elisa实验中的洗板方法，小鼠心肌肌钙蛋白Ⅰ（cTn-Ⅰ）使用说明书
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小鼠（Mouse）心肌肌钙蛋白Ⅰ（cTn-Ⅰ）ELISA检测试剂盒使用说明书检测原理试剂盒采用双抗体一步夹心法酶联免疫吸附试验（ELISA）。往预先包被心肌肌钙蛋白Ⅰ（cTn-Ⅰ）抗体的包被微孔中，依次加入标本、标准品、HRP标记的检测抗体，经过温育并彻底洗涤。用底物TMB显色，TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成Z终的黄色。颜色的深浅和样品中的心肌肌钙蛋白Ⅰ（cTn-Ⅰ）呈正相关。用酶标仪在450nm波长下测定吸光度（OD值），计算样品浓度。样品收集、处理及保存方法1.血清：使用不含热原和内毒素的试管，操作过程中避免任何细胞刺激，收集血液后，3000转离心10分钟将血清和红细胞迅速小心地分离。2.血浆：EDTA、柠檬酸盐或肝素抗凝。3000转离心30分钟取上清。3.细胞上清液：3000转离心10分钟去除颗粒和聚合物。4.组织匀浆：将组织加入适量生理盐水捣碎。3000转离心10分钟取上清。5.保存：如果样本收集后不及时检测，请按一次用量分装，冻存于-20℃，避免反复冻融，在室温下解冻并确保样品均匀地充分解冻。自备物品1.酶标仪（450nm）2.高精度加样器及枪头：0.5-10uL、2-20uL、20-200uL、200-1000uL3.37℃恒温箱操作注意事项1.试剂盒保存在2-8℃，使用前室温平衡20分钟。从冰箱取出的浓缩洗涤液会有结晶，这属于正常现象，水浴加热使结晶完全溶解后再使用。2.实验中不用的板条应立即放回自封袋中，密封（低温干燥）保存。3.浓度为0的S0号标准品即可视为阴性对照或者空白；按照说明书操作时样本已经稀释5倍，Z终结果乘以5才是样本实际浓度。4.严格按照说明书中标明的时间、加液量及顺序进行温育操作。5.所有液体组分使用前充分摇匀。试剂盒组成名称96孔配置48孔配置备注微孔酶标板12孔×8条12孔×4条无标准品0.3mL*6管0.3mL*6管无样本稀释液6mL3mL无检测抗体-HRP10mL5mL无20×洗涤缓冲液25mL15mL按说明书进行稀释底物A6mL3mL无底物B6mL3mL无终止液6mL3mL无封板膜2张2张无说明书1份1份无自封袋1个1个无注：标准品（S0-S5）浓度依次为：0、15、30、60、120、240pg/mL试剂的准备20×洗涤缓冲液的稀释：蒸馏水按1：20稀释，即1份的20×洗涤缓冲液加19份的蒸馏水。洗板方法1.手工洗板：甩尽孔内液体，每孔加满洗涤液，静置1min后甩尽孔内液体，在吸水纸上拍干，如此洗板5次。2.自动洗板机：每孔注入洗液350μL，浸泡1min，洗板5次。操作步骤1.从室温平衡20min后的铝箔袋中取出所需板条，剩余板条用自封袋密封放回4℃。2.设置标准品孔和样本孔，标准品孔各加不同浓度的标准品50μL；3.样本孔先加待测样本10μL，再加样本稀释液40μL；空白孔不加。4.除空白孔外，标准品孔和样本孔中每孔加入辣根过氧化物酶（HRP）标记的检测抗体100μL，用封板膜封住反应孔，37℃水浴锅或恒温箱温育60min。5.弃去液体，吸水纸上拍干，每孔加满洗涤液，静置1min，甩去洗涤液，吸水纸上拍干，如此重复洗板5次（也可用洗板机洗板）。6.每孔加入底物A、B各50μL，37℃避光孵育15min。7.每孔加入终止液50μL，15min内，在450nm波长处测定各孔的OD值。结果判断绘制标准曲线：在Excel工作表中，以标准品浓度作横坐标，对应OD值作纵坐标，绘制出标准品线性回归曲线，按曲线方程计算各样本浓度值。试剂盒性能1.准确性：标准品线性回归与预期浓度相关系数R值，大于等于0.9900。2.灵敏度：Zdi检测浓度小于1.0pg/mL。3.特异性：不与其它可溶性结构类似物交叉反应。4.重复性：板内、板间变异系数均小于15%。5.贮藏：2-8℃，避光防潮保存。6.有效期：6个月免责声明1.试剂盒仅供研究使用，不得用于临床实验或人体实验，否则所产生的一切后果，由实验者承担，本公司概不负责。2.严格按照说明书操作，实验者违反说明书操作，后果由实验者承担。FORFORRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTICPROCEDURES.MousecardiactroponinⅠ（cTn-Ⅰ）ELISAKitinstructionIntendeduseThiscTn-ⅠELISAkitisintendedLaboratoryforResearchuseonlyandisnotforuseindiagnosticortherapeuticprocedures.TheStopSolutionchangesthecolorfrombluetoyellowandtheintensityofthecolorismeasuredat450nmusingaspectrophotometer.InordertomeasuretheconcentrationofcTn-Ⅰinthesample,thiscTn-ⅠELISAKitincludesasetofcalibrationstandards.ThecalibrationstandardsareassayedatthesametimeasthesamplesandallowtheoperatortoproduceastandardcurveofOpticalDensityversuscTn-Ⅰconcentration.TheconcentrationofcTn-IinthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.SamplecollectionandstoragesSerum-Useaserumseparatortubeandallowsamplestoclotfor30minutesbeforecentrifugationfor10minutesatapproximately3000×g.Removeserumandassayimmediatelyoraliquotandstoresamplesat-20℃or-80℃.Avoidrepeatedfreeze-thawcyclesPlasma-CollectplasmausingEDTAorheparinasananticoagulant.Centrifugesamplesfor30minutesat3000×gat2-8℃within30minutesofcollection.Storesamplesat-20℃or-80℃.Avoidrepeatedfreeze-thawcycles.Cellculturesupernatesandotherbiologicalfluids-Removeparticulatesbycentrifugationandassayimmediatelyoraliquotandstoresamplesat-20℃or-80℃.Avoidrepeatedfreeze-thawcycles.Note:Thesamples首lebecentrifugateddequatelyandnohemolysisorgranulewasallowed.Materialsrequiredbutnotsupplied1.Standardmicroplatereader（450nm）2.PrecisionpipettesandDisposablepipettetips.3.37℃incubatorPrecautions1.Donotsubstitutereagentsfromonekittoanother.Standard,conjugateandmicroplatesarematchedforoptimalperformance.Useonlythereagentssuppliedbymanufacturer.2.Donotremovemicroplatefromthestoragebaguntilneeded.Unusedstrips首ldbestoredat2-8°Cintheirpouchwiththedesiccantprovided.3.Mixallreagentsbeforeusing.Removeallkitreagentsfromrefrigeratorandallowthemtoreachroomtemperature（20-25°C）MaterialssuppliedName96determinations48determinationsMicroelisastripplate12*8strips12*4stripsStandard0.3ml*6tubes0.3ml*6tubesSampleDiluent6.0ml3.0mlHRP-Conjugatereagent10.0ml5.0ml20XWashsolution25ml15mlChromogenSolutionA6.0ml3.0mlChromogenSolutionB6.0ml3.0mlStopSolution6.0ml3.0mlClosureplatemembrane22Usermanual11Sealedbags11Note:Standard（S0→S5）concentrationwasfollowedby:0,15,30,60,120,240pg/ml.Reagentpreparation20×washsolution:DilutewithDistilledordeionizedwater1:20.Assayprocedure1.Prepareallreagentsbeforestartingassayprocedure.ItisrecommendedthatallStandardsandSamplesbeaddedinduplicatetotheMicroelisaStripplate.2.Addstandard:SetStandardwells,testingsamplewells.Addstandard50μltostandardwell.3.AddSample:Addtestingsample10μlthenaddSampleDiluent40μltotestingsamplewell;Blankwelldoesn’taddanyting.4.Add100μlofHRP-conjugatereagenttoeachwell,coverwithanadhesivestripandincubatefor60minutesat37°C.5.Aspirateeachwellandwash,repeatingtheprocessfourtimesforatotaloffivewashes.Washbyfil领eachwellwithWashSolution（400μl）usingasquirtbottle,manifolddispenserorautowasher.Completeremovalofliquidateachstepisessentialtogoodperformance.Afterthelastwash,removeanyremainingWashSolutionbyaspiratingordecanting.Inverttheplateandblotitagainstcleanpapertowels.6.AddchromogensolutionA50μlandchromogensolutionB50μltoeachwell.Gentlymixandincubatefor15minutesat37°C.Protectfromlight.7.Add50μlStopSolutiontoeachwell.Thecolorinthewells首ldchangefrombluetoyellow.Ifthecolorinthewellsisgreenorthecolorchangedoesnotappearuniform,gentlytaptheplatetoensurethoroughmixing.8.ReadtheOpticalDensity（O.D.）at450nmusingamicrotiterplatereaderwithin15minutes.Calculationofresults1.Thisstandardcurveisusedtodeterminetheamountinanunknownsample.ThestandardcurveisgeneratedbyplottingtheaverageO.D.（450nm）obtainedforeachofthesixstandardconcentrationsonthevertical（Y）axisversusthecorrespondingconcentrationonthehorizontal（X）axis.2.First,calculatethemeanO.D.valueforeachstandardandsample.AllO.D.values,aresubtractedbythemeanvalueofthezerostandardbeforeresultinterpretation.Constructthestandardcurveusinggraphpaperorstatisticalsoftware.3.Todeterminetheamountineachsample,firstlocatetheO.D.valueontheY-axisandextendahorizontallinetothestandardcurve.Atthepointofintersection,drawaverticallinetotheX-axisandreadthecorrespondingconcentration.4.Anyvariationinoperator,pipettingandwashingtechnique,incubationtimeortemperature,andkitagecancausevariationinresult.Eachuser首ldobtaintheirownstandardcurve.5.Thesensitivitybythisassayis1.0pg/ml6.StandardcurveStorage：2-8℃.validity：sixmonths.FORRESEARCHUSEONLY;NOTFORTHERAPEUTICORDIAGNOSTICAPPLICATIONS!PLEASEREADTHROUGHENTIREPROCEDUREBEFOREBEGINNING!
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