Digital Imaging as a Detection Method for a Fluorescent Protease Assay in 96-Well and Miniaturized Assay Plate Formats.

The demand to increase throughput in HTS programs, without a concomitant addition to costs, has grown significantly during the past few years. One approach to handle this demand is assay miniaturization, which can provide greater throughput, as well as significant cost savings through reduced reagent costs. Currently, one of the major challenges facing assay miniaturization is the ability to detect the assay signal accurately and rapidly in miniaturized formats. Digital imaging is a detection method that can measure fluorescent or luminescent signals in these miniaturized formats. In this study, an imaging system capable of detecting the signal from a fluorescent protease assay in multiple plate formats was used to evaluate this detection method in an HTS environment. A direct comparison was made between the results obtained from the imaging system and a fluorescent plate reader by screening 8,800 compounds in a 96-well plate format. The imaging system generated similar changes in relative signal for each well in the screen, identified the same active compounds, and yielded similar IC50 values as compared to the plate reader. When a standard protease inhibitor was evaluated in 96-, 384-, 864-, and 1536-well plates using imaging detection, similar IC50 values were obtained. Furthermore, similar dose-response curves were generated for the compound in 96- and 384-well assay plates read in a plate reader. These results provide support for digital imaging as an accurate and rapid detection method for high-density microtiter plates.