Any chemical reaction produces both, light and heat. A
burning candle is an example of such reaction. When a candle is lit, its flame both glows and becomes hot. It is much less common for a chemical reaction
to produce light without heat. The light from such reactions is called cool light, because it is created without heat. Reactions that produce light
without heat are called chemiluminescent reactions.
Chemiluminescence is the generation of electromagnetic radiation as light by the release of energy from a chemical reaction. While the light can, in
principle, be emitted in the ultraviolet, visible or infrared region, those emitting visible light are the most common. They are also the most interesting
Principle: In any immunoassay, where the chemiluminescence is used as the label to detect the analyte, the reaction occurs as follows:
- The antigens (in the patient samples ie. the analyte and the antigen in the reagent) and the antibodies, which are labeled with the chemiluminescent
label or tag, are allowed to react with each other and form antigen-antibody complex.
- The next step involves the separation of bound and free reactants. This separation depends upon the type of separation system used. The bound form that
is retained at the end of the separation step contains the antigen that is bound to the antibody labeled with the chemiluminescent tag.
- To these antigens bound to the chemiluminescent substance, some substrate is added in order to initiate the chemiluminescence reaction. In some assays
this initiation of chemiluminescence is achieved by changing the nature of the reaction mixture.
- The chemiluminescence that is emitted is measured by means of the photomultiplier tube and luminometer. On the basis of the emission of
chemiluminescence, the concentration of the analyte is estimated.
Chemiluminescence is used for in vitro diagnostics. The primary use of luminescence is in molecular biology applications for the detection of proteins
and nucleic acids on gels, and in the visualization of expressed proteins in cells. The antibody based system provides target specificity and versatility.
The enzyme component provides signal amplification. One reason accounting for the growing popularity of chemiluminescent assays is their exquisite
detection sensitivity. Measurement of light intensity is relatively simple, requiring only a photomultiplier or photodiode and the associated electronics
to convert and record signals. The lack of inherent background and the ability to easily measure very low and very high light intensities with simple
instrumentation provide a large potential dynamic range of measurement.