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Human High Sensitivity
C-Reactive Protein(hs-CRP)
ELISA Kit
(96 T)
? This immunoassay kit allows for the in vitro quantitative determination of human
CRP 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
C-reactive protein (CRP) is a protein found in the blood in response to
inflammation (an acute-phase protein). CRP is synthesized by the liver in
response to factors released by fat cells (adipocytes). It is a member of the
pentraxin family of proteins. CRP is a member of the class of acute-phase
reactants as its levels rise dramatically during inflammatory processes
occurring in the body. This increment is due to a rise in the plasma
concentration of IL-6, which is produced predominantly by macrophages
as well as adipocytes. CRP binds to phosphocholine on microbes. It is
thought to assist in complement binding to foreign and damaged cells and
enhances phagocytosis by macrophages, which express a receptor for
CRP. It is also believed to play another important role in innate immunity, as
an early defense system against infections. CRP rises up to 50,000-fold in
acute inflammation, such as infection. It rises above normal limits within 6
hours, and peaks at 48 hours. Its half-life is constant, and therefore its
level is mainly determined by the rate of production (and hence the severity
of the precipitating cause). Serum amyloid A is a related acute-phase
marker that responds rapidly in similar circumstances. To measure the CRP
level, a "high-sensitivity" CRP or hs-CRP test needs to be performed and
analyzed by a laboratory. This is an automated blood test designed for
greater accuracy in measuring low levels of CRP, which allows the
physician to assess cardiovascular risk.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an
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antibody specific to CRP. Standards or samples are then added to the
appropriate microtiter plate wells with a HRP-conjugated antibody
preparation specific for CRP 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 CRP, HRP-conjugated antibody will
exhibit a change in color. 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 CRP in the samples is then determined by comparing the
O.D. of the samples to the standard curve.
DETECTION RANGE
0.625 ng/ml-40 ng/ml. The standard curve concentrations used for the
ELISA’s were 40 ng/ml, 20 ng/ml, 10 ng/ml,5 ng/ml, 2.5 ng/ml, 1.25 ng/ml,
0.625ng/ml.
SPECIFICITY
This assay recognizes recombinant and natural human CRP. No significant
cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of human CRP is typically less than 0.156
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 4 x 20 ml
HRP-conjugate Diluent 1 x 10 ml
HRP-conjugate 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 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. 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 for 30min.
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.
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2. 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 40 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 (40
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.
3. HRP-conjugate Dilute to the working concentration using
HRP-conjugate Diluent(1:100), respectively. The suggested 100-fold
dilution can be achieved by adding 10 uL HRP-conjugate to 990uL of
HRP-conjugate Diluent for 1ml working solution.
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 immediately or aliquot and store samples at -20° C.
Avoid repeated freeze-thaw cycles.
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? 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.
Avoid repeated freeze-thaw cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay.
If samples generate values higher than the highest standard, dilute the samples
with the Sample Diluent and repeat the assay.
SAMPLE PREPARTION
Normal human serum samples require a 1000-fold dilution into Sample
Diluent. The suggested 1000-fold dilution can be achieved by adding 5μl
sample to 95μl of Sample Diluent. Complete the 1000-fold dilution by
adding 5μl of this solution to 245μl of Sample Diluent. The recommended
dilution factor is for reference only. The optimal dilution factor should be
determined by users according to their particular experiments.
As CRP levels increase widely, the patient serum samples are
recommended to dilute 1:1000-1:30000 times before test. The
recommended dilution factor is for reference only. The optimal dilution
factor should be determined by users according to their particular
experiments.
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.
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1. Add 100μl Sample Diluent serves as the zero standard, Add 100μl of
Standard or Sample per well. Cover with the adhesive strip. Incubate for
60min at 37°C.
2. 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.
3. Add 100μl of HRP-conjugate working solution to each well. Incubate
for 60min at 37°C. HRP-conjugate working solution may appear cloudy.
Warm up to room temperature and mix gently until solution appears
uniform.
4. Repeat the aspiration and wash five times as before.
5. Add 90μl of TMB Substrate to each well. Incubate for 20 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.
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
CRP 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.
? 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 Immunoassay, the possibility of
interference cannot be excluded.
TECHNICAL HINTS
? When mixing or reconstituting protein solutions, always avoid foaming.
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? 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 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.