期刊
JOURNAL OF VIROLOGICAL METHODS
卷 173, 期 1, 页码 108-114出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jviromet.2011.01.015
关键词
Cell viability assay; Foot-and-mouth disease; Cytopathic effect; Virus neutralization test; Antiviral compound screening
资金
- European Community [226556, EC-EPIZONE FOOD-CT-2006-016236]
- Belgian Federal Public Service of Health, Food Chain Safety and Environment [RF 6203]
- CODA-CERVA-VAR
- Center for Drug Design and Discovery (CD3)
- Center for Innovation and Stimulation of Drug Discovery (CISTIM) from the KULeuven
Cell-based assays are still used widely in foot-and-mouth disease (FMD) research, despite the existence of a wide variety of molecular techniques. The aim of this study was to validate an automated, quantitative spectrometric reading to replace the time-consuming and subjective microscopic (MIC) evaluation of the FMD virus-induced cytopathic effect (CPE). Therefore, the diagnostic performance of two commercial cell viability assays (CellTiter 96 (R) AQueous One Solution Cell Proliferation Assay (MTS) and CellTiter-Blue (R) Cell Viability Assay (CTB), both from Promega, Leiden, The Netherlands) was evaluated. Following optimization of the assay protocols and using the MIC results as a reference standard, the absorbance-read MIS assay, the fluorescence-read CTB assay and the absorbance-read CTB (CTBabs) assay demonstrated similar high sensitivities (97%, 99% and 98%, respectively), specificities (100%, 98% and 99%, respectively), accuracy measures (0.99, 0.98 and 0.98, respectively), precision measures (1.00, 0.98 and 0.99, respectively) and Cohen kappa agreement indices (0.97, 0.97 and 0.96, respectively) for detecting CPE in cell cultures. Due to its performance, cost effectiveness and ease of use, the CTBabs assay was selected for further evaluation of its ability to detect virus neutralization and to screen antiviral compounds. The CTBabs assay had 99% sensitivity and 100% specificity for the detection of neutralizing antibodies in sera from cattle infected with FMDV and in sera from unvaccinated, uninfected cattle and resulted in a mean Z'-factor of 0.85 for antiviral compound test plates. The CTBabs assay is now used routinely in the Belgian FMD reference laboratory for serological testing and high-throughput antiviral compound screening. (C) 2011 Elsevier B.V. All rights reserved.
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