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MesoScaleDiscovery®p85p110PDKAktmTORp70S6KPPPPPI3-KinasePTENPPPPPPIP2PIP3PPPPRTK(ADA)MesoScaleDiscovery2TM®®12ABMULTI-ARRAYPlateRu(bpy)33+Ru(bpy)32+*Ru(bpy)32+TPATPA.+TPA.-H+e-e-LIGHTLIGHTAvidin(orStreptavidin)-Biotin3ExcellentYes1HighYes10sng/mL3-4logs5-25µL10XMorePoorNo3-4GoodYes100sng/mL1-2logs25-100µLBetterFreeDrugToleranceDetectionofLowAffinityAntibodiesFewerWashesHigh-ThroughputDirectConjugationofStableLabelImprovedSensitivityIncreasedDynamicRangeReducedSampleVolumeHigherBindingCapacity[ng/mL]100,00010,0000.011010,0001,000MSDELISA1x10+6+ELISA+MSDSignal/BackgroundADAConcentration[ng/ml]MSDassayshowscomparablesensitivitytoELISA,withalargerdynamicrangeandasimplehomogenousincubation.Reference:Moxness,M.,Tatarewicz,S.,Weeraratne,D.,Murakami,N.,Wullner,D.,Mytych,D.,Jawa,V.,Koren,E.,Swanson,S.J.(2005)ImmunogenicityTestingbyElectrochemiluminescentDetectionforAntibodiesDirectedagainstTherapeuticHumanMonoclonalAntibodies.ClinicalChemistry.51:1983-1985.ELISAMSDSULFO-TAGlabeleddruganti-drugantibodybiotin-labeleddrugMSDSAplateGoodMaybe2-3GoodNo10sng/mL3-4.5logs2-10fold10XMorePoorNo3-5GoodNo100sng/mL1-2logsBetterFreeDrugToleranceDetectionofLowAffinityAntibodiesFewerWashesHigh-ThroughputDirectConjugationofStableLabelImprovedSensitivityIncreasedDynamicRangeReductioninReagentConsumptionHigherBindingCapacityELISAMSDanti-humanSULFO-TAGantibodyanti-drugantibodyproteindrug1,0001001010.01101,000MSDBridgingAssayProtocol1.Combinebiotin-drug,sTAG-drugandsampleinpolypropyleneplateandincubatefor1hourtoovernight.2.Transfersolutiontopre-blockedstandardstreptavidinMSDplate.Incubatefor1hour.3.Washassayplate;addReadBufferT;readplateonSECTORTMinstrument.MSDSandwichImmunogenicityAssayProtocol1.Coatplatewithdrugat0.05to5pmoleperwellandincubatefor1hourtoovernight.2.Add150µL/wellofBlockingSolutionandincubatefor1hour.3.Washplate.Add25µLofsample.4.(Optionalwash).Add25µLofdetectionantibody.5.Washassayplate;addReadBufferT;readplateonSECTORinstrument.4101,0000.1100101SULFO-TAGlabeledDruganti-drugantibodybiotin-labeleddrugMSDPlate(avidinorstreptavidin)Druginterferenceinimmunogenicityassaysfromfreedruginpatientsamplescancausefalsenegativesandsuppressedsignal.AssaysdevelopedonMSD’srobusttechnologyplatformdemonstrateimproveddrugtoleranceoverELISAmethodsformanyreasons.TheimprovedsensitivityoftheMSDplatformproduceshighersignalandlowerbackgrounds,leadingtogreatersignaltobackgroundratios,detectionoflowlevelsofdrug-antidrugantibodycomplexes.TheuniquecarbonsurfaceofMSDplatesaffordsa10-100foldincreaseinsurfacecapacityoverELISAplates.Longersolutionphaseincubationscoupledwithhigherlevelsofbiotinylatedorlabeleddrugcanbeutilizedtobiastheantibodyassociationwiththelabeledmaterialwithoutsignificantlyincreasingbackground.Improvedassaysensitivityallowsforlargersampledilution,furtherreducingdruginterference.MSDassaysaretolerantofsamplepre-treatmentsusedtoreducedruginterferenceincludingacid/baseneutralizationandmaterialssuchassalts,surfactants,anddenaturingagents.WefeatureMSDbridgingassaysbelowtohighlightthedrugtoleranceoftheMSDplatformascomparedtotraditionalELISAassays.Visit(uptoovernight),significantlyreducesdruginterferenceeffects.Thisoccursthroughthedissociationofthenativedrugfromtheantibodyduringtheextendedincubation,freeingtheantibodyforassociationwithbiotinylatedorlabeleddrug.Anexampleofabridgingimmunogenicityassayisshownwithdifferentlevelsoffreedrugaddedtothesample(samplematrixwasneathumanserum).-LOQ30ng/mL-Noeffectonassayforfreedrugconcentrationsupto300ng/mL-Assaycantolerateupto3µg/mLfreedrugat100ng/mLofanti-drugantibodyMSDassayshaveimproveddrugtoleranceoverELISA-Highersensitivitywithlargersampledilution-Increasedplatesurfacecapacity-Longersolutionphaseincubation-Robustassaystolerantofsamplepre-treatmentsFreeDrugConcentrationSignal/BackgroundAnti-DrugAntibodyConcentration[ng/mL]0ng/mL3ng/mL30ng/mL300ng/mL3,000ng/mL30,000ng/mLDrugToleranceMSDBridgingAssayProtocol1.Combinebiotin-drug,SULFO-TAGdrugandsampleinpolypropyleneplateandincubate1hourtoovernight.2.Transfersolutiontopre-blockedstandardstreptavidinMSDplate.Incubatefor1hour.3.Washassayplate;addReadBufferT;readplateonSECTORinstrument.5DrugToleranceComparisontoELISAforFreeDrugToleranceComparisonofProtocolswithNoFreeDrugPresentSignal/BackgroundADAinWellDuringSampleIncubation(ng/mL)101001,00010,0000.11101001,000Protocol1:HomogenousProtocol2:ELISA-Like10,0001Sample,Capture,DetectioninPolypropylenePlateIncubateonMSDPlateWashReadPlateSampleDetectionWashReadPlateWashWashCaptureELISA-likeFormatELISA-likeFormatMSDHomogenousFormatMSDHomogenousFormatMSDHomogenousProtocolwithInterferingFreeDrugSignal/Back