Chemiluminescence

Chemiluminescence is the production of light by a chemical reaction in which a substrate molecule is converted to product with concomitant release of a single photon of light energy. A rapid homogeneous chemiluminescent telomerase hybridization protection assay amenable to HTS was described [62].

1. Luciferase Reporter Assays

Bioluminescence is chemiluminescence that occurs within an organism. Bioluminescent reporters of genetic transcription provide rapid quantitative analysis of many cellular events that are important in drug discovery, such as receptor function, signal transduction, gene expression, and protein-protein interaction. Firefly luciferase, a 62 K protein, is the most widely used luminescent reporter enzyme for studying gene regulation and expression. As no endogenous luciferase activity is found in mammalian cells, the background is very low. Bioluminescence is generated at 560 nm when luciferase converts the substrate luciferin to oxyluci-ferin in the presence of ATP, and the half-life of the signal is a few seconds. Developments in the luciferase assay increased the half-life from seconds (flash) to minutes (enhanced flash) and finally to hours (glow) [62]. Homogeneous luciferase assays have been developed by adding reagent containing lysis buffer, enzyme stabilizer, and luciferin directly to the cells in culture media and measuring the chemiluminescence signal [63]. Luciferase kits for homogeneous assays are available as Steady-Glo from Promega, LucScreen from Tropix, Luc Lite from Packard and also from other manufacturers. (Luciferase assays will be elaborated in Chapter 7.)

2. Instrumentation

Chemiluminescence can be measured in dedicated microplate luminometers (Wallac, Tropix, and other manufacturers) or the scintillation counters TopCount NXT (Packard) and MicroBeta (Wallac) or CLIPR (Molecular Devices) or the multimode readers Victor 1420, Analyst, and Polarstar.

3. Other Chemiluminescence Reporters

Some other common reporter genes, such as P-galactosidase and secreted alkaline phosphatase, are assayed conventionally with colorimetric reagents, and the sensitivity has been increased with the use of fluorescence substrates. The sensitivity of these enzymes has been remarkably enhanced (by at least 3 orders) with chemi-luminescent reporter gene assay reagents, galacto-star and galacto-light/plus for P-galactosidase, and phospha-light for secreted alkaline phosphatase from Tropix.

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