ELISA, which stands for Enzyme-Linked Immunosorbent Assay, is a widely used technique in immunology to detect the presence of various antibodies or antigens in a sample. ELISA techniques have proved their supremacy over Radioimmunoassay (RIA) with time. ELISA uses antibodies or antigens which are covalently bound to enzymes to detect specific antigen, antibody or protein rather than radioisotopes which are much more dangerous to handle. The principle behind ELISA is that enzyme-linked to an antigen or antibody converts the substrate added into the chemiluminescent or fluorescent product. This chemiluminescence or fluorescence is detected using an appropriate instrument. The ELISA method can also provide information on the amount of antigen, antibody or protein by measuring the amount of chemiluminescence of fluorescence. ELISA technique has its variants such as Indirect ELISA, Sandwich ELISA and Competitive ELISA. Let us briefly consider them one by one.
Indirect ELISA:
The technique is used to detect the presence of an antibody and the concentration of the antibody in a solution. For this purpose, antigen-specific for an antibody of interest is coated onto a microtiter well plate. Serum or sample containing the antibody of interest is then added into a well. Primary antibody conjugated with the antigen. After washing out non-specifically bound antibodies, a secondary antibody specific for the primary antibody is added. A secondary antibody is conjugated to an enzyme. Finally, a substrate for the enzyme is added, converted into a chemiluminescent or fluorescent product, which is then detected.
Sandwich ELISA:
The technique is used to detect the presence as well as the concentration of antigen in a sample. An antibody specific for the antigen of interest is immobilized in a microtiter well plate in this technique. The sample containing the antigen is then added, and the non-specifically bound antigen is washed away. A secondary antibody linked to the enzyme but specific for a different epitope of antigen is then added. Washing is done to remove non-specifically bound antibody. In the last step, a substrate for the enzyme is added, converted into a chemiluminescent or fluorescent product, which is then detected.
Competitive ELISA:
This technique is an extremely sensitive technique to determine the amount of antigen in a solution. In competitive ELISA, antibodies are first incubated with an antigen, and antigen-antibody complexes are allowed to form. This antigen-antibody mixture is then added to a microtiter well plate coated with antigen. The more antigen present in the initial solution-phase sample, the less free antibody will be available to bind to the antigen-coated well. After the washing step, a secondary antibody specific for primary antibody and conjugated with enzyme is added, and the amount of antibody is detected by adding substrate. In the competitive assay, the higher the concentration of antigen in the original sample, the lower the final signal.
