Luminescence assays are becoming more popular in high throughput screening (HTS) laboratories with the luciferase reporter gene being the most common. As with other assays that are adapted to HTS, improvements have been made to the luciferase assay to make it better suited to the requirements of HTS. For the luciferase reporter gene, these improvements included stabilization of the enzyme, increasing the half-life of the luminescence signal to 5 h, and eliminating separation steps (centrifugation and aliquot transfer) after cell lysis. The improved assay, LucLite, is homogeneous and is measured directly in the cell culture media. In addition to reagent improvements, a temperature-controlled, multidetector microplate counter, TopCount, can quickly and accurately measure luminescence signals.

A new microplate scintillation and luminescence counter (TopCount™ HTS, Packard) has been developed for counting both 96- and 384-well microplates. The first assay methods tested in the 384-well plate were the scintillation proximity assay and the luciferase reporter gene assay. The binding curve and luciferase induction were identical in either plate format. The signal-to-noise ratio (maximum binding divided by nonspecific binding) in the 384-well plate is equal to or greater than those measured in the 96-well format resulting in a comparable assay window. Reagent pipetting for SPA, including the scintillating beads, was performed with the MultiPROBETm 104 Automated Liquid Handling System (Packard, Meridan, CT). This instrument has both the positioning and pipetting accuracy to support the 384-well microplate; therefore, making complete automation practical. With little or no optimization, both the SPA and the luciferase reporter gene assay could be miniaturized to 50 ,Il volumes or less.

The TopCount Microplate Scintillation Counter and the Matrix 9600 Direct Beta Counter are microplate compatible instruments developed to meet the needs of investigators using radioisotope assays adapted for very high throughput. This paper describes these instruments and their application to receptor binding assays. When combined with the appropriate sample handling equipment and filter media, use of these multi-detector instruments improves sample handling efficiency and shortens overall counting time. The assay protocols including filtration through glass fiber mats and membrane filters have been investigated. Results obtained from these new instruments are compared to standard techniques using conventional liquid scintillation and gamma counting.

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