VMC 221: Veterinary Immunology and Serology (1+1)

SANDWICH ELISA

• Antigen can be detected or measured by a sandwich ELISA. The antibody (capture antibody) is immobilized on a microtitre plate well.
• A sample containing antigen is added and allowed to react with the immobilized antibody.
• After the well is washed, a second enzyme linked antibody (detecting antibody) specific for a different epitope on the antigen is added and allowed to react with the bound antigen.
• After any free secondary antibody is removed by washing, substrate is added and the colored reaction product is measured. This is the direct sandwich ELISA .
• In the double sandwich ELISA, the detecting antibody (specific antibody) can be from a different species than the capture antibody.
• The antispecies secondary antibody linked with enzyme bind to the detecting antibody and colour develops after substrate is added.

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