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（leptin)小鼠瘦素（leptin)Elisa试剂盒
厦门慧嘉生物经营ELISA试剂盒及抗体、细胞因子、生化试剂、耗材等生物试剂产品。诚信经营，价格实惠，服务周到，质量有保证。咨询电话:18906011628 0592-6020891 QQ:1048735792 http://www.biohj.com/download.aspx(说明书下载网站) 该说明书是PDF格式转化的，固排版有所变化， 欢迎老师QQ或电话或邮箱索取原版说明书
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Mouse Leptin （（LEP））ELISA kit
（（ ））
Catalog No. CSB-E04650m
(96T)
This immunoassay kit allows for the in vitro quantitative determination of mouse
LEP concentrations in cell culture supernates, 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.
CUSABIO BIOTECH CO., Ltd.
http://www.cusabio.com/ http://www.cusabio.cn/
E-mail: cusabio@cusabio.com cusabio@cusabio.cn
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INTRODUCTION
Leptin (from the Greek leptos, meaning thin) is a protein hormone with
important effects in regulating body weight, metabolism and reproductive
function. The protein is approximately ~16 kDa in mass and encoded by the
obese (ob) gene. Leptin is expressed predominantly by adipocytes, which
fits with the idea that body weight is sensed as the total mass of fat in the
body. Smaller amounts of leptin are also secreted by cells in the epithelium
of the stomach and in the placenta. Leptin receptors are highly expressed in
areas of the hypothalamus known to be important in regulating body weight,
as well as in T lymphocytes and vascular endothelial cells.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an
antibody specific to LEP. Standards or samples are then added to the
appropriate microtiter plate wells with a biotin-conjugated polyclonal
antibody preparation
specific for LEP. and Avidin conjugated to Horseradish Peroxidase (HRP) is
added 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 LEP., biotin-conjugated antibody and enzyme-conjugated
Avidin 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 LEP. in the samples is then determined by comparing the
O.D. of the samples to the standard curve.
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DETECTION RANGE
0.16 ng/ml-10 ng/ml. The standard curve concentrations used for the
ELISA’s were 10 ng/ml, 5 ng/ml, 2.5 ng/ml, 1.25 ng/ml, 0.62 ng/ml, 0.32
ng/ml, 0.16 ng/ml
SPECIFICITY
This assay recognizes recombinant and natural mouse LEP No significant
cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of mouse LEP is typically less than 0.04
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
Standard 2
Sample Diluent 1 x 20 ml
Biotin-antibody Diluent 1 x 10 ml
HRP-avidin Diluent 1 x 10 ml
Biotin-antibody 1 x 120μl
HRP-avidin 1 x 120μl
1 x 20 ml
Wash Buffer
(25×concentrate)
TMB Substrate 1 x 10 ml
Stop Solution 1 x 10 ml
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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 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. 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
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 10 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 (10 ng/ml). The Sample Diluent serves as the zero standard
(0 ng/ml).
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3. Biotin-antibody Dilute to the working concentration specified on the
vial label using Biotin-antibody Diluent(1:100), respectively.
4. HRP-avidin Dilute to the working concentration specified on the vial
label using HRP-avidin 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.
Pipettes and pipette tips.
Deionized or distilled water.
Squirt bottle, manifold dispenser, or automated microplate washer.
SAMPLE COLLECTION AND STORAGE
Cell Culture Supernates Remove particulates by centrifugation and
assay immediately or aliquot and store samples at -20° C. Avoid
repeated freeze-thaw cycles.
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 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.
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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. Add 100μl of Standard, Blank, or Sample per well. Cover with the
adhesive strip. Incubate for 2 hours at 37° C.
2. Remove the liquid of each well, don’t wash.
3. Add 100μl of Biotin-antibody working solution to each well. Incubate
for 1 hour at 37°C. Biotin-antibody working solution may appear
cloudy. Warm up to room temperature and mix gently until solution
appears uniform.
4. 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.
5. Add 100μl of HRP-avidin working solution to each well. Cover the
microtiter plate with a new adhesive strip. Incubate for 1 hour at 37° C.
6. Repeat the aspiration and wash three times as step 4.
7. Add 90μl of TMB Substrate to each well. Incubate for 30 minutes at
37°C. Keeping the plate away from drafts and other temperature
fluctuations in the dark.
8. Add 50μl of Stop Solution to each well. If color change does not
appear uniform, gently tap the plate to ensure thorough mixing.
9. 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
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
LEP. 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.
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 Quantikine Immunoassay, the
possibility of interference cannot be excluded.
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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.
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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