Several fluorescence, chemiluminescence, luminescence, and radioactive based assays that are one-step assays that do not require wash and multiple incubations or filtrations and washes are detailed in this chapter. There is not a single format that can completely replace all the assays for various targets in drug screening. Radioactive assays are generally very sensitive assays, but handling problems and the generation of radioactive waste are big concerns. In the radioactive assays based on SPA, FLASHplate, Cytostar, and LEADseeker assays, the radioactivity generated is reduced. SPA assays have been gaining in popularity in spite of being radioactive methods. Nonradioactive methods like fluorescence based methods are getting more attention. The FP assay format is comparatively simple as only one labeled molecule is needed. HTRF requires two labeled molecules, and if some generic labeled molecules can be utilized, an assay can be developed faster. Both FP and HTRF formats are very robust and sensitive assays and have the added advantage that these are ratiometric methods, hence there is less interference from colored compounds. When tyrosine kinases and serine kinase were compared in FP, FRET, and SPA formats, all three techniques produced very similar IC50 values for some peptide substrates, but the FP assay was found to be faster, cheaper, and more sensitive and robust than the SPA assay .
The HTS lab should concentrate on a few technologies that are amenable to a wide variety of assays and also adaptable to high density plates rather than diversifying on too many platforms. It will consume a lot of time and money (instrumentation) to test all the technologies available in optimizing each assay. Some assays can be done with equal efficiency by more than one format, and it will be advantageous to go with an assay format that is already tested in the laboratory. Unless the new technologies offer substantial improvements over the existing platforms, it is not cost-effective to switch. Several new assay technologies based on fluorescence confocal microscopy, fluorescence correlation spectroscopy, fluorescence imaging, electrochemiluminescence, AlphaScreen, FMAT, and SPR methods are being developed to increase the throughput to adopt to HTS and uHTS. Microassays using microchip technologies along with mi-crofluidic array and imaging technologies are gaining importance in drug discovery.
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