Abstract
A dengue nonstructural protein 1 (NS1) antigen capture enzyme-linked immunosorbent assay (ELISA)-based tissue culture infectious dose-50 (TCID50) test (TCID50-ELISA) was developed as an alternative to the standard plaque assay for titrating dengue virus. Virus titers obtained by TCID50-ELISA were comparable to those obtained by the plaque assay and by the traditional TCID50-cytopathic effect (CPE) test (TCID50-CPE), with a better reproducibility and a lower coefficient of variation. Quantitative comparison of TCID50-ELISA and TCID50-CPE resulted in a correlation coefficient of 0.976. Moreover, this new method showed a wider application to C6/36, Vero E6, BHK-21, and Vero cells compared with other titration methods. In summary, the novel TCID50-ELISA method described here provides a more reliable and more accurate alternative compared to the plaque assay and TCID50-CPE for titration of dengue virus.
Highlights
Over the past 60 years, dengue fever and dengue haemorrhagic fever have become increasingly serious public health problems in the tropics and subtropics due to overpopulation, ever-increasing regional and international travel, as well as global warming [1]
The 6 day incubation appeared to be sufficient for the infected cells to produce enough Nonstructural protein 1 (NS1) protein to be detected by enzyme-linked immunosorbent assay (ELISA), and this assay should be applicable to cultures at anytime between 6 and 20 days
There were no significant differences between the CVs from the four cell lines when tested by tissue culture infectious dose-50 assay (TCID50)-ELISA or by TCID50-cytopathic effect (CPE) (Friedman test; P = 0.825 and P = 0.552, respectively). All these results showed that the TCID50-ELISA assay has a higher degree of reproducibility and is applicable to a wider range of cell lines than the plaque assay and the TCID50CPE assay
Summary
Over the past 60 years, dengue fever and dengue haemorrhagic fever have become increasingly serious public health problems in the tropics and subtropics due to overpopulation, ever-increasing regional and international travel, as well as global warming [1]. A variety of methods for titrating DENV have been developed, including classical assays, the plaque assay and the tissue culture infectious dose-50 assay (TCID50), and immunofluorescence-based assays such as fluorescence-activated cell sorting (FACS) assay and fluorescent focus assay [4,5,6,7]. Most primary clinical isolates do not form clear plaques or have a visible cytopathic effect (CPE) on cell monolayers Both of these assays require manual microscope examination daily, which is time consuming and labour intensive. A novel TCID50 assay was developed, which employs this dengue NS1 antigen capture ELISA for the accurate and objective titration of DENV
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