Describe the principles of ELISA and contrast the three types

Question

What will be an ideal response?

Answer 1

Enzyme-linked immunosorbent assays (ELISAs) are widely-used diagnostic tests. ELISA relies on the specific interaction between an antibody and the antigen it binds to. All variations of ELISA use a reporter enzyme linked to a detection antibody. The detection antibody binds to its specific antigen (which sometimes is another antibody). After unbound antibody is removed by washing, the chemical substrate for the reporter enzyme is added. Typically, the enzyme-substrate pair is chosen to generate a colorimetric or chemiluminescent reaction which can be visually observed or detected with a device called a plate reader. ELISAs can be run in bulk in 96-well plates or can be designed in a variety of single-use devices such as a home pregnancy test or the "rapid-Strep" used in clinics and doctors' offices. There are three ELISA "formats" but ELISAs can be designed for virtually any antigen, and hundreds of different ELISAs are commercially available. Direct ELISA allows for identification of an antigen in a sample in three basic steps: binding antigens to a well surface, addition of the detector antibody with bound enzyme, followed by the enzyme substrate. Direct ELISA is not recommended for crude samples. Indirect ELISA is often used to detect the presence of a specific antibody in a patient sample. With the indirect ELISA, the antigen is already bound to the plate. The test sample containing unknown antibodies is added, followed by a secondary detection antibody which is designed to bind to the antibody being measured. Finally, the enzyme substrate is added. A sandwich ELISA allows detection of an antigen in a sample but is more sensitive than the direct ELISA. The wells come with a capture antibody already loaded. The patient sample is added, and if the antigen is present, it will bind to the capture antibody. The enzyme-linked detection antibody is added, sandwiching the antigen between the two antibodies. Finally, the enzyme substrate is added. All forms of ELISA depend specifically on the binding of the specific antigen to its antibody. If the antigen being sought is not in the sample, antibody cannot bind and will be washed away prior to the addition of the enzyme substrate.