Summary
Highlights
ELISA (Enzyme-Linked Immunosorbent Assay) is a plate-based technique for detecting and quantifying soluble substances such as proteins. It involves immobilizing an antigen, which is then specifically detected by enzyme-coupled antibodies. These enzymes convert a colorless substrate into a measurable colorful product.
In an ELISA, an antibody is attached to the bottom of a well, specifically designed to bind a protein of interest. A second, soluble, enzyme-conjugated antibody also specific for the target protein is then added. When a substrate is introduced, the enzyme converts it to a colorful product that can be measured.
There are several variants of ELISA. In direct ELISA, the antigen is immobilized directly, and an enzyme-linked antibody binds to it. Indirect ELISA uses a primary antibody to bind the antigen, followed by a secondary enzyme-linked antibody that binds the primary antibody. The most common type is the sandwich ELISA, where a primary antibody captures the antigen, and a second primary antibody specifically binds the same antigen.
Regardless of the ELISA type, the last antibody binding step is always conjugated to an enzyme, confirming the binding of the target substance.
An example of a sandwich ELISA involves two wells, both coated with primary antibody specific for protein X. One well receives a sample containing the protein, and the other serves as a control without the target protein. After initial binding and a wash step, enzyme-linked antibodies are added, binding to the antigen in the sample well. A final wash is performed, and then a colorless substrate is added. The enzyme in the sample well converts the substrate into a measurable, colorful product, while the control well remains colorless, allowing for quantification using spectrophotometry.