An investigation into the performance of bioluminescence-based measurements

Abstract

This work describes an amplification system, which can greatly increase the amount of ATP available for detection by bioluminescent methods, increasing the sensitivity. The system makes use of an enzyme, adenylate kinase (AK), present in virtually all living cells. The sensitivity of the assay was increased by l0 to 100-fold. A suitable extractant needed to be developed, which had the role of allowing intracellular AK to be detected and assayed, but which was not harmful to the enzyme or ATP, and did not interfere with the assay in any way. The assay required ADP as a substrate, and contaminant ATP from commercial ADP preparations needed to be removed beforehand. Initially, HPLC was used to do this on an analytical scale but was developed into a large-scale preparative procedure employing ion-exchange chromatography. The ATP contamination was reduced to a concentration of less than 1 x 10-6 %. Similarly, commercial luciferase enzymes for the detection of ATP contain contaminating AK, and several approaches were developed to remove this, including Iso-electric Focusing and low-ionic strength precipitation. Over 98% of contaminant AK was successfully removed. Industrial applications of the system were successfully proven, such as incorporation of the assay into a continuous flow luminometer, equipped with a timed 'delay loop' to allow ATP levels to be amplified prior to detection with luciferase in a 'flowcell' detection system. The assay has also been shown to be of significant use in the brewing industry, for the assessment of yeast quality. Current methods involve the assay of protease activity, which is time consuming, expensive, and of poor sensitivity. The AK assay was found to provide an extremely sensitive method for the determination of yeast leakage and autolysis, allowing selection of yeast for re-pitching as part of yeast management in the brewing sector.