ELISA or Enzyme Linked Immunosorbent Assay is one of the most used and trusted Ligand Binding Assay (LBA). This plate-based assay technique is often employed to detect and quantify analytes in large molecule bioanalytical techniques.
When using the ELISA method, the antigen must be immobilized on a solid surface. It is then complexed with an antibody linked to an enzyme. A highly specific antibody-antigen interaction is necessary for proper detection.
ELISA is widely used for detecting and quantifying peptides, proteins, antibodies, and hormones. This has diverse applications in PK, TK, and Biomarker testing. Several variants find use in the method, including direct, indirect, and sandwich.
A Tour Of ELISA Method Development
Enzyme Linked Immunosorbent Assay is based on colorimetric or chemiluminescent reaction. Its results usually lead to colored end products that quantify the analyte present. These are typically performed on 96-well polystyrene plates, though it is also possible to use 384-well plates.
The detection enzyme is of particular importance. The most commonly used enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Enzymes convert the substrate into a detectable product. In these cases, the enzyme-conjugated antibodies result in a colored product detected through chromogenic, chemifluorescent and chemiluminescent imaging.
Each of these substrates has its strengths and weaknesses. For example, chromogenic substrates are not as sensitive as other options. However, they enable direct visualization and are helpful for kinetic studies.
A key requirement for the success of the ELISA method is that the antigen must be immobilized on a solid surface. This is easier said than done and sometimes can lead to a conformational change in the analyte or antibody. Any such change can prevent or compromise detection. Proper strategies for immobilization are necessary.
ELISA Assay Development Formats And Strategies
ELISA assay is a solid-phase assay. As previously mentioned, the key to success lies in proper immobilization of the antigen. Several ELISA formats are available for use as applicable and desirable for detection. The major formats are:
Direct ELISA
This format requires fewer steps and results in faster detection. Though there is a lower chance of error in this method, it suffers from higher background noise. This is caused by the antigen immobilization not being specific. The format is less flexible and best used for analyzing the immune response to an antigen.
Indirect ELISA
Indirect ELISA follows a two-step detection process and has the antigen adsorbed into the plate. In the first step, the primary antibody binds to an antigen. For the second step, an enzyme-conjugated secondary antibody is used.
This format is flexible, economical, and has a high sensitivity. It suffers from the risk of background noise due to the secondary antibody. It’s also a more time-consuming process compared to the direct format.
Sandwich ELISA
Sandwich ELISA is the most powerful and commonly used ELISA assay validation format. It is flexible, robust, and has high sensitivity and high flexibility. The format uses matched antibody pairs, the capture antibody, and the detection antibody.
These antibodies bind the analyte between themselves. The format is great for complex samples but has the disadvantage of presenting some difficulty in antibody optimization.
Competitive ELISA
Competitive ELISA is also known as competition/inhibition ELISA or blocking ELISA. It is the most complex ELISA assay development format, but is consistent, flexible, robust, and needs no sample processing. The format is most commonly used when the small antigen has only one epitope or antibody binding site.
