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

================  蚂蚁淘在线  ================

免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容

版权声明：未经蚂蚁淘在线授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘在线”。违反上述声明者，本网将追究其相关法律责任。