Benchtop assay

In most cases, in consultation with the HTS group, the research area laboratory will develop a benchtop assay that is at least compatible with the HTS format of choice for their target. This tends to facilitate project transitions and provides a tool that the research area laboratory will use later to follow up hits and develop SAR. In other cases, the HTS assay development group will assume all responsibility for assay development. The formality of the transfer of the project from the research area to the HTS group varies between organizations, but the outcomes are quite similar. All of the details of the prototype assay are reviewed by both teams, and, where applicable, reagents, protocols, and even plates or pipette tips are exchanged. 

The overall process and critical activities for robotic implementation of automated HTS from intake of the researcher s benchtop assay through to the identification of a validated, optimized lead series of compounds around a bona fide chemical scaffold are shown in Fig. 2. The downstream steps of hit confirmation/ validation and structure-activity relationship (SAR) elucidation and hit to lead (HTL) have been included to emphasize that the same robotic assay used for production HTS can and should be used by the HTS team to perform high-volume hit confirmation and first-line automated dose-response analysis of confirmed hits to obtain quantitative potency (IC50) rankings to elucidate the nascent SAR of emergent hit series... 

Rg. 2 Schematic pipeline diagram with major steps from intake of a benchtop assay through to generation of a lead series. This chapter reviews the processes up to HTS execution. The sourcing of critical reagent in a test development lot and production lot for HTS is indicated above the assay adaptation, development and validation chevron by blue and light red bricks. The chemical library is represented by the cylindrical tank below the HTS chevron... 

It is important to point out that while most of the burden and expertise for impact are borne by the proposing researcher, it is often the chemists on the overall drug discovery team that have the appropriate domain expertise to evaluate the competitive landscape. It is also important for the assay development team to review and verify the assay development and optimization data of the benchtop assay. Sometimes the fundamental assumptions made are faulty, for example that the steady-state conditions are not met for an enzyme assay (i.e., nonlinear progress curves with too high a level of substrate consumption), that endpoint measurements were taken when a progress curve had already plateaued (this is a surprisingly common error, whose impact has been analyzed) [see Fi f. 3 in [4] for simulation, discussion of reasons for nonlinearity with respect to substrate depletion, and workable off-linearity for HTS], or that substrate concentration-to-7Cm ratio is set inappropriately and so will not allow detection of the desired types of inhibitors... 

Even if the benchtop assay appears a suitable starting point for direct automation-compatible HTS assay development, it behooves the assay development team to obtain starting material from the assay provider and to replicate, at least manually, that the assay performs according to the assay provider s protocol. Table 4 provides a comparison of some of the mechanical and technical parameters that need to be adjusted in the process of adapting a benchtop assay into an HTS (automatable robotic) format. The general guidelines and practical principles are as follows ... 

Problems that may arise with spectrophotometric assays may be due to turbid samples, inadequate mixing, or poor design of controls/blanks. Turbid samples should be clarified by centrifugation (a simple benchtop centrifuge at -5000 x g should be adequate) before reading the absorbance of the clear supernatant. 

Comparison of parameters between laboratory benchtop and HTS assays... 

Benchtop stability (sometimes also referred to as short-term stability) involves evaluation of the stability of the therapeutic during the execution of the assay, for example, testing the limits of time the samples can be on the bench during processing, testing whether the samples can be pretreated (MRD performed) and stored overnight under refrigerated conditions, and so on [25]. 

Immobilization of Urease on Sweetzyme Pellets For co-immobilization of urease on the Sweetzyme pellets, 500 ml of 1 g/1 urease solution and 2 g of Sweetzyme pellets was added to a 1-1 beaker [35]. The beaker was left on the benchtop at room temperature for 24 h. The pellets were separated from the solution by decanting and gravity filtration and dried on a paper towel at room temperature for 24 h or until dry. Co-immobilized pellets were stored at 4 °C until use. Activity of immobilized urease was measured at pH 7.5 and 25°C using a standard assay procedure that measures the rate of ammonia liberation [43]. The urease activities obtained with our immobilization procedure were in the range of 550-577 U/g pellets, where a unit liberates 1 pmol of ammonia per minute under the assay conditions. 

The stems and roots of 10 plants from each of three containers from each group are prepared for 30% homogenate of plant material in buffer (0.01-M Tris-HCl pH 7.8, 7.5-mM PMSF, 2.5-mM EDTA, 325-pM bestatin, 3.5-pM E-64, 2.5-pM leu-peptin, and 0.75-pM aprotinin). The homogenate is centrifuged at 4°C and 12,000g in a benchtop centrifuge for 2h, and the supernatant is stored at -80°C for the GST and GPX assay. 

Wallace D, Hildesheim A, Pinto LA. Comparison of benchtop microplate beta counters with the traditional gamma counting method for measurement of chromium-51 release in C5dotoxic assays. Clin Diagn Lab Immunol 2004 11 255-260. 

For a GC-MS assay using benchtop instruments, the method has excellent sensitivity. Sensitivity is lowest for testosterone with a signal-to-noise ratio (S/ N) of 2.4 for 10 pg, and highest for 5a-androstane-3a,17p diol with a S/N of 17 for 10 pg. For the steroids studied, the intra- and interassay coefficients of variation are between 2.7 and 7.6%, respectively. Accuracy was determined by spiking plasma with known amounts of steroids. The agreement between the values found and the amounts added was excellent with relative errors less than 7.5%. Standard curves were prepared by analysis of a selection of reference analyte/internal standard mixtures of known concentrations. All standard plots were linear. 

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