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人生長激素（GH）ELISA試劑盒

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  Human Growth Hormone（HGH）

                           ELISA KIT

                      Catalog No. CSB-E04567h

                                    （96T）

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

    HGH 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

HGH      is  Human     Growth    Hormone,     a  natural   hormone      produced    in  the

pituitary gland of the brain. HGH is considered "the key" hormone because

it controls so many functions. It's responsible for youth, vitality, energy and

all   of   the   health   benefits   we   associate   with   youth.   Dr.   Daniel   Rudman's

study     in  the   New     England     Journal    Of   Medicine     demonstrated       the

remarkable ability to reverse the effects of aging upon the human body with

the   employment   of   HGH   -   Human   Growth   Hormone!   Due   in   part   to   his

efforts, Dr. Rudmans's study saw the effects of HGH upon overweight men

between the ages of 61 and 80 years of age.

HGH reduces body fat The men did not alter their personal habits of eating,

smoking,      or  exercise,   yet  with   the  consumption      of  HGH,    they   lost  an

average of 14% of their body fat, while gaining an average of 8.8% lean

muscle mass. Their skin became firmer and they experienced a localized

increase in bone density. Over all, HgH appeared to reverse the effects of

aging by 10-20 years!!! HGH is prescribed and administered by a doctor in

the form of injections. But a nonprescription form is also available over the

counter and through mail order.

HGH   promotes   growth   in   children   and   plays   an   important   role   in   adult

metabolism.      The    body   secretes    the   hormone,     in  decreasing     amounts,

throughout      our  lifetimes.  The    amount    of  hormone     in the   body    can   be

measured by levels of IGF-1 （Insulin Growth Factor）. Growth hormone has

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a profound effect on all the cells of the body, more than any other hormone

because it is the cell generator.

HGH is the "master hormone" controlling many organs and body functions

and   is   directly   responsible   for   stimulating   tissue  repair,   cell   replacement,

brain   functions,   and   enzyme   function!   It’s   human   growth   hormone   that

grows the cells, bones, muscles, and organs, and it is the decreasing level

of human growth hormone after age 30 that slowly robs us of our "youth."

PRINCIPLE OF THE ASSAY

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

and    Avidin.   Standards    or  samples     are  then   added    to the   appropriate

microtiter plate wells with a biotin-conjugated HGH antibody and incubated.

Then     Horseradish     Peroxidase     （HRP）-conjugated        antibody    preparation

specific for HGH are added and incubated. Substrate solutions are added to

each well. 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 HGH in the samples is then determined by comparing the

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

DETECTION RANGE

2.5 ng/ml-50 ng/ml. The standard curve concentrations used for the ELISA’s

were 50 ng/ml, 25ng/ml,10 ng/ml, 5 ng/ml，2.5 ng/ml.

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SPECIFICITY

This   assay   recognizes    human    HGH.    No  significant  cross-reactivity  or

interference was observed.

SENSITIVITY

The minimum detectable dose of human HGH is typically less than 1 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

              Standards （S1-S4）                           5x 1 ml

              Biotin-antibody                             1 x 6 ml

              HRP-conjugate                              1 x 10 ml

                                                         1 x 20 ml

              Wash Buffer

                                                     （10×concentrate）

              Substrate A                                 1 x 7 ml

              Substrate B                                 1 x 7 ml

              Stop Solution                               1 x 7 ml

   Standard       Standard1    Standard2    Standard3     Standard4    Standard5

Concentration

                     2.5             5           10           25            50

    （ng/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. 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 200 ml of Wash Buffer.

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.

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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.

     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.

     Avoid repeated freeze-thaw cycles.

Note: Grossly hemolyzed samples are not suitable for use in this assay.

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    50μl   of Standard      or  Sample       per  well.   Then    add   50μl    of

     Biotin-antibody to   each   well.   Standards   need   test   in   duplicate.   Mix

     well and then incubate for 30 min at 37°C.

2.    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.

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3.    Add 50μl of HRP-conjugate to each well. Mix well and then incubate for

     30 min at 37°C.

4.    Wash the plate as before.

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

     Incubate for 15 minutes at 18-25°C. Keeping the plate away from drafts

     and other temperature fluctuations in the dark.

6.    Add   50μl   of  Stop   Solution   to   each   well.   If   color   change   does   not

     appear uniform, gently tap the plate to ensure thorough mixing.

7.    Determine the optical density of each well within 10 minutes, using a

     microplate reader set to 450 nm.

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

HGH 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  Quantikine    Immunoassay,       the

    possibility of interference cannot be excluded.

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.

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      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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                  人生長激素人生長激素（GH）酶聯免疫分析酶聯免疫分析

                  人生長激素人生長激素             酶聯免疫分析酶聯免疫分析

                          試劑盒使用說明書試劑盒使用說明書

                          試劑盒使用說明書試劑盒使用說明書

本試劑盒僅供研究使用本試劑盒僅供研究使用

本試劑盒僅供研究使用本試劑盒僅供研究使用

產品編號產品編號：：CSB-E04567h

產品編號產品編號：：

檢測范圍檢測范圍：：2.5 ng/ml – 50 ng/ml

檢測范圍檢測范圍：：

zui低檢測限zui低檢測限：：1 ng/ml

zui低檢測限zui低檢測限：：

特異性特異性：：本試劑盒可檢測人HGH，且與其他相關蛋白無交叉反應。

特異性特異性：：

有效期有效期：：6 個月

有效期有效期：：

預期應用預期應用：：ELISA 法定量測定人血清、血漿或其它相關生物液體中HGH

預期應用預期應用：：

含量。

說明說明

說明說明

1.  濃洗滌液低溫保存會有鹽析出，稀釋時可在水浴中加溫助溶。

2.  剛開啟的酶聯板孔中可能會含有少許水樣物質，此為正常現象，不會對實

    驗結果造成任何影響。

實驗原理實驗原理

實驗原理實驗原理

     用*化牛血清白蛋白和親和素包被微孔板，制成固相載體，將已知

濃度的HGH 的標準品、標本加入微孔板中，使其與*標記的HGH 抗體

同時溫育，洗滌后，加入辣根過氧化物酶標記的抗體，再經過溫育和洗滌后

用底物顯色。顏色的深淺和樣品中的HGH  的濃度成比例關系。用酶標儀在

450nm 波長下測定吸光度 （OD 值），計算樣品濃度。

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試劑盒組成及試劑配制試劑盒組成及試劑配制

試劑盒組成及試劑配制試劑盒組成及試劑配制

1.  酶聯板酶聯板（Assay plate ）：                                      一塊（96 孔）。

    酶聯板酶聯板

2.  標準品標準品（Standard）：                                             5×1ml/瓶。

    標準品標準品

 Standard 1       Standard 2      Standard 3    Standard 4       Standard 5

  2.5 ng/ml         5 ng/ml        10ng/ml        25 ng/ml        50 ng/ml

3.  酶結合物酶結合物（HRP-Conjugate）：                                     1×10ml/瓶。

    酶結合物酶結合物

4.  *抗體*抗體（（Biotin-antibody））                                 1×6ml/瓶。

    *抗體*抗體（（                   ））

5.  底物溶液底物溶液A （（Substrate A）：）                                    1×7ml/瓶。

    底物溶液底物溶液 （（               ））

6.  底物溶液底物溶液B （（Substrate B）：）                                    1×7ml/瓶。

    底物溶液底物溶液 （（               ））

7.  濃洗滌液濃洗滌液（（Wash Buffer1×20ml/瓶 使用時每瓶用蒸餾水溶解到500ml。

    濃洗滌液濃洗滌液（（

8.  終止液終止液（（Stop Solution）：）                                      1×7ml/瓶。

    終止液終止液（（                ））

需要而未提供的試劑和器材需要而未提供的試劑和器材

需要而未提供的試劑和器材需要而未提供的試劑和器材

1.  標準規格酶標儀

2.  高速離心機

3.  電熱恒溫培養箱

4.  干凈的試管和Eppendof 管

5.  系列可調節移液器及吸頭，一次檢測樣品較多時，用多通道移液器

6.  蒸餾水，容量瓶等

標本的采集及保存標本的采集及保存

標本的采集及保存標本的采集及保存

1.  血清：全血標本請于室溫放置2 小時或4℃過夜后于1000g 離心20 分鐘，

    取上清即可檢測，或將標本放于-20℃或-80℃保存，但應避免反復凍融。

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2.  血漿：可用EDTA 或肝素作為抗凝劑，標本采集后30 分鐘內于2   -   8°C

    1000 g 離心15 分鐘，或將標本放于-20℃或-80℃保存，但應避免反復凍

    融。

注：注：以上標本置以上標本置4℃℃保存應小于保存應小于1 周，周，-20℃℃或或-80℃℃均應密封保存均應密封保存，，-20℃℃不應超過不應超過1 個個

注注：：以上標本置以上標本置 ℃℃保存應小于保存應小于 周周，， ℃℃或或 ℃℃均應密封保存均應密封保存，， ℃℃不應超過不應超過 個個

月，月，-80℃℃不應超過不應超過2  個月個月；標本溶；標本溶血會影響zui后檢測結果血會影響zui后檢測結果，因此溶血標本不宜進行檢，因此溶血標本不宜進行檢

月月，， ℃℃不應超過不應超過 個月個月；；標本溶標本溶血會影響zui后檢測結果血會影響zui后檢測結果，，因此溶血標本不宜進行檢因此溶血標本不宜進行檢

測。測。

測測。。

操作步驟操作步驟

操作步驟操作步驟

1.  將各種試劑至室溫 〔18-25℃〕平衡半小時，取濃縮洗滌液，根據當批檢

    測數量，用蒸餾水上1：10 稀釋，混勻后備用。

2.  將酶標板取出，分別向孔中加入50ul 標準品、標本，再分別加入50ul 生

    物素化抗體，震動10-20 秒混勻，置37℃孵育30 分鐘。

3.  手工洗板，棄去孔內液體。洗滌液注滿各孔，靜置10 秒甩干，重復三次

    后拍干；洗板機洗板，選擇洗滌三次程序，洗板后拍干。

4.  每孔加入100ul 酶結合物，震動10-20 秒混勻，置37℃孵育30 分鐘。

5.  手工洗板，棄去孔內液體。洗滌液注滿各孔，靜置10 秒甩干，重復三次

    后拍干；洗板機洗板，選擇洗滌三次程序，洗板后拍干。

6.  每孔加顯色劑A 液50μl，顯色劑B 液50μl，振蕩混勻后，18-25℃避光

    顯色15 分鐘，每孔加終止液50μl。

7.  用酶標儀讀數，取波長450nm，先用空白孔調零點，然后測定各孔OD

    值。

數據處理數據處理

數據處理數據處理

1.  手工作圖：用雙對數坐標紙，以標準品濃度為橫軸，以對應的0D 值為縱

    軸，畫出平滑曲線或直線，在曲線上按照待測血清OD 值找到對應的濃度

    值。

2.  計算機：使用線性擬合功能，應將標準品S1-S5  的濃度取對數（Log（濃

    度））作為X，將對應的OD 值減去空白對照孔OD 值后取對數（Log（OD

    值-NSB））作為Y，進行線性擬合。再從擬合線上計算出待測血清濃度。

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注意事項注意事項

注意事項注意事項

1.  從冷藏環境中取出的試劑盒內全部瓶裝試劑及所需預包被板條應置室溫

 （18-25℃）平衡30 分鐘后方可使用，余者應及時封好口，放回2-8℃中避

光保存，以備后用。

2.  使用前試劑應搖勻。

3.  結果判斷須在反應終止后10 分鐘內完成。

4.  不同批號的試劑不可混用。

5.  加樣時應注意避免所用各試劑及樣品之間的交又污染。

6.  操作時，試劑盒內每種試劑各使用一個吸頭，每一種標準品使用一個吸頭，

    每一個樣品各使用一個吸頭，吸頭一次性使用。

7.  每次測試必須重新制作標準曲線，上次實驗標準曲線不可重復使用。

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Notes

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