Principle and Application of Chemiluminescence Immunoassay (CLIA)

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Basic Principles of Chemiluminescence Immunoassay

Chemiluminescence immunoassay includes two systems: immunoassay and chemiluminescence analysis. Immunoassay system uses chemiluminescent substances or enzymes as markers, which are directly labeled on antigens or antibodies and react with antigens to form antigen-antibody immune complexes. Chemiluminescence analysis system is a kind of luminescent substrates added with oxidants or enzymes after the end of immune reaction. The chemiluminescent substances are oxidized by oxidants to form an intermediate in the excitation state, which emits photons to release energy to return to the stable ground state. The luminescent intensity can be measured by the luminescent signal measuring instrument. According to the relationship between chemiluminescent markers and luminescent intensity, the content of the tested substance can be calculated by standard curve.

Chemiluminescence immunoassay

Chemiluminescence immunoassay (CLIA) is a kind of immunoassay method which uses chemiluminescent agents to directly label antibodies or antigens. At present, luminol and acridine ester chemiluminescent agents are the most common markers.

Chemiluminescence Immunoassay with Luminol Labels:

The luminescence of luminol is oxidation reaction luminescence. In alkaline solution, luminol can be oxidized and luminescent by many oxidants, among which H2o2 is the most commonly used. Some enzymes or inorganic catalysts need to be added because of the slow rate of luminescence reaction. The main enzymes are horseradish peroxidase (HRP), and inorganic enzymes include O 3, halogen, Fe 3, Cu2, Co2 and their complexes. In the early stage, it was mainly used for the determination of inorganic substances and small organic molecules. The sensitivity was reduced due to the decrease of light intensity after labeling. It has been found that the addition of Some Phenols and their derivatives, amines and their derivatives and phenylboronic acid derivatives in the luminescent system can significantly enhance the luminescence of the system. The luminescent intensity can be increased by 1000 times, and the "background" luminescence can be significantly reduced, and the luminescent time can also be prolonged. With the use of these enhancers, chemiluminescent immunoassay (CLIA) has been widely used in protein and nucleic acid analysis.

Chemiluminescence immunoassay with acridine ester labels:

Because acridine ester is not very stable in CLIA, more stable acridine ester derivatives have been synthesized. Under the condition of H2o2 and OH, acridine esters can emit light rapidly, and the quantum yield is very high. For example, the quantum yield of acridine aromatic esters can reach 0.05. The acridine esters can be used as markers for immunoassay. The luminescence system is simple and fast, without adding catalysts, and the labeling efficiency is high, and the background is low. These characteristics have aroused great interest of analysts and diagnosticians.

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