Chemiluminescence immunoassay is not only the fastest developing and popularizing immunoassay method, but also the most advanced labeled immunoassay technology. Its sensitivity and accuracy are several orders of magnitude higher than enzyme immunoassay and fluorescence method. It can completely replace radioimmunoassay and completely eliminate enzyme-linked immunoassay. It mainly has the advantages of high sensitivity, strong specificity, low reagent price, stable reagent and long validity period (6-18 months), stable and rapid method, wide detection range, simple operation and high degree of automation.
The chemiluminescent reagent used as a marker shall meet the following conditions:
Luminol and acridine ester luminescent agents are commonly used labeling luminescent agents, which meet the following conditions:
1. Can participate in chemiluminescence reaction.
2. It can form a stable conjugate reagent after coupling with antigen or antibody.
3. After coupling, high quantum effect and reaction kinetics are still retained.
4. The physicochemical properties, especially immune activity, of the labeled substances should not be changed or rarely changed.
Classification of markers used in chemiluminescence immunoassay
1 Direct chemiluminescent agent
Direct chemiluminescent agent refers to the direct labeling of antigen or antibody with chemiluminescent agent, which mainly includes the following types.
① When acridine ester is oxidized by H2O2 under alkaline conditions, it emits light with a wavelength of 470nm and has high luminous efficiency. Its excited state product N-methyl acridone is the illuminant of the luminous reaction system. Acridine ester luminescent agents can be directly used to label haptens and proteins. When used to label antibodies, the binding is stable, the production of light quanta is not reduced after binding, and high specific activity can be obtained, which is conducive to the establishment of two site chemiluminescence immunoassay. Acridine ester is an ideal luminescent substrate.
②Tripyridine ruthenium tripyridine ruthenium is an electrochemiluminescence agent. It and electron donor tripropylamine (TPA) can lose an electron on the surface of the anode electrode at the same time.
2 Enzymatic reaction luminescent agent: the luminescent agent (substrate) emits light by the catalysis of labeled enzymes (horseradish peroxidase and alkaline phosphatase).
①The luminescent agent catalyzed by horseradish peroxidase is luminol and its derivatives; In addition, isoluminol and its derivatives have chemiluminescence properties. Luminol is the first synthesized luminescent substance.
②The luminescent substrate catalyzed by alkaline phosphatase is amppd, amppd It is a new chemiluminescent agent. There are two important parts in its molecular structure. One is the dioxygen ring connecting benzene ring and adamantane, which can break and emit photons; The other is the phosphate group, which maintains the stability of the whole molecular structure.
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