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:

• Onlymethodavailabletospecificallymeasureepithelialapoptosisandnecrosisintheblood
• SpecificquantificationmeasurementtoolforapoptosisandnecrosisinCK18positivecells
• SandwichELISAina96wellplateinaconvenientready-to-useformat
• Easytoperform,onlyaminimumofpipettingstepsrequired
• Itcanbesplitupforuseatseveraloccasions
• CK18ispresentinsimple epithelialcellsonly,thusincreasingspecificityofmeasurementinepithelial-basedmodels
• RemarkablestABIlityoftheCK18proteincomplexesinthecirculation,thusprovidingstablestorageofserum/plasmasamplesandallowingformultiplefreezethawcycles.
• Minimalday-to-dayfluctuationsinhealthysubjects
• SuitabletousetogetherwithM30Apoptosense®ELISAforquantificationofapoptosis,necrosisandtotalcelldeath

CK18KitComposition:

Reagents,Packaging,Storage,andStability

• M5CoatedMicrostrips: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.