ThesandwichELISAmeasurestheamountofantigenbetweentwolayersofantibodies.Theantigenstobemeasuredmustcontainatleasttwoantigenicsites,capableofbindingtoantibody,sinceatleasttwoantibodiesactinthesandwich.Sosandwichassaysarerestrictedtothequantitationofmultivalentantigenssuchasproteinsorpolysaccharides.SandwichELISAsforquantitationofantigensareespeciallyvaluablewhentheconcentrationofantigensislowand/ortheyarecontainedinhighconcentrationsofcontaminatingprotein.

Procedure

1.Acaptureantibodyisfirstdilutedin0.1MBicarbonatebuffer,pH9.2andthen50µlisaddedtoeachwellofthemicrotiterplate.

2.TheantibodycoatedplateiscoveredwithParafinandincubatedinthecoldroomovernightinamoistboxcontainingawetpapertoweloratroomtemperatureandhumidityfortwohours.

3.Theplateisemptiedandtheunoccupiedsitesareblockedwith100µlofblockingbuffercontaining100mMphosphatebuffer,pH7.2,1BSA,0.5Tween-20and0.02Thimerosolfor30minatroomtemperature.

4.Theplateisemptiedandwashedthreetimeswithwashbuffer(100mMphosphatebuffer,150mMNaCl,0.2BSAand0.05Tween20).

5.Theantigensolutionisfirstdilutedinantigenbuffer(100mMphosphatebuffer,150mMNaCl)andthenaddedtotheplateinavolumeof50µlperwell.Theplateisincubatedatroomtemperaturefor45mintoonehour.

6.Theplateisemptiedagainandwashedthreetimeswithwashbuffer.

7.Theenzyme-labeledantibodyagainstantigenisdilutedappropriatelyin0.1MBicarbonatebuffer,pH9.2andthen50µlisaddedtoeachwellandincubatedatroomtemperaturefor30min.

8.Theplateisemptiedagainandwashedthreetimeswithwashbuffer.

9.Thecolordevelopmentsystemisaddedandthecolorintensitiesaremeasured.