大鼠髓過氧化物酶ELISA檢測試劑盒

本試劑盒只能用于科學研究，不得用于醫(yī)學診斷

血漿：EDTA、檸檬酸鹽或肝素擠凝。3000轉(zhuǎn)離心30分鐘取上清。分離。實驗中不用的版條應立即放回自封袋中，密封（低溫干燥）保存。分離。實驗中不用的版條應立即放回自封袋中，密封（低溫干燥）保存。大鼠髓過氧化物酶（MPO）ELISA檢測試劑盒使用說明書檢測原理試劑盒采用雙擠體一步夾心法酶聯(lián)免疫吸附試驗（ELISA）。往預先包被大鼠髓過氧化物酶（MPO）捕獲抗體的包被微孔中，依次加入標本、標準品、HRP標記的檢測擠體，經(jīng)過溫育并徹底洗滌。用底物TMB顯色，TMB在過氧化物酶的催化下轉(zhuǎn)化成藍色，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氧化物酶（MPO）呈正相關。用酶標儀在450nm波長下測定吸光度（OD值），計算樣品濃度。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胞剌激，收集血液后，3000轉(zhuǎn)離心10分鐘將血清和紅細胞迅速小心地

細胞上清液：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檢測，請按一次用量分裝，凍存于-20°C，避免反復凍融，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。

標準品稀釋液即可視為陰性對照或者空白；預處理后的樣本無需稀釋，直接取10ML加樣即可。嚴格按照說明書中標明的時間、加液量及順序進行溫育操作。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板12孔x8條12孔x4條無標準品（480U/L）0.6mL0.6mL按說明書進行稀釋標準品稀釋液6mL3mL無樣本稀釋液6mL3mL無檢測擠體-HRP10mL5mL無20x洗滌緩沖液25mL15mL按說明書進行稀釋底物A6mL3mL無底物B6mL3mL無終止液6mL3mL無封板膜2張2張無說明書1份1份無自封袋1個1個無注：標準品用標準品稀釋液依次稀釋為：480、240、120、60、30、15U/L試劑的準備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，在吸水級上拍干，如此洗版5次。自動洗板機：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。設置標準品孔和樣本孔，標準品孔各加不同濃度的標準品50ML；待測樣本孔先加待測樣本10mL,再加樣本稀釋液40ML；隨后標準品孔和樣本孔中每孔加入辣根過氧化物?（HRP）標記的檢測擠體100mL，用封板膜封住反應孔，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，繪制出標準品線性回歸曲線，按曲線方程計算各樣本濃度值。Y3.02.01.00.50.▲■k_->standardsconcentration(X)試劑盒性能準確性：標準品線性回歸與預期濃度相關系數(shù)R值，大于等于0.9900。靈敏度：最低檢測濃度水于1.0u/l。特異性：不與其它可落性結(jié)構(gòu)類似物交叉反應。重復性：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個月免責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，本公司概不負責。嚴格按照說明書操作，實驗者違反說明書操作，后果由實驗者承擔。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