1.Materials

(1)TSBuffer:10mMTrispH8.0150mMNaCl

(2)TSTBuffer:TSBufferwith0.05%Tween-20

(3)AlkalinePhosphataseBuffer:50mMNaCarbonate1mMMgCl2(BringtopH9.8with1NHCl)

(4)APSubstrate:p-nitrophenylphosphate(5mg/tablet)。SigmaCat.#N9389

(5)AP-conjugatedsecondaryAb:AP-Rabbitanti-mouseIgG(Zymed).ReconstituteinddH2Oasprescribedbymanufacturerandaliquotandstoreat-70°C.

(6)100mMEDTApH8.0

2.Protocol

(1)Add50μlofantigentoeachwelloftheELISAplate.Iusea1μg/mlofpurifiedSec4pmostantigenswillworkinthe1-10μg/mlrange,butyouneedtodeterminethisemperically.Allowtheantigentobindtothewellsatroomtemperaturefor1-2hoursorovernightat4°C.

(2)Discardantigensolutionbyflickingintosinkandthenplacingupside-downontoKimwipes.Wash1Xwith100μlTSTBuffer.Add200μlTSTwith2%BSAtoblock.Incubateatroomtemperaturefor1hour.

(3)Discardblocksolutionandadd50μlmonoclonalcellculturesupernatantsdirectlyoraddthesamevolumeofseradilutedinTSTw/1%BSA.Incubate1houratroomtemperature.

(4)Discardtheprimaryantibodysolutionandwash3XwithTST.

(5)Add50μlofdilutedsecondaryantibodyconjugatedtoAlkalinePhosphatase.Iusea1:500dilutionofrabbitanti-mouseIgG(Zymed)。Incubateatroomtemperaturefor1hour.About15minbeforetheendoftheincubationstartdissolvingthep-nitrophenylphosphatesubstratetablets.ForeachELISAplatedissolve1tabletin5mlofAPBuffer.

(6)Discardthesecondaryantibodysolutionandwash3XwithTST.

(7)Add50μlperwellofthep-nitrophenylphosphatesolutionandincubateatroomtemperaturefor10-30min.Stopthereactionsbyadding50μlof0.1MEDTApH8.0.ForscreeningmonoclonalsitisbesttoletthemgountilallofthepositivesareclearlyvisIBLe,howeverifyouwanttobequantitativeitisimportantnottoletthemgetintenselyyellowortheywillbeoffscale--theplatereaderismostaccuratewhenthewellshavejustbeguntoclearlyturnyellow.

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