大鼠(Rat)活性氧簇(ROS)ELISA試劑盒說(shuō)明書_圖文

1、本試劑盒只能用于科學(xué)研究,不得用于醫(yī)學(xué)診斷 大鼠(Rat活性氧簇(ROS ELISA 檢測(cè)試劑盒 使用說(shuō)明書檢測(cè)原理試劑盒采用雙抗體一步夾心法酶聯(lián)免疫吸附試驗(yàn)(ELISA 。往 預(yù)先包被活性氧簇(ROS 抗體的包被微孔中,依次加入標(biāo)本、標(biāo)準(zhǔn) 品、HRP標(biāo)記的檢測(cè)抗體,經(jīng)過(guò)溫育并徹底洗滌。用底物TMB顯色, TMB在過(guò)氧化物酶的催化下轉(zhuǎn)化成藍(lán)色,并在酸的作用下轉(zhuǎn)化成最終 的黃色。顏色的深淺和樣品中的活性氧簇(ROS 呈正相關(guān)。用酶標(biāo) 儀在450nm 波長(zhǎng)下測(cè)定吸光度(OD值 ,計(jì)算樣品濃度。樣品收集、處理及保存方法1. 血清:使用不含熱原和內(nèi)毒素的試管,操作過(guò)程中避免任何細(xì) 胞刺激, 收集血液后

2、, 3000轉(zhuǎn)離心 10分鐘將血清和紅細(xì)胞迅速小心 地分離。2. 血漿:EDTA、 檸檬酸鹽或肝素抗凝。 3000轉(zhuǎn)離心 30分鐘取上清。3. 細(xì)胞上清液:3000轉(zhuǎn)離心 10分鐘去除顆粒和聚合物。4. 組織勻漿:將組織加入適量生理鹽水搗碎。 3000轉(zhuǎn)離心 10分鐘 取上清。5. 保存:如果樣本收集后不及時(shí)檢測(cè),請(qǐng)按一次用量分裝,凍存 于-20, 避免反復(fù)凍融, 在室溫下解凍并確保樣品均勻地充分解凍。 自備物品1. 酶標(biāo)儀(450nm2. 高精度加樣器及槍頭:0.5-10uL、 2-20uL、 20-200uL、 200-1000uL3. 37恒溫箱1. 試劑盒保存在 2-8,使用前室溫平衡

3、 20分鐘。從冰箱取出的 濃縮洗滌液會(huì)有結(jié)晶,這屬于正常現(xiàn)象,水浴加熱使結(jié)晶完全溶解 后再使用。2. 實(shí)驗(yàn)中不用的板條應(yīng)立即放回自封袋中,密封(低溫干燥保 存。3. 濃度為 0的 S0號(hào)標(biāo)準(zhǔn)品即可視為陰性對(duì)照或者空白;按照說(shuō)明 書操作時(shí)樣本已經(jīng)稀釋 5倍,最終結(jié)果乘以 5才是樣本實(shí)際濃度。4. 嚴(yán)格按照說(shuō)明書中標(biāo)明的時(shí)間、加液量及順序進(jìn)行溫育操作。5. 所有液體組分使用前充分搖勻。上海篤瑪生物科技有限公司試劑盒組成名稱 96孔配置 48孔配置 備注 微孔酶標(biāo)板 12孔×8條 12孔×4條 無(wú)標(biāo)準(zhǔn)品 0.3mL*6管 0.3mL*6管 無(wú)樣本稀釋液 6mL 3mL 無(wú)檢測(cè)抗體

4、-HRP 10mL 5mL 無(wú)20×洗滌緩沖液 25mL 15mL 按說(shuō)明書進(jìn)行稀釋 底物 A 6mL 3mL 無(wú)底物 B 6mL 3mL 無(wú)終止液 6mL 3mL 無(wú)封板膜 2張 2張 無(wú)說(shuō)明書 1份 1份 無(wú)自封袋 1個(gè) 1個(gè) 無(wú)注:標(biāo)準(zhǔn)品(S0-S5濃度依次為:0、30、60、120、240、480U/ml試劑的準(zhǔn)備20×洗滌緩沖液的稀釋:蒸餾水按 1:20稀釋,即 1份的 20×洗滌 緩沖液加 19份的蒸餾水。洗板方法1. 手工洗板:甩盡孔內(nèi)液體,每孔加滿洗滌液,靜置 1min 后甩盡 孔內(nèi)液體,在吸水紙上拍干,如此洗板 5次。2. 自動(dòng)洗板機(jī):每孔注入洗

5、液 350L,浸泡 1min,洗板 5次。1. 從室溫平衡 20min 后的鋁箔袋中取出所需板條,剩余板條用自 封袋密封放回 4。 2. 設(shè)置標(biāo)準(zhǔn)品孔和樣本孔,標(biāo)準(zhǔn)品孔各加不同濃度的標(biāo)準(zhǔn)品 50L;3. 樣本孔先加待測(cè)樣本 10L,再加樣本稀釋液 40L;空白孔不 加。4. 除空白孔外,標(biāo)準(zhǔn)品孔和樣本孔中每孔加入辣根過(guò)氧化物酶 (HRP標(biāo)記的檢測(cè)抗體 100L,用封板膜封住反應(yīng)孔,37水浴鍋 或恒溫箱溫育 60min。5. 棄去液體,吸水紙上拍干,每孔加滿洗滌液,靜置 1min,甩去 洗滌液,吸水紙上拍干,如此重復(fù)洗板 5次(也可用洗板機(jī)洗板 。6. 每孔加入底物 A、B 各 50L,37避光

6、孵育 15min。7. 每孔加入終止液 50L,15min 內(nèi),在 450nm 波長(zhǎng)處測(cè)定各孔的 OD 值。結(jié)果判斷繪制標(biāo)準(zhǔn)曲線:在 Excel 工作表中,以標(biāo)準(zhǔn)品濃度作橫坐標(biāo),對(duì)應(yīng) OD 值作縱坐標(biāo),繪制出標(biāo)準(zhǔn)品線性回歸曲線,按曲線方程計(jì)算各樣 本濃度值。上海篤瑪生物科技有限公司試劑盒性能1. 準(zhǔn)確性:標(biāo)準(zhǔn)品線性回歸與預(yù)期濃度相關(guān)系數(shù) R 值,大于等于 0.9900。2. 靈敏度:最低檢測(cè)濃度小于 1.0U /ml。3. 特異性:不與其它可溶性結(jié)構(gòu)類似物交叉反應(yīng)。4. 重復(fù)性:板內(nèi)、板間變異系數(shù)均小于 15%。5. 貯藏:2-8,避光防潮保存。6. 有效期:6個(gè)月 免責(zé)聲明1. 試劑盒僅供研

7、究使用,不得用于臨床實(shí)驗(yàn)或人體實(shí)驗(yàn),否則所 產(chǎn)生的一切后果,由實(shí)驗(yàn)者承擔(dān),本公司概不負(fù)責(zé)。2. 嚴(yán)格按照說(shuō)明書操作,實(shí)驗(yàn)者違反說(shuō)明書操作,后果由實(shí)驗(yàn)者 承擔(dān)。 上海篤瑪生物科技有限公司FOR RESEARCH USE ONLY.NOT FOR USE IN DIAGNOSTIC PROCEDURES.Rat reactive oxygen species (ROSELISA Kit instructionIntended useThis ROS ELISA kit is intended Laboratory for Research use only and is not for use i

8、n diagnostic or therapeutic procedures.The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450nm using a spectrophotometer. In order to measure the concentration of ROS in the sample, this ROS ELISA Kit includes a set of calibration standards. The ca

9、libration standards are assayed at the same time as the samples and allow the operator to produce a standard curve of Optical Density versus ROS concentration. The concentration of ROS in the samples is then determined by comparing the O.D. of the samples to the standard curve.Sample collection and

10、storagesSerum -Use a serum separator tube and allow samples to clot for 30minutes before centrifugation for 10minutes at approximately 3000×g.Remove serum and assay immediately or aliquot and store samples at -20 or -80.Avoid repeated freeze-thaw cyclesPlasma -Collect plasma using EDTA or hepar

11、in as an anticoagulant. Centrifuge samples for 30minutes at 3000×gat 2-8 within 30minutes of collection. Store samples at -20or -80. Avoid repeated freeze-thaw cycles.Cell culture supernates and other biological fluids -Remove particulates by centrifugation and assay immediately or aliquot and

12、store samples at -20or -80. Avoid repeated freeze-thaw cycles.Note:The samples shoule be centrifugated dequately and no hemolysis or granule was allowed.Materials required but not supplied1. Standard microplate reader(450nm2. Precision pipettes and Disposable pipette tips.3. 37 incubatorPrecautions1

13、. 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上海篤瑪生物科技有限公司should be stored at 2-8°Cin their

14、 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°CMaterials suppliedName 96determinations 48determinationsMicroelisa stripplate 12*8strips12*4stripsSample Diluent 6.0ml 3.0mlHRP-Conjugat

15、e reagent 10.0ml 5.0ml20X Wash solution 25ml 15mlChromogen Solution A 6.0ml 3.0mlChromogen Solution B 6.0ml 3.0mlStop Solution 6.0ml 3.0mlClosure plate membrane 22User manual 11Sealed bags 11Standard (S0 S5 concentration was followed by:0,30,60,120,240,480 U/mlReagent preparation20×washsolution

16、:Dilutewith Distilled or deionized water 1:20.Assay procedure1. Prepare all re a g e n t s 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 50lt

17、o standard well.3. Add Sample:Add testing sample 10lthen add Sample Diluent 40lto testing sample well; Blank well doesnt add anyting.4. Add 100lof HRP-conjugate reagent to each well, c over with an adhesive strip and incubate for 60minutes at 37°C.5. Aspirate each well and wash, repeating the p

18、rocess four times for a total of five washes. Wash by filling each well with Wash Solution (400l 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

19、 decanting. Invert the plate and blot it against clean paper towels.6. Add chromogen solution A 50land chromogen solution B 50lto each well. Gently mix and incubate for 15minutes at 37°C.Protect from light.7. Add 50lStop Solution to each well. The color in the wells should change from blue to y

20、ellow. If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.上海篤瑪生物科技有限公司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 averag

21、e 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