A Sensitive Cell-Based Assay to Measure the Doxycycline Concentration in Biological Samples
Open accessAbstract
Doxycycline (DOX) is widely used as a pharmacological agent and as an effector molecule in inducible gene expression systems. For most applications, it is important to determine whether the DOX concentration reaches the level required for optimal efficacy. We developed a sensitive bioassay for measuring the DOX concentration in biological samples. We used a modified HeLa cell line with the luciferase reporter gene under the control of the DOX-inducible Tet-On system for regulation of gene expression. These HeLaDOX cells constitutively express a novel variant of the rtTA transcriptional activator protein that is highly DOX-sensitive. Incubation of the cells with a DOX-containing biological sample triggers luciferase expression, which can be quantitated by standard methods. This bioassay is sensitive, with a DOX detection limit of 22 ng/ml in plasma. The assay was used to determine the DOX concentration in plasma derived from DOX-treated rhesus macaques and mice. Furthermore, we found that the DOX concentration in murine cerebrospinal fluid is 31-fold lower than the concurrent plasma DOX level. This bioassay for the quantification of DOX concentration in biological samples has several advantages over high-performance liquid chromatography-based and microbiological assays: (1) multiple samples can be assayed in a single experiment; (2) only small sample volumes are required; (3) the assay has a low detection limit; and (4) the assay can be performed in any cell culture laborator…
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The high dox-sensitivity of V16 allowed the authors to develop a sensitive bioassay for measuring dox concentration in biological samples.
“The high dox-sensitivity of V16 also allowed us to develop a sensitive bioassay for measuring the dox concentration in biological samples [72].”
From Tet-On Systems For Doxycycline-inducible Gene Expression (2016) · cites this paper as [72]
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References 30 · Cited by 20
- Authors
- Kleibeuker, W., Zhou, X., Centlivre, M., Legrand, N., Page, M., Almond, N., Berkhout, B., & Das, A. T.
- Affiliations
- Amsterdam UMC Location University of Amsterdam (NL)
- National Institute for Biological Standards and Control (GB)
- Corresponding author
- Das, Atze T.
- Venue
- Human Gene Therapy
- Publisher
- SAGE Publications
- Published
- 2009-04-02
- Volume
- 20
- Issue
- 5
- Pages
- 524-530
- ISSN
- 1043-0342 (ISSN-L)
- 1557-7422
- OpenAlex
- W1999296717
- Cited by
- 20
- References
- 30
- Citations by year
- 2024: 1 · 2020: 1 · 2018: 2 · 2017: 2 · 2016: 4 · 2015: 3 · 2014: 1 · 2013: 1 · 2012: 3
- FWCI
- 0.67
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Human Gene Therapy (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Data Archiving and Networked Services (DANS) (submittedVersion)
- Pure Amsterdam UMC (submittedVersion)
- Funders
- Bill and Melinda Gates Foundation
- International AIDS Vaccine Initiative
- Nederlandse Organisatie voor Wetenschappelijk Onderzoek
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › bioluminescence and chemiluminescence research
- Physical Sciences › Chemistry › Analytical Chemistry › Analytical Methods in Pharmaceuticals
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Keywords
- doxycycline, Tet-On system, luciferase reporter, cell-based assays, plasma drug concentration, cerebrospinal fluid drug concentration, rhesus monkeys, mice, inducible gene expression
- MeSH terms
- Animals, Biological Assay, DNA-Binding Proteins, Doxycycline, HeLa Cells, Humans, Luciferases, Macaca mulatta, Mice, Mutant Strains, Gene Expression, Genes, Reporter, Mice, Limit of Detection
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