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人血管緊張素轉(zhuǎn)化酶（ACE）ELISA試劑盒

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  Human Angiotensin converting

           enzyme,ACE ELISA Kit

                      Catalog No. CSB-E04489h

                                   （96 T）

    This immunoassay kit allows for the in vitro quantitative determination of human

    ACE concentrations in 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.

PRINCIPLE OF THE ASSAY

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The microtiter plate provided in this kit has been pre-coated with

an   antibody   specific   to   ACE.   Standards   or   samples   are   then

added      to   the    appropriate     microtiter    plate    wells    with   a

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

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

DETECTION RANGE

3.12 U/ml-200 U/ml. The standard curve concentrations used for

the   ELISA’s   were   200   U/ml,   100   U/ml,   50   U/ml,   25   U/ml,   12.5

U/ml, 6.25 U/ml, 3.12 U/ml.

SPECIFICITY

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This assay recognizes recombinant and natural human ACE. No

significant cross-reactivity or interference was observed.

SENSITIVITY

The   minimum   detectable   dose   of   human   ACE   is   typically   less

than 0.78 U/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

STORAGE

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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.

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

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

    Diluent. This reconstitution produces a stock solution of 200

    U/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 （200 U/ml）.

    The Sample Diluent serves as the zero standard （0 U/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.    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: 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.

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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 five times as step 4.

7.   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.

8.   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.

9.   Determine the optical density of each well within 30 minutes,

    using a microplate reader set to 450 nm.

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）

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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 ACE 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   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.

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   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.

TECHNICAL HINTS

   Centrifuge vials before opening to collect contents.

   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.

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   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.