title: Development of epitope-blocking ELISA for universal detection of antibodies to human H5N1 influenza viruses.
abstract: BACKGROUND
  Human infections with highly pathogenic H5N1 avian influenza viruses have generally been confirmed by molecular amplification or culture-based methods. Serologic surveillance has potential advantages which have not been realized because rapid and specific serologic tests to detect H5N1 infection are not widely available.
  
  
  RESULTS
  Here we describe an epitope-blocking ELISA to detect specific antibodies to H5N1 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (5F8) that binds to an epitope comprising amino acid residues 274-281 (CNTKCQTP) in the HA1 region of H5 hemagglutinin. Database search analysis of publicly available sequences revealed that this epitope is conserved in 100% of the 163 H5N1 viruses isolated from humans. The sensitivity and specificity of the epitope-blocking ELISA for H5N1 were evaluated using chicken antisera to multiple virus clades and other influenza subtypes as well as serum samples from individuals naturally infected with H5N1 or seasonal influenza viruses. The epitope-blocking ELISA results were compared to those of hemagglutinin inhibition (HI) and microneutralization assays. Antibodies to H5N1 were readily detected in immunized animals or convalescent human sera by the epitope-blocking ELISA whereas specimens with antibodies to other influenza subtypes yielded negative results. The assay showed higher sensitivity and specificity as compared to HI and microneutralization.
  
  
  CONCLUSIONS
  The epitope-blocking ELISA based on a unique 5F8 mAb provided highly sensitive and 100% specific detection of antibodies to H5N1 influenza viruses in human sera.
authors.affiliation: Animal Health Biotechnology, Temasek Life Sciences Laboratory, National University of Singapore, Singapore, Singapore.
authors.forename: Mookkan
authors.initials: M
authors.lastname: Prabakaran
authors.forename: Hui-Ting
authors.initials: HT
authors.lastname: Ho
authors.forename: Nayana
authors.initials: N
authors.lastname: Prabhu
authors.forename: Sumathy
authors.initials: S
authors.lastname: Velumani
authors.forename: Milene
authors.initials: M
authors.lastname: Szyporta
authors.forename: Fang
authors.initials: F
authors.lastname: He
authors.forename: Kwai-Peng
authors.initials: KP
authors.lastname: Chan
authors.forename: Li-Mei
authors.initials: LM
authors.lastname: Chen
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Matsuoka
authors.forename: Ruben O
authors.initials: RO
authors.lastname: Donis
authors.forename: Jimmy
authors.initials: J
authors.lastname: Kwang
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral / Q000097:blood*; D001717:Birds; D017124:Conserved Sequence; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes; D006388:Hemagglutinins / Q000276:immunology; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology*; D005585:Influenza in Birds / Q000175:diagnosis; D007251:Influenza, Human / Q000175:diagnosis; D009976:Orthomyxoviridae Infections / Q000175:diagnosis; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000939:Epitopes; D006388:Hemagglutinins