大鼠髓過氧物酶(MPO)ELISA檢測試劑盒

本試劑盒只能用于科學(xué)研究，不得用于醫(yī)學(xué)診斷大ifii氧化物II（MPO）ELISA檢需試劑盒使用說明書檢測原理試劑盒采用雙抗體一步夾心法酶聯(lián)免疫吸附試驗（ELISA）o往預(yù)先色被大鼠髓過氧化物酶（MPO）捕獲抗體的色被微孔中，依歡Jffl入標(biāo)本、標(biāo)準(zhǔn)品、HRP標(biāo)記的檢測抗體，經(jīng)過溫育并御底洗滌。用底物TME顯色，TMB在過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。顧色的深淺和樣品中的大鼠髓過氧化物1（MP0）呈正相關(guān)。用酶標(biāo)儀在450mn玻長下測定吸光度（0D值）,汁算樣品濃度。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，操作過程中避免任何細(xì)胞剌激，收集血液后，3000轉(zhuǎn)離心10分鉀將血清和紅細(xì)胞迅速小心地分離。血漿：EDTA、檸檬酸鹽或肝素抗凝。3000轉(zhuǎn)離心30分鉀取上清。細(xì)胞上清液：3000轉(zhuǎn)離心10分鉀去除顆應(yīng)和聚合物。組級勻漿：將組級Jffl入適量生理鹽水搗碎。3000轉(zhuǎn)離心10分鉀取上清。保存：如果樣本收集后不及時檢測，靖按一歡用量分裝，凍存于-20°C,避免反夏凍融，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Jffl樣器及槍頭：0.5-10uL、2-20uL.20-200uL.200-1000uL37°C恒溫箱操作注意事項試劑盒保存在2-8°C,使用前室溫平衡20分鉀。從冰箱取出的濃縮洗滌液會有結(jié)晶，這屬于正?，F(xiàn)象，水浴Jffl熱使結(jié)晶完全溶解后再使用。實驗中不用的板條應(yīng)立1?0自封袋中，密封（低溫干燥）保存。標(biāo)準(zhǔn)品稀釋液即可視為陰性對照或者空白；預(yù)址理后的樣本尢需稀釋，直接?lOpLl樣即可。嚴(yán)格按照說明書中標(biāo)明的時間、Jffl液量及順序進(jìn)彳丁溫育操作。所有液體組分使用前充分搖勻。號劑盒組成名稱96孔配置48HK置備注微孑LII標(biāo)板12JLx8條12JLx4條尢標(biāo)準(zhǔn)品（480U/L）0.6mL0.6mL按說明書進(jìn)行稀釋標(biāo)準(zhǔn)品稀釋液6mL3mL尢樣本稀釋液6mL3mL尢檢測抗tt-HRP10mL5mL尢20x洗滌緩沖液25mL15mL按說明書進(jìn)行稀釋底物A6mL3mL尢底物B6mL3mL尢終止液6mL3mL無封板膜2張2張尢說明書1份lift無自封袋1個1個無注：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號劑的準(zhǔn)備20x洗滌緩沖液的稀釋：蒸錨水按1：20ft釋，即1份的20x洗滌緩沖液Jffl19份的蒸錨水。洗檢方法手工洗板：甩盡孔內(nèi)液體，毎孔Jffl滿洗滌液，靜置lmin后甩盡孔內(nèi)液體，在吸水紙上抽干，如此洗板5次。自動洗板機：毎孔注入洗液350pL，浸泡lmin,洗板5次。操作步驟從室溫平S20minB的鉗箔袋中取出所需板條，剩余板條用自封袋密封?04°CO設(shè)置標(biāo)準(zhǔn)品孔和樣本孔，標(biāo)準(zhǔn)品孔各Jffl不同濃度的標(biāo)準(zhǔn)品50pL;3?彳寺測樣本孔先Jffl彳寺測樣本lOpL,再Jffl樣本稀釋液40pL;I?后標(biāo)準(zhǔn)品孔和樣本孔中毎JLJfflA辣根ii氧化物IS(HRP)標(biāo)記的檢測抗tt100ML,用封板膜封住反應(yīng)孔，37°C水浴鍋或恒溫箱溫育60mino棄去液體，吸水紙上抽干，毎孔Jffl滿洗滌液，靜置lmin,甩去洗滌液，吸水紙上抽干，如此重夏洗板5&：(也可用洗板機洗板)。毎孔Jffl入底物A、B各50|aL,37°C避光孵育15mino每孔加入終止液50uL,15min內(nèi)，在450nm波長處測定各孔的試劑盒性能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，按曲線方程計算各樣本濃度值。蚌1anil呂Tibconcentradon(X)

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：最低檢測濃度小于1.0U/L。特異性：不與其它可溶性結(jié)構(gòu)類似物交叉反應(yīng)。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。有效期：6個月免責(zé)聲明試劑盒僅供研究使用，不得用于臨床實驗或人體實驗，否則所產(chǎn)生的一切后果，由實驗者承擔(dān)，本公司概不負(fù)責(zé)。嚴(yán)格按照說明書操作，實驗者違反說明書操作，后果由實驗者承擔(dān)。FORRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTICPROCEDURES.

Plasma-CollectplasmausingEDTAorheparinasananticoagulant.CentrifugeRatmyeloperoxidase(MPO)ELISAKitinstructionIntendeduseThisMPOELISAkitisintendedLaboratoryforResearchuseonlyandisnotforuseindiagnosticortherapeuticprocedures.TheStopSolutionchangesthecolorfrombluetoyellowandtheintensityofthecolorismeasuredat450nmusingaspectrophotometer.InordertomeasuretheconcentrationofMPOinthesample,thisMPOELISAKitincludesasetofcalibrationstandards.ThecalibrationstandardsareassayedatthesametimeasthesamplesandallowtheoperatortoproduceastandardcurveofOpticalDensityversusMPOconcentration.TheconcentrationofMPOinthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.SamplecollectionandstoragesSerum-Useaserumseparatortubeandallowsamplestoclotfor30minutesbeforecentrifugationfor10minutesatapproximately3000Xg.Removeserumandassayimmediatelyoraliquotandstoresamplesat-20°Cor-80°C.Avoidrepeatedfreeze-thawcycles

samplesfor30minutesat3000Xgat2-8Cwithin30minutesofcollection.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.Assayprocedureappearuniform,gentlytaptheplatetoensurethoroughmixing.Prepareallreagentsbeforestartingassayprocedure.ItisrecommendedthatallStandardsandSamplesbeaddedinduplicatetotheMicroelisaStripplate.Addstandard:SetStandardwells,testingsamplewells.Addstandard50卩1tostandardwell.AddSample:Addtestingsample1OglThenaddsamplediluent40]iltotestingsamplewell;Blankwelldoesn'taddanyting.Add100]ilofHRP-conjugatereagenttoeachwell,coverwithanadhesivestripandincubatefor60minutesat37°C.Aspirateeachwellandwash,repeatingtheprocessfourtimesforatotaloffivewashes.WashbyfillingeachwellwithWashSolution(400卩1)usingasquirtbottle,manifolddispenserorautowasher.Completeremovalofliquidateachstepisessentialtogoodperformance.Afterthelastwash,removeanyremainingWashSolutionbyaspiratingordecanting.Inverttheplateandblotitagainstcleanpapertowels.AddchromogensolutionA50plandchromogensolutionB50pltoeachwe11.Gentlymixandincubatefor15minutesat379.Protectfromlight.Add50卩1StopSolutiontoeachwell.Thecolorinthewellsshouldchangefrombluetoyellow.IfthecolorinthewellsisgreenorthecolorchangedoesnotReadtheOpticalDensity(O.D.)at450nmusingamicrotiterplatereaderwithin15minutes.CalculationofresultsThisstandardcurveisusedtodeterminetheamountinanunknownsample.ThestandardcurveisgeneratedbyplottingtheaverageO.D.(450nm)obtainedforeachofthesixstandardconcentrationsonthevertical(Y)axisversusthecorrespondingconcentrationonthehorizontal(X)axis.First,calculatethemeanO.D.valueforeachstandardandsample.AllO.D.values,aresubtractedbythemeanvalueofthezerostandardbeforeresultinterpretation.Constructthestandardcurveusinggraphpaperorstati