Development of bioluminescent reagents for kinetic analysis and some applications in clinical chemistry

Sammanfattning: Firefly luciferase and a bacterial luciferase system have been used to develop analytical methods suitable for use in clinical analysis.The influence of the level of NADH:FMN oxidoreductase on the kinetics of light emission in the bacterial luciferase system was studied. The results were used to define reaction conditions for a constant reaction rate, i.e. a constant level of light emission.A bacterial luciferase reagent based on these principles was developed, using purified bacterial luciferase and NADH:FMN oxidoreductase. During this work a new assay for determination of bacterial luciferase activity was developed.The bacterial luciferase reagent was used in a kinetic assay for total oile acids in serum. A kinetic method for assay of creatine kinase isoenzymes in serum was developed by use of an already existing firefly luciferase reagent and an inhibiting antibody.The bioluminescent assays of bile acids and creatine kinase were found to be more precise in the lower range, have lower detection limits, wider dynamic ranges and to be more rapid than corresponding established spectrophotometric methods.