Enzyme-linked immunosorbent assays (ELISAs) are often used to measure antibody or antigen binding activity, either in a pure solution of a single protein, or in a mixed solution of multiple proteins such as serum.
ELISAs perform this measurement via conversion of the binding activity of an antibody or antigen to a measurable spectrophotometric signal. ELISAs are a common assay in industrial and research applications, especially in diagnostics and quality control. This technique was first introduced in the 1970s to replace the use of radioactive isotopes protein detection.
The ELISA technique leverages the specificity of antibodies in the detection of protein. More specifically, an enzyme is covalently linked with a target-specific antibody (either polyclonal or more reliable monoclonal antibodies). In the presence of the corresponding antigen, the antibody-enzyme is bound, any unbound antibody-enzyme complexes are removed via a washing step, and a substrate which reacts with the enzyme to produce a colored product is introduced in order to measure the antibody-antigen binding. ELISAs are typically performed in a 96-well plate and are sufficiently sensitive to detect a nanogram or less of a target antigen. Two common ELISA types used are indirect ELISA and sandwich ELISA.
An indirect ELISA is utilized in the detection of antibodies. These ELISAs are performed by first coating a surface with a known amount of antigen, and then a solution with an unknown quantity of antibody (possibly patient serum) is added to the well in order to bind it to the antigen. Detection is performed with the addition of enzyme-linked antibodies (chosen due to their specificity for the antibody being measured), which are allowed to bind, and unbound antibodies are removed by washing. Substrate is added to induce coloration in the enzyme, indicating the presence of the target antibody. One application of this type of ELISA is in the test for HIV infection.
Sandwich ELISAs are used to detect and quantify a particular antigen, even in small quantities. An antibody specific to the antigen being measured is coated on the bottom of a well. The solution containing the antigen is added to the well and allowed to bind to the antigen. A second, different antibody that also binds the target antigen and has an enzyme linked to it is added to the well, allowed to bind, and then excess antibody is washed off. The addition of substrate causes the presence of coloration measured by spectrophotometry, and the extent of reaction is proportional to the quantity of antigen present.
Other types of ELISA are possible to test different varieties of ligand binding or measure other biological reactions. The ELISA is a powerful tool for both industrial and research applications including diagnostics and biotechnological quality control.