Biomedical subjects

Wilma Keighley

Publications and source records attributed to Wilma Keighley.

4 recordsLinked to original sources

PubMed2005

Evaluating real-life high-throughput screening data.

High-throughput screening (HTS) is the result of a concerted effort of chemistry, biology, information technology, and engineering. Many factors beyond the biology of the assay influence the quality and outcome of the screening process, yet data analysis and quality control are often focused on the analysis of a limited set of control wells and the calculated values derived from these wells. Taking into account the large number of variables and the amount of data generated, multiple views of the screening data are necessary to guarantee quality and validity of HTS results. This article does not aim to give an exhaustive outlook on HTS data analysis but tries to illustrate the shortfalls of a reductionist approach focused on control wells and give examples for further analysis.

Biological Assay↗

PubMed2004

Development and automation of a 384-well cell fusion assay to identify inhibitors of CCR5/CD4-mediated HIV virus entry.

This article describes the automation of an in vitro cell-based fusion assay for the identification of novel inhibitors of receptor mediated HIV-1 entry. The assay utilises two stable cell lines: one expressing CD4, CCR5 and an LTR-promoter/beta-galactosidase reporter construct, and the other expressing gp160 and tat. Accumulation of beta-galactosidase can only occur following fusion of these two cell lines via the gp160 and receptor mediators, as this event facilitates the transfer of the tat transcription factor between the two cell types. Although similar cell fusion systems have been described previously, they have not met the requirements for HTS due to complexity, throughput and reagent cost. The assay described in this article provides significant advantage, as (a) no transfection/infection events are required prior to the assay, reducing the potential for variability, (b) cells are mixed in solution, enhancing fusion efficiency compared to adherent cells, (c) miniaturization to low volume enables screening in 384-well plates; and (d) online cell dispensing facilitates automated screening. This assay has been employed to screen approximately 650,000 compounds in a singleton format. The data demonstrate that the assay is robust, with a Z' consistently above 0.6, which compares favourably with less complex biochemical assays.

Animals↗

PubMed2003

Measuring intracellular calcium fluxes in high throughput mode.

The measurement of intracellular calcium fluxes in real time is widely applied within the pharmaceutical industry to measure the activation of G-protein coupled receptors (GPCRhyp;s), either for pharmacological characterisation or to screen for new surrogate ligands. Initially restricted to G(q) coupled GPCRs, the introduction of promiscuous and chimeric G-proteins has further widened the application of these assays. The development of new calcium sensitive dyes and assays has provided sensitive, homogeneous assays which can be readily applied to high throughput screening (HTS). In this paper we describe the full automation of this assay type using a fluorometric imaging plate reader (FLIPR ) integrated into a Beckman/Sagian system to establish a simple robotic system that is well suited for the current medium throughput screening in this area of lead discovery. Using a recently completed HTS we discuss important determinants for FLIPR based screening, highlight some limitations of the current approach, and look at the requirements for future automated systems capable of keeping up with expanding compound files.

Automation↗

PubMed2002

High-throughput screening for ion channel modulators.

Ion channels present a group of targets for major clinical indications, which have been difficult to address due to the lack of suitable rapid but biologically significant methodologies. To address the need for increased throughput in primary screening, the authors have set up a Beckman/Sagian core system to fully automate functional fluorescence-based assays that measure ion channel function. They apply voltage-sensitive fluorescent probes, and the activity of channels is monitored using Aurora's Voltage/Ion Probe Reader (VIPR). The system provides a platform for fully automated high-throughput screening as well as pharmacological characterization of ion channel modulators. The application of voltage-sensitive fluorescence dyes coupled with fluorescence resonance energy transfer is the basis of robust assays, which can be adapted to the study of a variety of ion channels to screen for both inhibitors and activators of voltage-gated and other ion channels.

Automation↗