title: The development of a novel serotyping-NS1-ELISA to identify serotypes of dengue virus.
abstract: BACKGROUND
  Dengue virus (DENV), which causes mosquito-borne disease dengue hemorrhagic fever (DHF), consists of four serotypes co-circulating in endemic areas. Currently, DENV serotypes can be identified by laborious virus isolation followed by immunofluorescent assay and sophisticated RT-PCR.
  
  
  OBJECTIVE
  To establish a new assay designated as "serotyping-NS1-ELISA" to detect the NS1 protein and to identify DENV serotypes simultaneously.
  
  
  METHODS
  The monoclonal antibodies (Mabs) against NS1 of each DENV serotype were produced and characterized for their serotype-specificity. To develop serotyping-NS1-ELISA, the selected serotype-specific anti-NS1 Mabs were applied to detect the NS1 antigen, which was previously captured by a flavivirus cross-reactive anti-NS1 Mab. Serotyping accuracy of the developed assay was validated with NS1 from DENV-infected cell culture supernatants and from well-characterized clinical specimens.
  
  
  RESULTS
  Of 30 anti-NS1 Mabs, 1 serotype-specific anti-NS1 Mab to each DENV serotype was selected based on NS1 capture ELISA results for developing the serotyping-NS1-ELISA. Using DENV-infected cell culture supernatants for validation, the selected antibodies were shown to be capable of differentiating four DENV serotypes. When acute phase plasma from DENV-infected patients was used for validation, 65 out of 85 specimens (76.5% overall sensitivity) were positive to one of the four serotypes developed in our assay. Interestingly, identification of DENV serotypes by our serotyping-NS1-ELISA was 100% accurate for DENV1, 3 and 4 and 82.4% for DENV2 as compared with standard RT-PCR. Assay specificity was 100% (90/90).
  
  
  CONCLUSIONS
  The developed serotyping-NS1-ELISA provides an alternative for simultaneous detection of DENV NS1 and identification of its serotype in acute patients' specimens. The assay would be applicable for dengue diagnosis and epidemiological studies.
authors.affiliation: Medical Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok, Thailand. chunyapk@biotec.or.th
authors.forename: Chunya
authors.initials: C
authors.lastname: Puttikhunt
authors.forename: Tanapan
authors.initials: T
authors.lastname: Prommool
authors.forename: Nathaporn
authors.initials: N
authors.lastname: U-thainual
authors.forename: Prapapun
authors.initials: P
authors.lastname: Ong-ajchaowlerd
authors.forename: Kroong
authors.initials: K
authors.lastname: Yoosook
authors.forename: Chutithorn
authors.initials: C
authors.lastname: Tawilert
authors.forename: Thaneeya
authors.initials: T
authors.lastname: Duangchinda
authors.forename: Aroonroong
authors.initials: A
authors.lastname: Jairangsri
authors.forename: Nattaya
authors.initials: N
authors.lastname: Tangthawornchaikul
authors.forename: Prida
authors.initials: P
authors.lastname: Malasit
authors.forename: Watchara
authors.initials: W
authors.lastname: Kasinrerk
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D003429:Cross Reactions / Q000276:immunology; D003716:Dengue Virus / Q000145:classification* / Q000276:immunology / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D012703:Serotyping / Q000379:methods*; D019595:Severe Dengue / Q000821:virology; D017361:Viral Nonstructural Proteins / Q000032:analysis* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; C082515:NS1 protein, dengue-1 virus; D017361:Viral Nonstructural Proteins