SandwichELISA

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

Procedure

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

2.TheantibodycoatedplateiscoveredwithParafinandincubatedinthecoldroomovernightinamoistboxcontainingawetpapertoweloratroomtemperatureandhumidityfortwohours.

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

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

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

6.Theplateisemptiedagainandwashedthreetimeswithwashbuffer.

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

8.Theplateisemptiedagainandwashedthreetimeswithwashbuffer.

9.Thecolordevelopmentsystemisaddedandthecolorintensitiesaremeasured.

相关仪器试剂设备

• 酶标仪
• 洗板机
• 化学发光检测仪
• ELISA检测试剂盒
• ELISPOT检测
• ELISA试剂盒
• 抗体
• 酶标板