title: Development of a duplex Fluorescent Microsphere Immunoassay (FMIA) for the detection of antibody responses to influenza A and newcastle disease viruses.
abstract: Highly pathogenic avian influenza virus (HPAI) and virulent forms of avian paramyxovirus-1 (APMV-1) cause serious illnesses in domestic poultry, both of which are reportable to the World Organization of Animal Health (OIE). The clinical presentation of avian influenza (AI) and APMV-1 infections are difficult to differentiate, emphasizing the importance of rapid and sensitive serologic assays that are able to distinguish them. Currently, a variety of serological assays are used for the serologic diagnosis of both diseases, but these assays are not used in multiplex formats. In this study, development of a duplex fluorescent microsphere immunoassay (FMIA) based on Luminex xMAP Technology is described. The assay employs MagPlex magnetic microspheres that are covalently coated with recombinant avian influenza virus nucleoprotein and APMV-1 nucleocapsid antigens produced in a baculovirus insect cell expression system. The assay is able to detect AIV antibodies against all existing hemagglutinin (H1-H16) subtypes and simultaneously detect antibodies against APMV-1. In the process of this assay development different bead coupling conditions were compared. The assay has the capability of detecting serum antibodies from chickens and turkeys and optimization was accomplished by using 2462 chicken and 446 turkey field and experimental sera and had a comparable detection capability with currently used assays in the laboratory. Assay threshold values were calculated with Receiver Operating Characteristic Analysis (ROC) in non-parametric analysis due to a highly skewed data distribution; this analysis resulted in AIV nucleoprotein relative diagnostic sensitivity and specificity of 99.7%, and 97.3% respectively. The APMV-1 nucleocapsid relative diagnostic sensitivity and specificity were 95.4%, and 98.5% respectively.
authors.affiliation: Department of Animal Science, University of Manitoba, Winnipeg, Manitoba R3T 2 N2, Canada; National Centre for Foreign Animal Diseases, Winnipeg, Manitoba, Canada. Electronic address: umpinetm@myumanitoba.ca.
authors.forename: Mathieu M
authors.initials: MM
authors.lastname: Pinette
authors.affiliation: Department of Animal Science, University of Manitoba, Winnipeg, Manitoba R3T 2 N2, Canada; Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island C1A 4P3, Canada. Electronic address: jrodriguez@upei.ca.
authors.forename: Juan C
authors.initials: JC
authors.lastname: Rodriguez-Lecompte
authors.affiliation: National Centre for Foreign Animal Diseases, Winnipeg, Manitoba, Canada. Electronic address: john.pasick@inspection.gc.ca.
authors.forename: John
authors.initials: J
authors.lastname: Pasick
authors.affiliation: Animal Health Laboratory, University of Guelph, Guelph, Ontario, Canada. Electronic address: dojkic@isd.uoguelph.ca.
authors.forename: Davor
authors.initials: D
authors.lastname: Ojkic
authors.affiliation: National Centre for Foreign Animal Diseases, Winnipeg, Manitoba, Canada. Electronic address: marsha.leith@inspection.gc.ca.
authors.forename: Marsha
authors.initials: M
authors.lastname: Leith
authors.affiliation: National Centre for Foreign Animal Diseases, Winnipeg, Manitoba, Canada. Electronic address: matthew.suderman@inspection.gc.ca.
authors.forename: Matthew
authors.initials: M
authors.lastname: Suderman
authors.affiliation: Department of Animal Science, University of Manitoba, Winnipeg, Manitoba R3T 2 N2, Canada; National Centre for Foreign Animal Diseases, Winnipeg, Manitoba, Canada. Electronic address: yohannes.berhane@inspection.gc.ca.
authors.forename: Yohannes
authors.initials: Y
authors.lastname: Berhane
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology*; D000917:Antibody Formation; D015153:Blotting, Western; D002645:Chickens; D005453:Fluorescence; D007118:Immunoassay / Q000379:methods*; D009980:Influenza A virus / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D005585:Influenza in Birds / Q000097:blood / Q000175:diagnosis / Q000276:immunology; D008863:Microspheres; D009521:Newcastle Disease / Q000097:blood / Q000175:diagnosis / Q000276:immunology; D009522:Newcastle disease virus / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012372:ROC Curve; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D015203:Reproducibility of Results; D061987:Sf9 Cells; D018411:Spodoptera; D014422:Turkeys
chemical_list: D000914:Antibodies, Viral; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins
keywords: APMV-1; Avian Influenza; FMIA; Immunoassay; Luminex; Newcastle Disease