Enzyme-linked immunosorbent assay (ELISA)
Enzyme-linked immunosorbent assay (ELISA) utilizes an enzyme system to show specific combination of an antigen with its antibody. It is a method of quantifying an antigen immobilized on a solid surface. ELISA uses a specific antibody with a covalently coupled enzyme. The amount of antibody that binds the antigen is proportional to the amount of antigen present, which is determined by spectrophotometrically measuring the conversion of a clear substance to a colored product by the coupled enzyme. The ELISA technique was first conceptualized and developed by Peter Perlmann and Eva Engvall at Stockholm University, Sweden.
Enzyme system of ELISA consists enzyme which is labeled to a specific antibody or antigen and a chromogenic substrate which is added after antigen-antibody reaction. The substrate is hydrolysed by the enzyme attached to antigen-antibody complexes. An ELISA test uses components of the immune system (such as IgG or IgM antibodies) and chemicals for the detection of immune responses in the body. The ELISA test involves an enzyme (a protein that catalyzes a biochemical reaction). It also involves an antibody or antigen (immunologic molecules). Examples of the uses of an ELISA test includes to diagnose infections such as HIV (human immunodeficiency virus) and some allergic diseases like food allergies. ELISA tests are also known as an immunosorbent assay.
Following the antigen– antibody reaction, chromogenic substrate specific to the enzyme (o- phenyldiamine dihydrochloride for peroxidase, p-nitrophenyl phosphate for alkaline phosphatase, etc.) is added. The substrate is acted upon (usually hydrolysed) by enzyme attached to antigen-antibody complex to give color change. The color in reaction can be read visually or The reaction is detected by reading the optical density (estimated colorimetrically) using microassay plate reader i.e. ELISA reader. Usually, a standard curve based on known concentrations of antigen or antibody is prepared from which the unknown quantities are calculated.
The antigen or antibody is coated on solid surface such as in plastic tube or well of microtiter plate. Thus, after the antigen and antibody have combined (Antigen-antibody complex formed) they remain firmly attached to solid surface during subsequent washing stages.
Enzyme immunoassays (EIAs) can be used for detection of either antigens or antibodies in serum and other body fluids of the patient. In EIA techniques, antigen or antibody labeled with enzymes are used. Alkaline phosphatase, Horseradish peroxidase, and β-galactosidase are the enzymes used in the EIA tests.
There are different types of ELISAs available for the detection and quantitation of either the antigen or antibodies in serum and other body fluids. There are four types of ELISA tests:
Antigen is attached to a polystyrene plate. Enzyme-labeled antibody is added that can react with the antigen and a substrate that can be measured.
Antigen is attached to a polystyrene plate. Addition of primary antibody followed by an enzyme-labeled antibody that can react with both the primary antibody and substrate.
A capture antibody is attached to the polystyrene plate, then antigen is added that specifically attaches or captures the antigen. A second antibody, also specific for the antigen but not the same as the capture antibody is added and “sandwiches” the antigen. This second antibody is then followed by an enzyme-labeled antibody specific for the second antibody that can react with a substrate that can be measured.
This test is like the sandwich ELISA but involves the addition of competing antibodies or proteins when the second antibody is added. This results in a decrease in the substrate signal that is generated. This test is considered to give good, highly specific results.