Miniaturization of a hepatitis C virus RNA polymerase assay using a -102 degrees C cooled CCD camera-based imaging system.
Innovations in detection technologies have allowed us to develop a novel assay in 1536-well plate format and assess the advantages of screen miniaturization compared with conventional high-throughput compound screening in 96- or 384-well plates. An HCV RNA polymerase assay has been miniaturized in 1536-well plates by using a new detection technology known as LEADseeker homogeneous imaging system. It uses a -102 degrees C cooled charge-coupled device (CCD) camera and newly designed scintillation proximity microparticles. The miniaturized assay used europium-doped streptavidin-coated yttrium oxide (YO(x)) or polystyrene (PS) microspheres to capture biotin-labeled [(3)H]RNA product transcripts. Beads in proximity to the radioisotope convert the emitted beta(-) particles into photons having wavelengths in the red region of the visible spectrum, optimal for detection by the CCD camera. Because the camera collects light from all wells of the plate simultaneously, 1536-well plates are imaged as rapidly as 384-well plates, on the order of 10 min per plate. The assay has a signal to background of approximately 20-fold, satisfactory for high-throughput robotics screening. The enzyme kinetics and potency of a known inhibitor were similar to those obtained from the conventional assay using scintillation proximity assay (SPA) beads and a scintillation plate counter. Furthermore, the newly developed microbeads (emitting at 610 to 620 nm) are less prone to quenching effects caused by yellow-colored compounds, than conventional SPA beads or scintillation fluid (emitting at 400 to 480 nm region). Thus, the LEADseeker imaging system is a useful new tool for miniaturization of assays for high-throughput screening.
Department of Automated Biotechnology, Merck & Company, 502 Louise Lane, North Wales, Pennsylvania 19454, USA. wei_zheng@merck.com
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Zheng
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Carroll
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Inglese
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Graves
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Howells
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Strulovici
D003080:Cold Temperature; D012321:DNA-Directed RNA Polymerases / Q000032:analysis* / Q000037:antagonists & inhibitors; D003952:Diagnostic Imaging / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016174:Hepacivirus / Q000201:enzymology*; D020128:Inhibitory Concentration 50; D012588:Scintillation Counting / Q000379:methods; D015398:Signal Transduction
D004791:Enzyme Inhibitors; D012321:DNA-Directed RNA Polymerases