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Development of a Fluorescent-Microsphere Immunoassay for Detection of Antibodies Specific to Equine Arteritis Virus and Comparison with the Virus Neutralization Test

Maxwell H. Gluck Equine Research Center, Department of Veterinary Science,¹ College of Public Health,³ Livestock Disease Diagnostic Center, University of Kentucky, Lexington, Kentucky 40546,⁴ Wadsworth Center, New York State Department of Health, Albany, New York 12201²

Received 26 September 2007/ Returned for modification 23 October 2007/ Accepted 5 November 2007

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.

Published ahead of print on 21 November 2007.