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J Immunol Methods. 2014 Feb 17. pii: S0022-1759(14)00049-0. doi: 10.1016/j.jim.2014.02.004. [Epub ahead of print]
Development of a Duplex Fluorescent Microsphere Immunoassay (FMIA) for the Detection of Antibody Responses to Influenza A and Newcastle Disease Viruses.
Pinette MM1, Rodriguez-Lecompte JC2, Pasick J3, Ojkic D4, Leith M5, Suderman M6, Berhane Y7.
Author information
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 xMAP Luminex 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 2,462 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.
Crown Copyright ? 2014. Published by Elsevier B.V. All rights reserved.
KEYWORDS:
Antibodies, Avian influenza, Baculovirus Insect Cell Expression System, Chickens, Fluorescent Immunoassay, Luminex, Newcastle disease, Serologic diagnosis, Turkey
PMID:
24556589
[PubMed - as supplied by publisher]
Development of a Duplex Fluorescent Microsphere Immunoassay (FMIA) for the Detection of Antibody Responses to Influenza A and Newcastle Disease Viruses.
Pinette MM1, Rodriguez-Lecompte JC2, Pasick J3, Ojkic D4, Leith M5, Suderman M6, Berhane Y7.
Author information
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 xMAP Luminex 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 2,462 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.
Crown Copyright ? 2014. Published by Elsevier B.V. All rights reserved.
KEYWORDS:
Antibodies, Avian influenza, Baculovirus Insect Cell Expression System, Chickens, Fluorescent Immunoassay, Luminex, Newcastle disease, Serologic diagnosis, Turkey
PMID:
24556589
[PubMed - as supplied by publisher]
