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小鼠（T4）小鼠甲狀腺素（T4）ELISA試劑盒說明書

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           Mouse thyroxine  （（T4））

                                                      （（ ））

                            ELISA Kit

                      Catalog No. CSB-E05083m

                                 （96 tests）

    This  immunoassay   kit  allows  for  the  in  vitro  rapid detection  of  Mouse  T4

    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.

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INTRODUCTION

Thyroxine,   or  3,5,3',5'-tetra-iodothyronine   （often  abbreviated  as

T4）, a form of thyroid hormones is the major hormone secreted

by    the    follicular  cells   of   the   thyroid    gland.    Thyroxine      is

synthesized       via  the   iodination    and    covalent    bonding     of  the

phenyl   portions  of tyrosine residues found in  an  initial  peptide,

thyroglobulin,     which    is  secreted    into   thyroid   granules.    These

iodinated   diphenyl   compounds   are   cleaved   from   their   peptide

backbone upon being stimulated by thyroid stimulating hormone.

More in the T3 and T4 section of thyroid.

T4 is transported in blood, with 99.95% of the secreted T4 being

protein   bound,   principally   to   thyroxine-binding   globulin   （TBG）,

and, to a lesser extent, to transthyretin and serum albumin. T4 is

involved   in   controlling   the   rate   of   metabolic   processes   in   the

body   and   influencing   physical   development.   Administration   of

thyroxine      has    been     shown      to   significantly     increase    the

concentration of nerve growth factor in the brains of adult mice.

Thyroxine is a prohormone and a reservoir for the active thyroid

hormone   triiodothyronine   （T3）   which   is   about   four   times   more

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potent. T4 is converted in the tissues by deiodinases, including

thyroid hormone iodine peroxidase （TPO）, to T3. The "D" isomer

is called "Dextrothyroxine" and is used as a lipid modifying agent.

The half-life of thyroxine once released into the blood circulatory

system is about 1 week.

PRINCIPLE OF THE ASSAY

This     assay      employs      the    competitive      inhibition    enzyme

immunoassay   technique.   A   antibody   specific   to   T4   has   been

pre-coated onto a microplate. Standards or samples are added

to the appropriate microtiter plate wells with biotin-conjugated T4

and     incubated.    A   competitive     inhibition  reaction    is  launched

between   T4   （Standards   or   samples）   and   Biotin-conjugated   T4

with the pre-coated antibody specific for T4. The more amount of

T4 in samples, the less antibody bound by Biotin-conjugated T4.

Then     Avidin   conjugated   to   Horseradish   Peroxidase          （HRP）    is

added   to   each   microplate   well   and   incubated.   The   substrate

solutions   are   added   to   the   wells,   respectively.   And   the   color

develops   in   opposite   to   the   amount   of   T4   in   the   sample.   The

color   development   is   stopped   and   the   intensity   of   the   color   is

measured.

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DETECTION RANGE

The   standard   curve   concentrations   used   for   the   ELISA’s   were

320 ng/ml, 160 ng/ml, 80 ng/ml, 40 ng/ml, 20 ng/ml.

SPECIFICITY

This    assay    recognizes     T4.   No   significant   cross-reactivity    or

interference was observed.

MATERIALS PROVIDED

      Reagent                                             Quantity

      Assay plate                                              1

      Standards （S1-S5）                                        5

      HRP-avidin                                           1 x 6 ml

      Conjugate                                            1 x 6 ml

                                                          1 x 15 ml

      Wash Buffer

                                                     （20×concentrate）

      Substrate A                                          1 x 6 ml

      Substrate B                                          1 x 6 ml

      Stop Solution                                        1 x 6 ml

          Standard               S1        S2        S3        S4       S5

   Concentration（ng/ml）         20         40        80       160      320

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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.

TECHNICAL HINTS

1.   Bring all reagents and plate to room temperature for at least

    30 minutes before use. Unused wells need store at 2-8℃and

    avoid sunlight.

2.    Wash Buffer        If crystals have formed in the concentrate,

   warm to room temperature and mix gently until the crystals

    have compley dissolved. Dilute 15 ml of Wash Buffer

    Concentrate into deionized or distilled water to prepare 300 ml

    of Wash Buffer.

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3.   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.

4.   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.

5.   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.

6.   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.

Precaution: The Stop Solution provided with this kit is an acid solution. Wear

           eye, hand, face, and clothing protection when using this material.

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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

    Serum       Use     a  serum     separator    tube   （SST）    and    allow

    samples   to   clot   for   30   minutes   before   centrifugation   for   15

    minutes at 1000 x g. Remove serum and assay immediay

    or   aliquot   and   store   samples     at  -20°  C.  Avoid    repea ted

    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.

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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.   Set a Blank well without any solution. Add 50μl of Standard

      or Sample per well. Standard need test in duplicate.

  2.   Add 50μl of Conjugate to each well （not to Blank well）, Mix

      well and then incubate for 1 hour at 37°C.

  3.   Fill   each   well   with   Wash   Buffer   （about   200μl）,  stay  for   10

      seconds   and   Spinning.   Repeat   the   process   for   a   total   of

      three   washes.   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.

  4.   Add 50μl of HRP-avidin to each well. Incubate for 30mins

      at 37°C.

  5.   Repeat the aspiration and wash five times as step 4.

  6.   Add 50μl of Substrate A and Substrate B to each well, mix

      well.   Incubate   for   15   minutes   at   37°C.   Keeping   the   plate

      away from drafts and other temperature fluctuations in the

      dark.

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  7.   Add 50μl of Stop Solution to each well.

  8.   Determine the optical density of each well within 10 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,   Blank,   and

sample      and    subtract    the   optical    density    of  Blank.    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 T4 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.

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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

    Immunoassay,         the   possibility   of   interference     cannot    be

    excluded.

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