Abstract:
:Time-resolved fluorescence (TRF) assay formats are frequently used technologies in high-throughput screening. In this article, we have characterised the novel Plate::Vision(2) 96-microlens array reader (Carl Zeiss Jena GmbH, Germany) and compared it to the novel LEADseeker Generation IV multimodality imaging system (LEADseeker Gen IV; Amersham Biosciences UK Ltd., UK) for applications in the TRF mode. In europium measurements using the TRF mode, the Plate::Vision displayed a limit of detection for europium of approximately 3 pM, which was comparable to two established TRF readers, the Discovery and the Victor V (both PerkinElmer Life Sciences Inc., USA). The LEADseeker's limit of detection only extended down to europium concentrations of approximately 10 pM in these experiments. For TRF resonance energy transfer (TR-FRET) experiments, a europium-biotin (Eu-biotin) conjugate was titrated with a streptavidin-allophycocyanin (SA-APC) conjugate. The Plate::Vision produced Z' values larger than 0.5 for the acceptor fluorophor emission with concentrations of Eu-biotin as low as 3 nM combined with 175 pM SA-APC. To achieve Z' values of at least 0.5 with the LEADseeker, concentrations of 10 nM Eu-biotin combined with SA-APC of at least 0.8 nM were required. In a drug screening application using TR-FRET, the energy transfer from a europium-labelled protein X (Eu-protein X) to a complex of biotinylated peptide Y with SA-APC was measured. Using the Plate::Vision, a Z' factor larger than 0.5 for the acceptor fluorophor emission was only obtained for a Eu-protein X concentration of at least 10 nM in combination with biotinylated peptide Y/SA-APC at saturating concentrations. Both the Plate::Vision and the LEADseeker show good quality results for applications in the TRF mode and enable an increased throughput based on their shortened measurement time in comparison to classic photomultiplier tube-based readers.
journal_name
Comb Chem High Throughput Screenjournal_title
Combinatorial chemistry & high throughput screeningauthors
Merk S,Lietz A,Kroner M,Valler M,Heilker Rdoi
10.2174/138620704772884814subject
Has Abstractpub_date
2004-02-01 00:00:00pages
45-54issue
1eissn
1386-2073issn
1875-5402journal_volume
7pub_type
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journal_title:Combinatorial chemistry & high throughput screening
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journal_title:Combinatorial chemistry & high throughput screening
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journal_title:Combinatorial chemistry & high throughput screening
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journal_title:Combinatorial chemistry & high throughput screening
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journal_title:Combinatorial chemistry & high throughput screening
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journal_title:Combinatorial chemistry & high throughput screening
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journal_title:Combinatorial chemistry & high throughput screening
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更新日期:2019-01-01 00:00:00
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journal_title:Combinatorial chemistry & high throughput screening
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