Enzyme Assay (ELISA)

Enzyme Linked Immuno-sorbent Assay (ELISA)

ELISA is an antigen antibody reaction. In 1971, ELISA was introduced by Peter Perlmann and Eva Engvall at Stockholm University in Sweden. It is a common laboratory technique which is usually used to measure the concentration of antibodies or antigens in blood.

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.

ELISA is a plate based assay technique which is used for detecting and quantifying substances such as peptides, proteins, antibodies and hormones. An enzyme conjugated with an antibody reacts with colorless substrate to generate a colored product. Such substrate is called chromogenic substrate.

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 hydrolyzed 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.



ELISAs are typically performed in 96-well polystyrene plates. The serum is incubated in a well, and each well contains a different serum. A positive control serum and a negative control serum would be included among the 96 samples being tested. Antibodies or antigens present in serum are captured by corresponding antigen or antibody coated on to the solid surface. After some time, the plate is washed to remove serum and unbound antibodies or antigens with a series of wash buffer. To detect the bound antibodies or antigens, a secondary antibodies that are attached to an enzyme such as peroxidase or alkaline phosphatase are added to each well. After an incubation period, the unbound secondary antibodies are washed off. When a suitable substrate is added, the enzyme reacts with it to produce a color. This color produced is measurable as a function or quantity of antigens or antibodies present in the given sample. The intensity of color/ optical density is measured at 450nm. The intensity of the color gives an indication of the amount of antigen or antibody.

Fig: 96 well plate ELISA Assay

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.


Types of ELISA

There are 3 types of ELISA on the basis of binding structure between the Antibody and Antigen.

  1. Indirect
  2. Sandwich
  3. Competitive


Download link: Enzyme Assay (ELISA).pdf