A microsphere immunoassay for detection of antibodies to avian influenza virus.
A microsphere immunoassay (MIA) was developed for the detection of serum antibodies to avian influenza virus. A recombinant influenza A nucleoprotein expressed in baculovirus was conjugated to microspheres and incubated with antibodies. High median fluorescent intensities (MFIs) were obtained with a monoclonal antibody and positive chicken sera. Chickens were inoculated with 10 strains of avian influenza virus representing different subtypes, including high and low pathogenic H5 and H7 subtypes. Three hundred and fifty-four samples from experimentally infected chickens and controls were tested with a competitive ELISA (cELISA) and the MIA. MFIs were converted to positive/negative (PN) ratios. The results of both tests, as percentage inhibition and PN ratio, showed a high correlation (R2 = 0.77). From the comparison data, a ratio of > or =4.5 was selected as the cut-off value for positivity in the MIA. Using this cut-off value, the sensitivity and specificity of the MIA relative to the cELISA when all discordant experimental samples were retested was 99.3 and 93.1%, respectively. The relative specificity increased to 94.7% when additional negative sera (n = 68) were tested. The MIA may be useful for surveillance testing and as a screening test for flocks infected with low pathogenic avian influenza virus and could be expanded for simultaneous detection of antibodies against other avian infectious disease agents.
Deregt
Dirk
D
Virology Section, Lethbridge Laboratory (Animal Diseases Research Institute), Canadian Food Inspection Agency, P.O. Box 640, Lethbridge, Alberta T1J 3Z4, Canada. deregtd@inspection.gc.ca
Furukawa-Stoffer
Tara L
TL
Tokaryk
Kara L
KL
Pasick
John
J
Hughes
Kimberley M Burton
KM
Hooper-McGrevy
Kathleen
K
Baxi
Shailja
S
Baxi
Mohit K
MK
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D001710:Biotin; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay; D005453:Fluorescence; D007118:Immunoassay / Q000379:methods*; D009980:Influenza A virus / Q000276:immunology*; D005585:Influenza in Birds / Q000276:immunology*; D008863:Microspheres; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity; D013223:Statistics as Topic; D019809:Streptavidin; D014758:Viral Core Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D000914:Antibodies, Viral; C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D001710:Biotin; D019809:Streptavidin