Enzyme-LinkedImmunosorbentAssay419419From:MedicalBiomethodsHandbookEditedby:J.M.WalkerandR.Rapley©HumanaPress,Inc.,Totowa,NJ30Enzyme-LinkedImmunosorbentAssayWilliamJ.Jordan1.IntroductionTheenzyme-linkedimmunosorbentassay(ELISA)istypicallyusedtodetectandquantifyantigenwithinbiologicalfluids.AmongtheELISA’sattributesareaveryhighlevelofsensi-tivityandrobustnessaswellasbeinganextremelycost-effectiveassaythatcanbeperformedwithonlybasiclaboratoryequipment.Havingbeendevelopedover30yrago(1),theELISAremainsanassayofchoiceformanyroutinemedicalandveterinarydiagnosticassays,aswellasbeingavitalassayinbasicscienceresearch.ApplicationsoftheELISAtechniquearemanyandvaried.Intheclinicallaboratory,thetestiscommonlyusedtodetectproteinsofpathogensor“markers”thatareindicativeofinfectionordisease.AcommonapplicationoftheELISAintestingforinfectionisintheidentificationofpathogen-specificantibodiesinserum(2).PossiblythebestknownoftheclinicaldiagnosticassayscurrentlyutilizingtheELISAarethosefordiagnosisofhumanimmunodeficiencyvirus(HIV)infection.However,thistechniqueisroutinelyusedfordiagnosisofmostofthemajorviral,andbacterialinfectionsincludinghepatitis,anthrax,andmalaria.OfthehugenumberofvariationsoftheELISA,themostcommonlyusedare(1)thesand-wichELISA,oftenreferredtoasthe“dual-antibodysandwich”orthe“dual-antibodycapture”ELISA,(2)theindirectELISA,and(3)thecompetitiveELISA.Subheading2outlinesthesebasictechniqueswithbriefexamplesoftheiruseintheclinic.2.RoutinelyUsedELISATechniques2.1.TheDual-AntibodySandwichELISAThedual-antibodysandwichELISA(DASELISA)isoneofthemostsensitiveandspecifictechniquesforquantifyingmoleculesinsolution.AnillustrationofthistechniqueisgiveninFig.1A.TheDASELISArequirestwoantibodiesthatrecognizeseparateepitopesontheanti-gentobemeasuredsuchthattheyareabletobindtothemoleculesimultaneously.The“cap-ture”antibody,whichisspecificforthesubstancetobemeasured,isfirstcoatedontoahigh-capacityprotein-bindingmicrotiterplate(anELISAplate).Followingthecoatingstage,anyvacantbind...
