ELISA Development and Optimization
There are many things to consider when developing an ELISA. The first is what ELISA format to use—direct, indirect, or sandwich. Formats differ in how the target antigen is captured and detected. Direct and indirect ELISA both immobilize the antigen on the assay plate and then use either a labeled primary antibody (direct) or primary antibody and labeled secondary antibody (indirect) to detect the antigen. Sandwich ELISA is considered the most robust format because the antigen is “sandwiched” between two primary antibodies (capture and detection) and then detected using a labeled secondary antibody (or labeled streptavidin, if the detecting antibody is biotinylated).
The following videos describe detection methods in sandwich ELISA, as well as an entirely different ELISA format called competitive ELISA.