32.15: Enzyme-Linked Immunosorbent Assay
In 1971, Peter Perlman and Eva Engvall developed an Enzyme-linked immunosorbent assay (ELISA or EIA). ELISA differs from western blot in that the assays are conducted in microtiter plates or in vivo rather than on an absorbent membrane.
There are many different types of ELISAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen. Enzyme-substrate reaction allows the antigen to be visualized or quantified. In ELISAs, the substrate for the enzyme is most often a chromogen, a colorless molecule that is converted into a colored end product. The most widely used enzymes are alkaline phosphatase and horseradish peroxidase, for which appropriate substrates are readily available. In some ELISAs, the substrate is a fluorogen, a nonfluorescent molecule that the enzyme converts into a fluorescent form. ELISAs that utilize a fluorogen are termed fluorescent enzyme immunoassays (FEIAs). Fluorescence can be detected by either a fluorescence microscope or a spectrophotometer.
There are several types of ELISA, based on differences in the format of detection and general workflow. In direct ELISA, antigens are immobilized in the well of a microtiter plate. An antibody that is specific for a particular antigen, and is conjugated to an enzyme, is added to each well. If the antigen is present, then the antibody will bind. After washing to remove any unbound antibodies, a chromogen is added.The presence of the enzyme converts the substrate into a colored end product.
Indirect ELISA is an extremely sensitive and flexible procedure. Here, in addition to a primary antibody, a secondary antibody is added for detection purposes. The secondary antibody quantifies how much antigen-specific antibody is present in the sample by the intensity of the color produced from the conjugated enzyme-chromogen reaction.
Sandwich ELISA is more specific and sensitive than direct and indirect ELISA. The goal is to use antibodies to precisely quantify the specific antigen present in a solution, such as the antigen from a pathogen, a serum protein, or hormone from blood or urine. The primary antibody captures the antigen and, following a wash, the polyclonal enzyme-conjugated secondary antibody is added. After a final wash, a chromogen is added, and the enzyme converts it into a colored end product. The amount of color produced (measured as absorbance) is directly proportional to the amount of enzyme, which in turn is directly proportional to the captured antigen. The complex workflow and several optimizations make this process error-prone.
For competitive ELISA, crude samples can be directly used. The experimental setup is highly flexible, wherein direct, indirect, or sandwich ELISAs can be adapted to a competitive format. The sample analyte concentration is determined by the signal interference. The "competition" comes from the fact that if a sample antigen is being tested, then the incubation of the sample with a primary antibody will result in lesser antibodies available to bind to the wells coated with the same antigen. Thus, the intensity of the signal produced in the well, due to the competitive binding, which is concentration dependent, becomes inversely correlated to the amount of sample antigen.
Depending on the purpose, the subtype of ELISA is chosen for a suitable outcome. For detecting large proteins comprising multiple epitopes, sandwich ELISA is most appropriate, whereas competitive ELISA is ideal for small protein detection. ELISA has significant applications in diagnostics, such as pregnancy testing, detecting food allergens, and identifying cancer biomarkers.