大鼠髓過(guò)氧化物酶(MPO)ELISA檢測(cè)試劑盒

1、分離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。本試劑盒只能用于科學(xué)研究，不得用于醫(yī)學(xué)診斷大鼠髓過(guò)氧化物酶（MPO） ELISA檢測(cè)試劑盒使用說(shuō)明書(shū)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) 先包被大鼠髓過(guò)氧化物酶（MPO）捕獲抗體的包被微孔中，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大鼠髓過(guò)氧化物酶（MPO） 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

2、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，3000轉(zhuǎn)離心10分鐘將血清和紅細(xì)胞迅速小心地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 C,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（450nm）2. 高精度加樣器及槍頭：0.5-10uL、2-20uL、20-200uL、200-1000uL3. 37C恒

3、溫箱操作注意事項(xiàng)1. 試劑盒保存在2-8 C,使用前室溫平衡20分鐘。從冰箱取出的 濃縮洗滌液會(huì)有結(jié)晶，這屬于正?，F(xiàn)象，水浴加熱使結(jié)晶完全溶解 后再使用。沖液加19份的蒸餾水6. 每孔加入底物A、B各50卩L，37C避光孵育15min稀釋?zhuān)苯尤?0卩L加樣即可4. 嚴(yán)格按照說(shuō)明書(shū)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試劑盒組成名稱(chēng)96孔配置48孔配置備注微孔酶標(biāo)板12孔X8條12孔X4條無(wú)標(biāo)準(zhǔn)品（480 U/L ）0.6mL0.6mL按說(shuō)明書(shū)進(jìn)行稀釋標(biāo)準(zhǔn)品稀釋液6mL3mL無(wú)樣本稀釋液6mL3mL無(wú) ：檢測(cè)抗體-HRP10mL5mL無(wú)20 X洗滌緩沖液25mL

4、15mL按說(shuō)明書(shū)進(jìn)行稀釋底物A6mL3mL無(wú) ：底物B6mL3mL無(wú)終止液6mL3mL無(wú)圭寸板膜2張2張無(wú)說(shuō)明書(shū)1份1份無(wú)自封袋1個(gè)1個(gè)無(wú)注：標(biāo)準(zhǔn)品用標(biāo)準(zhǔn)品稀釋液依次稀釋為：480、240、120、60、30、15 U/L試劑的準(zhǔn)備20 X洗滌緩沖液的稀釋?zhuān)赫麴s水按1: 20稀釋?zhuān)?份的20 X洗滌緩1. 手工洗板：甩盡孔內(nèi)液體，每孔加滿(mǎn)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)：每孔注入洗液 350卩L,浸泡1min,洗板5次。操作步驟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°C。2. 設(shè)置標(biāo)準(zhǔn)品孔

5、和樣本孔，標(biāo)準(zhǔn)品孔各加不同濃度的標(biāo)準(zhǔn)品50卩L;3. 待測(cè)樣本孔先加待測(cè)樣本10卩L，再加樣本稀釋液40卩L;4. 隨后標(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100卩L,用封板膜封住反應(yīng)孔，37C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(mǎn)洗滌液，靜置 1mi n，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）。Serum - Use a serum separator tube and allow samples to clot for 30 minutes7. 每孔加入終止液50卩L, 15min內(nèi)，在450nm

6、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(xiàn)：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(xiàn)性回歸曲線(xiàn)，按曲線(xiàn)方程計(jì)算各樣 本濃度值。試劑盒性能1. 準(zhǔn)確性：標(biāo)準(zhǔn)品線(xiàn)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1.0 U/L。3. 特異性：不與其它可溶性結(jié)構(gòu)類(lèi)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 C,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(shū)操作，實(shí)驗(yàn)者違

7、反說(shuō)明書(shū)操作，后果由實(shí)驗(yàn)者 承擔(dān)。FOR RESEARCH USE ONLY.NOT FOR USE IN DIAGNOSTIC PROCEDURES.Rat myeloperoxidase (MPO) ELISA Kit instruction Intended useThis MPO ELISA kit is inten ded Laboratory for Research use only and is not for use in diagnostic or therapeutic procedures. The Stop Solution changes the color fro

8、m blue to yellow and the inten sity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of MPO in the sample, this MPO ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples and allow th

9、e operator to produce a standard curve of Optical Density versus MPO concentration. The concentration of MPO in the samples is then determined by comparing the O.D. of the samples to the sta ndard curve.Sample collection and storagesbefore cen trifugati on for 10 minu tes at approximately 3000 g. Re

10、move seruMa nd assay immediately or aliquot and store samples at -20°C or -80 °C .Avoid repeatedfreeze-thaw cyclesPlasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes at 3000 g at 2-8<°C within 30 minutes of collection. Store samples

11、at -20C or -80 C . Avoid repeated freeze-thaw cycles.Cell culture supernates and other biological fluids Remove particulatesby centrifugation and assay immediately or aliquot and store samples at -20 C or -80 C . Avoid repeated freeze-thaw cycles.:三 Note:The samples shoule be cen trifugated dequatel

12、y and no hemolysisor granule was allowed.Materials required but not supplied1. Stan dard microplate reader(450nm)2. Precisi on pipettes and Disposable pipette tips.3. 37 C in cubatorshould be stored at 2-8 C in theirpouch with the desiccant provided.3. Mix all reage nts before using.Remove all kit r

13、eage nts from refrigerator and allow them to reach room temperature(20-25 C°Materials suppliedName96determi natio ns48 determ in ati onsMicroelisa stripplate12*8strips12*4stripsStandard ( 480 U/L)0.6ml0.6mlSta ndard dilue nt6.0ml3.0mlSample dilue nt6.0ml3.0mlHRP-Conjugate reage nt10.0ml5.0ml20X

14、 Wash solution25ml15mlChromoge n Soluti on A6.0ml3.0mlChromoge n Soluti on B6.0ml3.0mlStop Solution6.0ml3.0mlClosure plate membra ne22User manual11Sealed bags11Precautions1. Do not substitute reagents from one kit to another. Standard, conjugate and microplates are matched for optimal performa nee.

15、Use only the reage nts supplied by manu facturer.2. Do not remove microplate from the storage bag until needed. Unused stripsNote: Stan dard dilue nt with Stan dard dilue nt concen trati on was followed by:480、240、120、60、30、15 U/LReagent preparation20 <wash solution:Dilute with Distilled or deion

16、ized water 1:20.Assay procedure1. Prepare all reage nts before start ing assay procedure. It is recomme nded thatall Stan dards and Samples be added in duplicate to the Microelisa Stripplate.2. Add standard: Set Standard wells, testing sample wells. Add standard50 卩 l tosta ndard well.3. Add Sample:

17、 Add testing sample 1 0 卩 Then add sample diluent 4 0 l to testing sample well; Blank well doesn 'add anyting.4. Add 10 0(!of HRP-conjugate reage nt to each well, cover with an adhesive stripand incubate for 60 minutes at 37 C.°5. Aspirate each well and wash, repeat ing the process four tim

18、es for a total of five appear uniform, gen tly tap the plate to en sure thorough mixing.8. Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 min utes.Calculation of results1. This sta ndard curve is used to determ ine the amount in an unknown sample.The sta ndard cu

19、rve is gen erated by plott ing the average O.D. (450 nm) obtained for each of the six standard concentrations on the vertical (Y) axis versus the corresp onding concen trati on on the horiz on tal (X) axis.2. First, calculate the mean O.D. value for each standard and sample. All O.D. values, are sub

20、tracted by the mean value of the zero standard before result in terpretati on. Con struct the sta ndard curve using graph paper or statistical washes. Wash by filli ng each well with Wash Soluti on (400 M) using a squirt bottle, manifold dispenser or autowasher. Complete removal of liquid at each st

21、ep is essential to good performanee. After the last wash, remove any remaining Wash Soluti on by aspirat ing or deca nti ng. Invert the plate and blot it aga inst clea n paper towels.6. Add chromogen solution A 50m l and chromogen solution B 50mGen tly mix and in cubate for 15 mi nu tes at 37 C. Protect from light.7. Add 50 M Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is gree n or the