title: Development and evaluation of a DAS-ELISA for rapid detection of avian influenza viruses.
abstract: Rapid detection of avian influenza virus (AIV) infection is critical for control of avian influenza (AI) and for reducing the risk of pandemic human influenza. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for this purpose. The method employed a monoclonal antibody (MAb) as the capture antibody and rabbit polyclonal IgG labeled with horseradish peroxidase as the detector antibody, and both antibodies were against type-specific influenza A nucleoprotein (NP). The DAS-ELISA could detect minimally 2.5 ng of influenza viral protein in virus preparations treated with Triton X-100, which is equvilent to 2.5 x 10(2) EID50 virus particles. This DAS-ELISA could detect all 15n AIV subtypes (H1-H15) and did not cross react with other avian pathogens tested. The DAS-ELISA were directly compared with virus isolation (VI) in embryonated chicken eggs, the current standard of influenza virus detection, for 805 chicken samples. The DAS-ELISA results correlated with VI results for 98.6% of these samples, indicating a sensitivity of 97.4% and specificity of 100%. The method was further tested with H5N1 and H9N2 AIV experimentally infected chickens, ducks, and pigeons, as well as field samples obtained from central China in 2005. The DAS-ELISA method has demonstrated application potential as an AIV screening tool and as a supplement for virus isolation in Asia.
authors.affiliation: Unit of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei, 430070, P.R. China.
authors.forename: Anding
authors.initials: A
authors.lastname: Zhang
authors.forename: Meilin
authors.initials: M
authors.lastname: Jin
authors.forename: Fan fang
authors.initials: Ff
authors.lastname: Liu
authors.forename: Xuebo
authors.initials: X
authors.lastname: Guo
authors.forename: Qiaoyun
authors.initials: Q
authors.lastname: Hu
authors.forename: Li
authors.initials: L
authors.lastname: Han
authors.forename: Yadi
authors.initials: Y
authors.lastname: Tan
authors.forename: Huanchun
authors.initials: H
authors.lastname: Chen
mesh_terms: D000818:Animals; D002642:Chick Embryo; D002645:Chickens / Q000821:virology; D010856:Columbidae / Q000821:virology; D004372:Ducks / Q000821:virology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000662:veterinary; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology / Q000302:isolation & purification*; D053127:Influenza A Virus, H9N2 Subtype / Q000276:immunology / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000175:diagnosis* / Q000821:virology*; D011933:Reagent Kits, Diagnostic / Q000662:veterinary; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D011933:Reagent Kits, Diagnostic