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ElevatingELISPOTtoanExactScience
ELISPOT:fieldsofapplicationVisualizingthesecretoryproductsofindividualcells –detectingrarecells.Thetechniqueofchoiceinimmunemonitoring:BasicimmunologyInfectiousdiseasesAutoimmunityTumorimmunityTransplantationAllergiesVaccinedevelopment
AdvantagesofELISPOTvs.othersSensitivity:(detectionlimit1/1million,andless:severalhundredfoldmoresensitivethanELISA,BeadArray,Tetramer,ICS,mRNAetc).Functionalassay:Lowcost:Instrumentation,reagentsandlaborareverylowcostrelativetoflowbasedtechniques.Highthroughput:Singleinvestigatorcantestdozensofsamplesforhundredsofreactivateseachinasingleexperiment.Lesssample:Easeofuse:CTLcantrainexpertsin4days
KarulinandLehmann,J.Immunol.,2000,164:1862-1872.ICS(AC)andELISPOT(BD)havesimilarresultsfordetectingcytokine(co)expressioninsingleTcells
MajorStrength:Sensitivity:ELISPOTpermitsaccuratefrequencymeasurementsthe1/1,000,000cellrange–ICSandELISA(alsoCBA)reachdetectionlimitat1:1,000
-6/14cases(43%)positiveresponsesaremissedbypentamers:-Evenverystrongresponsescanbemissed-Numberscanbemisleading,e.g.,0.05%canbespecific,0.09%nonspecific*Nonspecificstaining
Proportionsdonotmatchup(seeboxesofsamecolor)
IFN-gPFNGzBMediumCEF
RapidlygrowingELISPOT
Problem
Performanceof11well-establishedELISPOTlaboratories
Singlecellresolution:AssayImageanalysis
CaptureantibodyEnzyme-coupleddetectionantibodySecretedcytokineAntigen-stimulatedcellChromogenBACDEPVDF-membraneEnzyme-catalyzedchromogenspotdevelopmentPlatecoatedwithdetectionantibodySecretingcell,e.g.antigen-stimulatedTcellsecretingcytokinePlate-boundsecretoryproduct(cytokine)aftercellsarewashedawayProduct-specificdetectionantibodywithlinkedenzymePrincipleoftheELISPOTassay
Inassay1:TheTypeofPlate
Inassay2:AntigenConcentration
Inassay3:The
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