Enzyme-Linked Immunosorbent Assay (ELISA)


Enzyme-Linked Immunosorbent Assay (ELISA)

The ELISA test, also known as enzyme immunoassay (EIA), contains an enzyme-antibody complex that can be used as a color tracer for antigen-antibody reactions. The enzymes used most often are horseradish peroxidase and alkaline phosphatase, both of which release a dye (chromogen) when exposed to their substrate. This technique also relies on a solid support such as a plastic microtiter plate that can adsorb (attract on its surface) the reactants.

The indirect ELISA

To detect antibodies in a serum sample, an indirect ELISA test is used. The final positive reaction is achieved, as with other indirect tests, through an antibody-antibody reaction. The indicator antibody is compounded into an enzyme that causes positive serum samples to change colour. A known antigen that is adsorbed to a well ‘s surface is the starting reactant. An unfamiliar serum is added to this. An enzyme-Ab reagent that can react with the unknown test antibody is placed into the well after rinsing. Then the substrate for the enzyme is added, and colour changes are scanned for the wells. The development of colour suggests that all the components reacted and that the antibody was present in the serum of the patient. This is the common screening test for HIV antibodies, various species of rickettsia, hepatitis A and C, cholera vibrio, and gastric ulcer-causing Helicobacter. A verification test (such as the Western blot for HIV) may be necessary because false positives can occur.

In direct ELISA (or sandwich) testsĀ 

A known antibody is adsorbed to the bottom of a well in direct ELISA (or sandwich) tests and incubated with a solution containing an unknown antigen. An enzyme-antibody indicator that may react with the antigen is added after excess unbound components have been rinsed off. It will attract the indicator-antibody if antigen is present and hold it in place. Next, the substrate for the enzyme is placed and incubated in the wells. The substrate will be hydrolyzed by enzymes attached to the antigen and will release a coloured dye. Thus, a positive result is any colour that develops in the wells. The lack of colour implies that the antigen was not present and that the enzyme-antibody complex was removed by subsequent rinsing.

For the detection of antibodies to hantavirus, rubella virus and toxoplasma, a direct technique is used. A newer technology utilizes electronic monitors that read out antigen-antibody reactions directly. These systems contain computer chips that sense the minute changes in electrical current released when an antibody binds to antigen without belaboring the technical aspects.
The potential for sensitivity is extreme; in a sample, quantities as small as 12 molecules of a substance are thought to be detectable. In another procedure, samples are incubated with antibody substrate molecules and then exposed to the alkaline phosphatase enzyme. The enzyme reacts with the substrate and causes visible light to be emitted if the antibody is bound. Machines or photographic films can detect light. It is likely that some of the newer ELISA kits that use reporting mechanisms (i.e. labels) that do not come from enzymes but come from light-emitting substances should not be called “enzyme-linked” assays, but the name sticks since the principles the same.

Enzyme-Linked Immunosorbent Assay (ELISA)


  1. Radioimmunoassay(RIA)


  1. https://www.ncbi.nlm.nih.gov/books/NBK555922/
  2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2366430/