In vitro Biochemical Assays

Fluorescence-based detection methods are the most commonly used readouts for HTS as these readouts are sensitive, usually homogeneous and can be readily miniaturised, even down to the single molecule level.7,8 Fluorescent signals can be detected by methods such as fluorescence intensity (FI), fluorescence polarisation (FP) or anisotropy (FA), fluorescence resonance energy transfer (FRET), time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence intensity life time (FLIM). Confocal single molecule techniques such as fluorescence correlation spectroscopy (FCS) and one- or two-dimensional fluorescence intensity distribution analysis (ID FID A, 2D FIDA) have been reported but are not commonly used. 

Whereas fluorescence is typically measured during non-separation, mix-and-read protocols offering high readout throughput, several approaches exist today that involve separation steps in front of the actual detection. In the Caliper Labchip technology, a multi-parallel microfluidic separation system coupled with fluorescence detection allows the monitoring of enzymatic reactions. Quantification of substrate and product of the enzymatic reaction, after separation from each other as well as from the test compound, minimises artefacts and offers ratiometric results, although with comparatively lower 

Fluorescent microvolume assay technology (FMAT ) is a bead-based or cell-based fluorescent technology for homogeneous ELISA-like assays. In FMAT, a laser beam is focused on the bottom of the assay well and the localised fluorescence intensity bound to beads (or cells) is detected as an area of intense fluorescence over the unbound and background fluorescence in solution. Different analytes can be detected with appropriate fluorophores and, by using different sized beads, the assay can be multiplexed to monitor multiple analytes.13 

Luminex is also a bead-based, non-separation technology using the Luminex colour-coded beads and detection systems (Luminex 100 IS or Luminex HT ). The readers used for this assay format are based on the principle of flow cytometry. The system enables assays to be multiplexed, i.e. allowing different analytes to be monitored simultaneously. The Luminex HT system is compatible with 96- and 384-well microplates14 but throughput of the reader is still a limiting factor for large-scale HTS. 

Luminescence detection has the advantage of very low background compared with fluorescent technologies, so assay sensitivity can be extremely high. 

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In vitro biochemical assays with purified proteins are developed to demonstrate the direct recognition of damage-induced DNA structures by checkpoint sensors (41). Various checkpoint proteins have been successfully expressed in bacteria, yeast. 

Several homogeneous in vitro biochemical assays based on these systems have been described for proteases [139, 140], kinases [141, 142] [143], IL-2-IL-2R inter-... 

For example, the evidence of medical utility that a patent offers may be slight. It suffices if some compounds of the invention prove weakly active merely in a particular in vitro biochemical assay using isolated animal cells. 

FUNCTIONAL ASSAYS FOR 5-HT,d RECEPTORS In vitro biochemical assays... 

The production of a high-quahty Drosophila embryonic cytoplasmic extract for use in protein purification or biochemistry is relatively easy, providing population cages are available that produce at least 5 g of embryos during a 3-hour period (for methods to maintain population cages, see Sisson, this volume). Smaller quantities of embryos can be used to produce extracts that are useftil for in vitro biochemical assays (see, e.g., Moritz et al. 1998), and thus it should be possible to make extracts from mutant stocks that could then be tested in vitro. Protocol 33.1 describes the collection and dechorionation of embryos for making cytoplasmic extracts. It is fairly easy to prepare even very concentrated cytoplasmic extracts from Drosophila embryos, with protein concentrations of 50-75 mg/ml (see Protocol 33.2). 

Rodgers KE, Leung N, Imamura T, et al. 1986. Rapid in vitro screening assay for immunotoxic effects of organophosphorus and carbamate insecticides on the generation of cytotoxic T lymphocyte responses. Pestic Biochem Physiol 26 292-301. 

Cytotoxicity Evaluated for confounding interpretation of in vitro efficacy assays, for predicting potential for human toxicity especially in liver but also if warranted by other safety assessments in bone marrow, kidney, neurons, immu-nocytes and so on. Also used for developing understanding of biochemical mechanisms of toxicity. HCA has been repeatedly demonstrated to be an effective tool in predictive toxicology. May also be used for certain translational safety biomarkers of toxicity [37]... 

Although there is less biochemical information on secretion in prokaryotes, genetic data indicate that the process is likely to be similar to that in eukaryotes. The development of in vitro translocation assays for bacterial systems (Muller and Blobel, 1984a,b Rhoads et al., 1984 Chen et al., 1985) should allow a more detailed analysis of the biochemistry of prokaryotic secretion in the near future. 

Biochemical Assays. In vitro biochemical methods are based on the fact that TSH will release from thyroids previously labeled with by incubation of guinea pig thyroid slices with the labeled iodine (Bll). El Kabir (El) improved the method by treating the guinea pigs with a goitrogen prior to removal of the thyroids. 

This empirical approach would predict, in the absence of any data related to interactions with a molecular target, that because j3-adre-noceptor antagonists lower blood pressure, then any compound that lowers blood pressure is by definition a j3-adrenoceptor antagonist, an absurd conclusion, but one that happened nonetheless. The 1960s saw the development of a number of in vitro biochemical screens that moved the measurement of the RL interaction a little closer to the molecular level. Nonetheless, the major challenge was to develop assays that measured the RL interaction independently of "downstream" events such as enzyme activation and second and third messenger systems. By such means, the ability of a compound to bind to a receptor could be determined on the basis of the SAR and thus provide the chemist with a more direct means to model the RL interaction. 

Potent and specific MDM2 inhibitors such as Nutlin-3 [33] and MI-219 [50, 51] have provided an opportunity to examine the details of their cellular mechanism of p53 activation. Consistent with in vitro biochemical binding assays, potent MDM2 inhibitors are capable of blocking the MDM2-p53 protein-protein interaction in cells. They induce accumulation of p53 protein but do not increase the transcription... 

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