Chemiluminescence immunoassay dioxetanes

Chemiluminescence represents emitted light as a result of a certain chemical reaction. Some examples of chemiluminescent reagents are shown in Figure 2. Luminol, acridinium esters, and dioxetanes are the most frequently used labels for chemiluminescence immunoassays (CLIAs). 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Dioxetanes, labeled with triggers sensitive to the alkaline-phosphatase enzyme, serve as highly sensitive chemiluminescent probes in numerous bioassays. Current applications include immunoassays, membrane-based detection of proteins and nucleic acids, and microplate-based and array-based nucleic-acid detection. ... 

Several other examples of 1,2-dioxetane derivatives containing easily oxidizable groups have been reported and the high singlet quantum yield observed in their decomposition was attributed to the occurrence of the intramolecular CIEEL sequence Based on this concept, Schaap and coworkers have introduced the concept of induced chemiluminescence, which is very relevant for investigations into firefly luciferin bioluminescence and has led to the development of chemiluminescent probes widely used in immunoassays (Section N. 

Chemiluminescence assays are ultrasensitive (attomole to zeptomole detection limits) and have wide dynamic ranges. They are now widely used in automated immunoassay and DNA probe assay systems, (e.g., acridinium ester and acri-dinium sulfonamide labels and 1,2-dioxetane substrates for alkaline phosphatase labels and the enhanced-luminol reaction for horseradish peroxidase labels [see Chapter 9]). 

Signal antibodies have most often been radiolabeled (for immunoradiometric assays [IRMA]) with ( 25j) 7,62,i%,344,4i4 labeled with a chemiluminescent (for immunochemilummo-metric assays [ICMA]) compound, such as acridinium ester, or an enzyme (enzyme-linked immunosorbent assay [ELISA] or enzyme immunoassay [EIA]), such as ALP, converting a substrate (1,2-dioxetane phosphate) to a chemiluminescent product. 

As of 2004, the College of American Pathologists Interlaboratory Survey listed five manufacturers as providing first generation noncompetitive immunoassays for intact PTH. Most of these commercially available methods are on fully automated immunoassay analyzers using chemiluminescence detection including ALP with 1,2-dioxetane phosphate, acridinium ester, or electrochemiluminescence (ruthenium chelate). Signal antibody is less commonly radiolabeled with L... 

Various 1,2-dioxetane derivatives have been synthesized and reported. Among them, AMPPD (Adamantyl Methoxy Phenyl Phosphoryl Dioxetane) is a well known dioxetane bearing a spiroadamantyl group at the 3-position and a phenol phosphate at the 4-position, which is now used for chemiluminescence enzyme immunoassay (CLEIA) using alkaline phosphatase with highly sensitive detection. 

Using the substrate of 1,2-dioxetane derivative in combination with an enhancer, we applied a chemiluminescence enzyme immunoassay (CLEIA) for TSH (Thyroid Stimulating Hormone). 

In conclusion, our substrate comprised of a new 1,2-dioxetane derivative and an enhancer was found to have excellent characteristics with a high thermal stability and a very high light yield which can be well applied in clinical usage. CLEIA using new chemiluminescence substrate showed highly sensitive immunoassay. 

Albrecht, S., Ehle, H., Schollberg, K., Bublitz, R., and Horn, A., Chemiluminescent enzyme immunoassay of human growth hormone based on adamantyl dioxetane phenyl phosphate substrate. In Bioluminescence and Chemiluminescence Current Status (P. E. Stanley and L. J. Kricka, eds.), pp. 115-118. Wiley, Chichester, 1991. 

Brestel, E. P., Co-oxidation of luminol by hypochlorite and hydrogen peroxide. Implications for neutrophil chemiluminescence. Biochem. Biophys. Res. Commun. 126, 482-488 (1985). Brolin, S., and Wetteimark, G., Bioluminescence Analysis. VCH Verlag, Weinheim, 1992. Bronstein, L, Chemiluminescent 1,2-dioxetane-based enzyme substrates and their applications. In Luminescence Immunoassay and Molecular Applications (K. Van Dyke and R. Van Dyke, eds.), pp. 255-274. CRC Press, Boca Raton, FL, 1990. 

B23. Bronstein, 1., Edwards, B., and Voyta, J. C., 1,2-dioxetanes Novel chemiluminescent enzyme substrates. Applications to immunoassays. J. Biolumin. Chemilumin. 4, 99-111 (1989). 

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