRP-Conjugate to each well (Not to Blank!).
Incubate for 1 hour at 37°C.
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3. Aspirate each well and wash, repeating the process three
times for a total of three washes. Wash by filling each well
with ddH2O (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
solution 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.
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 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.
6. 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.
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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 CTGF 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 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.
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.
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To ensure accurate results, proper adhesion of plate sealers
during incubation steps is necessary.
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.
