The enzyme-linked immunosorbent assay (ELISA) is a labeled immunoassay that is considered the gold standard of immunoassays. This immunological test is highly sensitive and is used to detect and quantify substances, including antibodies, antigens, proteins, glycoproteins, and hormones. Detection of these products is accomplished by complexing antibodies and antigens to produce a measurable result. An antibody is a type of protein produced by an individual’s immune system.
This type of protein has specific regions that bind antigens. An antigen is a protein that can come from some foreign source and, when bound to an antibody, induces a cascade of events through the body’s immune system. This interaction is used in ELISA tests and allows specific protein antigens and antibodies to be identified with only small amounts of a test sample. ELISA tests are used to diagnose HIV infection, pregnancy tests, and blood type, among others. This article will discuss the basic principles, procedures, and clinical significance of ELISA.
Although many ELISA variants have been developed and used in different situations, they all depend on the same basic elements:
- Coating/capture: direct or indirect immobilization of antigens on the surface of polystyrene microplate wells.
- Plate blocking: addition of irrelevant protein or another molecule to cover all unsaturated surface binding sites in microplate wells.
- Probing/detection: incubation with antigen-specific antibodies that affinity bind antigens.
- Signal measurement: detection of the signal generated through the direct or secondary label on the specific antibody.
The most commonly used enzyme markers are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes have also been used; these include β-galactosidase, acetylcholinesterase, and catalase. A large selection of substrates for performing ELISAs with an HRP or AP conjugate is commercially available. The choice of substrate depends on the sensitivity of the assay required and the instrumentation available for signal detection (spectrophotometer, fluorometer, or luminometer).
ELISA formats: direct, indirect, and sandwich ELISA
There are several formats used for ELISA. These are classified into direct, indirect, or sandwich detection and capture methods. The key step is the immobilization of the antigen of interest, either by direct adsorption to the test plate or indirectly through a capture antibody that has adhered to the plate. The antigen is then detected directly (labeled primary antibody) or indirectly (as a labeled secondary antibody).
The most widely used Enzyme-Linked Immunoassay format is the sandwich ELISA assay, which immobilizes and indirectly detects the presence of the target antigen. This type of capture assay is called a “sandwich” assay because the analyte to be measured is bound between two primary antibodies, each of which detects a different epitope on the antigen: the capture antibody and the detection antibody. The ELISA sandwich format is widely used due to its sensitivity and specificity.
Direct versus indirect ELISA detection strategies
Amongst the standard assay formats discussed and illustrated above, where differences in both capture and detection were the concern, it is important to differentiate between particular strategies that exist specifically for the detection step. Regardless of the method by which an antigen is captured on the plate (by direct adsorption to the surface or via a pre-coated “capture” antibody, as in an ELISA sandwich), it is the detection step (either as direct detection or indirect) ) which largely determines the sensitivity of an ELISA.
The direct detection method uses a primary antibody labeled with a reporter enzyme or a label that reacts directly with the antigen. Direct detection can be performed with an antigen that is immobilized directly on the assay plate or with the capture assay format. Direct detection, although not widely used in ELISA, is quite common for immunohistochemical staining of tissues and cells.
The indirect detection method uses a labeled secondary antibody or biotin-streptavidin complex for amplification and is the most popular format for ELISA. The secondary antibody has specificity for the primary antibody. In a sandwich ELISA, it is critical that the secondary antibody be specific for the detection of the primary antibody only (and not the capture antibody) or the assay will not be specific for the antigen.
In general, this is accomplished by using primary and capture antibodies from different host species (eg, mouse IgG and rabbit IgG, respectively). For sandwich assays, it is beneficial to use secondary antibodies that have been cross-adsorbed to eliminate any secondary antibodies that may have an affinity for the capture antibody.