Miniaturization of absorbance assays using the fluorescent properties of white microplates

Zuck, Paul; O'Donnell, Gregory; Cassaday, Jason; Chase, Peter; Hodder, Peter; Strulovici, Berta; Ferrer, Marc

doi:10.1016/j.ab.2005.04.029

Cited by 33 publications

(26 citation statements)

References 11 publications

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“…1A). 23 They observed that when white, opaque microtiter plates are irradiated at 430 nm, they emit fluorescence in a broad range between approximately 450 and 600 nm (Fig. 1B).…”

Section: Resultsmentioning

confidence: 94%

“…1C). Zuck and colleagues found that this strategy improves sensitivity because both the fluorescence emission and excitation are absorbed by the chromophore 23. Further, the Walt group has explored the mechanistic underpinnings of a similar process, and they found that both radiative and non-radiative energy transfer events contribute to improved sensing 29…”

Section: Resultsmentioning

confidence: 99%

“…Unfortunately, our attempts to further miniaturize the absorbance platforms to low volume 384-well microtiter plates were frustrated by significantly decreased sensitivity. Guided by the work of Zuck and coworkers,23 we explored whether sensitivity could be enhanced using energy transfer methodology. We were particularly interested in studying DnaK, as this chaperone has been implicated as an emerging anti-bacterial target 4.…”

Section: Introductionmentioning

confidence: 99%

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High-Throughput Screen for Escherichia coli Heat Shock Protein 70 (Hsp70/DnaK): ATPase Assay in Low Volume by Exploiting Energy Transfer

et al. 2010

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Members of the heat shock protein 70 (Hsp70) family of molecular chaperones are emerging as potential therapeutic targets. Their ATPase activity has classically been measured using colorimetric phosphate-detection reagents, such as quinaldine red (QR). While such assays are suitable for 96-well plate formats, they typically lose sensitivity when attempted in lower volume due to path length and meniscus effects. These limitations and Hsp70’s weak enzymatic activity have combined to create significant challenges in high throughput screening. To overcome these difficulties, we have adopted an energy transfer strategy that was originally reported by Zuck et al. (Anal. Biochem. 2005, 342:254–259). Briefly, white 384-well plates emit fluorescence when irradiated at 430 nm. In turn, this intrinsic fluorescence can be quenched by energy transfer with the QR-based chromophore. Using this more sensitive approach, we tested 55,400 compounds against DnaK, a prokaryotic member of the Hsp70 family. The assay performance was good (Z′ ~ 0.6, CV ~8%) and at least one promising new inhibitor was identified. In secondary assays, this compound specifically blocked stimulation of DnaK by its co-chaperone, DnaJ. Thus, this simple and inexpensive adaptation of a colorimetric method might be suitable for screening against Hsp70-family members.

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“…1A). 23 They observed that when white, opaque microtiter plates are irradiated at 430 nm, they emit fluorescence in a broad range between approximately 450 and 600 nm (Fig. 1B).…”

Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

High-Throughput Screen for Escherichia coli Heat Shock Protein 70 (Hsp70/DnaK): ATPase Assay in Low Volume by Exploiting Energy Transfer

et al. 2010

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“…Traditionally, colorimetric assays are not readily scalable to small volumes required for miniaturization into high dense plates [21]. A method was described to miniaturize colorimetric assays to 1,536-well format using fluorescence properties of white solid-bottom microplates [22]. In this fluorescence- quench absorbance assay, absorbance is measured by the decrease of fluorescent signal from white material of the plate at an excitation or emission wavelength that matches the absorbance maximum of the colored reaction product.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, the reaction is performed in clear-bottom (transparent) microplates to measure trans-absorbance. Since white plastic solid-bottom plates have fluorescence properties [22], when an excitation light is directed to white solid-bottom microplates, emission light can be detected by epi-absorbance ( A ). The green arrow represents the fluorescence signal emitted from of solid-bottom of the well of a plate.…”

Section: Resultsmentioning

confidence: 99%

Novel Patient Cell-Based HTS Assay for Identification of Small Molecules for a Lysosomal Storage Disease

et al. 2011

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Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs), inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS) assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA) activity found in patients with metachromatic leukodystrophy (MLD), a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t) cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS), detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S) cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC) acts as “plate fluorescence quencher” in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an opportunity to identify therapeutic small molecules in a disease-cellular environment where potentially disrupted pathways are exposed and available as targets.

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High‐Throughput Screening for Small‐Molecule Drug Discovery

Parker¹,

Zhang²

2011

Pharmaceutical Sciences Encyclopedia

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The aim of this chapter will be to review some of the high‐throughput screening (HTS) processes in current drug discovery, including assay technologies and screening strategies, the steps taken in assay development and validation, the screening process, and commonly used quality control measures. Also highlighted in this chapter are the analysis of HTS data and some of the approaches used for data mining for finding lead series. Finally, the chapter will reviews some of the emerging trends occurring in the science of biomolecular screening.

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Miniaturization of absorbance assays using the fluorescent properties of white microplates

Cited by 33 publications

References 11 publications

High-Throughput Screen for Escherichia coli Heat Shock Protein 70 (Hsp70/DnaK): ATPase Assay in Low Volume by Exploiting Energy Transfer

High-Throughput Screen for Escherichia coli Heat Shock Protein 70 (Hsp70/DnaK): ATPase Assay in Low Volume by Exploiting Energy Transfer

Novel Patient Cell-Based HTS Assay for Identification of Small Molecules for a Lysosomal Storage Disease

High‐Throughput Screening for Small‐Molecule Drug Discovery

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