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.

Diapharma的COVID-19血清学COVID-19血清学测试可测量因SARS-CoV-2（引起COVID-19的病毒）感染而产生的抗SARS-CoV-2抗体的数量。COVID-19血清学测试表明，某人最近可能已暴露于SARS-CoV-2病毒。重要的是要注意，COVID-19血清学检测并不表明存在活跃的SARS-CoV-2感染。对于基于人群的COVID-19研究和基本的SARS-CoV-2研究项目，COVID-19血清学测试是一种有价值的研究工具。这包括疫苗试验，治疗药物试验，流行病学研究和患病率分析。免疫学，传染病和凝血领域的COVID-19研究也可能发现SARS-CoV-2抗体检测有益和适用。SARS-CoV-2的刺突蛋白（S）和核壳蛋白（N蛋白或NP）结构域通常在COVID-19血清学测试中用作重组抗原。S蛋白包含一个受体结合域（RBD），该域与宿主细胞上的ACE2细胞表面受体结合，从而促进SARS-CoV-2进入细胞。N蛋白高度保守，大量表达，并在SARS-CoV-2病毒转录和复制过程中发挥功能。针对N蛋白的抗体显示出最高的敏感性，并在感染后最早的时间检测到。尽管在感染后的较晚时间检测到针对RBD的抗体，但建议抗RBD抗体与病毒的中和相关，因此是免疫力的有力指标。 免疫球蛋白类别（IgM，IgA和IgG）使抗体能够识别并结合SARS-CoV-2抗原。IgM抗体是SARS-CoV-2感染后第一个被检测到的抗体。IgA抗体是在感染后的早期阶段检测到的，并且存在于呼吸道上皮和胃肠系统的粘液和唾液中。进一步建议，IgA抗体在针对SARS-CoV-2的第一道防线中起作用，这可能对从呼吸道清除SARS-CoV-2至关重要。在SARS-CoV-2感染后的较晚时间检测到IgG抗体。IgG抗体在血清中的保留时间最长，使其成为赋予免疫力的有趣指标。 测量COVID-19抗体的血清学检测方法有多种类型：快速诊断检测（RDT），ELISA检测和中和检测。RDT在最短的时间内测量定性数据（阳性或阴性），通常在护理时使用。这意味着RDT指示样品中存在COVID-19抗体，但未测量检测到的抗体的准确数量。ELISA血清学测试可以定性或定量，需要使用实验室设备和试剂，并且需要几个小时才能完成。COVID-19 ELISA分析利用重组SARS-CoV-2蛋白结构域作为抗原来结合样品中的抗体。接下来，标记的抗人免疫球蛋白抗体结合抗体-抗原复合物。最后，酶标记物能够读出COVID-19抗体水平（图1）。中和测定法定量测量样品中抗体中和细胞培养物中活的SARS-CoV-2病毒的能力。该方法精确地定量了赋予保护性免疫和抑制病毒复制所需的抗体数量。中和测定需要几天的时间才能完成，需要严格的生物学安全预防措施。 图1：COVID-19 ELISA血清学检测产品名称方法捕获抗原免疫球蛋白读出Technozyme®抗SARS-CoV-2 RBD IgG ELISA试剂盒ELISA，开放系统刺突蛋白的受体结合结构域（RBD）IgG抗体定量的Technozyme®抗SARS-CoV-2 NP IgG ELISA试剂盒ELISA，开放系统核衣壳蛋白（N蛋白）IgG抗体定量的金标准诊断SARS-CoV-2 IgG ELISA测试试剂盒ELISA，开放系统核衣壳蛋白（N蛋白）IgG抗体定性的金标准诊断SARS-CoV-2 IgA ELISA检测试剂盒ELISA，开放系统核衣壳蛋白（N蛋白）抗体定性的