The ELISA (Enzyme-Linked Immuno Sorbent Assay) is an analytic biochemistry assay that uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a substance, usually an antigen. It allows to detect very small amount of substances.
The analyte is immobilized on the surface of a solid phase (typically a microplate) with covalent or low energy binding. The solid phase can be directly the container (polymer) or antibodies adsorbed on the polymer. The advantage is that the binding of the analyte to a solid phase allows the washing of the container and at the end to amplify the detection of the analyte with an enzymatic reaction.
Direct ELISA
Principle
1. Empty container (microplate well)
2. Analyte is added and binds to the solid phase
3. Antibody with binded enzyme is added. The container is wasched to eliminate the not binded antibodies.
4. The enzyme substrate is added. It is transformed with the enzyme to detectable product (like with a spectrophotometer). As the enzyme can transform a lot of substrate molecule, there is here an amplification of the detection.
Competitive ELISA
Principle.
1. Antigen is coated to the solid phase
2. Sample is added to the container (microplate well)
3. Antibody with binded enzyme is added. There is a competition between the coated and free antigene to the antibodies. The container is wasched to eliminate not coated components. In some cases, there is no disponibility of antibody with conjugated enzyme. In this case a second antibodiy with enzyme is added. This second antibody binds to the first antibody.
4. The enzyme substrate is added. It is transformed with the enzyme to detectable product (like with a spectrophotometer). As the enzyme can transform a lot of substrate molecule, there is here an amplification of the detection.
The measurement of Aflatoxin is a good exemple of such ELISA test.
The result can be qualitative (presence or absence of the analyte) or quantitative. In this case a calibration curve with known analyte concentrations has to be determined (spectrophotometer).
