A sensitive cell-based assay to measure the doxycycline concentration in biological samples.
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 laboratory.
Kleibeuker
Wendy
W
Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center of the University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.
Zhou
Xue
X
Centlivre
Mireille
M
Legrand
Nicolas
N
Page
Mark
M
Almond
Neil
N
Berkhout
Ben
B
Das
Atze T
AT
D000818:Animals; D001681:Biological Assay; D004268:DNA-Binding Proteins / Q000235:genetics; D004318:Doxycycline / Q000032:analysis* / Q000097:blood / Q000134:cerebrospinal fluid / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects*; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D057230:Limit of Detection; D008156:Luciferases / Q000235:genetics; D008253:Macaca mulatta; D051379:Mice; D008817:Mice, Mutant Strains
D004268:DNA-Binding Proteins; C499308:Rag2 protein, mouse; D008156:Luciferases; D004318:Doxycycline