本試(shì)劑盒只能用於科學研究(jiū)，不得用於醫學診斷

植物（Plant）果膠(jiāo)酯酶（Pectinesterase）

ELISA檢(jiǎn)測試(shì)劑盒

使(shǐ)用說(shuō)明(míng)書(shū)

檢(jiǎn)測原理

試(shì)劑盒採用雙抗體一步夾心法(fǎ)酶聯免疫吸(xī)附試(shì)驗（ELISA） 。往(wǎng)預(yù)先包被果膠(jiāo)酯酶（Pectinesterase）抗體的包被微孔中 ，依次加(jiā)入標本、標準品 、HRP標記的檢(jiǎn)測抗體，經(jīng)過溫育(yù)並徹-底洗滌 。用底物TMB顯色 ，TMB在過氧化(huà)物酶的催化(huà)下轉(zhuǎn)化(huà)成藍色，並在酸(suān)的作用下轉(zhuǎn)化(huà)成最(zuì)終(zhōng)的黃色 。顏色的深淺和(hé)樣品中的果膠(jiāo)酯酶（Pectinesterase）呈正相(xiàng)關 。用酶標儀在450nm 波長(zhǎng)下測定吸(xī)光度（OD 值） ，計(jì)算樣品活性。

樣品收集、處理及(jí)保存方法(fǎ)

1.  樣本不能含疊氮鈉（NaN3），因(yīn)為疊氮鈉（NaN3）是(shì)辣根過氧化(huà)物酶（HRP）的抑制劑。

2.  標本採集後盡早進(jìn)行(xíng)提取 ，提取按相(xiàng)關文獻進(jìn)行(xíng) 。

3.  植物萃取液或其它相(xiàng)關樣本 ：請1000 x g離心20分(fèn)鐘，取上清即可檢(jiǎn)測。

4.  保存 ：如果樣本收集後不及(jí)時檢(jiǎn)測，請按一次用量(liàng)分(fèn)裝，凍存於-20℃，避免反復凍融 ，在室(shì)溫下解凍並確保樣品均勻(yún)地充分(fèn)解凍 。

自備物品

1. 酶標儀（450nm）

2. 高精(jīng)度加(jiā)樣器及(jí)槍頭 ：0.5-10uL 、2-20uL、20-200uL、200-1000uL

3. 37℃恆(héng)溫箱

操(cāo)作注意(yì)事(shì)項

1.  試(shì)劑盒保存在2-8℃，使(shǐ)用前(qián)室(shì)溫平衡20分(fèn)鐘。從冰箱取出(chū)的濃縮洗滌液會(huì)有結(jié)晶，這屬於正常現象 ，水浴加(jiā)熱使(shǐ)結(jié)晶完-全(chéng)溶解後再使(shǐ)用。

2.  實驗中不用的板條應立即放回自封袋中 ，密封（低溫乾燥）保存 。

3.  濃度為0的S0號標準品即可視為陰性對照或者空白；按照說(shuō)明(míng)書(shū)操(cāo)作時樣本已經(jīng)稀釋5倍 ，最(zuì)終(zhōng)結(jié)果乘以5才是(shì)樣本實際濃度。

4.  嚴格按照說(shuō)明(míng)書(shū)中標明(míng)的時間、加(jiā)液量(liàng)及(jí)順序進(jìn)行(xíng)溫育(yù)操(cāo)作 。

5.  所有液體組分(fèn)使(shǐ)用前(qián)充分(fèn)搖(yáo)勻(yún)。

試(shì)劑盒組成

注：標準品（S0-S5）濃度依次為：0、5、10、20 、40、80 U/mL

試(shì)劑的準備

 20×洗滌緩衝液的稀釋：蒸餾水按1：20稀釋 ，即1份的20×洗滌緩衝液加(jiā)19份的蒸餾水 。

洗板方法(fǎ)

1.  手工洗板 ：甩盡孔內(nèi)液體 ，每孔加(jiā)滿洗滌液，靜(jìng)置1min後甩盡孔內(nèi)液體 ，在吸(xī)水紙上拍乾 ，如此洗板5次。

2.  自動洗板機 ：每孔注入洗液350μL，浸泡1min ，洗板5次。

操(cāo)作步驟

1.  從室(shì)溫平衡20min後的鋁箔袋中取出(chū)所需板條，剩餘板條用自封袋密封放回4℃。

2.  設置標準品孔和(hé)樣本孔，標準品孔各加(jiā)不同(tóng)濃度的標準品50μL ；

3.  樣本孔先加(jiā)待測樣本10μL，再加(jiā)樣本稀釋液40μL；空白孔不加(jiā) 。

4.  除空白孔外，標準品孔和(hé)樣本孔中每孔加(jiā)入辣根過氧化(huà)物酶（HRP）標記的檢(jiǎn)測抗體100μL ，用封板膜封住反應孔 ，37℃水浴鍋或恆(héng)溫箱溫育(yù)60min。

5.  棄去液體，吸(xī)水紙上拍乾，每孔加(jiā)滿洗滌液，靜(jìng)置1min ，甩去洗滌液，吸(xī)水紙上拍乾，如此重復洗板5次（也(yě)可用洗板機洗板）。

6.  每孔加(jiā)入底物A 、B各50μL ，37℃避光孵育(yù)15min。

7.  每孔加(jiā)入終(zhōng)止液50μL，15min內(nèi) ，在450nm波長(zhǎng)處測定各孔的OD值。

結(jié)果判斷

 繪制標準曲線：在Excel工作表中，以標準品濃度作橫坐標 ，對應OD值作縱坐標 ，繪制出(chū)標準品線性回歸曲線，按曲線方程計(jì)算各樣本濃度值 。

試(shì)劑盒性能

1.  準確性：標準品線性回歸與預(yù)期濃度相(xiàng)關係數(shù)R值 ，大於等(děng)於0.9900。

2.  靈敏度 ：最(zuì)-低檢(jiǎn)測濃度小於1.0 U/mL 。

3.  特異性：不與其它可溶性結(jié)構類(lèi)似物交(jiāo)叉反應。

4.  重復性 ：板內(nèi)、板間變異系數(shù)均小於15%。

5.  貯藏 ：2-8℃ ，避光防(fáng)潮保存 。

6.  有效期：6個月

免責聲明(míng)

1.   試(shì)劑盒僅供研究(jiū)使(shǐ)用 ，不得用於臨床實驗或人體實驗 ，否則所產生(shēng)的一切後果
，由實驗者承擔，本公司概(gài)不負責。

2.   嚴格按照說(shuō)明(míng)書(shū)操(cāo)作 ，實驗者違反說(shuō)明(míng)書(shū)操(cāo)作，後果由實驗者承擔。

FOR RESEARCH USE ONLY. 

NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Plant Pectinesterase ELISA Kit instruction

Intended use

This Pectinesterase ELISA kit is intended Laboratory for Research use only and is not for use in diagnostic or therapeutic procedures.The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of Pectinesterase in the sample , this Pectinesterase ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples and allow the operator to produce a standard curve of Optical Density versus Pectinesterase concentration. The concentration of Pectinesterase in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Sample collection and storages

1. Can’t detect the samples which contain NaN3, because NaN3 inhibits HRP activity of the horseradish peroxidase.

2.  Extract as soon as possible after Specimen collection , Extracted according to the relevant literature.

Cell culture supernates and plant exact fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw.

Materials required but not supplied

1.  Standard microplate reader(450nm)

2.  Precision pipettes and Disposable pipette tips.

3.  37 ℃ incubator

Precautions

1.  Do not substitute reagents from one kit to another. Standard, conjugate and microplates are matched for optimal performance. Use only the reagents supplied by manufacturer.

2.  Do not remove microplate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.

3.  Mix all reagents before using.

Remove all kit reagents from refrigerator and allow them to reach room temperature ( 20-25°C)

Materials supplied

Note: Standard (S0 → S5) concentration was followed by: 0 ,5,10,20,40 ,80 U/ml

Reagent preparation

20×wash solution:Dilute with Distilled or deionized water 1:20.

Assay procedure

1.  Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate.

2.  Add standard: Set Standard wells , testing sample wells. Add standard 50μl to standard well.

3.  Add Sample: Add testing sample 10μl then add Sample Diluent 40μl to testing sample well; Blank well doesn’t add anyting.

4.  Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37°C. 

5.  Aspirate each well and wash, repeating the process four times for a total of five washes. Wash by filling each well with Wash Solution (400μl) using a squirt bottle , manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash , remove any remaining Wash Solution by aspirating or decanting. Invert the plate and blot it against clean paper towels.

6.  Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.

7.  Add 50μl Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not

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

8.  Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.

Calculation of results

1. This standard curve is used to determine the amount in an unknown sample. The standard curve is generated by plotting the average O.D. (450 nm) obtained for each of the six standard concentrations on the vertical (Y) axis versus the corresponding concentration on the horizontal (X) axis.

2. First, calculate the mean O.D. value for each standard and sample. All O.D. values , are subtracted by the mean value of the zero standard before result interpretation. Construct the standard curve using graph paper or statistical software.

3. To determine the amount in each sample, first locate the O.D. value on the Y-axis and extend a horizontal line to the standard curve. At the point of intersection , draw a vertical line to the X-axis and read the corresponding concentration.

4. Any variation in operator, pipetting and washing technique, incubation time or temperature , and kit age can cause variation in result. Each user should obtain their own standard curve.

5. The sensitivity by this assay is 1.0 U/ml.

6. Standard curve

Storage ：  2-8℃.

validity ： six months.

FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!