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猪C肽(C-Peptide)ELISA Kit Porcine C-Peptide ELISA Kit
(96T)
? This immunoassay kit allows for the in vitro quantitative determination of Porcine
C-Peptide concentrations in serum, plasma.
? Expiration date six months from the date of manufacture
? FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with
an goat-anti-rabbit antibody. Standards or samples are then
added to the appropriate microtiter plate wells with a
HRP-conjugated C-Peptide and antibody preparation specific for
C-Peptide 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 C-Peptide in the samples is then
determined by comparing the O.D. of the samples to the
standard curve.
DETECTION RANGE
0.04 ng/ml-10 ng/ml. The standard curve concentrations used for
the ELISA’s were 10 ng/ml, 2.5 ng/ml, 0.625 ng/ml, 0.155 ng/ml,
0.04 ng/ml.
SPECIFICITY
This assay recognizes Porcine C-Peptide. No significant
cross-reactivity or interference was observed.
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SENSITIVITY
The minimum detectable dose of Porcine C-Peptide is typically
less than 0.025 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.
MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standards (S1-S5) 5 x 0.5 ml
HRP-conjugate 1 x 6 ml
Antibody 1 x 6 ml
Wash Buffer
1 x 15 ml
(20×concentrate)
Substrate A 1 x 7 ml
Substrate B 1 x 7 ml
Stop Solution 1 x 7 ml
Standard S 1 S 2 S 3 S 4 S 5
Concentration
(ng/ml)
0.04 0.155 0.625 2.5 10
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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.
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 completely dissolved. Dilute 15 ml of Wash Buffer
Concentrate into deionized or distilled water to prepare 300 ml
of Wash Buffer.
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3. 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.
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. To ensure accurate results, proper adhesion of plate sealers
during incubation steps is necessary. Sealers can not be
reused.
6. 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.
7. 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.
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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.
? 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. Avoid repeated freeze-thaw cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay.
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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. Standards need test in duplicate.
2. Add 50μl of HRP-conjugate to each well (not to Blank well),
then add 50μl Antibody to each 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 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.
5. Add 50μl of Stop Solution to each well. If color change does
not appear uniform, gently tap the plate to ensure thorough
mixing.
6. Determine the optical density of each well within 10 minutes,
using a microplate reader set to 450 nm.
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CALCULATION OF RESULTS
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 x-axis against the
concentration on the y-axis and draw a best fit curve through the
points on the graph. The data may be linearized by plotting the
log of the C-Peptide 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.
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? 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.