JournalofVisualizedExperimentswww.jove.comCopyright©2012CreativeCommonsAttribution-NonCommercialLicenseOctober2012|68|e4247|Page1of6VideoArticleHighThroughputSequentialELISAforValidationofBiomarkersofAcuteGraft-Versus-HostDiseaseBryanFiema*1,AndrewC.Harris*1,AurelieGomez1,PraechompooPongtornpipat1,KellyLamiman1,MarkT.VanderLugt1,SophiePaczesny11PediatricBloodandMarrowTransplantProgram,UniversityofMichigan*TheseauthorscontributedequallyCorrespondenceto:SophiePaczesnyatsophiep@umich.eduURL:http://www.jove.com/video/4247DOI:doi:10.3791/4247Keywords:Medicine,Issue68,ELISA,SequentialELISA,Cytokine,Bloodplasma,biomarkers,proteomics,graft-versus-hostdisease,Smallsample,QuantificationDatePublished:10/31/2012Citation:Fiema,B.,Harris,A.C.,Gomez,A.,Pongtornpipat,P.,Lamiman,K.,VanderLugt,M.T.,Paczesny,S.HighThroughputSequentialELISAforValidationofBiomarkersofAcuteGraft-Versus-HostDisease.J.Vis.Exp.(68),e4247,doi:10.3791/4247(2012).AbstractUnbiaseddiscoveryproteomicsstrategieshavethepotentialtoidentifylargenumbersofnovelbiomarkersthatcanimprovediagnosticandprognostictestinginaclinicalsettingandmayhelpguidetherapeuticinterventions.Whenlargenumbersofcandidateproteinsareidentified,itmaybedifficulttovalidatecandidatebiomarkersinatimelyandefficientfashionfrompatientplasmasamplesthatareevent-driven,offinitevolumeandirreplaceable,suchasattheonsetofacutegraft-versus-hostdisease(GVHD),apotentiallylife-threateningcomplicationofallogeneichematopoieticstemcelltransplantation(HSCT).HerewedescribetheprocessofperformingcommerciallyavailableELISAsforsixvalidatedGVHDproteins:IL-2Rα5,TNFR16,HGF7,IL-88,elafin2,andREG3α3(alsoknownasPAP1)inasequentialfashiontominimizefreeze-thawcycles,thawedplasmatimeandplasmausage.ForthisprocedureweperformtheELISAsinsequentialorderasdeterminedbysampledilutionfactorasestablishedinourlaboratoryusingmanufacturerELISAkitsandprotocolswithminoradjustmentstofacilitateoptimalsequentialELISAperformance.Theresultingplasmabiomarkerconcentrationscanthenbecompiledandanaly...
