A Comparison of Assay Performance Measures in Screening Assays: Signal Window, Z′ Factor, and Assay Variability Ratio

Iversen, Philip W.; Eastwood, Brian J.; Sittampalam, G. Sitta; Cox, Karen L.

doi:10.1177/1087057105285610

Cited by 212 publications

(208 citation statements)

References 7 publications

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“…In addition to the Z′ factor, the coefficient of variation (CV) is a dimensionless parameter that defines the extent of variability. 27 A small variability in the data (CV < 10%) suggests a robust assay. The calculated CVs with HDAC1, HDAC2, HDAC3, and HDAC6 were 9.7%, 10%, 9.2%, and 5.2%.…”

Section: Design Of the Hdac Activity Assaymentioning

confidence: 99%

Development of an ELISA-Based HDAC Activity Assay for Characterization of Isoform-Selective Inhibitors

2015

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Histone deacetylase (HDAC) proteins are promising targets for cancer treatment, with several HDAC inhibitors used clinically as anticancer drugs. Most HDAC inhibitors nonspecifically interact with all or many of the 11 HDAC isoforms. Isoform-selective HDAC inhibitors would be useful tools to dissect the individual functions of HDAC proteins in cancer formation, in addition to potentially displaying effective anticancer properties. We report here a robust HDAC activity assay for screening selective HDAC inhibitors, which is inspired by the traditional enzyme-linked immunosorbent assay (ELISA). The key feature of the ELISA-based HDAC activity assay is use of mammalian cell-derived HDAC isoforms instead of recombinant proteins. Importantly, the assay was validated with several known HDAC inhibitors. The ELISAbased HDAC activity assay will facilitate the characterization of isoform-selective HDAC inhibitors against mammalian cell-derived HDAC proteins, which will enhance HDAC-centered cancer research and provide a foundation for anticancer drug development.

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Section: Design Of the Hdac Activity Assaymentioning

confidence: 99%

Development of an ELISA-Based HDAC Activity Assay for Characterization of Isoform-Selective Inhibitors

2015

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“…The % CV is the variation of measurements relative to the mean and is a statistical measure used to estimate assay performance. 19 A Z′ factor > 0.5 is often interpreted as an indicator of acceptable assay quality. 20 For any assay, if CV is > 0.17 (17%), the Z′ factor will not reach 0.5.…”

Section: Introductionmentioning

confidence: 99%

Development of an Alamar Blue™ Viability Assay in 384-Well Format for High Throughput Whole Cell Screening of Trypanosoma brucei brucei Bloodstream Form Strain 427

Sykes¹,

Avery²

2009

The American Journal of Tropical Medicine and Hygiene

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Abstract. There is an urgent need for new compounds for the drug development pipeline for treatment of patients with African sleeping sickness. One approach for identifying such compounds is by high throughput screening (HTS) of compound collections. For time and cost considerations, there is a need for the development of an assay that uses at least 384-well formats. To our knowledge, there are currently no viability assays for whole cell screening of trypanosomes in the 384-well plate format. We have developed and optimized an Alamar Blue viability assay in a 384-well format for Trypanosoma brucei brucei bloodstream form strain 427 (BS427). The assay had a Z′ > 0.5 and tolerated a final dimethylsulfoxide concentration of 0.42%. Drug sensitivity was compared with those reported from previously developed 96-well methods and was found to be comparable. The sensitivity and cost benefit of the Alamar Blue assay make it an excellent candidate for HTS application.

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“…The Z'-factor is a widely accepted statistical parameter for evaluation and validation of HTS assays [36,37]. The Z'-factor describes an assay's ability to distinguish between the mean values for a positive and negative control, taking into account the data variability for both sample sets.…”

Section: Resultsmentioning

confidence: 99%

A continuous assay for monitoring the synthetic and proofreading activities of multiple aminoacyl-tRNA synthetases for high-throughput drug discovery

Grube

Roy

2017

RNA Biology

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Aminoacyl-tRNA synthetases (aaRSs) catalyze the aminoacylation of tRNAs to produce the aminoacyl-tRNAs (aa-tRNAs) required by ribosomes for translation of the genetic message into proteins. To ensure the accuracy of tRNA aminoacylation, and consequently the fidelity of protein synthesis, some aaRSs exhibit a proofreading (editing) site, distinct from the aa-tRNA synthetic site. The aaRS editing site hydrolyzes misacylated products formed when a non-cognate amino acid is used during tRNA charging. Because aaRSs play a central role in protein biosynthesis and cellular life, these proteins represent longstanding targets for therapeutic drug development to combat infectious diseases. Most existing aaRS inhibitors target the synthetic site, and it is only recently that drugs targeting the proofreading site have been considered. In the present study, we developed a robust assay for the high-throughput screening of libraries of inhibitors targeting both the synthetic and the proofreading sites of up to four aaRSs simultaneously. Thus, this assay allows for screening of eight distinct enzyme active sites in a single experiment. aaRSs from several prominent human pathogens (i.e., Mycobacterium tuberculosis, Plasmodium falciparum, and Escherichia coli) were used for development of this assay.

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A Comparison of Assay Performance Measures in Screening Assays: Signal Window, Z′ Factor, and Assay Variability Ratio

Cited by 212 publications

References 7 publications

Development of an ELISA-Based HDAC Activity Assay for Characterization of Isoform-Selective Inhibitors

Development of an ELISA-Based HDAC Activity Assay for Characterization of Isoform-Selective Inhibitors

Development of an Alamar Blue™ Viability Assay in 384-Well Format for High Throughput Whole Cell Screening of Trypanosoma brucei brucei Bloodstream Form Strain 427

A continuous assay for monitoring the synthetic and proofreading activities of multiple aminoacyl-tRNA synthetases for high-throughput drug discovery

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