（leptin）小鼠瘦素（leptin）Elisa試劑盒

----------------------- Page 1-----------------------

  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/

           : cusabio@cusabio.com        cusabio@cusabio.cn

                                       1

----------------------- Page 2-----------------------

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 approximay ~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.

                                             2

----------------------- Page 3-----------------------

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

                                        3

----------------------- Page 4-----------------------

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   compley

     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）.

                                             4

----------------------- Page 5-----------------------

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 immediay 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 immediay 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 immediay 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.

                                          5

----------------------- Page 6-----------------------

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.

                                              6

----------------------- Page 7-----------------------

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.

                                             7

----------------------- Page 8-----------------------

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.

                                             8