Chemiluminescence: Sensitive detection technology for reporter gene assays

Abstract

A series of enzyme-activated chemiluminescence-based assays of reporter gene expression, useful in many biomedical applications, has been developed. The chemiluminescence detection systems for β-galactosidase, β- glucuronidase (GUS), and secreted placental alkaline phosphatase (SEAP) reporter enzymes are all based on use of 1,2-dioxetane substrates. This detection technology also permits the combined luminescence detection of two different reporter enzymes in the same tube, e.g., a dual assay for β- galactosidase and luciferase. The sensitivity of these chemiluminescence assays is several orders of magnitude greater than that of conventional colorimetric or fluorometric detection methods; e.g. the detection limit for β-galactosidase by the chemiluminescence assay is 8 fg and by a fluorometric assay is 2 pg. Furthermore, chemiluminescence enables detection of β- galactosidase, GUS, and SEAP enzyme concentrations over a dynamic range of more than five to six orders in magnitude. These assays offer highly sensitive, quantitative, rapid, nonisotopic detection of reporter enzymes that are widely used in both mammalian cells and plant cells.