大鼠髓过氧化物酶ELISA检测试剂盒

本试剂盒只能用于科学研究，不得用于医学诊断2. 血浆：EDTA、柠檬酸盐或肝素抗凝。3000转离心30分钟取上清。离。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。大鼠髓过氧化物酶（MPO） 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 先包被大鼠髓过氧化物KMPO ）捕获抗体的包被微孔中，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 转化成最终的黄色。颜色的深浅和样品中的大鼠髓过氧化物酶（MPO） 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 -20C，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. 37C恒温箱操作注意事项1试剂盒保存在2-8 C，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。释，直接取10mL加样即可。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x8 条12 孔x4 条无标准品（480U/L ）0.6mL0.6mL按说明书进行稀释标准品稀释液6mL3mL无样本稀释液6mL3mL无检测抗体-HRP10mL5mL无20x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：标准品用标准品稀释液依次稀释为：480、240、120、60、30、15U/L试剂的准备20x洗涤缓冲液的稀释：蒸馏水按1： 20稀释，即1份的20x洗涤缓 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350 ML,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4C。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50mL；3. 待测样本孔先加待测样本10 uL,再加样本稀释液40 ML；4随后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100uL，用封板膜封住反应孔，37C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）。冲液加19份的蒸馏水。6每孔加入底物A、B各50 uL, 37 C避光孵育15mino7每孔加入终止液50ML, 15min内，在450nm波长处测定各孔的OD 试剂盒性能值。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0D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。standards concentration (X)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：最低检测浓度小于1.0u/l。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：2-8C，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。FORRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTICPROCEDURES.Ratmyeloperoxidase(MPO)ELISAKitinstructionIntendeduseNote：ThesamplesshoulebecentrifugateddequatelyandnohemolysisorgranulewasalloThisMPOELISAkitisintendedLaboratoryforResearchuseonlyandisnotforuseindiagn osticortherapeuticprocedures.TheStopSolutionchangesthecolorfrombluetoyellowan dtheintensityofthecolorismeasuredat450nmusingaspectrophotometer.Inordertomeas uretheconcentrationofMPOinthesample,thisMPOELISAKitincludesasetofcalibratio nstandards.Thecalibrationstandardsareassayedatthesametimeasthesamplesandallowt heoperatortoproduceastandardcurveofOpticalDensityversusMPOconcentration.Thec oncentrationofMPOinthesamplesisthendeterminedbycomparingtheO.D.ofthesample stothestandardcurve.SamplecollectionandstoragesSerum-Useaserumseparatortubeandallowsamplestoclotfor30minutesbeforecentrifu gationforl0minutesatapproximately3000xg.Removeserumandassayimmediatelyoral iquotandstoresamplesat-20Cor-80C.Avoidrepeatedfreeze-thawcyclesPlasma-CollectplasmausingEDTAorheparinasananticoagulant.Centrifugesamplesf or30minutesat3000xgat2-8 C within30minutesofcollection.Storesamplesat-20C or-8 0 C .Avoidrepeatedfreeze-thawcycles.Cellculturesupernatesandotherbiologicalfluids-Removeparticulatesbycentrifugationandassayimmediatelyoraliquotandstoresamplesat-20C or-80 C .Avoidrepe atedfreeze-thawcycles.Awed.Materialsrequiredbutnotsupplied1.Standardmicroplatereader(450nm)2. PrecisionpipettesandDisposablepipettetips.3.37 C incubatorPrecautions1. Donotsubstitutereagentsfromonekittoanother.Standard,conjugateandmicroplatesar ematchedforoptimalperformance.Useonlythereagentssuppliedbymanufacturer.2. Donotremovemicroplatefromthestoragebaguntilneeded.Unusedstripsshouldbestore dat2-8Cintheirpouchwiththedesiccantprovided.3. Mixallreagentsbeforeusing.Removeallkitreagentsfromrefrigeratorandallowthemtoreachroomtemperature(20-25C)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.Assayprocedure1. Prepareallre a gent sbeforestartingassayprocedure.ItisrecommendedthatallStandar dsandSamplesbeaddedinduplicatetotheMicroelisaStripplate.2. Addstandard:SetStandardwells,testingsamplewellsAddstandard50pltostandardweappearuniform,gentlytaptheplatetoensurethoroughmixing.8.ReadtheOpticalDensity(O.D.)at450nmusingamicrotiterplatereaderwithin15minutes.Calculationofresults1. Thisstandardcurveisusedtodeterminetheamountinanunknownsample.Thestanda ll.3. AddSample:Addtestingsample10ylThenaddsamplediluent40yltotestingsamplewel l;Blankwelldoesntaddanyting.4. Add100plofHRP-conjugatereagenttoeachwell,coverwithanadhesivestripandincuba tefor60minutesat37C.5. Aspirateeachwellandwash,repeatingtheprocessfourtimesforatotaloffivewashes.Wa shbyfillingeachwellwithWashSolution(40gl)usingasquirtbottle,manifolddispensero rautowasher.Completeremovalofliquidateachstepisessentialtogoodperformance.Afte rthelastwash,removeanyremainingWashSolutionbyaspiratingordecanting.Invertthepl ateandblotitagainstcleanpapertowels.6. AddchromogensolutionA50ylandchromogensolutionB50pltoeachwell.Gentlymix andincubatefor15minutesat37C.Protectfromlight.7. Add50plStopSolutiontoeachwell.Thecolorinthewellsshouldchangefrombluetoyell ow.IfthecolorinthewellsisgreenorthecolorchangedoesnotrdcurveisgeneratedbyplottingtheaverageO.D.(450nm)obtainedforeachofthesixs tandardconcentrationsonthevertical(Y)axisversusthecorrespondingconcentratio nonthehorizontal(X)axis.2. First,calculatethemeanO.D.valueforeachstandardandsample.AllO.D.values,are subtractedbythemeanvalueofthezerostandardbeforeresultinterpretation.Constru ctthestandardcurveusinggraphpaperorstatisticalsoftware.3. Todeterminetheamountineachsample,firstlocatetheO.D.valueontheY-axisandex tendahorizontallinetothestandardcurve.Atthepointofintersection,drawaverticalli netotheX-axisandreadthecorrespondingconcentration.4. Anyvariationinoperator,pipettingandwashingtechnique,incubationtimeortempe rature,andkitagecancausevariationinresult.Eachusershouldobtaintheirownstand ardcurve.Storage： 2-8C.validity： sixmonths.FORRESEARCHUSEONLY;NOTFORTHERAPEUTICORDIAGNOSTICAPPLICATIONS!PLEASEREADTHROUGHENTIREPROCEDUREBEFOREBEGINNING!5. Thesensitivitybythisassayisl.OU/L6. Standardcurvestandards concentration (X)