Description:The M30Apoptosense®CK18Kit (ELISA)isauniquetoolforreliableandreproducIBLequantitativedetectionofapoptosisinepithelialkeratin18(K18) (cytokeratin18[CK18])positivecells.ThespecificityisreliablebecausetheM30Apoptosense®ELISAsolelyquantifiestheaccumulationofsolublecaspase-cleavedkeratin18,aproductofapoptosis.Theassayisintendedforsubjectserumorplasma. Anotherusefulapplicationisthedetectionoftumorapoptosisinmousexenograftmodelsusingplasmasamples.M30Apoptosense®isanELISAassaybasedontheM30monoclonalantibody(Leersetal.,1999).Thisantibodyspecificallybindscaspase-cleavedkeratin18(ccK18,caspase-celavedcytokeratin18,ccCK18orK18-Asp396)butdoesnotbindnative,uncleaved,K18.TheM30Apoptosense®assaythereforerepresentsaspecificquantitativetestforapoptosisinepithelialcells(Häggetal.2002).Allreagentsareprovidedinaconvenientready-for-useformat.Theseparablestripsprovidetheflexibilityforyourexperimentaldesign.Becauseofthecriticalimportanceofapoptosis,numerousapplicationfieldsexists,butparticularly:Hepatology:Researchshowsthatapoptosisisincreasedinliverdisease.CK18ishighlyabundantandcriticalfornormallivers.Apoptosiscanbemeasuredinliverdiseasesubjects.Oncology:MeasureschemotherapyinducedapoptosisinepithelialcarcinomainresearchToxicology:Suitableforstudieswhenevaluatingdifferentpharmaceuticalagents’impactonlivercelldeathNote:M30Apoptosense®ELISAwaspreviouslysoldundercatalog#P10010.CK18KitAdvantages:OnlymethodavailabletospecificallymeasureepithelialapoptosisinthebloodSpecificquantificationmeasurementtoolforapoptosisinCK18positivecellsSandwichELISAina96wellplateinaconvenientready-to-useformatEasytoperform,onlyaminimumofpipettingstepsrequiredItcanbesplitupforuseatseveraloccasionsCK18ispresentinsimple epithelialcellsonly,thusincreasingspecificityofmeasurementinepithelial-basedmodelsRemarkablestABIlityoftheCK18proteincomplexesinthecirculation,thusprovidingstablestorageofserum/plasmasamplesandallowingformultiplefreezethawcycles.Minimalday-to-dayfluctuationsinhealthysubjectsSuitabletousetogetherwithM65®ELISAforquantificationofapoptosis,necrosisandtotalcelldeathCK18KitComposition:M5CoatedMicrostrips:Onemicroplate,12stripswith8wellseach,96drywells intotal.ThewellsarecoatedwithmousemonoclonalK18antibodyM5.The microplateissealedinanaluminiumbag,whichcontainsadesiccatingdevice.If notallthestripsareused,resealthebagandkeepthedesiccatingdeviceinside. Readyforuse!M30Conjugate:Concentrate(24×conc).Onevialcontaining0.4mLofmouse monoclonalM30antibody(anti-K18Asp396neo-epitope)conjugatedwith horserADIshperoxidase(HRP)inphosphatebufferwithproteinstabilizers. Preservativeadded.ShouldbedilutedwithM30ConjugateDilutionBuffer. Note:Donotexposetolight.M30ConjugateDilutionBuffer:Onevialcontaining11mLofphosphatebuffer withproteinstabilizersfordilutionoftheM30Conjugate.Preservativeadded. Bluecolored.M30StandardA–G:StandardAcontaining2mLofphosphatebufferwithfetal calfserum(FCS).StandardB–G,0.5mLeach,containingstandardmaterialin phosphatebufferwithFCS.ThevaluesofStandardA–Gare0,75,150,250,500, 750and1000U/L,respectively.Preservativeadded.Yellowcolored.Readyfor use!StandardAcanbeusedfordilutionsofsamples>1000U/L.M30ControlLow&High:Twovialscontaining0.5mLofreactivecomponents inphosphatebufferwithFCS.ThevaluesofM30ControlLowandM30Control Higharestatedontherespectivevials.Preservativeadded.Yellowcolored. Readyforuse!WashTablet:Onetabletfor500mLofpreparedwashsolution.Dissolvethe WashTabletin500mLoffreshdeionizedwater.TMBSubstrate:Onebottlecontaining22mLofTMB(3,3’,5,5’-Tetramethylbenzidine) Solution.Note!Donotexposetolight!Readyforuse!StopSolution:Onevialcontaining7mLof1.0Msulphuricacid.Readyforuse!SealingTape:One(1)sheet.MeasurementPrinciple:TheM30ApoptosenseELISAisasolid-phasesandwichenzymeimmunoassay. Standards,controlsandsamplesreactwithasolidphasecaptureantibodyM5 directedagainstCK18andtheHRP-(horseradishperoxidase)conjugatedM30 antibodydirectedagainsttheCK18Asp396neo-epitope.Unboundconjugateis removedbyawashingstep.TMBSubstrateisadded.Thecolordevelopment isstoppedandtheabsorbanceisread.Theresultingcolorisdirectly proportionaltotheconcentrationoftheanalyte. Byplottingastandardcurvefromknownconcentrationsversusmeasured absorbance,theamountofantigeninthesamplecanbecalculated.The concentrationoftheantigenisexpressedasunitsperliter(U/L).Thekitcontainscalibratorsandcontrols,andcanbeusedinanylaboratory.Calibratorsandcontrolscontainrecombinantpeptides,whichincludeM5andM30epitopes.Thetestcanprovidearesultwithin5 hours.StandardELISAequipmentisusedandthustheM30kitoffersandeasytouseformatthatshouldallowwideaccessibilityfortesting.InXenograftModelsTheM30 Apoptosense®ELISA detectshuman,butnotmouse/rat,CK18.ThedetectionofCK18inthebloodofamousecarryingahumantumorxenograftisthereforeduetoapoptosisofthehumantumorcells. Olofssonet.al(CancerBioMarkers,2009)usedaslightlymodifiedprotocolallowingforlessvolume(12.5µlsample)andusedanadditionalblockingagentaddedtotheconjugate(ie,HRPPlus).Background: DevelopmentoftheM30antibodyintoanELISArepresentedthefirstresearchtooltomeasureaspecificapoptosisproductintheblood.TheM30Apoptosense®ELISAmeasuresthelevelsofsolubleccK18fragmentscontainingtheK18Asp396neo-epitope.Afterinductionofapoptosisinepithelialtissues,ccK18increasesareobservedintheblood.Caspase-cleavedK18fragmentsareremarkablystableinhumanserum,furtheraddingtoconvenienceandtheeasytousemethodofthisELISA.