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AntibodiesOnline/Cortisone (COR) ELISA Kit/ABIN577661/5 x 96 tests188bio精品生物—专注于实验室精品爆款的电商平台 - 蚂蚁淘旗下精选188款生物医学科研用品
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AntibodiesOnline/Cortisone (COR) ELISA Kit/ABIN577661/5 x 96 tests
Antigen
Cortisone(COR)
Reactivity
Hormone
Alternatives
Kitswithalternativereactivityto:
3Human
2AllSpecies
2Cow(Bovine)
2General
2Hormone
2Monkey
1Chicken
1Dog(Canine)
1Goat
1GuineaPig
1Horse(Equine)
1Mouse(Murine)
1Pig(Porcine)
1Rabbit
1Rat(Rattus)
1Sheep(Ovine)
1VariousSpecies
MethodType
SandwichELISA
MinimumDetectionLimit
59.6pg/mL
Application
ELISA
Options
Bulkdiscount
Supplier
SupplierProductNo.
PurposeTheDetectX®CortisoneChemiluminescentImmunoassaykitisdesignedtoquantitativelymeasureCortisonepresentinextracteddriedfecalsamples,urine,saliva,andserumsamples.
BrandDetectX®
SampleTypeSerum,Saliva,Urine,Fecal
AnalyticalMethodQuantitative
DetectionMethodChemiluminescent
SpecificitySpeciesIndependent.SamplesTypesvalidated:DriedFecalExtracts,Urine,Saliva,andSerum
Sensitivity10.6pg/mL
CharacteristicsTheCortisoneChemiluminescentImmunoassaykitisdesignedtoquantitativelymeasureCortisonepresentinsamples.Acortisonestandardisprovidedtogenerateastandardcurvefortheassay.Afteratwohourincubationtheplateiswashedandthechemiluminescentsubstrateisadded.Thesubstratereactswiththeboundcortisone-peroxidaseconjugatetoproducelight.Thegeneratedlightisdetectedinamultilabelmicrotiterplatereader.Cortisoneandcortisolconcentrationsvaryduetotheactivityoftwo11ß-hydroxysteroiddehydrogenases(11-HSD).Whilemosttissueshavetheabilitytoexpresseitherenzyme,11ß-HSD1isfoundprimarilyintheliverwhereitconvertscortisonetocortisolwhile11ß-HSD2isfoundintissuessuchasthekidneywherecortisolreceptorbindingisrequired.11ß-HSD2deactivatescortisoltocortisone,prohibitingreceptoractivation.Thisglucocorticoidshuttlehelpstoinitiateandregulatetheanti-inflammatoryresponse.Monitoringtheratioofcortisone:cortisolhasapplicationsindiabetes,obesity,metabolicsyndrome,osteoporosis,andchronicfatiguesyndromeinadditiontoadrenaldiseases.Cortisoneandcortisolconcentrationsexhibitapredictablediurnalpatternandcanbemeasuredinextracteddriedfeces,orinserum,plasma,salivaandurine.Arecentpublicationhassuggestedthatsalivarycortisoneisagoodsurrogatemarkerforserumcortisol.
ComponentsCoatedWhite96WellPlatesAwhiteplasticmicrotiterplate(s)withbreak-apartstripscoatedwithgoatanti-rabbitIgG.1Or5Each
CortisoneStandardCortisoneat1,000ng/mLinaspecialstabilizingsolution.50μLOr125μL
DetectX®CortisoneCLIAAntibodyArabbitpolyclonalantibodyspecificforcortisone.3mLOr13mL
DetectX®CortisoneCLIAConjugateConcentrateAcortisone-peroxidaseconjugateconcentrateinaspecialstabilizingsolution.1mLOr3.5mL
ConjugateDiluentContainsspecialstabilizersandadditives.3mLOr13mL
AssayBufferConcentrateA5Xconcentratethatmustbedilutedwithdeionizedordistilledwater.28mLOr55mL
DissociationReagent1mLOr5mLNOTE:DissociationReagentistobeusedonlywithSerumsamples.
WashBufferConcentrateA20Xconcentratethatshouldbedilutedwithdeionizedordistilledwater.30mLOr125mL
SubstrateSolutionA6mLOr28mL
SubstrateSolutionB6mLOr28mL
PlateSealer1Or5Each
MaterialnotincludedDistilledordeionizedwater.
Repeaterpipetwithdisposabletipscapableofdispensing25μLand100μL.
Amicroplateshaker.96wellmicroplatereadercapableofreadingglowchemiluminescence.
Alistofsomemodelsofsuitablereaderscanbefoundonourwebsiteatwww.ArborAssays.com/resources/lit.asp.
AllluminometersreadRelativeLightUnits(RLU).
TheseRLUreadingswillvarywithmakeormodelofplatereader.
ThenumberofRLUsobtainedisdependantonthesensitivityandgainofthereaderused.
Ifyouareunsureofhowtoproperlyconfigureyourreadercontactyourplatereadermanufacturerorcarryoutthefollowingprotocol:Dilute5μLoftheCortisoneCLIAConjugateConcentrateinto995μLofdeionizedwater.
Pipet5μLofdilutedconjugateintoawhitewellandadd100μLofpreparedCLIAsubstrate(seepage8fordetails).
Thiswellwillgiveyouanintensityclosetothemaximumbindingsignalfortheassay.
Adjustthegain,integrationtimeorsensitivitysothatyourreaderisgivingclosetoitsmaximumsignal.
ToproperlyanalyzethedatasoftwarewillberequiredforconvertingrawRLUreadingsfromtheplatereaderandcarryingoutfourparameterlogisticcurve(4PLC)fitting.
AlternativeNameCortisone
BackgroundCortisone(C21H28O5,KendallsCompoundE)wasidentifiedbyMason,MyersandKendallin1936asCompoundEextractedfrombovinesuprarenalglandtissuethathadthequalitativebutnotquantitativeactivityofcortin.Thepresenceofmultiplecortin-likecompoundsledtheauthorstospeculatethatthestudyofCompoundEwouldrevealthenatureofcortin1.CompoundEisnowcalledcortisoneandthemoreactiveCompoundF,cortisol,andtheconcentrationsofthesetwoglucocorticoidsvaryduetotheactivityoftwo11ß-hydroxysteroiddehydrogenases(11-HSD)2,3.Whilemosttissueshavetheabilitytoexpresseitherenzyme,11ß-HSD1isfoundprimarilyintheliverwhereitconvertscortisonetocortisolwhile11ß-HSD2isfoundintissuessuchasthekidneywherecortisolreceptorbindingisrequired.11ß-HSD2deactivatescortisoltocortisone,prohibitingreceptoractivation.Thisglucocorticoid"shuttle"helpstoinitiateandregulatetheanti-inflam-matoryresponse,makingcortisoneoneofthemodern"wonderdrugs".Monitoringtheratioofcortisone:cortisolhasapplicationsindiabetes,obesity,metabolicsyndrome,osteoporosis,andchronicfatiguesyndromeinadditiontoadrenaldiseases4-7.Cortisoneandcortisolconcentrationsexhibitapredictablediurnalpatternandcanbemeasuredinextracteddriedfeces,orinserum,plasma,salivaandurine.Arecentpublication8hassuggestedthatsalivarycortisoneisagoodsurrogatemarkerforserumcortisol
ResearchAreaEndocrinesystem,Hormones
ApplicationNotesThisassayhasbeenvalidatedforurine,saliva,andserumsamples.
Ithasalsobeenvalidatedfordriedfecalextractsamples.
Samplescontainingvisibleparticulateshouldbecentrifugedpriortousing.
Moderatetoseverelyhemolyzedsamplesshouldnotbeusedinthiskit.
Cortisoneisidenticalacrossallspeciesandweexpectthiskitshouldmeasurecortisonefromsourcesotherthanhuman.
Theendusershouldevaluaterecoveriesofcortisoneinothersamplesbeingtested.
AssayTime2h
PlatePre-coated
ProtocolThiskitmeasurestotalcortisoneinserumandplasmaandinextractedfecalsamples.
Acortisonestandardisprovidedtogenerateastandardcurvefortheassayandallsamplesshouldbereadoffthestandardcurve.
Standardsordilutedsamplesarepipettedintoawhitemicrotiterplatecoatedwithanantibodytocapturerabbitantibodies.
Acortisone-peroxidaseconjugateisaddedtothestandardsandsamplesinthewells.
Thebindingreactionisinitiatedbytheadditionofapolyclonalantibodytocortisonetoeachwell.
Afteratwohourincubationtheplateiswashedandthechemiluminescentsubstrateisadded.
Thesubstratereactswiththeboundcortisone-peroxidaseconjugatetoproducelight.
Thegeneratedlightisdetectedinamicrotiterplatereadercapableofreadingluminescence.
Theconcentrationofthecortisoneinthesampleiscalculated,aftermakingsuitablecorrectionforthedilutionofthesample,usingsoftwareavailablewithmostplatereaders.
ReagentPreparation

Allowthekitreagentstocometoroomtemperaturefor30minutes.
Werecommendthatallstandardsandsamplesberuninduplicatetoallowtheendusertoaccuratelydeterminecorticos-teroneconcentrations.
Ensurethatallsampleshavereachedroomtemperatureandhavebeendilutedasappropriatepriortorunningtheminthekit.
AssayBufferDiluteAssayBufferConcentrate1:5byaddingonepartoftheconcentratetofourpartsofdeion-izedwater.
Oncedilutedthisisstableat4°Cfor3months.
WashBufferDiluteWashBufferConcentrate1:20byaddingonepartoftheconcentratetonineteenpartsofdeionizedwater.
Oncedilutedthisisstableatroomtemperaturefor3months.
CortisoneConjugateThesuppliedCortisoneConjugateConcentrateshouldbediluted1:4withtheConjugateDiluent.
OncedilutedtheCortisoneconjugateisstableforonemonthwhenstoredat4°C.
StandardPreparationLabelninetesttubesas#1through#9.
Pipet490μLofAssayBufferintotube#1and250μLintotubes#2to#9.
Carefullyadd10μLofthecortisonestocksolutiontotube#1andvortexcomplete-ly.
Take250μLofthecortisonesolutionintube#1andaddittotube#2andvortexcompletely.
Re-peattheserialdilutionsfortubes#3through#9.
Theconcentrationofcortisoneintubes1through9willbe20,000,10,000,5,000,2,500,1,250,625,312.5156.3,and78.1pg/mL.
UseallStandardswithin2hoursofpreparation.

SamplePreparation

SerumsamplesneedtobetreatedwiththesuppliedDissociationReagent.Additionofthisre-agentwillyieldthetotalcortisoneconcentrationinserum.DissociationReagentistobeusedonlywithSerumorPlasmasamples.Freecortisonecanbemeasuredinsalivaandurinesamplesasdirectedbelow.DriedFecalSamplesWehaveadetailedExtractionProtocolavailableonourwebsiteat:http://www.ArborAssays.com/resources/lit.asp.TheethanolconcentrationinthefinalAssayBuf-ferdilutionaddedtothewellshouldbe<5%.SalivaSamplesSalivasamplesshouldbefrozenandthawed,thencentrifugedat14,000rpmfor15minutes.Thesupernatantshouldbediluted1:5to1:10withthesuppliedAssayBufferpriorrunningintheassay.SeeourSalivaSampleHandlingInstructionsat:http://www.arborassays.com/assets/saliva-sample-protocol.pdf.UrineSamplesUrinesamplesshouldbediluted≥1:100withthesuppliedAssayBufferpriorrunningintheassay.SerumandPlasmaSamplesAllowtheDissociationReagent(DR)towarmcompletelytoRoomTemperaturebeforeuse.Wesuggestpipeting5μLofDRinto1mLEppendorftubes.Add5μLofserumorplasmatotheDRinthetube,vortexgentlyandincubateatroomtemperaturefor5minutesorlonger.Dilutewith490μLofsuppliedAssayBuffer.This1:100dilutioncanbedilutedfurtherwithAssayBuffer.Finalserumandplasmadilutionsshouldbe≥1:100.NOTE:DissociationReagentistobeusedonlywithSerumandPlasmasamples.

AssayProcedure
  1. Usetheplatelayoutsheetonthebackpagetoaidinpropersampleandstandardidentification.Determinethenumberofwellstobeusedandreturnunusedwellstothefoilpouchwithdesiccant.Sealtheziplocplatebagandstoreat4ºC.
    2.Pipet50μLofsamplesorstandardsintowellsintheplate.
    3.Pipet75μLofAssayBufferintothenon-specificbinding(NSB)wells.
    4.Pipet50μLofAssayBufferintowellstoactasmaximumbindingwells(B0or0pg/mL).
    5.Add25μLoftheDetectX®CortisoneConjugatetoeachwellusingarepeaterpipet.
    6.Add25μLoftheDetectX®CortisoneAntibodytoeachwell,excepttheNSBwells,usingarepeaterpipet.
    7.Gentlytapthesidesoftheplatetoensureadequatemixingofthereagents.Covertheplatewiththeplatesealerandshakeatroomtemperaturefor2hours.Iftheplateisnotshakensignalsboundwillbeapproximately45 %lower.
    8.Aspiratetheplateandwasheachwell4timeswith300μLwashbuffer.Taptheplatedryoncleanabsorbenttowels.
    9.Add100μLofthemixedChemiluminescentSubstratetoeachwell,usingarepeaterpipet.10.Incubatetheplateatroomtemperaturefor5 minuteswithoutshaking.11.Readtheluminescencegeneratedfromeachwellinamutimodeorchemiluminescentplatereaderusinga0.1secondreadtimeperwell.Thechemiluminescentsignalwilldecreaseabout40 %over60 minutes.12.Usetheplatereadersbuilt-in4PLCsoftwarecapabilitiestocalculateCortisoneconcentrationforeachsample.
CalculationofResults

AllluminometersreadRelativeLightUnits(RLU).
TheseRLUreadingswillvarywithmakeormodelofplatereader.
AveragetheduplicateRLUreadingsforeachstandardandsample.
Cre-ateastandardcurvebyreducingthedatausingthe4PLCfittingroutineontheplatereader,aftersubtractingthemeanRLUsfortheNSB.
Thesampleconcentrationsobtained,calculatedfromthe%B/B0curve,shouldbemultipliedbythedilutionfactortoobtainneatsamplevalues.
Or,usetheMyAssays™onlinetoolfromhttp://www.myassays.com/arbor-assays-cortisone-chemiluminescent-clia-kit.assaytocalculatethedata.
QRcodeforDataAnalysis:*TheMyAssayslogoisaregisteredtrademarkofMyAssaysLtd.typicaldataSampleMeanRLUNetRLU%B/B0CortisoneConc.(pg/mL)NSB7,6600-Standard1290,990283,33022.0720,000Standard2366,485358,82527.9510,000Standard3443,125435,46533.925,000Standard4525,230517,57040.312,500Standard5626,385618,72548.191,250Standard6755,005747,34558.21625Standard7901,865894,20569.65312.5Standard81,032,5401,024,88079.83156.25Standard91,147,0601,139,40088.7578.1B01,291,5001,283,8401000Sample1384,985377,32529.397,879Sample2947,150939,49073.18237.5Alwaysrunyourownstandardcurveforcalculationofresults.
Donotusethisdata.
ConversionFactor:100pg/mLofcortisoneisequivalentto277.6pM.

RestrictionsForResearchUseonly
PrecautionofUseAswithallsuchproducts,thiskitshouldonlybeusedbyqualifiedpersonnelwhohavehadlabo-ratorysafetyinstruction.
Thecompleteinsertshouldbereadandunderstoodbeforeattemptingtousetheproduct.
Theantibodycoatedplateneedstobestoreddesiccated.
Thesilicagelpackincludedinthefoilziplocbagwillkeeptheplatedry.
Thesilicagelpackwillturnfrombluetopinkiftheziplochasnotbeenclosedproperly.
Thiskitutilizesaperoxidase-basedreadoutsystem.
Buffers,includingothermanufacturersWashBuffers,containingsodiumazidewillinhibitcolorproductionfromtheenzyme.
Makesureallbuffersusedforsamplesareazidefree.
EnsurethatanyplatewashingsystemisrinsedwellwithdeionizedwaterpriortousingthesuppliedWashBufferaspreparedonPage8.
Storage4°C,RT
StorageCommentAllcomponentsofthiskitshouldbestoredat4°Cuntiltheexpirationdateofthekit.
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