Miniaturization of a Hepatitis C Virus RNA Polymerase Assay Using a −102°C Cooled CCD Camera-Based Imaging System

Zheng, Wei; Carroll, Steven S.; Inglese, James; Graves, Robert J.; Howells, Leighton; Strulovici, Berta

doi:10.1006/abio.2001.4991

Cited by 23 publications

(15 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…SPA beads are available in PVT or YSi for the PMT-based readers and in PS or YOx for the CCD-based readers. Although yttrium-based beads have a higher sensitivity, 57,74 55 Wang et al 56 Zheng et al 57 Glycogen synthase kinase 68,74 In many cases the bead sensitivity is not an issue for assay development, and PS or PVT beads can be used more easily. A major cause of low sensitivity can be the background caused by unspecific binding of radiolabeled reagents to the beads, combined with low expression level of receptor or low enzyme turnover, and these issues cannot always be surmounted with the use of yttrium-based scintillants.…”

Section: Scintillation Materialsmentioning

confidence: 98%

“…57,89,90 First, because the CCD imagers collected a digital "photograph" of the entire MTP, all of the wells were read simultaneously, with an exposure time of about 2 min. These instruments' software is used to quantify the brightness of emission from each well (see Fig.…”

Section: Scintillation Readersmentioning

confidence: 99%

“…[83][84][85][86] The next major advancement was the development of red-shifted scintillation beads (imaging beads [GE Healthcare BioSciences, Piscataway, NJ]) or red-shifted FlashPlate ® microplates (PerkinElmer Life Sciences) along with supercooled charge-coupled device (CCD)-based imagers (LEADseeker™ [GE Healthcare Bio-Sciences] and ViewLux™ [PerkinElmer Life Sciences]). 45,57,74,[87][88][89][90] This allowed for the parallel reading of 384 or 1,536 wells simultaneously, reducing plate reading time by 10-20-fold and resulting in a lowered risk of artifacts due to color quenching in the yellow range. Any well-density MTP could now be read within 2-5 min depending upon the signal.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Scintillation Proximity Assays in High-Throughput Screening

Glickman

Schmid

Ferrand

2008

ASSAY and Drug Development Technologies

101

View full text Add to dashboard Cite

Scintillation proximity assays (SPAs) have become a powerful tool for high-throughput screening (HTS) because they can measure the activity and binding of very diverse classes of drug targets. By applying the basic principles of ligand-receptor binding and enzyme kinetics, it is possible to build a large variety of miniaturized, high-throughput assays and screen millions of compounds. SPAs are enabled by the diversity of radiolabeled molecules and affinity tags that are commercially available. These synthetic radiotracers allow for minimal disturbance of the natural binding interactions. This article will present a comprehensive review of the technique and provide detailed information on its applications related to HTS, highlighting the major uses and giving some suggestions for future research.

show abstract

Section: Scintillation Materialsmentioning

confidence: 98%

Section: Scintillation Readersmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Scintillation Proximity Assays in High-Throughput Screening

Glickman

Schmid

Ferrand

2008

ASSAY and Drug Development Technologies

101

View full text Add to dashboard Cite

show abstract

“…Other accessory replication proteins involved in the process of both leading-and lagging-strand DNA synthesis may also be incorporated into this system, thus further increasing its usefulness for the identification of antagonists of the DNA replication machine. Last, most scintillation proximity assays monitoring polymerase activity use biotinylated nucleotide substrates (13,14), followed by capture onto streptavidin-coated SPA beads. In this assay unmodified substrates may be used, eliminating any confounding factors associated with using nonnatural substrates.…”

Section: Specificity For Dnag Primasementioning

confidence: 99%

Homogenous Assays for Escherichia coli DnaB-Stimulated DnaG Primase and DnaB Helicase and Their Use in Screening for Chemical Inhibitors

Zeng

Yang

Kao

et al. 2002

Analytical Biochemistry

View full text Add to dashboard Cite

“…The first-generation LEADseeker platform, the radiometric LEADseeker, has already demonstrated the benefits that imaging offers over conventional PMT-based systems for radioactive-based screening. 14 Here, in addition to comparing fluorophore/quencher pairs, we assess the potential advantages that imaging provides over PMT-based detection systems for fluorescence-based HTS protease assays.…”

mentioning

confidence: 99%

Evaluation of an Imaging Platform during the Development of a FRET Protease Assay

et al. 2003

View full text Add to dashboard Cite

Synthetic peptide substrates labeled with a fluorescent donor and quenching moiety flanking an enzyme cleavage site provide a reliable method for monitoring enzyme activity. The dye pair Mca/Dnp has been widely used for this purpose, but poor solubility characteristics, combined with fluorescence emission in the region of the spectrum associated with interference from bi-ologicals and library compounds, can limit the usefulness of Mca/Dnp substrates in a high-throughput screening (HTS) environment. Peptide Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2is a matrix-metalloproteinase 3 (MMP-3) enzyme substrate that the authors have labeled with a CyDye pair, Cy3/Cy5Q. The Mca/Dnp- and CyDye-labeled substrates were compared during the development of an MMP-3 inhibitor assay. The results obtained showed that although the peptide substrates behaved similarly throughout the development of the MMP-3 assay, during a test screen of 934 compounds randomly selected from a collection of more than 70,000 compounds, the CyDye substrate was considerably more reliable. Screen Z factor values of 0.84 and 0.15 were obtained using the CyDye and Mca/Dnp peptides respectively, and the authors found that although < 1% of the test compounds were auto-fluorescent at Cy3 wavelengths, > 10% could not be screened using the Mca/Dnp substrate because of compound auto-fluorescence and interference. During this study, the authors used a PMTbased fluorescence plate reader and at the same time evaluated a charged couple device (CCD)—based imaging platform specifically optimized for use with CyDye reagents. The imaging platform gave improved read accuracy and faster plate processing times compared with the PMT reader. Overall, the results presented here highlight the potential benefit of employing the red-shifted CyDye reagents and imaging technology during the development and execution of HTS protease screens. (Journal of Biomolecular Screening 2003:72-80)

show abstract

Miniaturization of a Hepatitis C Virus RNA Polymerase Assay Using a −102°C Cooled CCD Camera-Based Imaging System

Cited by 23 publications

References 11 publications

Scintillation Proximity Assays in High-Throughput Screening

Scintillation Proximity Assays in High-Throughput Screening

Homogenous Assays for Escherichia coli DnaB-Stimulated DnaG Primase and DnaB Helicase and Their Use in Screening for Chemical Inhibitors

Evaluation of an Imaging Platform during the Development of a FRET Protease Assay

Contact Info

Product

Resources

About