Immunoassays dioxetane enzyme substrates

I. Bronstein, B. Edwards, J.C. Voyta, 1,2-Dioxetanes—novel chemi-luminescent enzyme substrates—applications to immunoassays, J. Chemilum. Biolum. 4 (1989) 99-111. 

Although they have not been used as labels in immunoassays, dioxetane substrates for p-galactosidase have been synthesized and characterized (B18, B23, S9). Current data are sparse, but it would appear that the enzyme is less detectable... 

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). 

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). 

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. 

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). 

For each type of assay, the label or tag involved is listed along with typical substrates, type of absorbance, and type of instrument required. RIA radioimmunoassay IRMA immunoradiometric assay CPM counts per minute ELISA enzyme linked immunosorbent assay EIA enzyme immunoassay TMB 5,5 tetramethylbenzidine HPA p hydroxyphenylacetic acid AMPPD 4 methoxy 4 (3 phosphatephenyl) spiro(l,2 dioxetane) 3,2 adamantane p NPP p nitrophenyl phosphate 4 MUP 4 methylumbelliferyl phosphate ONPG o nitrophenyl P galactopyranoside MUG 4 methylumbelliferyl P D galactopyrano side AMPGD 3 (4 methoxyspiro( 1,2 dioxetane 3,2 tricyclo(3.3.1.1(3,7))decan) 4 yl)phenylgalacto pyranoside ECL electrochemiluminescence. 

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. 

---