本試劑盒只能用于科學(xué)研究，不得用于醫(yī)學(xué)診斷

血漿：EDTA、檸檬酸鹽或肝素?cái)D凝。3000轉(zhuǎn)離心30分鐘取上清。分離。實(shí)驗(yàn)中不用的版條應(yīng)立即放回自封袋中，密封（低溫干燥）保存。分離。實(shí)驗(yàn)中不用的版條應(yīng)立即放回自封袋中，密封（低溫干燥）保存。大鼠髓過(guò)氧化物酶（MPO）ELISA檢測(cè)試劑盒使用說(shuō)明書檢測(cè)原理試劑盒采用雙擠體一步夾心法酶聯(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)擠體，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、處理及保存方法血清：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)胞剌激，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

細(xì)胞上清液：3000轉(zhuǎn)離心10分鐘去除顆粒和聚合物。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。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。自備物品酶標(biāo)儀（450nm）高精度加樣器及槍頭：0.5-10UL、2-20uL、20-200uL、200-1000UL37°C恒溫箱操作注意事項(xiàng)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，這屬于正常現(xiàn)象，水浴加熱使結(jié)晶完全溶解后再使用。

標(biāo)準(zhǔn)品稀釋液即可視為陰性對(duì)照或者空白；預(yù)處理后的樣本無(wú)需稀釋，直接取10ML加樣即可。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。所有液體組分使用前充分搖勻。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x8條12孔x4條無(wú)標(biāo)準(zhǔn)品（480U/L）0.6mL0.6mL按說(shuō)明書進(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)擠體-HRP10mL5mL無(wú)20x洗滌緩沖液25mL15mL按說(shuō)明書進(jìn)行稀釋底物A6mL3mL無(wú)底物B6mL3mL無(wú)終止液6mL3mL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)品用標(biāo)準(zhǔn)品稀釋液依次稀釋為：480、240、120、60、30、15U/L試劑的準(zhǔn)備20x洗滌緩沖液的稀釋：蒸餾水按1：20稀釋，即1份的20x洗滌緩洗板方法手工洗板：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，在吸水級(jí)上拍干，如此洗版5次。自動(dòng)洗板機(jī)：每孔注入洗液350ML，浸泡1min,洗板5次。操作步驟從室溫平衡20min后的鋁箔袋中取出所需版條，剩余版條用自封袋密封放回4°C。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50ML；待測(cè)樣本孔先加待測(cè)樣本10mL,再加樣本稀釋液40ML；隨后標(biāo)準(zhǔn)品孔和樣本孔中每孔加入辣根過(guò)氧化物?（HRP）標(biāo)記的檢測(cè)擠體100mL，用封板膜封住反應(yīng)孔，37°C水浴鍋或恒溫箱溫育60min。沖液加19份的蒸餾水。每孔加入底物A、B各50mL,沖液加19份的蒸餾水。每孔加入底物A、B各50mL,37°C避光孵育15min。每孔加入終止液50兒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。Y3.02.01.00.50.▲■k_->standardsconcentration(X)試劑盒性能準(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。靈敏度：最低檢測(cè)濃度水于1.0u/l。特異性：不與其它可落性結(jié)構(gòu)類似物交叉反應(yīng)。重復(fù)性：板內(nèi)、版間變異系數(shù)均小于15%。貯藏：2-8°C,避光防潮保存。有效期：6個(gè)月免責(zé)聲明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)。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)。FORRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTICPROCEDURES.Ratmyeloperoxidase(MPO)ELISAKitinstructionIntendeduseThisMPOELISAkitisintendedLaboratoryforResearchuseonlyandisnotforuseindiagnosticortherapeuticprocedures.TheStopSolutionchangesthecolorfrombluetoyellowandtheintensityofthecolorismeasuredat450nmusingaspectrophotometer.InordertomeasuretheconcentrationofMPOinthesample,thisMPOELISAKitincludesasetofcalibrationstandards.ThecalibrationstandardsareassayedatthesametimeasthesamplesandallowtheoperatortoproduceastandardcurveofOpticalDensityversusMPOconcentration.TheconcentrationofMPOinthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.SamplecollectionandstoragesSerum-Useaserumseparatortubeandallowsamplestoclotfor30minutesbeforecentrifugationfor10minutesatapproximately3000Xg.Removeserumandassayimmediatelyoraliquotandstoresamplesat-20°Cor-80°C.Avoidrepeatedfreeze-thawcyclesPlasma-CollectplasmausingEDTAorheparinasananticoagulant.Centrifugesamplesfor30minutesat3000Xgat2-8Cwithin30minutesofcollection.Storesamplesat-20Cor-80C.Avoidrepeatedfreeze-thawcycles.Cellculturesupernatesandotherbiologicalfluids-Removeparticulatesbycentrifugationandassayimmediatelyoraliquotandstoresamplesat-20Cor-80C.Avoidrepeatedfreeze-thawcycles.Note:Thesamplesshoulebecentrifugateddequatelyandnohemolysisorgranulewasallowed.MaterialsrequiredbutnotsuppliedStandardmicroplatereader(450nm)PrecisionpipettesandDisposablepipettetips.37CincubatorPrecautionsDonotsubstitutereagentsfromonekittoanother.Standard,conjugateandmicroplatesarematchedforoptimalperformance.Useonlythereagentssuppliedbymanufacturer.Donotremovemicroplatefromthestoragebaguntilneeded.Unusedstripsshouldbestoredat2-8°Cintheirpouchwiththedesiccantprovided.

Mixallreagentsbeforeusing.Removeallkitreagentsfromrefrigeratorandallowthemtoreachroomtemperature(20-25°C)MaterialssuppliedName96determinations48determinationsMicroelisastripplate12*8strips12*4stripsStandard(480U/L)0.6ml0.6mlStandarddiluent6.0ml3.0mlSamplediluent6.0ml3.0mlHRP-Conjugatereagent10.0ml5.0ml20XWashsolution25ml15mlChromogenSolutionA6.0ml3.0mlChromogenSolutionB6.0ml3.0mlStopSolution6.0ml3.0mlClosureplatemembrane22Usermanual11Sealedbags11Note:StandarddiluentwithStandarddiluentconcentrationwasfollowedby:480、240、120、60、30、15U/LReagentpreparation20xwashsolution:DilutewithDistilledordeionizedwater1:20.Prepareallreagentsbeforestartingassayprocedure.ItisrecommendedthatallStandardsandSamplesbeaddedinduplicatetotheMicroelisaStripplate.Addstandard:SetStandardwells,testingsamplewells.Addstandard50gltostandardwell.AddSample:Addtestingsample10glThenaddsamplediluent40gltotestingsamplewell;Blankwelldoesn’taddanyting.Add100glofHRP-conjugatereagenttoeachwell,coverwithanadhesivestripandincubatefor60minutesat37°C.Aspirateeachwellandwash,repeatingtheprocessfourtimesforatotaloffivewashes.WashbyfillingeachwellwithWashSolution(400gl)usingasquirtbottle,manifolddispenserorautowasher.Completeremovalofliquidateachstepisessentialtogoodperformance.Afterthelastwash,removeanyremainingWashSolutionbyaspiratingordecanting.Inverttheplateandblotitagainstcleanpapertowels.AddchromogensolutionA50glandchromogensolutionB50gltoeachwell.Gentlymixandincubatefor15minutesat37°C.Protectfromlight.AssayprocedureAdd50glStopSolutiontoeachwell.Thecolorinthewellsshouldchangefrombluetoyellow.Ifthecolorinthewellsisgreenorthecolorchangedoesnotappearuniform,gentlytaptheplatetoensurethoroughmixing.AssayprocedureReadtheOpticalDensity(O.D.)at450nmusingamicrotiterplatereaderwithin15minutes.CalculationofresultsThisstandardcurveisusedtodeterminetheamountinanunknownsample.ThestandardcurveisgeneratedbyplottingtheaverageO.D,(450nm)obtainedforeachofthesixstandardconcentrationsonthevertical(Y)axisversusthecorrespondingconcentrationonthehorizontal(X)axis.First,calculatethemeanO.D.valueforeachstandardandsample.AllO.D.values,aresubtractedbythemeanvalueofthezerostandardbeforeresultinterpretation.Constructthestanda