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（TGF-β1）大鼠轉化生長因子β1（TGF-β1）Elisa試劑盒說明書

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Rat Transforming Growth factor β1

                   （TGF-β1）ELISA kit

                      Catalog No. CSB-E04727r

                                   （96 T）

?  This   immunoassay   kit   allows   for  the in   vitro   quantitative  determination   of  rat

    TGF-β1 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 TGF-β1. Standards or samples are then

added      to   the   appropriate      microtiter   plate    wells   with    a

biotin-conjugated antibody preparation specific for TGF-β1 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 TGF-β1, 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 TGF-β1 in the samples is then determined by 

comparing the O.D. of the samples to the standard curve.

DETECTION RANGE

6.25 pg/ml-400  pg/ml. The standard curve concentrations used

for the ELISA’s were 400 pg/ml, 200 pg/ml, 100 pg/ml,50 pg/ml,

25 pg/ml, 12.5 pg/ml, 6.25 pg/ml.

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SPECIFICITY

This assay recognizes rat TGF-β1. No significant cross-reactivity

or interference was observed.

SENSITIVITY

The minimum detectable dose of rat TGF-β1 is typically less than

1.56 pg/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                           2 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. The

    test kit may be used throughout the expiration date of the kit,

    provided     it  is  stored   as  prescribed     above.    Refer    to  the

    package label for the expiration date.

2.  Opened test plate should be stored at 2-8?C in the aluminum

   foil bag with desiccants to minimize exposure to damp air. The

    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.

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2.  Standard       Centrifuge   the   standard   vial   at   6000-10000rpm

    for   30s.   Reconstitute   the  Standard  with   1.0   ml   of  Sample

    Diluent.     This   reconstitution     produces     a  stock   solution    of

    400pg/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

    （400pg/ml）. The Sample Diluent serves as the zero standard

    （0 pg/ml）. Prepare fresh for each assay. Use within 4 hours

    and discard after use.

3.  Biotin-antibody         Centrifuge the vial before opening.  Dilute

    to     the    working     concentration       using    Biotin-antibody

    Diluent（1:100）, respectively.

4.  HRP-avidin        Centrifuge the vial before opening. Dilute to the

    working      concentration      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.

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? Pipettes and pipette tips.

? Deionized or distilled water.

? Squirt   bottle,   manifold   dispenser,   or   automated   microplate

    washer.

? An incubator which can provide stable incubation conditions

    up to 37°C±0.5°C.

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 immediay or

    aliquot   and  store   samples   at  -20°C.   Centrifuge   the   sample

    again     after   thawing     before    the   assay.   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   immediay   or   aliquot   and

    store  samples   at  -20°C.   Centrifuge   the   sample   again   after

    thawing before the assay. 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.

All   the   reagents  should   be   added   directly   to   the  liquid  level   in   the   well.   The

pipette should avoid contacting the inner wall of the well.

1.   Recommend   to   dilute   the   serum   or   plasma   samples   with

     Sample   Diluent（1:200）   before   test. The   suggested   200-fold

     dilution   can   be   achieved   by   adding  5μl  sample  to  95μl  of 

     Sample   Diluent.   Complete   the   200-fold   dilution   by   adding

    25μl   of   this   solution   to   225μl   of   Sample   Diluent.          The

     recommended           dilution   factor   is   for  reference      only.   The

     optimal     dilution    factor    should     be   determined       by    users

     according to their particular experiments.

2.  Add 100μl of Standard, Blank, or Sample per well. Cover with 

    the adhesive strip. Incubate for 2 hours at 37°C.

3.   Remove the liquid of each well, don’t wash. 

4.  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.

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5.  Aspirate   each   well   and   wash,   repeating   the   process   three

    times for a total of three washes. Wash: Fill each well with

    Wash  Buffer  （200μl）  and  let  it  stand  for  2  minutes,  then 

    remove      the   liquid  by   flicking  the  plate   over   a  sink.   The

    remaining drops are removed by patting the plate on a paper

    towel. Complete removal of liquid at each step is essential to

    good performance.

6.  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.

7.  Repeat the aspiration and wash five times as step 5.

8.  Add 90μl of TMB Substrate to each well. Incubate for 10-30

    minutes   at   37°C.  Keeping   the   plate   away   from   drafts   and

    other temperature fluctuations in the dark.

9.  Add 50μl of  Stop Solution to each well   when the first four

    wells    containing     the   highest    concentration      of  standards

    develop obvious blue color. If color change does not appear

    uniform, gently tap the plate to ensure thorough mixing.

10.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 TGF-β1 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.

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? Do not mix or substitute reagents with those from other lots or

    sources.

? It    is  important    that  the  Standard    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 Standard 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

? Centrifuge vials before opening to collect contents.

? When mixing or reconstituting protein solutions, always avoid

    foaming.

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? 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 or light blue until

    added to the plate. Keep Substrate  Solution protected from

    light. Substrate Solution should change from colorless or light

    blue 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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