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人胰高血糖素样肽1(GLP-1)ELISA Kit Human glucagon-like peptide-1
(GLP-1)ELISA Kit
(96T)
? This immunoassay kit allows for the in vitro quantitative determination of human
GLP-1 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
antibody specific to GLP-1. Standards or samples are then added
to the appropriate microtiter plate wells with a Horseradish
Peroxidase (HRP)-conjugated antibody preparation specific for
GLP-1 and incubated. Then substrate solution A and B are added
to each well. Only those wells that contain GLP-1, 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 GLP-1 in the
samples is then determined by comparing the O.D. of the samples
to the standard curve.
DETECTION RANGE
2.29ng/ml-40ng/ml. The standard curve concentrations used for
the ELISA’s were 40ng/ml, 22.86ng/ml, 9.14ng/ml, 4.57ng/ml,
2.29ng/ml
SPECIFICITY
This assay recognizes human GLP-1. No significant
cross-reactivity or interference was observed.
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SENSITIVITY
The minimum detectable dose of human GLP-1 is typically less
than 1.45ng/ml.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was
defined as the lowest concentration that could be differentiated
from zero.
MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standard(S0-S5) 6
HRP-conjugate 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 S0 S1 S2 S3 S4 S5
Concentration
(ng/ml)
0 2.29 4.57 9.14 22.86 40
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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 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.
REAGENT PREPARATION
1. Bring all reagents and plate to room temperature for at least 30
minutes before use. Unused wells need store at 2-8°C 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.
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 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. Reconstitute the every Standard（S1-S5） with 0.5 ml of
distilled water.
2. Set a Blank well without any solution. Add 50μl of Standard or
Sample per well. Standard need test in duplicate.
3. Add 50μl of HRP-conjugate to each well (not to Blank well).
Mix well and then incubate for 2 hour at 37°C.
4. Complete remove the liquid. Then 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.
5. Add 50μl of Substrate A and 50μl of 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.
6. Add 50μl of Stop Solution to each well. If color change does
not appear uniform, gently tap the plate to ensure thorough
mixing.
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7. Determine the optical density of each well within 10 minutes,
using a microplate reader set to 450 nm.
CALCULATION OF RESULTS
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 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 GLP-1 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.
TECHNICAL HINTS
? 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.
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? 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.