大鼠血管生成素Ⅱ(AngⅡ)酶聯(lián)免疫檢測

試劑盒使用說明書

使用前仔細閱讀本說明書。本酶聯(lián)免疫試劑盒是基于雙抗體夾心技術(shù)原理，來檢測大鼠血管生成素Ⅱ(AngⅡ)，只能用于研究用途，不得用于醫(yī)學(xué)診斷。

用    途：用于大鼠血清、血漿及相關(guān)液體樣本中血管生成素Ⅱ(AngⅡ)測定。

工作原理

本試劑盒采用的是生物素雙抗體夾心酶聯(lián)免疫吸附法（ELISA）測定樣品中大鼠血管生成素Ⅱ(AngⅡ)水平。向預(yù)先包被了血管生成素Ⅱ(AngⅡ)單克隆抗體的酶標(biāo)孔中加入血管生成素Ⅱ(AngⅡ)，溫育；溫育后，加入生物素標(biāo)記的抗AngⅡ抗體。再與鏈霉親和素-HRP結(jié)合，形成免疫復(fù)合物，再經(jīng)過溫育和洗滌，去除未結(jié)合的酶，然后加入底物A、B，產(chǎn)生藍色，并在酸的作用下轉(zhuǎn)化成zui終的黃色。顏色的深淺與樣品中血管生成素Ⅱ(AngⅡ)的濃度呈正相關(guān)。

試劑盒組成

需要而未提供的試劑和器材

1. 37℃恒溫箱。
2. 標(biāo)準(zhǔn)規(guī)格酶標(biāo)儀。
3. 精密移液器及一次性吸頭
4. 蒸餾水，
5. 一次性試管
6. 吸水紙

注意事項

1. 從2-8℃取出的試劑盒，在開啟試劑盒之前要室溫平衡至少30分鐘。酶標(biāo)包被板開封后如未用完，板條應(yīng)裝入密封袋中保存。
2. 各步加樣均應(yīng)使用加樣器，并經(jīng)常校對其準(zhǔn)確性，以避免試驗誤差
3. 嚴格按照說明書的操作進行，試驗結(jié)果判定必須以酶標(biāo)儀讀數(shù)為準(zhǔn).
4. 為避免交叉污染，要避免重復(fù)使用手中的吸頭和封板膜。
5. 不用的其它試劑應(yīng)包裝好或蓋好。不同批號的試劑不要混用。保質(zhì)前使用。
6. 底物B對光敏感，避免長時間暴露于光下。

洗板方法

手工洗板方法：甩掉酶標(biāo)板內(nèi)的液體；在實驗臺上鋪墊幾層吸水紙，酶標(biāo)板朝下用力拍幾次；將稀釋后的洗滌液至少0.35ml注入孔內(nèi)，浸泡1-2分鐘。根據(jù)需要，重復(fù)此過程數(shù)次。

自動洗板：如果有自動洗板機，應(yīng)在熟練使用后再用到正式實驗過程中

標(biāo)本要求

1．不能檢測含NaN3的樣品，因NaN3抑制辣根過氧化物酶的（HRP）活性。

2．標(biāo)本采集后盡早進行提取，提取按相關(guān)文獻進行，提取后應(yīng)盡快進行實驗。若不能馬上進行試驗，可將標(biāo)本放于-20℃保存，但應(yīng)避免反復(fù)凍融。

操作程序

1. 標(biāo)準(zhǔn)品的稀釋：(本試劑盒提供原倍標(biāo)準(zhǔn)品一支，用戶可按照下列圖表在小試管中進行稀釋。)

1. 根據(jù)代測樣品數(shù)量加上標(biāo)準(zhǔn)品的數(shù)量決定所需的板條數(shù)。每個標(biāo)準(zhǔn)品和空白孔建議做復(fù)孔。每個樣品根據(jù)自己的數(shù)量來定，能使用復(fù)孔的盡量做復(fù)孔。
2. 加樣：1）空白孔，空白對照孔不加樣品，生物素標(biāo)記的抗AngⅡ抗體，鏈霉親和素-HRP，只加顯色劑A&B和終止液，其余各步操作相同；2）標(biāo)準(zhǔn)品孔：加入標(biāo)準(zhǔn)品50ul，鏈霉親和素-HRP50ul(標(biāo)準(zhǔn)品中已事先整合好生物素抗體，故不加)；3）代測樣品孔：加入樣本40ul，然后各加入抗AngⅡ抗體10ul、鏈霉親和素-HRP50ul，蓋上封板膜，輕輕震蕩混勻，37℃溫育60分鐘。
3. 配液：將30倍濃縮洗滌液用蒸餾水30倍稀釋后備用。
4. 洗滌：小心揭掉封板膜，棄去液體，甩干，每孔加滿洗滌液，靜置30秒后棄去，如此重復(fù)5次，拍干。
5. 顯色：每孔先加入顯色劑A50ul，再加入顯色劑B50μl，輕輕震蕩混勻，37℃避光顯色15分鐘.
6. 終止：每孔加終止液50μl，終止反應(yīng)（此時藍色立轉(zhuǎn)黃色）。
7. 測定：以空白空調(diào)零，450nm波長依序測量各孔的吸光度（OD值）。 測定應(yīng)在加終止液后10分鐘以內(nèi)進行。
8. 根據(jù)標(biāo)準(zhǔn)品的濃度及對應(yīng)的OD值計算出標(biāo)準(zhǔn)曲線的直線回歸方程，再根據(jù)樣品的OD值在回歸方程上計算出對應(yīng)的樣品濃度。也可以使用各種應(yīng)用軟件來計算。

檢測范圍：15ng/L→600 ng/L。

保存：2-8℃。

有效期：6個月(2-8℃)。

Rat AngⅡ ELISA Kit

Instruction

This kit is only for scientific research, and shall not be used as a clinical diagnosis of use.

Purpose

This kit allows for the determination of AngⅡ concentrations in Rat serum, cell culture supernatant, and other biological fluids.

Principle

The kit assay Rat AngⅡ level in the sample，add Rat AngⅡ antibody to microtiter plate wells, after Incubating,add Biotinylated anti –AngⅡ-antibody , then Combined Streptavidin-HRP, become complex, after Incubating and washing Compley, Add TMB substrate solution,TMB substrate becomes blue color, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of AngⅡ in the samples is then determined by comparing the O.D. of the samples to the standard curve.

 Materials provided with the kit

Materials required but not supplied

1. 37 ℃ incubator

2. Standard microplate reader(450nm)

3. Precision pipettes and Disposable pipette tips.

4. deionized water.

5. Disposable Test tube

6 Absorbent paper

Important notes

1.       The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.

2.       add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error.

3.       Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.

4.       Use new disposal plastic pipette tips and Closure plate membrane for each standard, in order to avoid cross contamination.

5.       Do not mix reagents with those from other lots.

6.       The substrate evade the light preservation.

Specimen requirements

1. extract as soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can’t, specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.
2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.

Assay procedure

1. Dilute and add sample:Dilute Original density Standard as follow table:

2. according testing Sample numbers to define how many wells nedd, Standard and blank suggested Do holes.

3.add sample：1) blank wells: (blank comparison wells don’t add sample , Biotinylated anti –AngⅡ -antibody and Streptavidin-HRP ,other each step operation is same); 2) Standard wells: add Standard 50μl and Streptavidin-HRP 50μl; 3) testing Sample wells: add sample 40μl,then add anti –AngⅡ  -antibody 10μl , Streptavidin-HRP 50μl. closing plate with Closure plate membrane ,incubate for 60 min at 37℃.

4.Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.

5.washing：Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.

6.color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37℃

7.Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction(the blue color change to yellow color).

8.assay：take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 10min.

9. Calculate of result

Assay range

15ng/L→600 ng/L

Storage and validity

1．Storage：  2-8℃.

2．validity： six months.