- In enzyme immunoassay (or EIA) antibody or antigen conjugated with the enzyme (enzyme-labelled) take part.
- This test has many technical variations. The resulting enzymatic activity is revealed by reaction with an appropriate substrate. Horseradish peroxidase or alkaline phosphatase are used frequently for enzyme labelling.
- In immunological testing, the basic are homogenous and heterogeneous (or solid-phase) modifications of enzyme immunoassay. The latter is widespread, especially for specific antibodies determination. It is usually named as enzyme-linked immunosorbent assay (or ELISA).
- To detect antibodies here, the known antigens are attached to a solid phase (typically, to the bottom of plastic microdilution plate wells).
- Test serum dilutions are put into the wells. If Abs match the antigen, the immune complex is formed.
- After incubation serum excess is removed, and the wells are washed. Then the specimens are re-incubated with anti-immunoglobulin antibodies labelled with an enzyme (horseradish peroxidase).
- Anti-immunoglobulin-enzyme conjugate binds to antibodies present in the immune complex.
- After the next wash enzyme activity is evaluated by adding the specific substrate (hydrogen peroxide) and chromogen (e.g., o-phenylene-diamine).
- Chromogen is a colourless substrate substance that produces a color end-product when acted upon by an enzyme.
- The enzyme reaction is stopped by adding sulphuric acid.
- The brown color of the reaction medium appears, and the reaction is assessed with multichannel colorimetric analyzer (microplate reader). The optical density of samples is proportional to the amount of antibody bound.
- For the determination of an antigen by ELISA, the antigen-specific antibodies are first absorbed on the bottom of plastic wells of the solid phase.
- Then the antigen-containing clinical specimens are added and incubated with antibodies. If the specific antigen is present in the sample, it binds to specific antibodies creating an immune complex.
- After a thorough wash, the bound antigen is repeatedly treated with a new generation of specific antibodies usually from another species origin. This is known as the “sandwich” version of the ELISA test.
- The remaining steps of this test (like the addition of species-specific antiglobulin conjugate and application of other reagents) are virtually the same as for conventional ELISA technique.
- ELISA test is very sensitive (as RIA, see below) but unlike RIA it doesn’t require special expensive equipment or safety measures for technical personnel.