Symposium 1997: Abstract for KV Wood

Bioluminescence assays are generally recognized for their exceptional sensitivity, but in fact their commercial success has been based largely on their rapidity and simplicity. This fact is particularly important for drug discovery where very high volume assay throughput is necessary.

Although relative changes in gene expression can be quantified within seconds using the firefly luciferase, the luminescence signal of the conventional assay persists only briefly necessitating a reagent injector for reliable quantitation.

Subsequent assays designs, most notably those including CoA in the reaction chemistry, extend signal lifetime providing increased sensitivity and eliminating the need for injectors in individual assays. These extended lifetime assays were nevertheless insufficient for 96-well or 384-well plates required in high throughput screening.

Although assays with very long lifetimes (approximately 5 hours) have been developed, these impose a severe penalty to assay sensitivity. However, careful assay design can minimize this penalty while retaining the extended signal lifetime. The simultaneous analysis of two expression events, or the inclusion of an internal control, can be achieved by combining the bioluminescence assays of firefly luciferase with Renilla luciferase.

Although this dual assay requires a reagent injector for 96-well plate analysis, it substantially increases throughput over conventional non-integrated co-reporter assays. Because firefly bioluminescence is dependent on ATP, quantification of cell viability is also possible, which has applications such as screening for antimicrobial agents. Cell viability can be measured either by using an integrated luciferase gene, or by adding a reagent containing luciferase. By either strategy, luciferases with increased stability have been created to allow greater assay reliability and compatibility with automated instrumentation.