High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds has resulted in a requirement for assays and instrumentation that are amenable to nonradioactive formats and that can be miniaturized. Homogeneous assays that minimize upstream automation of the individual assays 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-independent, and while it was designed specifically for homogeneous ligand binding and immunological assays, it is amenable to any assay utilizing a fluorescent cell or bead. The instrument fits on a standard laboratory bench and consists of a laser scanner that generates a 1 mm2 digitized image of a 100-μm deep section of the bottom of a microwell plate. The instrument is directly compatible with a Zymark Twister™ (Zymark Corp., Hopkinton, MA) for robotic loading of the scanner and unattended operation in HTS mode. Fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using data processing. Unbound flurophore comprising the background signal is ignored, allowing for the development of a wide variety of homogeneous assays. The use of FMAT for peptide ligand binding assays, immunofluorescence, apoptosis and cytotoxicity, and bead-based immunocapture assays is described here, along with a general overview of the instrument and software.
References
1.
Broach, J.R., Thorner, J. (1996). High-throughput screening for drug discovery. Nature384:14-16.
2.
Fernandes, P.B. (1998). Technological advances in high throughput screening. Curr. Opin. Chem. Biol.2;597-603.
3.
Rogers, M.V. (1997). Light on high-throughput screening: Fluorescence-based technologies. Drug Discovery Today2:156-160.
4.
Fox, S., Yund, A., Farr-Jones, S. (1998). Seeking innovation in high-throughput screening. Drug Discovery Dev32-36.
5.
Giuliano, K.A., and Taylor, D.L. (1998). Fluorescent-protein biosensors: New tools for drug discovery. Trends Biotechnol.16:135-140.
6.
Giuliano, K.A., DeBiasio, R.L., Dunlay, R.T., Gough, A., Volosky, J.M., Zock, J., Pavlakis, G.N., Taylor, D.L. (1997). Highcontent screening: A new approach to easing key bottlenecks in the drug discovery process. J. Biomolecular Screening2:249-259.
7.
Dietz, L.J., Dubrow, R.S., Manian, B.S., Sizto, N.L. (1996). Volumetric capillary cytometry: A new method for absolute cell enumeration. Cytometry23:177-186.
8.
Koller K.J., Whitehorn, E.A., Tate, E., Ries, T., Aguilar, B., Chernov-Rogan, T., Davis, A.M., Dobbs, A., Yen, M., Barrett, R.W. (1997). A generic method for the production of cell lines expressing high levels of 7-transmembrane receptors. Anal. Biochem.250:51-60.
9.
Tomei, L.D., Cope, F.O. (1994). Apoptosis II: The Molecular Basis of Apoptosis in Disease, Cold Srping Harbor Laboratory Press, Plainview, N.Y.
10.
Spector, D.L., Goldman, R.D., and Leinwand, L.A. (1998) Cells: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
11.
Swartzman, E.E., Miraglia, S.J., Mellentin-Michelotti, J., Evangelista, L., and Yuan, P.M. (1999). A homogeneous and multiplexed immunoassay for high throughput screening using fluorometric microvolume assay technology. Anal. Biochem.271:143-151.
12.
Rose, P.M., Fernandes, P., Lynch, J.S., Frazier, S.T., Fisher, S.M., Kodukula, K., Kienzle, B., Seethala, R. (1995). Cloning and functional expression of a cDNA encoding a human type 2 neuropeptide Y receptor. J. Biol. Chem.270:29038.
13.
Gerald, C., Walker, M.W., Vaysse, P.J., He, C., Branchek, T.A., Weinshank, R.L. (1995). Expression cloning and pharmacological characterization of a human hippocampal neuropeptide Y/peptide YY Y2 receptor subtype. J. Biol. Chem.270:26758-26761.
14.
Rudolf, K., Eberlein, W., Engel, W., Wieland, H.A., William, K.D., Entzeroth, M., Wienen, W., Beck-Sickinger, A.G., Doods, H.N (1994). The first highly potent and selective nonpeptide neuropeptide Y Y1 receptor antagonist: BIBP3226. Eur. J. Pharmacol.271:R11-R13.
15.
Doods, H.N., Wieland, H.A., Engel, W., Eberlein, W., William, K.D., Entzeroth, M., Wienen, W., Rudolf, K. (1996). BIBP 3226, the first selective neuropeptide Y1 receptor antagonist: A review of its pharmacological properties. Regul. Pept.65:71-77.
16.
Mellentin-Michelotti, J., Evangelista, L., Swartzman, E.E., Miraglia, S.J., Werner, W., Yuan, P.M. (1999). Determination of ligand-binding affinities for endogenous 7-transmembrane receptors using fluorometric microvolume assay technology. Anal. Biochem. (in press).
17.
Vermes, I, Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. (1995). A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods184:39-51.
18.
Hande, K.R. (1998). Etoposide: Four decades of development of a topoisomerase II inhibitor [in process citation]. Eur. J. Cancer34:1514-1521.
