title: A novel peptide ELISA for universal detection of antibodies to human H5N1 influenza viruses.
abstract: BACKGROUND
  Active serologic surveillance of H5N1 highly pathogenic avian influenza (HPAI) virus in humans and poultry is critical to control this disease. However, the need for a robust, sensitive and specific serologic test for the rapid detection of antibodies to H5N1 viruses has not been met.
  
  
  RESULTS
  Previously, we reported a universal epitope (CNTKCQTP) in H5 hemagglutinin (HA) that is 100% conserved in H5N1 human isolates and 96.9% in avian isolates. Here, we describe a peptide ELISA to detect antibodies to H5N1 virus by using synthetic peptide that comprises the amino acid sequence of this highly conserved and antigenic epitope as the capture antigen. The sensitivity and specificity of the peptide ELISA were evaluated using experimental chicken antisera to H5N1 viruses from divergent clades and other subtype influenza viruses, as well as human serum samples from patients infected with H5N1 or seasonal influenza viruses. The peptide ELISA results were compared with hemagglutinin inhibition (HI), and immunofluorescence assay and immunodot blot that utilize recombinant HA1 as the capture antigen. The peptide ELISA detected antibodies to H5N1 in immunized animals or convalescent human sera whereas some degree of cross-reactivity was observed in HI, immunofluorescence assay and immunodot blot. Antibodies to other influenza subtypes tested negative in the peptide-ELISA.
  
  
  CONCLUSIONS
  The peptide-ELISA based on the highly conserved and antigenic H5 epitope (CNTKCQTP) provides sensitive and highly specific detection of antibodies to H5N1 influenza viruses. This study highlighted the use of synthetic peptide as a capture antigen in rapid detection of antibodies to H5N1 in human and animal sera that is robust, simple and cost effective and is particularly beneficial for developing countries and rural areas.
authors.affiliation: Animal Health Biotechnology, Temasek Life Sciences Laboratory, National University of Singapore, Singapore, Singapore.
authors.forename: Sumathy
authors.initials: S
authors.lastname: Velumani
authors.forename: Hui-Ting
authors.initials: HT
authors.lastname: Ho
authors.forename: Fang
authors.initials: F
authors.lastname: He
authors.forename: Syed
authors.initials: S
authors.lastname: Musthaq
authors.forename: Mookkan
authors.initials: M
authors.lastname: Prabakaran
authors.forename: Jimmy
authors.initials: J
authors.lastname: Kwang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis*; D002645:Chickens / Q000276:immunology; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D005455:Fluorescent Antibody Technique; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000378:metabolism; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007114:Immunization; D015151:Immunoblotting; D053124:Influenza A Virus, H5N1 Subtype / Q000145:classification / Q000276:immunology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012680:Sensitivity and Specificity
chemical_list: D000914:Antibodies, Viral; D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007106:Immune Sera; D010455:Peptides; D011994:Recombinant Proteins