1,2‐Dioxetanes: Novel chemiluminescent enzyme substrates. Applications to immunoassays

Abstract

We have synthesized and studied two 1,2-dioxetane-based chemiluminescent enzyme substrates: 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD), and, 3-(2'-spiroadamantane)-4-methoxy-4-(3"-beta-D'-galactopyrano -yloxy)phenyl-1,2- dioxetane (AMPGD), which can be activated to chemiluminescence at 470 nm by alkaline phosphatase and beta-D-galactosidase, respectively. In addition, we observed that certain macromolecules enhance the luminescence of AMPPD. For example, the addition of 0.1% bovine serum albumin amplifies the luminescent signal and improves the detection limit for alkaline phosphatase by approximately one order of magnitude under certain conditions. This effect is due to the presence of a hydrophobic microenvironment provided by the enhancer which 'stabilizes' the dephosphorylated AMPPD emitter. Alkaline phosphatase catalysed chemiluminescence from AMPPD is constant for a prolonged period of time. Using AMPPD we were able to detect 0.01 attomole quantities of alkaline phosphatase immobilized on membrane supports and imaged on photographic film and, in solution, measured in a luminometer. AMPPD and AMPGD offer alternatives to colorimetric and fluorescent substrates for alkaline phosphatase and beta-D-galactosidase labels used in enzyme immunoassays. The simplicity and sensitivity of this chemiluminescent readout allowed the development of rapid clinical assays (e.g. beta-hCG, LH, TSH and others).

Inhibition of NF-κB Activation by Peptides Targeting NF-κB Essential Modulator (NEMO) Oligomerization

“…The lysate was centrifuged at 1000 ϫ g for 20 min at 4°C, and the supernatant was kept on ice before performing the ␤-galactosidase assay. Assays were performed on 30 l of the supernatant and in an assay mix containing 4 l of Galacton-star chemiluminescent substrate (29) and 200 l of reaction buffer (Clontech). The activity was measured with a plate luminometer (Berthold).…”

Bioluminescent PCR-RFLP Enzyme-Linked Immunosorbent Assay for Analysis of Vitamin D Receptor Gene Polymorphism

“…Recently, highly sensitive chemiluminescent (CL) and bioluminescent (BL) have become available for detecting alkaline phosphatase (ALP), β-D-galactosidase (β-gal), horse radish peroxidase (HRP) and luciferase which can be used as a label enzyme of EIA. These CL and BL assays use an adamantyl-1,2-dioxetane phosphate (AMPPD) 8 , 5-bromo-4-chrolo-3-indolyl phosphate 17 , and D-luciferin-o-phosphate 9 for ALP, and 3-(2-spiroadamantane)-4-methoxy-4-(3″-β-D-galactopyranosyl oxyphenyl)-1,2-dioxetane (AMPGD) 8 , and 5-bromo-4-chrolo-3-indolyl β-D-galactopyranoside for β-gal 18 as a substrate, respectively. We have already developed a bioluminescent assay of AK.…”

“…Highly sensitive chemiluminescent (CL) methods 8 and bioluminescent methods 9 have become available for detecting alkaline phosphatase and β-D-galactosidase labels used generally in ELISA. We have already developed a sensitive bioluminescent binding assay 10 and EIA using firefly luciferase and acetate kinase (AK) as a label enzyme 11,12 , respectively.…”

Peer Reviewed: Analytical Bioluminescence and Chemiluminescence

“…Very efficient CL substrates are available for HRP and AP, which are the label enzymes most commonly used in immunoassays. Using dioxetane phosphate substrates, researchers detected 10 -20 mol of AP (~6000 molecules), which is the lowest detection limit achieved so far in CL enzyme detection (10). Maeda et al reported a new BL enzyme immunoassay for hormones that uses acetate kinase as the label and measures ATP, produced by the label-catalyzed reaction, with the conventional luciferin/luciferase system with a detection limit of 1-2 ϫ 10 -20 mol (11).…”