Goat Anti-Mouse α-glucosidase

文档简介GoatAnti-Mouseα-glucosidaseStorage:2-8°CPackagesize:96determinationsPRINCIPLEOFTHEMETHODTheα-glucosidasekitisasolidphasephasesandwichenzymelinkedimmunosorbentassay(ELISA).Samples,includingstandardsofknownα-glucosidaseconcentrationsandunknownsarepipettedintothesewells.Duringthefirstincubation,theα-glucosidaseantigenandabiotinylatedmonoclonalantibodyspecificforα-glucosidasearesimultaneouslyincubated.Afterwashing,theenzyme(streptavidin-peroxydase)isadded.Afterincubationandwashingtoremovealltheunboundenzyme,asubstratesolutionwhichisactingontheboundenzymeisaddedtoinduceacolouredreactionproduct.Theintensityofthiscolouredproductisdirectlyproportionaltotheconcentrationofα-glucosidasepresentinthesamples.REAGENTSPROVIDEDANDRECONSTTTUTIONREAGENTS（Storeat2-8℃）1×96WELLS0.5×96WELLSRECONSTTTUTION96/48-wellsmicrotiterplates10.5Ready-to-usePlastivcover21Ready-to-useStandard:400nmol/L1Vials(0.6ml)0.5Vials(0.3ml)Seereagentspreparationonpage3Blankcontrol1Vials(1.0ml)1Vials(0.5ml)Ready-to-useStandardDiluent1Vials(4.0ml)1Vials(2.0ml)Ready-to-useBiotinylatedanti-α-glucosidase1Vials(6.0ml)1Vials(3.0ml)Ready-to-useStreptavidin-HRP1Vials(8.0ml)1Vials(4.0ml)Ready-to-useWashingBuffer1Vials(20ml)1Vials(10ml)50×concentrateSubstrateA1Vials(6.0ml)1Vials(3.0ml)Ready-to-useSubstrateB1Vials（6.0ml)1Vials(3.0ml)Ready-to-useStoppingSolution1Vials(6.0ml)1Vials(3.0ml)Ready-to-useSampleDiluent1Vials(12ml)1Vials(6.0ml)Ready-to-useGoatAnti-Mouseα-glucosidaseMATERIALREQUIREDBUTNOTPROVIDED?Distilledwater?Pipettes:10ul、50ul、100ul、200ul、1000ul。?Vortexmixerandmagneticstirrer.SAFETY?Forresearchuseonly?AvoidanyskincontactwithH2SO4andTMB.Incaseofcontact,washthoroughlywater.?Donoteat,drink,smokeorapplycosmeticswherekitreagentsareused.?Donotpipettebymouth.PROCEDURALNOTES/LAB.QUALITYCONTROL?Whennotinuse,kitcomponents首ldbestoredrefrigeratedorfrozenasindicatedonvialsorbottles.Allreagents首ldbewarmedtoroomtemperaturebeforeuse.Lyophilizedstandards首ldbediscardedafteruse.?Oncethedesirednumberofstripshasbeenremoved,immediatelyresealthebagtoprotecttheremainingstripsfromedterioration.?Coverorcapallreagentswhennotinuse.?Donotmisorinterchangereagentsbetweendifferentlots.?Donotusereagentsbeyondtheexpirationdateofthekit.?Useacleandisposableplasticpipettetipforeachreagent,standard,orspecimenadditioninordertoavoidcross-contamination,forthedispensingofH2SO4andsubstratesolution,avoidpipetteswithmetalparts.?Useacleanplasticcontainertopreparethewashingsolution.?Thoroughlymixthereagentsandsamplesbeforeusebyagitationorswir领.?Allresidualwashingliquidmustbedrainedfromthewellsbyefficientaspirationorbydecantationfollowedbytappingtheplateforcefullyonabsorbentpaper.Neverinsertabsorbentpaperdirectlyintothewells.?TheTMBsolutionislightsensitive.Avoidprolongedexposuretolight,also,avoidcontactoftheTMBsolutionwithmetaltopreventcolourdevelopment.WarningTMBistoxicavoiddirectcontactwithhands.Disposeoffproperly.Ifadarkbluecolourdevelopswithinafewminutesafterpreparation,thisindicatesthattheTMBsolutionhasbeencontaminatedandmustbediscarede.Readabsorbanceswithin1houraftercompletionoftheassay.?Whenpipettingreagents,maintainaconsistentorderofadditionfromwell-to-well.Thiswillensureequalincubationtimesforallwells.?Respectincubationtimesdescribedintheassayprocedure.SPECIMENCOLLECTION\PROCESSINGANDSTORAGE?Serum---Avoidanyinintentionalstimulationofthecellsbytheprocedure.Usepyrogen\endotoxinfreecollectingtubes.Serum首ldberemovedrapidlyandcarefullyfromtheredcellsafterclothing.Forthat,afterclothing,centrifugeatapproximately1000×gfor10minandremoveserum.?Plasma---EDTA\citrateandheparinplasmacanbeassayed.Spinsamplesat1000×gfor30minremoveparticulates.Harvestplasma.?Cellculturesupernatants---Removeparticulatesandaggregatesbyspinningatapproximately1000×gfor10min.?Storage---Ifnotanalyzedshortlyaftercollection,samples首ldbealiquoted(250-500ul)toavoidfreeze-thawcyclesandstoredfrozenat-70℃.Avoidmultiplefreeze-thawcyclesoffrozenspecimens.Whenpossible,avoiduseofbadlyhemolyzedorlipemicsera.Iflargeamountsofparticlesarepresent,this首ldberemovedpriortoassaybycentrifugationorfiltration.?Recommendation---Donotthawbyheatingat37℃or56℃.Thawatroomtemperatureandmakesurethatsampleiscompletelythawedandhomogenousbeforeassaying.PREPARATIONOFREAGENTS?Standards:Standardhavetobereconstituledwiththevolumeofstandardbufferdiluentindicatedonthevial.Thisreconstitutionproducesastocksolutionof400nmol/Lα-glucosidase.Allowstandardtostandfor5?minuteswithgentleswir领priortomakingdilutions.Serialdilutionsofstandardmustbemadebeforeeachassysandcannotbestored.400nmol/L（6Standard）Originaldensity50ul。200nmol/L（5Standard）100ul6Standard+100uldiludent100nmol/L（4Standard）100ul5Standard+100uldiludent50nmol/L（3Standard）100ul4Standard+100uldiludent25nmol/L（2Standard）100ul3Standard+100uldiludent12.5nmol/L（1Standard）100ul2Standard+100uldiludent0nmol/LBlankControl50ul。?Washingbuffer50×concentrate:Dilute50timesindistilledwater.ASSAYMETHOD?Beforeuse,mixallreagentsthoroughlywithoutmakingfoam.?Determinethenumberofmicrowellstripsrequiredtotestthedesirednumberofsamples,plusappropriatenumberofwellsneededforrunningblanksstandards.Eachsample,standardandblank首ldbeassayedinduplicate.Removesufficientmicrowellstripsfromthepouch.?Add50ulofstandarddiluenttostandardwellsB1,B2,C1,C2,D1,D2,E1,E2,F1,F2.Reconstitutestandardvialwiththeappropriatevolumeasdescribedinthechapterreagentspreparation.Preparation.Pipet100ulofstandardintowellsA1andA2(seeplateschemebelow).Transfer50ulfromA1andA2toB1andB2wells.Mixthecontentsbyrepeatedaspirationsandejections.Takecarenottoscratchtheinnersurfaceofmicrowells.RepatthisprocedurefromthewellsB1,B2towellsC1,C2andfromwellsC1,C2toD1,D2andsooncreatingtwoparallelrowsofα-glucosidasestandarddilutionsranging，Add50ulofstandarddiluenttotheblandwells.?Dilutesamples1:1distribing50ulofsampleinto50ulofdilluent,Add50ulofdilutedsampletowells..?Add50ulofdilutedbiotinylatedanti-α-glucosidasetoallwells.?Coverwithaplatevoverandincubatefor1hourat37℃.?Removethecoverandwashtheplateasfollows:⑴aspiratetheliquidfromeachwell,⑵dispensse0.3mlofwashingsolutionintoeachwell.⑶Aspirateagainthecontetofeachwellafter0.5minute.⑷Repeatsteps⑵and⑶threetimes.?Distribute60ulofstreptavidin-HRPsolutiontoallwells,includingblankwells.?Coverandincubate30minat37℃.?Removethecoverandemptywells,Washmicrowellstripsaccordingtostep,Proceedimmediatelytothenextstep.?Add50ulSubstrateAandSubstrateBtoeachwell。Incubatefor10minat37℃。?Theenzyme-substratereactionisstoppedbyquicklypipetting50ulofH2SO4.stopreagentintoeachwell,includingtheblankwells,tocompletelyanduniformlyinactivatetheenzyme.ResultsmustberedimmediatelyaftertheadditionofH2SO4.?Readabsorbanceofeachwellonaspectrophotometerusing450nmastheprimarywavelengthandoptionally620nm(610nmto650nmisacceptable)asthereferencewavelength.GoatAnti-Mouseα-glucosidaseSUGGESTEDPLATESCHEMEStandardconcentrations（nmol/L）A400400samplesamplesamplesamplesamplesamplesamplesamplesamplesampleB200200samplesamplesamplesamplesamplesamplesamplesamplesamplesampleC100100samplesamplesamplesamplesamplesamplesamplesamplesamplesampleD5050samplesamplesamplesamplesamplesamplesamplesamplesamplesampleE2525samplesamplesamplesamplesamplesamplesamplesamplesamplesampleF12.512.5samplesamplesamplesamplesamplesamplesamplesamplesamplesampleG00samplesamplesamplesamplesamplesamplesamplesamplesamplesampleHsamplesamplesamplesamplesamplesamplesamplesamplesamplesamplesamplesampleLIMITATIONSOFTHEPROCEDUREDonotextrapolatethestandardcurvebeyondthemaxstandardcurvepoint.Thedose-responseisnon-linearinthisregionandgoodaccruacyisdifficulttoobtain.CALCULATIONOFRESULTSTheminimumdetectableconcentrationinthisassayisestimatedtobe1.0nmol/L