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Human Adiponectin(ADP)
ELISA Kit
Catalog No. CSB-E07270h
(96 T)
? This immunoassay kit allows for the in vitro quantitative determination of human
ADP 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
Adiponectin,alternatively named Adipocyte Complement-Related
Protein of 30 kDa (Acrp30), shares structural similarity with
complement factor C1q and is a member of the family of defense
collagens. It is secreted exclusively by differentiated adipocytes
and circulates at high concentrations. Adiponectin has a modular
structure comprising an N-terminal collagenous domain with
multiple collagen triple helix repeats, followed by a C-terminal
C1q-like globular domain. The globular domain has similar folding
topology with tumor necrosis factor-α and assembles into
homotrimers. Higher order oligomeric adiponectins (hexamers and
higher molecular weight forms) are also formed via interactions
between the collagenous stalk. A truncated form of Adiponectin
containing only the globular domain (gAdiponectin or gAcrp30) can
be generated by proteolytic cleavage. The gAdiponectin as well as
all oligomeric forms of the full length Adiponectin are detected in
serum. Different isoforms of Adiponectin have been shown to
activate different signal transduction pathways. Conflicting
biological activities have been reported for the various isoforms.
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Two seven membrane-spanning Adiponectin receptors, designated
AdipoR1 and AdipoR2 have been identified. AdipoR1 is expressed
predominantly in muscle and functions as a high-affinity receptor
for gAdiponectin, but a very low-affinity receptor for the full length
Adiponectin. AdipoR2 binds both the full length and globulin
domain with intermediate affinity and is expressed primarily in liver.
Adiponectin is an anti-diabetic and anti-atherogenic hormone that
plays important roles in the regulation of lipid and glucose
metabolism.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an
antibody specific to ADP. Standards or samples are then added to
the appropriate microtiter plate wells with a biotin-conjugated
antibody preparation specific for ADP and Avidin conjugated to
Horseradish Peroxidase (HRP) is added to each microplate well
and incubated. Then a TMB (3,3’,5,5’ tetramethyl-benzidine)
substrate solution is added to each well. Only those wells that
contain ADP, biotin-conjugated antibody and enzyme-conjugated
Avidin will exhibit a change in color. The enzyme-substrate reaction
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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 ADP in the samples is then
determined by comparing the O.D. of the samples to the standard
curve.
DETECTION RANGE
0.47ng/ml-30 ng/ml. The standard curve concentrations used for
the ELISA’s were 30 ng/ml, 15 ng/ml, 7.5 ng/ml, 3.75 ng/ml,
1.88ng/ml, 0.94ng/ml, 0.47 ng/ml.
SPECIFICITY
This assay recognizes recombinant and natural human ADP. No
significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of human ADP is typically less than
0.12 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 2
Sample Diluent 1 x 20 ml
Biotin-antibody Diluent 1 x 10 ml
HRP-avidin Diluent 1 x 10 ml
Biotin-antibody 1 x 120μl
HRP-avidin 1 x 120μl
Wash Buffer
1 x 20 ml
(25×concentrate)
TMB Substrate 1 x 10 ml
Stop Solution 1 x 10 ml
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.
REAGENT PREPARATION
Bring all reagents to room temperature before use.
1. Wash Buffer If crystals have formed in the concentrate, warm
up to room temperature and mix gently until the crystals have
completely dissolved. Dilute 20 ml of Wash Buffer Concentrate
into deionized or distilled water to prepare 500 ml of Wash
Buffer.
2. Biotin-antibody Centrifuge the vial before opening. Dilute to
the working concentration using Biotin-antibody
Diluent(1:100), respectively.
3. HRP-avidin Centrifuge the vial before opening. Dilute to the
working concentration using HRP-avidin Diluent(1:100),
respectively.
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4. Standard Centrifuge the standard vial at 6000-10000rpm for
30s. Reconstitute the Standard with 1.0 ml of Sample Diluent.
This reconstitution produces a stock solution of 30 ng/ml. Allow
the standard to sit for a minimum of 15 minutes with gentle
agitation prior to making serial dilutions. The undiluted standard
serves as the high standard (30 ng/ml). The Sample Diluent
serves as the zero standard (0 ng/ml). Prepare fresh for each
assay. Use within 4 hours and discard after use.
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.
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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 g. Remove serum and assay immediately 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 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. Add 100μl of Standard, Blank, or Sample per well. Cover with
the adhesive strip. Incubate for 2 hours at 37°C.
2. Remove the liquid of each well, don’t wash.
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3. Add 100μl of Biotin-antibody working solution to each well.
Incubate for 1 hour at 37°C. Biotin-antibody working solution
may appear cloudy. Warm up to room temperature and mix
gently until solution appears uniform.
4. Aspirate each well and wash, repeating the process three times
for a total of three washes. Wash: Fill each well with Wash
Buffer (200μl) and let it stand for 2 minutes, then remove the
liquid by flicking the plate over a sink. The remaining drops are
removed by patting the plate on a paper towel. Complete
removal of liquid at each step is essential to good performance.
5. Add 100μl of HRP-avidin working solution to each well. Cover
the microtiter plate with a new adhesive strip. Incubate for 1
hour at 37°C.
6. Repeat the aspiration and wash five times as step 4.
7. Add 90μl of TMB Substrate to each well. Incubate for 10-30
minutes at 37°C. Keeping the plate away from drafts and other
temperature fluctuations in the dark.
8. 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.
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9. 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.
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 ADP 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.
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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 Standard 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 Standard 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
Quantikine Immunoassay, the possibility of interference cannot
be excluded.
TECHNICAL HINTS
? Centrifuge vials before opening to collect contents.
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? 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.
? 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.
