An enzyme-linked immunosorbent assay (ELISA) is a common test used in immunology to detect antigens or antibodies. Antigens provoke an immune system reaction — in other words, they cause illnesses. ELISA is used to detect many bacterial and viral antigens, including human immunodeficiency virus (HIV), malaria, cholera, measles, and mumps. A slightly different ELISA protocol can be used to detect antibodies to these and many other pathogens. An ELISA protocol is the step-by step procedure for performing a specific ELISA.
Though the ELISA protocol varies slightly, depending on the type of ELISA being performed, the basic concept is the same for all of them. ELISA depends on enzyme-linked antibodies, also called antiglobulins. A signal molecule attached to these antiglobulins causes a substrate added during the assay to change color, if the desired antigen or antibody is present. The amount of antigen or antibody present is proportional to the amount of color change, which can be measured with an electronic plate reader.
There are three main types of ELISA. A direct ELISA is usually used to detect antigens rather than antibodies. This is the simplest ELISA protocol, as the enzyme-linked antibody binds directly to the desired antigen. Substrate is then added to determine the quantity of antigen present.
An indirect ELISA is used to detect antibodies, while another type of indirect ELISA — called a sandwich ELISA — can be used to detect antigens. A sandwich ELISA protocol requires two antibodies, called the capture and detection antibodies, to bind to the desired antigen. An antiglobulin is added, which binds to the detection antibody, and the substrate is added. The indirect ELISA follows a similar protocol, but uses a capture antigen rather than a capture antibody in order to detect the desired antibody.
Competitive ELISA is often used for detecting antigens that are present in low concentrations, because it is a very sensitive assay. In contrast to the other ELISA protocols, competitive ELISA uses an enzyme-linked antigen instead of an antibody, and a slightly different quantification method. In it, the sample containing the antigen to be detected and the enzyme-linked antigen are both added to an antibody, and the two antigens compete for antibody-binding sites. When the substrate is added, the color change is greater, with a higher ratio of bound enzyme-linked antigens to bound sample antigens. This means that higher color change is detected at lower concentrations of the sample antigen, which is what makes this method so sensitive.
