Enzyme Linked ImmunoSorbant Assay (ELISA)

Enzyme immunoassay combine the specificity of antibodies with the sensitivity of simple spectrophotometric enzyme assays by using antibodies or antigens coupled to an easily assayed enzyme that also possesses a high turnover number. ELISA is replacing RIA, despite the latter already being established, extensively automated and sometimes more sensitive.

ELISA may be used for assaying antigens by either a competitive or a double antibody method and for assaying a specific antibody by an indirect method. All these methods require the preparation of a calibration curve during the assay.

The ELISA divided into three types, they are

a) Competitive method b) Double-antibody method c) Indirect method

a) Competitive method: A mixture of a known amount of enzyme - labelled antigen and an unknown amount of unlabelled antigen is allowed to react with a specific antibody to attach to a solid phase. After the complex has been washed with buffer, the enzyme substrate is added and the enzyme activity measured. The difference between this value and that of a simple lacking unlabelled antigen is a measure of the concentration of unlabelled antigen.

Major disadvantage: this method is that each antigen may require a different method to couple it to the enzyme; this is not so for the double-antibody method.

b) Double-antibody method: The unknown antigen solution is reacted with specific antibody attached to a solid phase, washed and treated with enzyme labelled antibody (directed against a different epitope, if monoclonal antibodies). After a further wash the enzyme substrate is added. The amount of enzyme activity measured under standard conditions directly proportional to the amount of antigen present. Advantage: It is that only one procedure required coupling the enzyme to all preparations.

c) Indirect method: The method may be used to measure antibody levels. The putative antiserum is reacted with specific antigen attached to a solid phase. Any specific antibody molecules bind to the antigen and all other material is washed away. Exposure of the complex to enzyme labelled anti-immunoglobulin antibody results in binding to any specific antibody molecules adsorbed from the original serum. The complex is washed and the substrate for the enzyme added, resulting in activity proportional to the amount of specific antibody in the serum.

 

ELISA, in which enzyme labelled antibody binds an antigen, leading to substrate breakdown and colour change, can be used to detect primary antigen-antibody reactions

ELISA uses an enzyme for labelling rattier than a radioisotope. Like the radioisotopes, the enzyme is covalently coupled to the antibody, in principle, this is the only difference between ELISA and RIA. ELISA measures bound enzyme activity rather than bound counts per minutes. Quantification requires measuring the colour intensity of the colored products generated by the enzyme and added substrate. The intensity of the colour is equivalent to the amount of labeled antibody bound to antigen. ELISA has replaced RIA in many clinical and basic science laboratories. RIAs are potentially hazardous and tedious, require bookkeeping, and involve radioisotopes. These reasons combined with the commercial availability of plate readers that can be measure the absorbance of 96 wells in less than a minute, account for ELISA's growing popularity. ELISA and RIA are similar in sensitivity. Theoretically, ELISA can be more sensitive than RIA because each enzyme molecule can generate hundreds of thousands of colored product molecules that can be measured. In contrast, ^12S I molecule decays only once. Once application of ELISA is to detect the presence and titre of specific antibody for the AIDS virus.

Applications of ELISA:

• Whilst ELISA can be used for the assay of virtually any antigen, hapten or antibody, it is used predominantly in clinical biochemistry laboratories to measure, for example, IgG, and IgE, oncofetal proteins, hematological factors, immune complexes and hormones such as insulin, oestrogen and human chorionic gonadotropin.
• Examples of its use in the study of infectious diseases include the detection of bacterial toxins, Candida albicans, rotaviruses, herpes simplex viruses and the hepatitis B surface antigen.
• ELISA has also been used extensively for the assay of antibodies in infectious diseases including: anti-viral antibodies, e.g. to Epstein-Barr virus and rubella virus; anti-bacterial antibodies, e.g. to Brucella, Rlckettsia and Salmonella species, anti-fungal antibodies, e.g. to Aspergillus and Candida species, anti-parasite antibodies, e.g. to Plasmodium, Schistosoma and Trypanosoma species, and autoantibodies, e.g. anti-DMA and anti thyroglobulin.