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人神经特异性烯醇化酶(NSE)ELISA Kit Human Neuron-Specific Enolase
(NSE)ELISA Kit
(96T)
? This immunoassay kit allows for the in vitro quantitative determination of human NSE
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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INTRODUCTION
Neuron Specific Enolase (NSE) is the most abundant form of the
glycolytic enolase found in adult neurons and is thought to serve as
a growth factor in neurons. Of the three enolase subunits (α, β, and
γ), NSE is a dimer composed of two γ subunits.
NSE is useful in studying neuronal differentiation and is, therefore,
a valuable tool for visualizing the entire neuron and endocrine
systems. Serum levels of NSE have been associated with such
disease states as Alzheimer’s, Huntington’s Chorea,
neuroblastoma, head trauma, neuroendocrine malignancies, and
small cell carcinomas of the lung.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an
antibody specific to NSE. Standards or samples are then added to
the appropriate microtiter plate wells with a HRP-conjugated
antibody preparation specific for NSE 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 NSE,
HRP-conjugated antibody will exhibit a change in color. The
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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 NSE in the samples is then determined by
comparing the O.D. of the samples to the standard curve.
DETECTION RANGE
1.56 ng/ml-100 ng/ml. The standard curve concentrations used for
the ELISA’s were 100 ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml, 6.25
ng/ml, 3.12 ng/ml, 1.56 ng/ml.
SPECIFICITY
This assay recognizes human NSE. No significant cross-reactivity
or interference was observed.
SENSITIVITY
The minimum detectable dose of human NSE is typically less than
0.39 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 2 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. 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.
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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. Standard Reconstitute the Standard with 1.0 ml of Sample
Diluent. This reconstitution produces a stock solution of 100
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 (100 ng/ml). The Sample
Diluent serves as the zero standard (0 ng/ml).
3. HRP-conjugate Dilute to the working concentration using
HRP-conjugate Diluent(1:100), respectively.
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.
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? 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.
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 100μl of Standard or
Sample per well. Cover with the adhesive strip. Incubate for
30min at 37°C.
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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 30min 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. 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.
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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 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 NSE 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.