Description:TheM65®ELISAmeasuressolublekeratin18(K18)(cytokeratin18[CK18])releasedfromdyingcellsandcanbeused intheresearchof overallcelldeath(duetoapoptosisandnecrosis)ofepithelialcells.TheM65®ELISAcan beusedtogetherwiththeM30Apoptosense®assay inresearch.AsbothassaysarecalibratedagainsttheidenticalreferencethecombinationofM30Apoptosense®andM65®ELISAshouldallowforresearchdeterminationoftherelativecontributionofapoptosistothetotaldegreeofcancercelldeath.CK18KitAdvantages:OnlymethodavailabletospecificallymeasureepithelialapoptosisandnecrosisinthebloodSpecificquantificationmeasurementtoolforapoptosisandnecrosisinkeratin18positivecellsSandwichELISAina96wellplateinaconvenientready-to-useformatEasytoperform,onlyaminimumofpipettingstepsrequiredItcanbesplitupforuseatseveraloccasionsCK18ispresentinsimple epithelialcellsonly,thusincreasingspecificityofmeasurementinepithelial-basedmodelsRemarkablestABIlityoftheCK18proteincomplexesinthecirculation,thusprovidingstablestorageofserum/plasmasamplesandallowingformultiplefreezethawcycles.Minimalday-to-dayfluctuationsinhealthysubjectsSuitabletousetogetherwithM30Apoptosense®ELISAforquantificationofapoptosis,necrosisandtotalcelldeathCK18KitComposition:Reagents,Packaging,Storage,andStabilityM65CoatedMicrostrips:Onemicroplate,12stripswith8wellseach, 96drywellsintotal.ThewellsarecoatedwithmousemonoclonalK18 antibodyM6.Themicroplateissealedinanaluminiumbag,whichcontains adesiccatingdevice.Ifnotallthestripsareused,resealthebagandkeep thedesiccatingdeviceinside.Readyforuse!M65HRPConjugate:Concentrate(24×conc).Onevialcontaining0.4mL ofmousemonoclonalM5antibody(anti-K18)conjugatedwithhorserADIsh peroxidase(HRP)inaphosphatebufferwithproteinstabilizers.Preservative added.ShouldbedilutedwithM65ConjugateDilutionBuffer. Note!Donotexposetolight!M65ConjugateDilutionBuffer:Onevialcontaining12mLofphosphate bufferwithproteinstabilizersfordilutionoftheM65HRPConjugate. Preservativeadded.Bluecoloured.Readyfor use!M65StandardA–G:StandardAcontaining4mLofphosphatebufferwith fetalcalfserum(FCS).StandardB–G,0.5mLeach,containingstandard materialinphosphatebufferwithFCS.ThevaluesofStandardA–Gare0, 125,250,500,750,1200and2000U/L,respectively.Preservativeadded.Yellowcolored.Readyforuse!Serum/plasmasamples>2000U/Lcanbediluted1+1withStandardA,butdilutionwithpooledhumanserumis recommended(seesection“PerformanceCharacteristics”).M65ControlLow&High:Twovialscontaining0.5mLofreactivecomponents inphosphatebufferwithFCS.ThevaluesofM65ControlLow andHigharestatedontherespectivevials.Preservativeadded.Yellow colored.Readyforuse!WashSolution:Onevialcontaining50mLofconcentrated(10×conc) WashSolution.Dilutewith450mLoffreshdeionisedwaterbeforeuse.PhosphatebufferwithTween®20.Preservativeadded.TMBSubstrate:Onebottlecontaining22mLofTMB(3,3’,5,5’-Tetramethylbenzidine) Solution.Note!Donotexposetolight!Readyforuse!StopSolution:Onevialcontaining7mLof1.0Msulphuricacid.Readyforuse!SealingTape:One(1)sheet.MeasurementPrinciple:TheM65®ELISAisasolid-phasesandwichenzymeimmunoassay.Standards, controlsandsamplesreactwithasolidphasecaptureantibodyM6 directedagainstK18andtheHRP(horseradishperoxidase)conjugated M5antibodydirectedagainstadifferentepitopeofK18.Unboundconjugate isremovedbyawashingstep.TMBsubstrateisadded.Thecolordevelopmentisstoppedandtheabsorbanceisread.Theresultingcolor isdirectlyproportionaltotheconcentrationoftheanalyte. Byplottingastandardcurvefromknownconcentrationsversusmeasured absorbance,theamountofantigeninthesamplecanbecalculated.Theconcentrationoftheantigenisexpressedasunitsperliter (U/L).Note:Pleasecontactusatinfo@Diapharma.comifvalidatingtheELISAwithspikinganddilutabilitystudies.Spikingexperiments–Werecommendnottospikewiththestandardincludedinthekit.Forspikingexperiments,pleaseinquireaboutaspecialspikingsample.DilutionwithStandardA–Forthedilutionofhumanbloodsamples,werecommenddilutionofthesamplesabovethestandardcurvewithpooledhumanserum.           InXenograftModelsTheM30 Apoptosense®ELISA detectshuman,butnotmouse/rat,CK18.ThedetectionofCK18inthebloodofamousecarryingahumantumorxenograftisthereforeduetoapoptosisofthehumantumorcells. Olofssonet.al(CancerBioMarkers,2009)usedaslightlymodifiedprotocolallowingforlessvolume(12.5µlsample)andusedanadditionalblockingagentaddedtotheconjugate(ie,HRPPlus).Background:CK18isanintracellularproteinexpressedathighlevelsbymanytypesofepithelialcells.MostCK18moleculeswillforminsolublefilamentsinthecell,butapoolofsolubleCK18canalsobedemonstrated.Duringcelldeath,thecellularcontentofCK18willbereleasedintotheextracellularcompartment.ResearchmeasurementsofextracellularsolubleCK18willthereforereflectepithelialcelldeath“byanycause”(duetoapoptosisandnecrosis).CK18iscleavedbycaspasesduringapoptosis,andcaspase-cleavedfragmentswillbereleasedtotheextracellularcompartment.Therelativeproportionofextracellularcaspase-cleavedCK18(ccKC18)comparedtototalCK18willthereforereflecttherelativeproportionofapoptosistototalcelldeathresearch(different“M30:M65ratios”).TheM65®ELISAusestwoanti-CK18mousemonoclonalantibodiesoftheIgGtypeandisprimarilyintendedtobeusedtogetherwiththeM30 Apoptosense®assayforassessmentofresearchintumorcelldeathusinghumanserumsamples.M30 Apoptosense® ELISAspecificallymeasuresaneo-epitopeformedbycaspase-cleavageofCK18atAsp396(CK18Asp396-NEM30neo-epitope)andwillreflectapoptosisofepithelialcells.Theunitsofthetwoassayshavebeencalibratedagainsttheidenticalstandardmaterialtoallowthecalculationofaratiobetweencaspase-cleavedandtotalCK18(“M30:M65ratio”).Inductionofapoptosisinculturedcellswillresultinreleaseofcaspase-cleavedCK18andinrelativelyhighM30:M65ratios,whereasinductionofnecrosiswillalmostexclusivelyresultinreleaseofCK18moleculesthatarenotcaspase-cleavedandinalowM30:M65ratio.TheM30:M65ratiowillthereforereflectthemodeofcelldeathofepithelialcells.