人肌营养不良蛋白（DMD）试剂盒（ELISA）

HumanDMDELISAKitForthequantitativeinvitrodeterminationofHumanDystrophinconcentrationsinserum-plasma-celiacfluid-tissuehomogenate-bodyfluidFORLABORATORYRESEARCHUSEONLY.NOTFORUSEINDIAGNOSTICPROCEDURES.Thispackageinsertmustbereadinitsentiretybeforeusingthisproduct.ELISAENZYMELINKEDIMMUNOSORBENTASSAY

IDDSEANDTESTPRINCIPLEThisDMDELISAkitisintendedLaboratoryforResearchuseonlyandisnotforuseindiagnosticortherapeuticprocedures.TheStopSolutionchangesthecolorfrombluetoyellowandtheintensityofthecolorismeasuredat450nmusingaspectrophotometer.InordertomeasuretheconcentrationofDMDinthesample,thisDMDELISAKitincludesasetofcalibrationstandards.ThecalibrationstandardsareassayedatthesametimeasthesamplesandallowtheoperatortoproduceastandardcurveofOpticalDensityversusDMDconcentration.TheconcentrationofDMDinthesamplesisthendeterminedbyparingtheO.D.ofthesamplestothestandardcurve.SAMPLECOLLECTIONANDSTORAGESSerum-Useaserumseparatortubeandallowsamplestoclotfor2hoursatroomtemperatureorovernightat4℃beforecentrifugationfor20minutesatapproximately2000×g.Removeserumandassayimmediatelyoraliquotandstoresamplesat-20℃.Plasma-Collectplasmausingheparinasananticoagulant.Centrifugesamplesfor30minutesat2000×gat2-8℃within30minutesofcollection.Storesamplesat-20℃.Avoidrepeatedfreeze-thawcycles.Cellculturesupernates,tissuehomogenateandotherbiologicalfluids-Removeparticulatesbycentrifugationandassayimmediatelyoraliquotandstoresamplesat-20℃.Avoidrepeatedfreeze-thawcycles.Note:Thesamplesshoulebecentrifugateddequatelyandnohemolysisorgranulewasallowed.MATERIALSREQUIREDBUTNOTSUPPLIED1.37℃incubator2.Standardmicroplatereadercapabeofeaungabobanceat450n3.Precisionpipettes,disposablepipettetipsandAbsorbentpaper4.Distilledordeionizedwater

REAGENTSPROVIDEDAllreagentsprovidedarestoredat2-8°C.Refertotheexpirationdateonthelabel.Name96determinations48determinationsMICROTITERPLATE8*12strips8*6stripsSTANDARD（6vial）0.3ml/vial0.3ml/vialSAMPLEDILUENT6.0ml3.0mlENZYMECONJUGATE10.0ml5.0mlWASHSOLUTION25ml15mlSUBSTRATEA6.0ml3.0mlSUBSTRATEB6.0ml3.0mlSTOPSOLUTION6.0ml3.0mlClosureplatemembrane22Usermanual11Sealedbags11Note:1.Standardconcentrationwasfollowedby:8,4,2,1,0.5,0ng/mL.2.If

samples

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Diluent

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assay.PRECAUTIONSDonotsubstitutereagentsfromonekitlottoanother.Standard,conjugateandmicrotiterplatesarematchedforoptimalperformance.Useonlythereagentssuppliedbymanufacturer.Allowkitreagentsandmaterialstoreachroomtemperature(20-25°C)beforeuse.Donotusewaterbathstothawsamplesorreagents.Donotusekitponentsbeyondtheirexpirationdate.Useonlydeionizedordistilledwatertodilutereagents.Donotremovemicrotiterplatefromthestoragebaguntilneeded.Unusedstripsshouldbestoredat2-8°Cintheirpouchwiththedesiccantprovided.Usefreshdisposablepipettetipsforeachtransfertoavoidcontamination.Donotmixacidandsodiumhypochloritesolutions.Serumandplasmashouldbehandledaspotentiallyhazardousandcapableoftransmittingdisease.Disposableglovesmustbewornduringtheassayprocedure,sincenoknowntestmethodcanofferpleteassurancethatproductsderivedfromRatbloodwillnottransmitinfectiousagents.Therefore,allbloodderivativesshouldbeconsideredpotentiallyinfectiousandgoodlaboratorypracticesshouldbefollowed.Allsamplesshouldbedisposedofinamannerthatwillinactivateviruses.LiquidWaste:Addsodiumhypochloritetoafinalconcentrationof1.0%.Thewasteshouldbeallowedtostandforaminimumof30minutestoinactivatethevirusesbeforedisposal.SubstrateSolutioniseasilycontaminated.Ifbluishpriortouse,donotuse.SubstrateBcontain20%acetone,keepthisreagentawayfromsourcesofheatorflame.Removeallkitreagentsfromrefrigeratorandallowthemtoreachroomtemperature(20-25°C).REAGENTPREPARATIONANDSTORAGEWashSolution(1X)-Dilute1volumeofWashsolution(20X)with19volumesofdeionizedordistilledwater.WashSolutionisstablefor1monthat2-8°C.ASSAYPROCEDURE1.2.Add50μlofStandardorSampletotheappropriatewells.Blankwelldoesn’taddanyting.3.Add100μlofEnzymeconjugatetostandardwellsandsamplewellsexcepttheblankwell,coverwithanadhesivestripandncubaeor60minutesat34.WashtheMicrotiterPlate4times.ManualWashing-Removeincubationmixturebyaspiratingcontentsoftheplateintoasinkorproperwastecontainer.Usingasquirtbottle,filleachwellpletelywithWashSolution(1X),thenaspiratecontentsoftheplateintoasinkorproperwastecontainer.Repeatthisprocedureforatotaloffourtimes.Afterfinalwash,invertplate,andblotdrybyhittingplateontoabsorbentpaperorpapertowelsuntilnomoistureappears.Note:Holdthesidesoftheplateframefirmlywhenwashingtheplatetoassurethatallstripsremainsecurelyinframe.AutomatedWashing-Aspirateallwells,thenwashplatesfourtimesusingWashBuffer(1X).Alwaysadjustyourwashertoaspirateasmuchliquidaspossibleandsetfillvolumeat350μL/well/wash.Afterfinalwash,invertplate,andblotdrybyhittingplateontoabsorbentpaperorpapertowelsuntilnomoistureappears.5.AddSubstrateA50μlandSubstrateB50μltoeachwell.Gentlymixandincubatefor15minutesat37°C.Protectfromlight6.Add50μlStopSolutiontoeachwell.Thecolorinthewellsshouldchangefrombluetoyellow.Ifthecolorinthewellsisgreenorthecolorchangedoesnotappearuniform,gentlytaptheplatetoensurethoroughmixing.7.ReadheOpcalDenyO.D.)at450nmungacoerpaeeaderhn15nue.CALCULATIONOFRESULTSThisstandardcurveisusedtodeterminetheamountinanunknownsample.ThestandardcurveisgeneratedbyplottingtheaverageO.D.(450nm)obtainedforeachofthesixstandardconcentrationsonthevertical(X)axisversusthecorrespondingconcentrationonthehorizontal(Y)axis.First,calculatethemeanO.D.valueforeachstandardandsample.AllO.D.Valuesaresubtractedbythemeanvalueofthebalnkwellbeforeresultinterpretation.Constructthestandardcurveusinggraphpaperorstatisticalsoftware.Todeterminetheamountineachsample,firstlocatetheO.D.valueontheY-axisandextendahorizontallinetothestandardcurve.Atthepointofintersection,drawaverticallinetotheX-axisandreadthecorrespondingconcentration.Anyvariationinoperator,pipettingandwashingtechnique,incubationtimeortemperature,andkitagecancausevariationinresult.Eachusershouldobtaintheirownstandardcurve.Intra-assayCV(%)islessthan10%andInter-assayCV(%)islessthan15%.Assayrange:0.25ng/mL–8ng/mL.7.Sensitivity:TheminimumdetectabledoseofHumanDMDistypicallylessthan0.1ng/mL.8.Cross-reactivity:ThisassayrecognizesrebinantandnaturalHumanDMD.Nosignificantcross-reactivityorinterferencewasobserved.9.Storage:2-8℃(Usefrequently);sixmonths(-20℃)。10.StandardcurveFORRESEARCHUSEONLY;NOTFORTHERAPEUTICORDIAGNOSTICAPPLICATIONS!PLEASEREADTHROUGHENTIREPROCEDUREBEFOREBEGINNING!

人肌营养不良蛋白（DMD）试剂盒（ELISA）使用说明书本试剂盒用于体外定量检测血清、血浆、组织、细胞上清及相关液体样本中人肌营养不良蛋白（DMD）的含量。有效期：6个月保存条件：2-8℃实验原理试剂盒采用双抗体一步夹心法酶联免疫吸附试验（ELISA）。往预先包被人肌营养不良蛋白（DMD）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人肌营养不良蛋白（DMD）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。样本处理及要求1.血清：全血标本请于室温放置2小时或4℃过夜后于1000g离心20分钟，取上清即可检测，或将标本放于-20℃或-80℃保存，但应避免反复冻融。

2.血浆：可用EDTA或肝素作为抗凝剂，标本采集后30分钟内于2-8°C1000g离心20分钟，或将标本放于-20℃或-80℃保存，但应避免反复冻融。

3.细胞培养物上清或其它生物标本：1000g离心20分钟，取上清即可检测，或将标本放于-20℃或-80℃保存，但应避免反复冻融。

注：标本溶血会影响最后检测结果，因此溶血标本不宜进行此项检测。

需要而未提供的试剂和器材酶标仪（450nm）高精度加样器及枪头：0.5-10uL、2-20uL、20-200uL、200-1000uL37℃恒温箱蒸馏水或去离子水试剂盒组成名称96孔配置48孔配置备注微孔酶标板8孔×12条8孔×6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个无备注：1.标准品浓度依次为：8、4、2、1、0.5、0ng/mL2.经过大量正常标本检验，