Flow cytometry compared with indirect immunofluorescence for rapid detection of dengue virus type 1 after amplification in tissue culture
Journal
Journal of Clinical Microbiology
Journal Volume
39
Journal Issue
10
Pages
3672-3677
Date Issued
2001
Author(s)
Abstract
Dengue virus (DV) was detected early in infected mosquito C6/36 cells by using indirect immunofluorescence (IF) in conjunction with flow cytometry. Three fixation-permeabilization methods and three DV serotype 1 (DEN-1)-specific monoclonal antibodies, 8-8 (anti-E), 16-4 (anti-NS1), and 15F3-1 (anti-NS1), were evaluated for the detection of DEN-1 in infected C6/36 cells. We found that these three monoclonal antibodies were capable of detecting DV in C6/36 cells as early as 24 h postinoculation by using a conventional indirect IF stain. Both 8-8 and 16-4 detected DV earlier and showed a greater number of DV-positive cells than 15F3-1. In flow cytometry, 3% paraformaldehyde plus 0.1% Triton X-100 with 16-4, the best fixation-permeabilization method for testing DV, showed higher sensitivity (up to 1 PFU) than indirect IF stain. The higher sensitivity of 16-4 in detecting DEN-1 was found with both IF and flow cytometry. Flow cytometry, which had a sensitivity similar to that of nested reverse transcription-PCR, was more sensitive in detecting DV in the infected mosquito cells 10 h earlier than the conventional IF stain. When clinical specimens were amplified in mosquito C6/36 cells and then assayed for DV using flow cytometry and conventional virus isolation at day 7 postinfection, both methods had 97.22% (35 out of 36) agreement. Moreover, among 12 positive samples which were detected by conventional culture method, the flow cytometry assay could detect DV in 58.33% (7 out of 12) of samples even at day 3 postinfection. In conclusion, both monoclonal antibodies 8-8 and 16-4 can be used for the early detection of DEN-1-infected C6/36 cells, with 16-4 (anti-NS1) being the best choice for the rapid diagnosis of DV by both the IF staining and flow cytometry methods.
SDGs
Other Subjects
monoclonal antibody; paraformaldehyde; animal cell; article; controlled study; Dengue virus; flow cytometry; immunofluorescence; nonhuman; priority journal; reverse transcription polymerase chain reaction; tissue culture; virus detection; Animals; Antibodies, Monoclonal; Antibodies, Viral; Antigens, Viral; Cells, Cultured; Culicidae; Dengue; Dengue Virus; Disease Outbreaks; Flow Cytometry; Fluorescent Antibody Technique; Humans; Taiwan; Time Factors; Tissue Fixation; Virus Cultivation; Animalia; Dengue virus; Dengue virus type 1; RNA viruses
Type
journal article
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