Description:

TheM65®ELISAmeasuressolublekeratin18(K18)(cytokeratin18[CK18])releasedfromdyingcellsandcanbeused intheresearchof overallcelldeath(duetoapoptosisandnecrosis)ofepithelialcells.TheM65®ELISAcan beusedtogetherwiththeM30Apoptosense®assay inresearch.AsbothassaysarecalibratedagainsttheidenticalreferencethecombinationofM30Apoptosense®andM65®ELISAshouldallowforresearchdeterminationoftherelativecontributionofapoptosistothetotaldegreeofcancercelldeath.

CK18KitAdvantages:

• Onlymethodavailabletospecificallymeasureepithelialapoptosisandnecrosisintheblood
• Specificquantificationmeasurementtoolforapoptosisandnecrosisinkeratin18positivecells
• SandwichELISAina96wellplateinaconvenientready-to-useformat
• Easytoperform,onlyaminimumofpipettingstepsrequired
• Itcanbesplitupforuseatseveraloccasions
• CK18ispresentinsimple epithelialcellsonly,thusincreasingspecificityofmeasurementinepithelial-basedmodels
• RemarkablestABIlityoftheCK18proteincomplexesinthecirculation,thusprovidingstablestorageofserum/plasmasamplesandallowingformultiplefreezethawcycles.
• Minimalday-to-dayfluctuationsinhealthysubjects
• SuitabletousetogetherwithM30Apoptosense®ELISAforquantificationofapoptosis,necrosisandtotalcelldeath

CK18KitComposition:

Reagents,Packaging,Storage,andStability

• M65CoatedMicrostrips: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.

InXenograftModels

TheM30 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.