Development of a fluorescent-microsphere immunoassay for detection of antibodies specific to equine arteritis virus and comparison with the virus neutralization test.
The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5(1-255)], M(1-162), and N(1-110)), as well as partial sequences of these structural proteins (GP5(1-116), GP5(75-112), GP5(55-98), M(88-162), and N(1-69)) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia coli were covalently bound to fluorescent polystyrene microspheres and analyzed with the Luminex xMap 100 instrument. Of the eight recombinant proteins, the highest concordance with the virus neutralization test (VNT) results was obtained with the partial GP5(55-98) protein. The MIA was validated by testing a total of 2,500 equine serum samples previously characterized by the VNT. With the use of an optimal median fluorescence intensity cutoff value of 992, the sensitivity and specificity of the assay were 92.6% and 92.9%, respectively. The GP5(55-98) MIA and VNT outcomes correlated significantly (r = 0.84; P < 0.0001). Although the GP5(55-98) MIA is less sensitive than the standard VNT, it has the potential to provide a rapid, convenient, and more economical test for screening equine sera for the presence of antibodies to EAV, with the VNT then being used as a confirmatory assay.
Go
Yun Young
YY
Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, Kentucky 40546, USA.
Wong
Susan J
SJ
Branscum
Adam J
AJ
Demarest
Valerie L
VL
Shuck
Kathleen M
KM
Vickers
Mary L
ML
Zhang
Jianqiang
J
McCollum
William H
WH
Timoney
Peter J
PJ
Balasuriya
Udeni B R
UB
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis* / Q000276:immunology; D018174:Arterivirus Infections / Q000276:immunology / Q000662:veterinary* / Q000821:virology; D007240:Equartevirus / Q000276:immunology* / Q000302:isolation & purification; D005455:Fluorescent Antibody Technique / Q000191:economics / Q000379:methods*; D006734:Horse Diseases / Q000276:immunology* / Q000821:virology*; D006736:Horses; D007118:Immunoassay / Q000191:economics / Q000379:methods*; D008863:Microspheres; D008969:Molecular Sequence Data; D009500:Neutralization Tests / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D015678:Viral Structural Proteins / Q000235:genetics / Q000276:immunology
D000914:Antibodies, Viral; D011994:Recombinant Proteins; D015678:Viral Structural Proteins