An enzyme-linked immunosorbent assay (ELISA) is typically performed to detect the presence and/or amount of a target protein of interest within an experimental sample. Detection of the target protein is made possible by antibodies, which make the ELISA an immunoassay. Through a series of incubation and washing steps, these antibodies, which are frequently linked, or conjugated, to an enzyme, will detect protein coating the bottom of a well on a microtiter plate. When exposed to a substrate, antibody-bound enzyme will cause a color change, thereby indicating the presence of the protein-of-interest in the sample.
In this video, the theory behind how ELISAs work is explained, including a discussion of both primary and secondary antibody binding and the importance of blocking steps. Theory is followed by practice, as the video progresses to an explanation of the step-by-step procedure. Finally, variations of the standard ELISA such as the sandwich and competitive ELISAs are introduced, and real world applications of this method, such as in over-the-counter pregnancy tests are explained.
Procédure
The ELISA, or enzyme-linked immunosorbent assay, is a widely used method for determining the presence or absence of a specific target protein. Via a series of washing and binding steps, an antibody conjugated, or linked, to an enzyme will recognize a target protein at the bottom of a 96-well plate. When substrate is added to the sample, an enzymatic reaction will occur, causing a color change that allows the identification and quantification of the target protein. Before we discuss…