Enzyme-linked Immunosorbent Assay (ELISA)
ELISA is based on a specific antigen-antibody reaction and usually involves the immobilization of antibodies or antigens in a 96 or 384 well plate. The basic steps of ELISA:
Immobilization of target proteins/antigens on the surface of a microplate
Washing unbound/excess proteins/antigens from the plate
Addition of a labeled antibody which will subsequently bind to the target antigen/protein present in the plate
Washing unbound (excess) antibody from the plate
Addition of enzyme-specific substrates which will react with the enzyme and yield a colored product, which can be measured colorimetrically using a microplate reader.
Horseradish peroxidase (HRP) or alkaline phosphatase are common enzymes used in ELISA, while substrates include tetramethylbenzidine (TMB) and 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid ( ABTS). Duplicate or triplicate sampling is generally preferred and different sample concentrations are used to ensure a biologically acceptable detection range.
To quantify the concentration of the target antigen, a standard curve is generated using known concentrations of the antigen. Then, the optical density (light absorption of the enzyme-substrate reaction product) obtained from the colorimetric test is plotted on the standard curve to accurately measure the target antigen level in the biological sample.
A target protein/antigen is immobilized on the surface of the plate, then an enzyme-labeled antibody against the target molecule is added.
The colorimetric detection is carried out after addition of a suitable substrate. This format is easy and takes less time. However, there is a high experimental background due to binding of all target antigens to the surface, in addition to difficulties with primary antibody labeling.
A target protein/antigen immobilized on the surface of the plate is incubated with a primary antibody that is directed against the target molecule. Then, an enzyme-labeled secondary antibody generated against the primary antibody is used for detection and quantification. Although this format is more sensitive than the direct ELISA, the detection of false positives is high due to the crossing of secondary antibodies.
Two antibodies (capture antibody and detection antibody) directed against different epitopes (a specific antibody binding site of an antigen) of a target protein/antigen are required for this ELISA format.
The procedure involves the immobilization of an antibody (capture antibody) against the target antigen in the microplate; add a biological sample containing the target antigen to the plate, which will then bind to the immobilized antibody present in the plate; adding an enzyme-labeled antibody (detection antibody) which will then bind to the target antigen present in the plate; and adding enzyme-specific substrates to the plate which will react with the enzyme and produce a colored product for detection. This format involves two antibodies detecting different epitopes of the target molecule which makes it very specific.
In this procedure, a reference antigen is immobilized on the surface of the plate and a biological sample pre-incubated with a specific amount of labeled antibody is added to the plate. The amount of antigen present in the sample will determine the amount of unbound or free antibody available to bind the reference antigen in the plate. This format is particularly suitable for low molecular weight targets.