Flow cytometry compared with indirect immunofluorescence for rapid detection of dengue virus type 1 after amplification in tissue culture.
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.
Kao
C L
CL
School and Graduate Institute of Medical Technology, College of Medicine, National Taiwan University, Taipei, Republic of China. clkao@ha.mc.ntu.edu.tw
Wu
M C
MC
Chiu
Y H
YH
Lin
J L
JL
Wu
Y C
YC
Yueh
Y Y
YY
Chen
L K
LK
Shaio
M F
MF
King
C C
CC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000032:analysis*; D002478:Cells, Cultured; D009033:Culicidae / Q000166:cytology; D003715:Dengue / Q000175:diagnosis* / Q000453:epidemiology / Q000821:virology; D003716:Dengue Virus / Q000302:isolation & purification*; D004196:Disease Outbreaks; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000379:methods; D006801:Humans; D013624:Taiwan / Q000453:epidemiology; D013997:Time Factors; D016707:Tissue Fixation / Q000379:methods; D014776:Virus Cultivation
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral