Homogeneous cell- and bead-based assays for high throughput screening using fluorometric microvolume assay technology

High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousa nds of compounds has resulted in a requirement for assays and instrumentati on that are amenable to nonradioactive formats and that can be miniaturized . Homogeneous assays that minimize upstream automation of the individual as says are also preferable, Fluorometric microvolume assay technology (FMAT) is a fluorescence-based platform for the development of nonradioactive cell - and bead-based assays for HTS, This technology is plate format-independen t, and while it was designed specifically for homogeneous ligand binding an d immunological assays, it is amenable to any assay utilizing a fluorescent cell or bead. The instrument fits on a standard laboratory bench and consi sts of a laser scanner that generates a 1 mm(2) digitized image of a 100-mu m deep section of the bottom of a microwell plate, The instrument is direc tly compatible with a Zymark Twister(TM) (Zymark Corp., Hopkinton, MA) for robotic loading of the scanner and unattended operation in HTS mode. Fluore scent cells or beads at the bottom of the well are detected as localized ar eas of concentrated fluorescence using data processing. Unbound flurophore comprising the background signal is ignored, allowing for the development o f a wide variety of homogeneous assays. The use of FMAT for peptide ligand binding assays, immunofluorescence, apoptosis and cytotoxicity, and bead-ba sed immunocapture assays is described here, along with a general overview o f the instrument and software.