ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique for detecting and measuring soluble molecules such as peptides, proteins, antibodies, and hormones. Other names for the same technique include enzyme immunoassay (EIA). In an ELISA, the antigen (target macromolecule) is immobilized on a solid surface (microplate) and then complexed with an antibody coupled to a reporting enzyme. The activity of the reporter enzyme is measured after incubation with the suitable substrate to produce a quantifiable result. A highly specific antibody-antigen interaction is the most important component of an ELISA. A highly specific antibody-antigen interaction is the most important component of an ELISA.
Because the ELISA reactants are immobilized on the microplate surface, it is simple to differentiate bound from non-bound material throughout the experiment. Because of its capacity to use high-affinity antibodies and wash away non-specific bound components, ELISA is a potent method for assessing particular analytes within a crude sample.
There are numerous types of ELISA that have been designed and developed and applied in various applications. However, they all share the same fundamental components.
Antigens can be immobilized to the surface of polystyrene microplate wells directly or indirectly.
Addition of an unrelated protein or other molecule to cover all of the unsaturated surface-binding sites of the microplate wells.
Fig: General steps of ELISA technique
Incubation with antigen-specific antibodies that attach to the antigens with high affinity.
Detection of the signal provided by the specific antibody’s direct or secondary tag.
Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most often utilized enzyme labels. Other enzymes that have been utilized include β-galactosidase, acetylcholinesterase, and catalase.
Commercially available substrates for performing ELISA with an HRP or AP conjugation include a wide range of options. The substrate used is determined by the necessary assay sensitivity and the instrumentation available for signal detection (spectrophotometer, fluorometer, or luminometer).
Fig: Detection Strategies of ELISA
ELISA tests are categorized into three categories based on the methods applied to bind antigen and antibodies, namely:
The crucial step is to immobilize the antigen of interest, which can be done directly on the test plate or indirectly via a capture antibody linked to the plate. The antigen is then detected directly (through labeled primary antibody) or indirectly (by labeled secondary antibody).
The key difference between direct and indirect ELISA is that the primary antibody is directly attached to the detection enzyme in direct ELISA, whereas a secondary antibody that is complementary to the primary antibody is conjugated with the detection enzyme in indirect ELISA.
Competitive ELISA is a type of ELISA in which analyte antigen and labeled antigen compete for a limited amount of particular antibody.
Fig: Direct, Indirect and Competitive ELISA
The sandwich ELISA assay format is the most often utilized ELISA assay type because it both indirectly 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. Due to its sensitivity and specificity, the sandwich ELISA format is widely employed.
Fig: Sandwich ELISA
ELISA (enzyme-linked immunosorbent assay) is a widely used analytical technique that detects and quantifies specific proteins or antibodies in a sample. It is commonly used in research and diagnostic applications, and has many applications in various fields.
Medical diagnostics: ELISA is used to detect the presence of specific proteins or antibodies in samples taken from patients, such as blood or urine. This can be used to diagnose various diseases, such as HIV, hepatitis, and autoimmune disorders.
Food safety: ELISA is used to detect contaminants, such as pesticides and bacterial toxins, in food samples.
Environmental testing: ELISA is used to detect the presence of specific pollutants or contaminants in air, water, and soil samples.
Drug development: ELISA is used to measure the effectiveness of drugs in clinical trials and to monitor their levels in the body.
Research: ELISA is used in various research applications, such as the study of protein-protein interactions, the identification of specific proteins in samples, and the measurement of protein levels in cells or tissues.
