
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.
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(1)优级纯试剂 亦称保证试剂,为一级品,纯度高,杂质极少,主要用于精密分析和科学研究,常以GR表示。
(2)分析纯试剂 亦称分析试剂,为二级品,纯度略低于优级纯,杂质含量略高于优级纯,适用于重要分析和一般性研究工作,常以AR表示。
(3)化学纯试剂 为三级品,纯度较分析纯差,但高于实验试剂,适用于工厂、学校一般性的分析工作,常以CP表示。
(4)实验试剂 为四级品,纯度比化学纯差,但比工业品纯度高,主要用于一般化学实验,不能用于分析工作,常以 LR表示。
以上按试剂纯度的分类法已在我国通用。根据化学工业部颁布的“化学试剂包装及标志”的规定,化学试剂的不同等级分别用各种不同的颜色来标志,见表1。
表1 我国化学试剂的等级及标志
bhclna2so4nano3na2co3
chclnaohna2co3nacl
dba(oh)2nahco3alcl3nahso4
刚入这行,谢谢大家
A.H2SO4Na2SO4AgNO3BaCl2.
B.NaOHNa2CO3NaHSO4MgCl2
C.CaCl2NaNO3MgSO4BaCl2
D.HNO3KOHKClK2CO3

