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大鼠（T）大鼠睪酮（T）ELISA試劑盒說明書

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   Rat Testoterone                          （（T））ELISA Kit

                                            （（））

                          Catalog No. CSB-E05100r

                                       （96T）

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

    testoterone 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.

                           CUSABIO BIOTECH CO., LTD.

                   /  /

                  : cusabio@  cusabio@

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INTRODUCTION

Testosterone      is  a  steroid   hormone     from    the  androgen     group.   In   mammals,

testosterone is primarily secreted in the testes of males and the ovaries of females,

although small amounts are also secreted by the adrenal glands. It is the principal

male sex hormone and an anabolic steroid.

In both men and women, testosterone plays a key role in health and well-being as

well as in sexual functioning. Examples include enhanced libido, increased energy,

increased   production   of   red   blood   cells   and   protection   against   osteoporosis.   On

average,   an   adult   human   male   body   produces   about   forty   to   sixty   times   more

testosterone     than   an  adult   female   body,    but  females    are,  from   a  behavioral

perspective     （rather  than   from    an  anatomical     or  biological   perspective）,    more

sensitive to the hormone. However the overall ranges for male and female are very

wide,    such    that  the  ranges    actually   overlap    at  the low    end    and   high   end

respectively.

PRINCIPLE OF THE ASSAY

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

goat-anti-rabbit antibody. Standards or samples are then added to the appropriate

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microtiter plate wells with a HRP-conjugated testoterone and antibody preparation

specific for testoterone and incubated. Then 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 testoterone in the samples is

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

DETECTION RANGE

0.1   ng/ml-25.6   ng/ml.   The   standard   curve   concentrations   used   for   the   ELISA’s

were 25.6 ng/ml, 6.4 ng/ml, 1.6 ng/ml, 0.39 ng/ml, 0.1 ng/ml.

SPECIFICITY

This   assay   recognizes   recombinant    and  natural  rat  testoterone.  No  significant

cross-reactivity or interference was observed.

SENSITIVITY

The minimum detectable dose of rat testoterone is typically less than 0.06 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.

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MATERIALS PROVIDED

               Reagent                                             Quantity

               Assay plate                                              1

                Standards （S1-S5）                                  5 x 0.5 ml

               HRP-conjugate                                        1 x 6 ml

               Antibody                                             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 x7 ml

   Standard          Standard 1    Standard 2     Standard 3     Standard 4     Standard 5

Concentration

                        0.1            0.39            1.6            6.4            25.6

    （ng/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. 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.

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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.

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.  To ensure accurate results, proper adhesion of plate sealers during incubation

    steps is necessary. Sealers can not be reused.

6.   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.

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7.   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.

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   repeated

     freeze-thaw cycles.

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     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. Standards need test in duplicate.

2.   Add 50μl of HRP-conjugate to each well （not to Blank well）, then add 50μl

     Antibody to each 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 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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5.   Add   50μl   of  Stop   Solution   to   each   well.   If   color   change   does   not   appear

     uniform, gently tap the plate to ensure thorough mixing.

6.   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, 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 testoterone 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   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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                   大鼠睪酮大鼠睪酮（T）酶聯(lián)免疫（T）酶聯(lián)免疫試劑盒試劑盒  

                   大鼠睪酮大鼠睪酮（T）（T）酶聯(lián)免疫酶聯(lián)免疫試劑盒試劑盒  

                               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(chǎn)品編號(hào)產(chǎn)品編號(hào)：CSB-E05100r 

產(chǎn)品編號(hào)產(chǎn)品編號(hào)

檢測(cè)范圍檢測(cè)范圍：：0.1 ng /ml -25.6 ng /ml

檢測(cè)范圍檢測(cè)范圍：：

zui低檢測(cè)限zui低檢測(cè)限：：0.06 ng/ml

zui低檢測(cè)限zui低檢測(cè)限：：

特異性特異性：：本試劑盒可同時(shí)檢測(cè)天然或重組的睪酮，且與其他相關(guān)蛋白基本

特異性特異性：：

無(wú)交叉反應(yīng)。

有效期有效期：6 個(gè)月 （2-8℃避光保存）

有效期有效期

預(yù)期應(yīng)用預(yù)期應(yīng)用：：ELISA 法定量測(cè)定大鼠血清，血漿及其它相關(guān)生物液體中睪酮

預(yù)期應(yīng)用預(yù)期應(yīng)用：：

含量。

說明說明

說明說明

1.  濃洗滌液低溫保存會(huì)有鹽析出，稀釋時(shí)可在水浴中加溫助溶。

2.  剛開啟的酶聯(lián)板孔中可能會(huì)含有少許水樣物質(zhì)，此為正?，F(xiàn)象，不會(huì)對(duì)實(shí)

    驗(yàn)結(jié)果造成任何影響。

實(shí)驗(yàn)原理實(shí)驗(yàn)原理

實(shí)驗(yàn)原理實(shí)驗(yàn)原理

     采用酶聯(lián)免疫競(jìng)爭(zhēng)法檢測(cè)睪酮含量。首先用羊抗兔包被微孔板，制備成

固相二抗，然后加入待測(cè)標(biāo)本、辣根過氧化物酶標(biāo)記睪酮以及抗睪酮抗體，

使之形成包被二抗-抗睪酮抗體- 睪酮 （HRP ）復(fù)合物，標(biāo)記睪酮的結(jié)合量與

標(biāo)本中的睪酮量成反比。經(jīng)顯色后在酶標(biāo)儀測(cè)定吸光值 （OD 值），通過計(jì)算

機(jī)或作圖擬合濃度-吸光度曲線，反算出待測(cè)標(biāo)本中睪酮含量。

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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(lián)酶聯(lián)板板（Assay plate ）：                                       一塊（96 孔）。

    酶酶聯(lián)聯(lián)板板

2.  標(biāo)準(zhǔn)品標(biāo)準(zhǔn)品（Standard）：                                            5×0.5ml/瓶。

    標(biāo)準(zhǔn)品標(biāo)準(zhǔn)品

 Standard 1       Standard 2      Standard 3     Standard 4      Standard 5

  0.1 ng/ml       0.39 ng/ml      1.6 ng/ml      6.4 ng/ml       25.6 ng/ml

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

    酶結(jié)合物酶結(jié)合物 （（                ））

4.  抗體抗體 （（Antibody ））                                             1×6ml/瓶。

    抗體抗體 （（         ））

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

    顯色劑顯色劑    （（           ））：：

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

    顯色劑顯色劑    （（           ））：：

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

    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 （（              ））

需要而未提供的試需要而未提供的試劑和器材劑和器材

需要而未提供的試需要而未提供的試劑和器材劑和器材

1.  標(biāo)準(zhǔn)規(guī)格酶標(biāo)儀

2.  高速離心機(jī)

3.  電熱恒溫培養(yǎng)箱

4.  干凈的試管和Eppendof 管

5.  系列可調(diào)節(jié)移液器及吸頭，一次檢測(cè)樣品較多時(shí)，用多通道移液器

6.  蒸餾水，容量瓶等

標(biāo)本的采集及保存標(biāo)本的采集及保存

標(biāo)本的采集及保存標(biāo)本的采集及保存

1.  血清：全血標(biāo)本請(qǐng)于室溫放置2 小時(shí)或4℃過夜后于 1000   x   g 離心20 分

    鐘，取上清即可檢測(cè)，或?qū)?biāo)本放于-20℃或-80℃保存，但應(yīng)避免反復(fù)凍

    融。

2. 血漿：可用EDTA 或肝素作為抗凝劑，標(biāo)本采集后30 分鐘內(nèi)于2 - 8° C 

    1000 x g 離心 15 分鐘，或?qū)?biāo)本放于-20℃或-80℃保存，但應(yīng)避免反復(fù)

    凍融。 

注：注：以上標(biāo)以上標(biāo)本置本置4℃保存應(yīng)小于℃保存應(yīng)小于 1 周，周，-20℃或℃或-80℃均應(yīng)密封保存℃均應(yīng)密封保存，，-20℃不應(yīng)超過℃不應(yīng)超過 1 個(gè)個(gè)

注注：：以上標(biāo)以上標(biāo)本置本置  ℃℃保存應(yīng)小于保存應(yīng)小于  周周，，   ℃℃或或   ℃℃均應(yīng)密封保存均應(yīng)密封保存，，   ℃℃不應(yīng)超過不應(yīng)超過  個(gè)個(gè)

月，月，-80℃不應(yīng)超過℃不應(yīng)超過 2  個(gè)月個(gè)月；標(biāo)本溶血會(huì)影響zui后檢測(cè)結(jié)果；標(biāo)本溶血會(huì)影響zui后檢測(cè)結(jié)果，因此溶血標(biāo)本不宜進(jìn)行，因此溶血標(biāo)本不宜進(jìn)行檢檢

月月，，   ℃℃不應(yīng)超過不應(yīng)超過   個(gè)月個(gè)月；；標(biāo)本溶血會(huì)影響zui后檢測(cè)結(jié)果標(biāo)本溶血會(huì)影響zui后檢測(cè)結(jié)果，，因此溶血標(biāo)本不宜進(jìn)行因此溶血標(biāo)本不宜進(jìn)行檢檢

測(cè)。測(cè)。

測(cè)測(cè)。。

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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(shí)，取濃縮洗滌液，根據(jù)當(dāng)批檢

    測(cè)數(shù)量，用蒸餾水上 1：20 稀釋，混勻后備用。

2.  將酶標(biāo)板取出，設(shè)一個(gè)空白對(duì)照孔、不加任何液體；每個(gè)標(biāo)準(zhǔn)點(diǎn)依次各設(shè)

    兩孔，每孔加入相應(yīng)標(biāo)準(zhǔn)品50ul；其余每個(gè)檢測(cè)孔直接加待測(cè)標(biāo)本50ul。

3.  每孔加入酶結(jié)合物 50ul            （空白對(duì)照孔除外），再按同樣的順序加入抗體

    50ul，充分混勻，貼上不干膠封片，置37℃溫育 1 小時(shí)。

4.  手工洗板，棄去孔內(nèi)液體。洗滌液注滿各孔，靜置 10 秒甩干，重復(fù)三次

    后拍干；洗板機(jī)洗板，選擇洗滌三次程序，洗板后拍干。

5.  每孔加顯色劑A 液 50 μl，顯色劑B 液 50 μl，振蕩混勻后，37℃避光顯

    色 15 分鐘，每孔加終止液50 μl。

6.  用酶標(biāo)儀讀數(shù)，取波長(zhǎng)450nm，先用空白孔調(diào)零點(diǎn)，然后測(cè)定各孔OD 值。

數(shù)據(jù)處理數(shù)據(jù)處理

數(shù)據(jù)處理數(shù)據(jù)處理

1.  手工作圖：用雙對(duì)數(shù)坐標(biāo)紙，以標(biāo)準(zhǔn)品濃度為橫軸，以對(duì)應(yīng)的0D 值為縱

    軸，畫出平滑曲線或直線，在曲線上按照待測(cè)血清OD 值找到對(duì)應(yīng)的濃度

    值。

2.  計(jì)算機(jī)：使用線性擬合功能，應(yīng)將標(biāo)準(zhǔn)品S1-S5 的濃度取對(duì)數(shù)（Log（濃度））

    作為 X，將對(duì)應(yīng)的 OD  值減去空白對(duì)照孔 OD  值后取對(duì)數(shù) （Log（OD  值

    -NSB））作為Y，進(jìn)行線性擬合。再?gòu)臄M合線上計(jì)算出待測(cè)血清濃度。

注意事項(xiàng)注意事項(xiàng)

注意事項(xiàng)注意事項(xiàng)

1.  從冷藏環(huán)境中取出的試劑盒內(nèi)全部瓶裝試劑及所需預(yù)包被板條應(yīng)置室溫

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

保存，以備后用。

2.  使用前試劑應(yīng)搖勻。

3.  結(jié)果判斷須在反應(yīng)終止后 10 分鐘內(nèi)完成。

4.  不同批號(hào)的試劑不可混用。

5.  加樣時(shí)應(yīng)注意避免所用各試劑及樣品之間的交又污染。

6.  操作時(shí)，試劑盒內(nèi)每種試劑各使用一個(gè)吸頭，每一種標(biāo)準(zhǔn)品使用一個(gè)吸頭，

    每一個(gè)樣品各使用一個(gè)吸頭，吸頭一次性使用。

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Notes

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