The enzyme-linked immunosorbent assay (ELISA) is a powerful method for detecting and quantifying a specific protein in a complex mixture. Originally described by Engvall and Perlmann (1971), the method allows the analysis of protein samples immobilized in microplate wells using specific antibodies. ELISAs are generally performed in 96- or 384-well polystyrene plates, which passively bind to antibodies and proteins. It is this binding and immobilization of reagents that makes ELISAs easy to design and perform. Having the ELISA reagents immobilized on the surface of the microplate makes it easier to separate bound from unbound material during the assay. This ability to use high-affinity antibodies and wash away non-specific bound materials makes ELISA a powerful tool for measuring specific analytes within a crude preparation.
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 (labelled primary antibody) or indirectly (as a labelled secondary antibody).
The most widely used ELISA assay 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 labelled 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 labelled 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.
Competitive ELISA and other formats
In addition to the standard direct and sandwich formats described above, there are several other ELISA styles:
Competitive ELISA is a strategy that is commonly used when the antigen is small and has only one epitope or antibody binding site. A variation of this method is to label the purified antigen instead of the antibody. The unlabelled antigen in the samples and the labelled antigen compete for binding to the capture antibody. A decrease in the signal from the purified antigen indicates the presence of the antigen in the samples compared to test wells with labelled antigens alone.
ELISPOT (enzyme-linked immunospot assay) refers to the ELISA-like capture and measurement of proteins secreted by cells that were seeded in PVDF membrane-backed microplate wells. It is a “sandwich” assay in which proteins are captured locally as they are secreted by seeded cells and detection is performed with a precipitating substrate. ELISPOT is like a western blot in the sense that the result is spotted on the surface of a membrane.
The cell ELISA is performed with cells that are plated and grown overnight in standard microplates. Once the cultured cells are fixed, permeabilized and blocked, the target proteins are detected with antibodies. This is an indirect trial, not a sandwich trial. Secondary antibodies are fluorescent (for direct measurement using a fluorescent plate reader or microscope) or enzyme-conjugated (for detection with a soluble substrate using a plate reader). ELISA is almost always performed using 96- or 384-well polystyrene plates and samples in solution (ie, biological fluids, culture media, or cell lysates). This is the platform discussed in the rest of this article.