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Take you to understand different chemiluminescence reagents
A technology that combines immune response and chemiluminescence reaction to detect antigen or antibody. It is to label the luminescent substance or enzyme on the antigen or antibody. After the immune reaction, add oxidant or enzyme substrate to emit light. By measuring the emitted light intensity, the concentration of the test substance is determined according to the standard curve. The main advantages of CLIA are high sensitivity, long validity period of markers, wide detection range, and full automation.
Luminous substrates for enzymatic reactions: Luminol, AMPPD, etc.
Direct chemiluminescent reagents: acridinium esters and derivatives
Electrochemiluminescence agent: ruthenium terpyridine
Luminescent substrates for enzymatic reactions:
It uses the catalytic action of the labeled enzyme to make the luminescent agent (substrate) emit light. This type of luminescent agent that needs to be catalyzed by the enzyme to emit light is called the enzymatic reaction luminescent agent.
Luminol's luminescence principle:
Luminol produces an electronically excited state intermediate 3-aminophthalic acid with luminescent properties in the presence of hydrogen peroxide (oxidant) and enzymes under alkaline conditions. During the transition from the excited state to the ground state, the energy released in the form of photons has a wavelength in the blue part of visible light.
Luminol-horseradish peroxidase labeled chemiluminescence immunoassay:
The analysis system uses horseradish peroxidase (HRP) to label the antigen (or antibody), which forms a solid-phase coated antibody after immunoreaction with the test substance sample and the solid-phase carrier in the reaction system-the antigen to be tested- Enzyme (HRP)-labeled antibody complex, which is added with luminol luminescence agent, hydrogen peroxide and luminescence enhancer to produce chemiluminescence.
The light-emitting principle of AMPPD:
AMPP is enzymatically hydrolyzed by AP (alkaline phosphatase) under alkaline conditions to produce a fairly stable AMP-D anion. It has a decomposition half-life of 2 to 30 minutes and emits continuous light with a wavelength of 470 nm. Its intensity reaches 15 minutes. At the peak, the light intensity remains relatively stable within 15 to 60 minutes.
AMPPD-Alkaline Phosphatase (AP) labeled chemiluminescence immunoassay:
The analysis system uses alkaline phosphatase to label the antigen (or antibody), and forms a solid-phase coated antibody-tested antigen-enzyme (AP) label after immunoreaction with the test substance sample in the reaction system and the solid-phase carrier For the antibody complex, AMPPD luminescent agent is added at this time, and alkaline phosphatase removes the phosphate group of AMPPD to emit light.
Direct chemiluminescence substrate:
The substrate does not require enzyme catalysis in the luminescence immunoassay process, and directly participates in the luminescence reaction. They have a unique group that produces luminescence in the chemical structure, and can directly label antigens or antibodies.
When it is oxidized by hydrogen peroxide under alkaline conditions, it emits light with a wavelength of 470nm and has high luminous efficiency. Its excited state product N-methylacridone is the luminous body of the luminescent reaction system.
Chemiluminescence immunoassay labeled with acridinium ester:
The antibody (antigen) is directly labeled with acridinium ester, and after an immune reaction with the corresponding antigen (antibody) in the test specimen, a solid-phase coating antibody-test antigen-acridinium ester-labeled antibody complex is formed. It is necessary to add oxidant (hydrogen peroxide) and sodium hydroxide to make it into an alkaline environment, and the acridine ester will decompose and emit light without a catalyst.