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小鼠（INS）小鼠胰島素（INS）ELISA試劑盒說明書

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      Mouse Insulin （INS）ELISA Kit

                       Catalog No. CSB-E05071m

                                      （96T）

     This immunoassay kit allows for the in vitro quantitative determination of mouse INS

    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

Insulin is a peptide hormone composed of 51 amino acid residues and has a

molecular weight of 5808 Da. It is produced in the islets of Langerhans in the

pancreas. Insulin's structure varies slightly between species of animal. Insulin

has extensive effects on both metabolism and several other body systems （eg,

vascular   compliance）.   Insulin   causes   most   of   the   body's   cells   to   take   up

glucose from the blood （including liver, muscle, and fat tissue cells）, storing it

as glycogen in the liver and muscle, and stops use of fat as an energy source.

When insulin is absent （or low）, glucose is not taken up by most body cells and

the   body   begins   to   use   fat   as   an   energy   source   （ie,   transfer   of   lipids   from

adipose tissue to the liver for mobilization as an energy source）. As its level is

a   central   metabolic   control   mechanism,   its   status   is   also   used   as   a   control

signal to other body systems （such as amino acid uptake by body cells）. It has

several other anabolic effects throughout the body.

PRINCIPLE OF THE ASSAY

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

specific   to   INS.   Standards   or   samples   are   then   added   to   the   appropriate

microtiter     plate   wells   with   a   Horseradish      Peroxidase      （HRP）-conjugated

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monoclonal       antibody   preparation     specific  for  INS    and   incubated.    Then

substrate   solution   A   and   B   are   added   to   each   well.  Only   those   wells   that

contain   INS,   HRP-conjugated   antibody   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 INS in the samples is then

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

DETECTION RANGE

8μIU/ml-140μIU/ml. The standard curve concentrations used for the ELISA’s

were 140μIU/ml,80μIU/ml,32μIU/ml, 16μIU/ml, 8μIU/ml

SPECIFICITY

This   assay   recognizes   recombinant   and   natural   mouse   INS.   No   significant

cross-reactivity or interference was observed.

SENSITIVITY

The minimum detectable dose of mouse INS is typically less than 5μIU/ml.

The sensitivity of this assay, or Lower Limit of Detection （LLD） was defined as

the lowest concentration that could be differentiated from zero.

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MATERIALS PROVIDED

              Reagent                                      Quantity

              Assay plate                                      1

              Standard（S -S ）                                  5

                           1  5

              HRP-conjugate                                 1 x 6 ml

                                                           1 x 15 ml

              Wash Buffer

                                                        （20×concentrate）

              Substrate A                                   1 x 7 ml

              Substrate B                                   1 x 7 ml

              Stop Solution                                 1 x 7 ml

                       Standard     Standard      Standard     Standard     Standard

     Standard

                            1            2             3            4            5

  Concentration

                            8             16           32           80          140

      （μIU/ml）

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   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.

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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

1.   Bring all reagents and plate to room temperature for at least 30 minutes

    before use. Unused wells need store at 2-8°C and av oid 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.

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

     well. Standard need test in duplicate.

2.    Reconstitute the every Standard with 0.5 ml of distilled water.

3.    Add 50μl of HRP-conjugate to each well （not to Blank well）. Mix well and

     then incubate for 2 hour at 37°C.

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4.    Complete remove the liquid. Then 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.

5.    Add 50μl of Substrate A and 50μl of 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.

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

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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  INS

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.

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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.

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        小鼠小鼠胰島素胰島素 （（Insulin））ELISA 快速檢測試劑盒快速檢測試劑盒

        小小鼠鼠胰島素胰島素 （（               ））           快速檢測試劑盒快速檢測試劑盒

                              使用說明書使用說明書

                              使用說明書使用說明書

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-E05071m

產品編號產品編號

檢測范圍檢測范圍：：8μIU /ml -140μIU /ml

檢測范圍檢測范圍：：

特異性特異性：：本試劑盒可同時檢測天然或重組的 INS，且與其他相關蛋白基本無

特異性特異性：：

交叉反應。

有效期有效期：6 個月 （2-8℃避光保存）

有效期有效期

預期應用預期應用：：ELISA 法定量測定小鼠血清，血漿及其它相關生物液體中INS 含

預期應用預期應用：：

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

     采用酶聯免疫法夾心法檢測胰島素含量。首先用一株單抗體包被微孔板，

制備成固相抗體，然后加入待測標本及辣根過氧化物酶標記的另一株單抗，使

之形成包被抗體-胰島素-酶標記抗體的復合物。經顯色后在酶標儀測定吸光值

 （OD 值），通過計算機或作圖擬合濃度-吸光度曲線，反算出待測標本中胰島素

含量。

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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 瓶（凍干品）。

    標準品標準品

 Standard 1       Standard 2     Standard 3      Standard 4      Standard 5

   8μIU/ml        16μIU/ml        32μIU/ml       80μIU/ml        140μIU/ml

3.  酶結合物酶結合物（（HRP-conjugate）：）                                     1×6ml/瓶。

    酶結合物酶結合物（（                 ））

4.  顯色劑顯色劑A （（Substrate A）：）：                                      1×7ml/瓶。

    顯色劑顯色劑 （（              ）：）：

5.  顯色劑顯色劑B （（Substrate B）：）：                                      1×7ml/瓶。

    顯色劑顯色劑 （（              ）：）：

6.  濃洗滌液濃洗滌液（（Wash Buffer ）：）  1×15ml/瓶，使用時每瓶用蒸餾水稀釋20 倍。

    濃洗滌液濃洗滌液（（              ））

7.  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

標本的標本的采集及保存采集及保存

標本的標本的采集及保存采集及保存

1.  血清：全血標本請于室溫放置2 小時或4℃過夜后于1000 x g 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。

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

    x 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。

3.  細胞培養物上清或其它生物標本：1000 x g 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。

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℃℃均應密均應密封保存封保存，，-20℃℃不應超過不應超過1 個月個月，，

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 ℃℃均應密均應密封保存封保存，， ℃℃不應超過不應超過 個月個月，，

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

   ℃℃不應超過不應超過 個月個月；；標本溶血會影響zui后檢測結果標本溶血會影響zui后檢測結果，，因此溶血標本不宜進行此項檢測因此溶血標本不宜進行此項檢測。。

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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1：20 稀釋，混勻后備用。

2.  標準品S1- S 5，*次使用前先用0.5ml 蒸餾水溶解，放置混勻后使用。

3.  將酶標板取出，設一個空白對照孔、不加任何液體；每個標準點依次各設兩

    孔，每孔加入相應標準品50ul；其余每個檢測孔直接加待測標本50ul。

4.  每孔加入酶結合物50ul  （空白對照孔除外），充分混勻，貼上不干膠封片，

    置37℃溫育2 小時。

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，37℃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。

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。

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Notes

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