title: Development of an antigen-capture ELISA for detection of H7 subtype avian influenza from experimentally infected chickens.
abstract: Emergence of highly pathogenic avian influenza H7N1 was due to mutation of low pathogenic avian influenza H7N1 strain, which caused outbreaks in Italy between 1999 and 2000, and resulted in complete mortality of infected poultry. This outbreak places increased importance on the early detection of H7N1 AIV. Here we describe the development of a detection method for H7N1 virus from infected chickens using a specific antigen-capture-ELISA (AC-ELISA). A panel of mAbs was developed against the surface antigen HA of H7N1 AIV strain A/chicken/Singapore/94. The mAbs were screened by immunofluorescence assays, ELISA and immunoblotting. Selected mAbs 5E5 and 8F10 were of isotypes IgM and IgG and were conformation- or linear epitope-specific, respectively. These mAbs were used as capture antibodies for AC-ELISA development. The detection limit was as little as 10(2)-10(3) TCID(50) units of virus derived from tissue culture supernatants. Virus from the tracheal swab samples of experimentally infected chickens was detected from days 3 to 7 post-infection using the AC-ELISA, with results being confirmed by RT-PCR. AIV subtypes H4N1, H5N3 H9N2 and H10N5 did not react in the AC-ELISA but were RT-PCR positive, indicating that this AC-ELISA is specific for H7N1 strains.
authors.affiliation: Animal Health Biotechnology, Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Singapore.
authors.forename: Sumathy
authors.initials: S
authors.lastname: Velumani
authors.forename: Qingyun
authors.initials: Q
authors.lastname: Du
authors.forename: Beau J
authors.initials: BJ
authors.lastname: Fenner
authors.forename: Mookkan
authors.initials: M
authors.lastname: Prabakaran
authors.forename: Lim Chee
authors.initials: LC
authors.lastname: Wee
authors.forename: Lin Yueh
authors.initials: LY
authors.lastname: Nuo
authors.forename: Jimmy
authors.initials: J
authors.lastname: Kwang
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D009980:Influenza A virus / Q000276:immunology / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000276:immunology / Q000821:virology*; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral