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閱讀：267發(fā)布時(shí)間：2013-4-23
Porcine Immunoglobulin M（IgM）
ELISA Kit
Catalog No. CSB-E06805p
（96T）
? This immunoassay kit allows for the in vitro quantitative determination of porcine
IgM concentrations in serum .
? Expiration date six months from the date of manufacture
? FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
1
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with porcine
IgM. Standards or samples are then added to the appropriate microtiter
plate wells with Horseradish Peroxidase （HRP） -conjugated antibody
preparation specific for porcine IgM， mix well and incubated. The more the
amount of porcine IgM in samples, the less HRP-conjugated antibody
preparation specific for porcine IgM bound by pre-coated porcine IgM.
Then a TMB （3,3',5,5' tetramethyl-benzidine） substrate solution is added to
each well. And the color develops in opposite to the amount of porcine IgM
in the sample. The color development is stopped and the intensity of the
color is measured.
DETECTION RANGE
0.28μg/ml-18 μg/ml. The standard curve concentrations used for the
ELISA’s were 18 μg/ml, 9μg/ml, 2.25 μg/ml, 0.56 μg/ml, 0.28 μg/ml.
SPECIFICITY
This assay recognizes porcine IgM. No significant cross-reactivity or
interference was observed.
SENSITIVITY
The minimum detectable dose of porcine IgM is typically less than
0.07μg/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.
2
MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standard 6×0.5ml
Sample Diluent 2 x 20 ml
HRP -conjugate 1 x 6 ml
Wash Buffer
1 x 20 ml
（25×concentrate）
TMB Substrate 1 x 10 ml
Stop Solution 1 x 10 ml
Standard S1 S 2 S3 S4 S5 S6
Concentration
（μg/ml）
0 0.28 0.56 2.25 9 18
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 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.
3
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 compley
dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or
distilled water to prepare 500 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.
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 immediay or aliquot and store samples at -20° C.
Avoid repeated freeze-thaw cycles.
? Recommend to dilute the serum samples with Sample Diluent（1:2000）
before test. The suggested 2000-fold dilution can be achieved by
adding 5μl sample to 195μl of Sample Diluent. Complete the 2000-fold
dilution by adding 5μl of this solution to 245μl of Sample Diluent. The
4
recommended dilution factor is for reference only. The optimal dilution
factor should be determined by users according to their particular
experiments.
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.
1. Add 50μl Standard or Sample to per well. Add 50ul HRP-conjugate to
each well immediay. Mix well with the pipette or shake the plate
gently for 60 seconds.
2. Then incubate for 30 minutes at 37° C.
3. Aspirate each well and wash, Wash by filling each well with Wash
Buffer （200μl） using a squirt bottle, multi-channel pipette, manifold
dispenser or autowasher. Repeating the process for a total of five time
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 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.
5. Add 50μl of Stop Solution to each well when the last four wells
containing the lowest 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 15 minutes, using a
microplate reader set to 450 nm.
5
CALCULATION OF RESULTS
Average the duplicate readings for each standard, control, and sample and
divide the average zero standard optical density. Create a standard curve
by reducing the data using computer software. As an alternative, construct
a standard curve by plotting the absorbance ratio 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
porcine IgM concentrations versus the ratio 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.
? 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 excluded.
6
7
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.
? 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.
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