Sandwich ELISA principle

Sandwich ELISA is a less common variant of ELISA, but is highly efficient in sample antigen detection. Moreover, many commercial ELISA pair sets are built on this sanwich ELISA.

The sandwich ELISA quantify antigens between two layers of antibodies (i.e. capture and detection antibody). The antigen to be measured must contain at least two antigenic epitope capable of binding to antibody, since at least two antibodies act in the sandwich. Either monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in Sandwich ELISA systems. Monoclonal antibodies recognize a single epitope that allows fine detection and quantification of small differences in antigen. A polyclonal is often used as the capture antibody to pull down as much of the antigen as possible. The advantage of Sandwich ELISA is that the sample does not have to be purified before analysis, and the assay can be very sensitive (up to 2 to 5 times more sensitive than direct or indirect ELISA), but lower than ELISpot.

Sandwich ELISA procedures can be difficult to optimize and tested match pair antibodies should be used. This ensures the antibodies are detecting different epitopes on the target protein so they do not interfere with the other antibody binding.

The principle is as follows: (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form.