Description:TheM65EpiDeath®ELISA kitmeasurestheconcentrationofsolubleCK18(K18)incellculturesupernatants,serumorplasmaforresearchstudies.CK18levelsreflecttheamountoftotalepithelialcelldeath,regardlessofthecauseofdeath.Whyarethere2M65ELISAs?TheM65®ELISAwasintroducedin2004andintendedforthedeterminationoftotalcelldeathinresearchofepithelialcells.AcombineduseofM65®ELISAandM30Apoptosense®ELISAfacilitatesthedeterminationofcelldeathmode(apoptosisornecrosis).TheM65EpiDeath®ELISAisbasedonthesamemonoclonalantibodiesastheM65®ELISA,except mAbM5isusedasthecaptureantibody(mAbM6isusedascaptureantibodyintheM65®ELISA).Reactionbuffershavealsobeenslightlymodified ThisCK18kitis manufactureddifferentlyandthus measuresthe(complexbound)CK18epitopesdifferentlythantheM65®ELISA,whichleadstoabetterdiscriminationbetweenlowornormalandhighlevels.Ithasabroadrangeaswell.TheM65EpiDeathELISAisrecommendedwheneveracustomerwantstoidentifycelldeathwithhighsensitivityandspecificityinresearch. TheM65ELISAisthe betterchoiceforcustomers intendingtodeterminethecelldeathmodewhenusingincombinationwiththeM30ApoptosenseELISA,especiallywhenusingamathematicalapproachtodescribethedistributionbetweenapoptosisandnecrosis.CK18KitAdvantages:OnlymethodavailabletospecificallymeasureepithelialapoptosisandnecrosisinthebloodSpecificquantificationmeasurementtoolforapoptosisandnecrosisinCK18positivecellsSandwichELISAina96wellplateinaconvenientready-to-useformatEasytoperform,onlyaminimumofpipettingstepsrequiredItcanbesplitupforuseatseveraloccasionsCK18ispresentinsimple epithelialcellsonly,thusincreasingspecificityofmeasurementinepithelial-basedmodelsRemarkablestABIlityoftheCK18proteincomplexesinthecirculation,thusprovidingstablestorageofserum/plasmasamplesandallowingformultiplefreezethawcycles.Minimalday-to-dayfluctuationsinhealthysubjectsSuitabletousetogetherwithM30Apoptosense®ELISAforquantificationofapoptosis,necrosisandtotalcelldeathCK18KitComposition:Reagents,Packaging,Storage,andStabilityM5CoatedMicrostrips:Onemicroplate,12stripswith8wellseach,96dry wellsintotal.ThewellsarecoatedwithmousemonoclonalK18antibodyM5. Themicroplateissealedinanaluminiumbag,whichcontainsadesiccating device.Ifnotallthestripsareused,resealthebagandkeepthedesiccating deviceinside.Readyforuse!M65EpiDeathConjugate:Concentrate(24×conc.).Onevialcontaining 0.4mLofmousemonoclonalM6antibody(anti-K18)conjugatedwith horserADIshperoxidase(HRP)inaphosphatebufferwithproteinstabilizers. Preservativeadded.ShouldbedilutedwithM65EpiDeathConjugate DilutionBuffer.Note!Donotexposetolight!M65EpiDeathConjugateDilutionBuffer:Onevialcontaining11mLof phosphatebufferwithproteinstabilizersfordilutionoftheM65EpiDeath Conjugate.Preservativeadded.Bluecolored.M65EpiDeathStandardA–H:StandardAcontaining2mLofphosphate bufferwithfetalcalfserum(FCS).StandardB–H,0.5mLeach,containing standardmaterialinphosphatebufferwithFCS.ThevaluesofStandard A–Hare0,200,400,800,1200,2000,3000,and5000U/L,respectively. Preservativeadded.Yellowcolored.Readyforuse!StandardAcanbeused fordilutionsofsamples>5000U/L.M65EpiDeathControlLow&High:Twovialscontaining0.5mLofreactive componentsinphosphatebufferwithFCS.ThevaluesofM65EpiDeath ControlLowandM65EpiDeathControlHigharestatedontherespective vials.Preservativeadded.Yellowcolored.Readyforuse!WashTablet:Onetabletfor500mLofpreparedwashsolution.Dissolvethe WashTabletin500mLoffreshdeionisedwater.TMBSubstrate:Onebottlecontaining22mLofTMB(3,3’,5,5’-Tetramethylbenzidine) Solution.Note!Donotexposetolight!Readyforuse!StopSolution:Onevialcontaining7mLof1.0Msulphuricacid.Readyfor use!SealingTape:One(1)sheet. MeasurementPrinciple:TheM65EpiDeath®ELISAisasolid-phasesandwichenzymeimmunoassay. Standards,controlsandsamplesreactwithasolidphasecaptureantibody M5directedagainstK18andtheHRP-(HorseradishPeroxidase)conjugated M6antibodydirectedagainstadifferentepitopeofK18.Unboundconjugateisremovedbyawashingstep.TMBSubstrateisadded.Thecolor developmentisstoppedandtheabsorbanceisread.Theresultingcoloris directlyproportionaltotheconcentrationoftheanalyte. Byplottingastandardcurvefromknownconcentrationsversusmeasured absorbance,theamountofantigeninthesamplecanbecalculated.The concentrationoftheantigenisexpressedasunitsperliter(U/L).Background:Cellsdiebyapoptosisornecrosis.Themodeofcelldeathdependsbothonthetypeofstimulus(e.g.thecytotoxicagentandtumouruponchemotherapy,orthemechanismofinjuryuponliverdiseases)andthetypeoftissue(e.g.thetumour).ThecombinationoftheM30ApoptosenseELISAandtheM65EpiDeathELISAquantifiesnotonlytheamountofcelldeath,butalsothecelldeathmode(apoptosisornecrosis)inbloodsamples(serumorplasma)orcellsupernatants.Cytotoxicagentsusedinoncologytrytoovercomethedisruptionoftheapoptoticprocessandstimulateapoptosis.Dependentonthetypeoftumourandthecytotoxicagent,necrosiscanbetriggeredinstead.AnapoptoticstimulusmayunderconditionsofdeficientcellularATPgenerationfailtoinducetheapoptoticprogramandcellsinsteadundergonecrosis.Whenresearchingtheliver,injury mayleadtocelldeathofhepatocytes–eitherbynecrosisorapoptosis.Keratin18(K18)isanintracellularproteinexpressedathighlevelsbymanytypesofepithelialcells.MostK18moleculeswillforminsolublefilamentsinthecell,butapoolofsolubleK18canalsobedemonstrated.Duringcelldeath,thecellularcontentofK18willbereleasedintotheextracellularcompartment.ResearchmeasurementsofextracellularsolubleK18willthereforereflectepithelialcelldeath“byanycause”(duetoapoptosisandnecrosis).K18iscleavedbycaspasesduringapoptosis,andcaspase-cleavedfragmentswillbereleasedtotheextracellularcompartment.Theconcentrationofextracellularcaspase-cleavedK18(ccK18)thereforereflectstheamountofapoptosis.FurThermore,theM30:M65ratioisanindicationoftheproportionofapoptosiscomparedtototalcelldeath.TheM65EpiDeath®ELISAusestwoanti-K18mousemonoclonalantibodiesoftheIgGtypeandisintendedforassessmentofepithelialcelldeathusinghumanserumorplasmaresearchsamples.TheM30Apoptosense®ELISAspecificallymeasuresaneo-epitopeformedbycaspase-cleavageofK18atAsp396(K18Asp396-NEorM30neo-epitope)andwillreflectapoptosisofepithelialcells.Theunitsofthetwoassayshavebeencalibratedagainsttheidenticalstandardmaterialtoallowthecalculationofaratiobetweencaspase-cleavedandtotalK18(“M30:M65ratio”).Inductionofapoptosisinculturedcellswillresultinreleaseofcaspase-cleavedK18andinrelativelyhighM30:M65ratios,whereasinductionofnecrosiswillalmostexclusivelyresultinreleaseofK18moleculesthatarenotcaspase-cleavedandinalowM30:M65ratio.