Firefly Luciferase Bioluminescence Research Articles

End-column chemiluminescence detector for capillary electrophoresis

A chemiluminescence detector is designed for capillary electrophoresis in which the signal is generated at the column outlet. As the analytes emerge from the column, they react with chemiluminescent reagents to produce visible light that is transported by a fiber optic to a photomultiplier tube. The luminol chemiluminescence and the firefly luciferase bioluminescence reactions are adapted for use with this type of detection scheme. The reactions yield concentrations detection limits of 2×10 -8 M for luminol and 5×10 -9 M for ATP, which are approximately 3 orders of magnitude lower than those obtained with absorbance

Histochemical representation of regional ATP in the brain using a firefly luciferase-immobilized membrane in a multilayer film format.

The enzymatic bioluminescence of firefly luciferase has been used in sensitive pictorial assays of ATP. We describe a method using a membrane with immobilized luciferase in a multilayer film format for the histochemical representation of brain ATP content. The multilayer film consisted of a transparent support, a reagent layer, and a pigment layer. The reagent layer contained all necessary reagents, including immobilized luciferase. The pigment layer was effective for high image resolution. An unfixed slice of frozen brain 16 microns thick was placed on the film. The chemical energy of brain ATP was converted into luminescent energy in the reagent layer and the bioluminescence emitted was recorded photographically with high spatial resolution. A close linear relationship was obtained between the optical density of the bioluminescent images and logarithmic plots of the brain ATP content. With this film, the regional ATP content in fine anatomical structures of gerbil brains was clearly demonstrated in both physiological and pathological states.

Brain-specific proteins: solid-phase immunobioluminescence assay for neuron-specific enolase in human plasma.

We raised specific antibodies in rabbits against pure alpha alpha- or gamma gamma-isoenzymes of enolase (EC 4.2.1.11) purified from human brain. After being specifically immunoabsorbed by the respective Sepharose-coupled enolase isoenzymes, the purified antibodies are desorbed by acidification and coated onto polystyrene tubes. Each sample containing the isoenzyme activity to be determined is incubated in the respective coated tube. The coating is then washed, and the reaction of the antibody-bound enzyme is initiated by adding 2-phosphoglycerate, ADP, and pyruvate kinase. The accumulation of ATP is measured by following the increase in light emission in the firefly luciferase bioluminescence system. The assay is as specific as the antibody used for coating. Its detection limit is about 5 X 10(-9) U, corresponding to about 0.1 pg or 10(-18) mol of enzyme protein per assay. Activities of enolase isoenzymes in human plasma can be evaluated separately, rapidly, and precisely. We used the assay to measure enolase isoenzyme activities in plasma of patients suffering from different types of malignant tumors.