DAY1 1.CoatNuncimmuno-moduleplatesovernight,inusualmanner. 2.Setupcompetitionassaybetweenantibodyandcompetingsubstance:- Preparea1:2serialdilutionofthecompetitivesubstancefrom480to0.4ng/ml.Add50ulofeachoftheabovedilutionsto25ulof3xstrengthtitreofantibody(i.e.asolution3xthetitregiving50%inhibitionondirectELISA),insealedtubes. 3.Incubateovernightatroomtemperature. 4.Setupcompetitionbetweenunknownconcentrationofcompetitivesubstanceandantibodyasabove. DAY2 1.Washplatewith4x200ul/wellPBS/Tween. 2.Add50ul/wellofantibodyandincubatefor2hrs. 3.Wash4xwith200ul/wellPBS/Tween. 4.Add50ul/wellsecondaryantibodyandincubatefor2hrs. 5.Wash4xwith200ulPBS/Tween. 6.Add50ulperoxidasesubstrate(TMB)asfordirectELISA. 7.ReadOD@450nm. 8.Plotstandardsagainstabsorbanceanddetermineunknownconcentrations.
