Dual Analyte Detection Using Tandem Flash Luminescence

A heterogeneous, dual analyte-binding assay which makes use of the flash luminescence from both aequorin and an acridinium-9-carboxamide label is presented. The signal generating species were triggered both differentially and sequentially using Ca2+ followed by basic peroxide. Both signals were resolved readily using a single photomultiplier tube without the need for multiwavelength detection. To demonstrate the tandem luminescence concept in a model assay system, dose–response curves for two analytes, biotinylated BSA and myoglobin, were generated using a competitive binding format. Because of the relatively short assay time and the well-resolved signals, this format will be useful in the development of dual analyte high-throughput assays.