Enzyme-linked immunosorbent assay (ELISA):
ELISA is one of the most common types of immunoassays designed to detect and quantify soluble substances, such as peptides, proteins, antibodies, and hormones. The ELISA technique is a solid-phase immunoassay system employing a plate-based assay that has the targeted antigen immobilized on a solid surface or microplate and then complexed with an antibody that is linked to a reporter enzyme. Detection of the targeted antigen is accomplished by measuring the activity of the reporter enzyme. The method enables the analysis of the antigen samples immobilized in the microplate wells using specific antibodies.
Typically, ELISAs are performed in 96-well or 384-well polystyrene plates, which passively bind antibodies and proteins. In general, the binding and immobilization of the reagents make most ELISAs relatively easy to design and perform. With the reactants immobilized on the microplate surface, it is much easier to separate bound from non-bound material during the performance of the ELISA method. The ELISA procedure is a powerful technique for measuring specific analytes because it allows for the use of high-affinity antibodies combined with the ability to wash away non-specific materials.
Although there are many variations of the ELISA technique, most assays have the following basic steps:
- Coating/capture - This involves the direct or indirect immobilization of antigens from the sample to be tested on the surface of polystyrene microplate wells. A matching antibody is applied over the surface so it can bind the antigen. In this step, the targeted antigen is immobilized on the solid surface microplate and is complexed with an antibody that is linked to a reporter enzyme.
- Plate blocking- This step involves the addition of irrelevant protein or other molecules to cover all unsaturated surface-binding sites of the microplate wells. The microplates are then washed to remove any unbound antibodies.
- Probing/detection–The next step involves the incubation with antigen-specific antibodies that affinity bind to the antigens.
- Signal measurement–The final step involves the detection and measurement of the signal generated by the reporter enzyme when the enzyme’s substrate is added. The signal generated is an indication of the binding of the antigen-specific antibody to the antigen and is typically measured using an instrument such as a spectrophotometer, fluorometer, or luminometer.
The most commonly used enzyme labels for ELISA procedures are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes, such as β-galactosidase, acetylcholinesterase, and catalase, have also been used. Using HRP or AP as the enzyme label, many substrates are commercially available for use in the ELISA technique. Typically, the choice of a particular substrate depends upon the required assay sensitivity and the instrumentation used to detect the signal, such as a spectrophotometer, fluorometer, or luminometer.
