Sandwich ELISA:
Of the various formats used for ELISA, the most widely used is the sandwich ELISA assay. This technique indirectly immobilizes and indirectly detects the presence of the target antigen. Sandwich ELISA is termed “sandwich” because the analyte or antigen to be measured is bound between two primary antibodies, a capture antibody and a detection antibody. Each antibody binds to and detects a different portion of the antigen. The sandwich ELISA format is highly used because it is very sensitive and specific.
In general, a "sandwich" ELISA involves the following steps:
Plate is coated with a capture antibody- Any nonspecific binding sites on the surface are blocked.
Sample is added, and any antigen present binds to capture antibody Plate is washed to remove unbound antigen.
Detecting antibody is added and binds to antigen The “sandwich” is now formed because the antigen is bound between two antibodies.
Enzyme-linked secondary antibody is added, and binds to detecting antibody Plate is washed to remove the unbound antibody-enzyme conjugates.
Substrate is added and is converted by enzyme to detectable forms such as a color or fluorescent or electrochemical signal The signal is measured to determine the presence and quantity of antigen.
