TheELISAprotocolsfordetectionoftheantibodybindingtoanantigen-coatedmicrotitreplatearestandardlaboratorytechniquesandwillnotbedescribedhere.WewilljustmentionthatmostrecombinantantibodyfragmentsaretypicallydetectedusingmonoclonalantibodiesdirectedagainstapeptidictagengineeredattheC-terminalextremityoftherecombinantantibody.Incertainexperimentalconditions,suchpeptidictagsmayundergoproteolyticcleavage,therebyloweringthesensitivityofantibodydetection.Reagentsthatbindtotheantibodymoleculewithoutimpairingantigenbinding(e.g.,proteinAorproteinL)maythereforebepreferable.Alternatively,theexperimentalschemedescribedbelowcanbeperformedinasimilarfashion,usingrADIolabeledantibodiesandradioacitvedetectionofantibody-mediatedantigenbinding.Theconcentrationofpurifiedantibodypreparationsistypicallydeterminedspectrophotometrically(1mg/mlantibodysolutionabsorbs1.4absorptionunitsat280nm).Ifnecessary(forexamplewhenusingsupernatants),theconcentrationofactiveantibodycanbedetectedwithastraightforwardELISAadaptationoftheprotocolmentionedaboveforthedeterminationofantibodyconcentrationbyband-shiftassay.
Coatwithantigen(inidenticalfashion)anappropriatenumberofwellsoftwomicrotitreplates.Preblockthewellswith3%MPBSfor2hoursatroomtemperature,thenwashwithPBS.Inparalleltubes,incubateanantibodysolution(atconcentrationsbelowKd,e.g.0.5nM)withincreasingconcentrationsofantigen(e.g.,rangingbetween0.1nMand1µM)inPBS[totalvolumeofeachreaction:100µl].After30minutesincubationatroomtemperature,apply90µlofthereactionmixturestothewellsofthefirstantigen-coatedmicrotitreplate(performtheexperimentinduplicateortriplicate),containing30µl10%MPBS.Incubatethereactionmixtureontheantigen-coatedplateforasuitablyshorttime(e.g.,10min.).Afterincubation,transferthereactionmixturestothesecondantigen-coatedmicrotitreplate.TheELISAassayusingthissecondplatewillnowbeperformedexactlyasforthefirstmicrotitreplate.ThepurposeofthesecondELISAassayistocheckthatonlyasmallfractionofthefreeantibodyiscapturedonthefirstmicrotitreplateand,therefore,noreadjustmentoftheequilibriumoccurredduringthefirstcapturestep.WashextensivelythefirstELISAplateandperformtheremainingstepsofanELISAprocedure,aimedatthedeterminationoftheantibodybindingtothecoatedantigen.DeveloptheELISAwithasuitablechromogenic,fluorogenicorchemiluminescentsubstrate,andmeasuretheindividualwellswithanappropriateELISAplatereader.ThehighestELISAsignalshouldbeobservedatlowconcentrationsofantigen.NoELISAsignalshouldbeobservedathighconcentrationsofantigen.Theconcentrationofantigenatwhichthehalf-maximalELISAsignalisdetectedcorrespondstothedissociationconstantKd.Alternatively,theKdvaluecanbeobtainedbyfittingtheELISAsignaloftheindividualwellstotheequation:Kd=[A][B]/[AB].
