The enzyme linked immunosorbent assay (ELISA) is a gold standard method for protein and antibody detection. It is routinely used for clinical lab work and is often used in research and development. The assay is based on the lock-and-key specificity between an antibody-antigen pair. Antibodies are naturally made by the adaptive immune system to recognize a specific piece of an antigenic protein. This is what allows the body to identify and fight invading pathogens so effectively. Since antibodies have such a fundamental role in most biosensing and diagnostic systems it is very easy to purchase them or have them custom-made to recognize a protein of interest. The ELISA assay is performed in 96- or 384-well plates that are treated to favorably adsorb antigen or antibody depending on the type of ELISA being performed. The ELISA assay is built up from the bottom of each well; it doesn’t float freely in the well. As a result, the unbound proteins are easily washed away and only the target remains to be quantified.
The four types of ELISA
The competitive ELISA is one of four methods. Each method is used for a different purpose. Some are good at detecting antigen while others are used to detect antibody. All of them require the use of a fluorescent, chemiluminescent, or colorimetric tag. These tags are called reporters; they are used to quantify the assay.
- Direct ELISA: The antigen is immobilized on the surface of the plate and is detected by an antibody conjugated to a reporter. A higher signal equals more target.
- Indirect ELISA: The antigen is immobilized on the surface of the plate and two antibodies are used for detection. The primary antibody binds to the antigen and the secondary antibody binds to the primary antibody. The secondary antibody is conjugated to a reporter, and it cannot recognize the antigen. Therefore, without any primary antibody the secondary will be washed away and there will be no signal to quantify. Therefore, the indirect ELISA is used to detect primary antibodies in a sample. A higher signal equals more target.
- Sandwich ELISA: An antibody is immobilized on the surface of the plate. The antigen is added and it binds to the antiboy Then, another antibody, which recognizes a different epitope of the antigen, is added to the well and it also binds to the antigen. Finally, a third antibody with a reporter is added to recognize the second antibody. This method is used to detect antigen in a sample. A higher signal equals more target.
- Competitive ELISA: A reference antigen is immobilized on the surface of the plate. The sample antigen is pre-incubated with labeled reporter antibody and added to the wells. Any free antibodies unbound with antigen during the pre-incubation step will be able to bind to the reference antigen. All other labeled antibodies will be unable to bind to the immobilized antigen and will be washed away. This method is used to detect antigen in a sample. A higher signal equals less target.