TY - JOUR
T1 - Development of a fluorescent-microsphere immunoassay for detection of antibodies specific to equine arteritis virus and comparison with the virus neutralization test
AU - Yun, Young Go
AU - Wong, Susan J.
AU - Branscum, Adam J.
AU - Demarest, Valerie L.
AU - Shuck, Kathleen M.
AU - Vickers, Mary L.
AU - Zhang, Jianqiang
AU - McCollum, William H.
AU - Timoney, Peter J.
AU - Balasuriya, Udeni B. R.
N1 - Publication details (e.g. title, author(s), publication statuses and dates) are captured on an “AS IS” and “AS AVAILABLE” basis at the time of record harvesting from the data source. Suggestions for further amendments or supplementary information can be sent to [email protected].
PY - 2008/1
Y1 - 2008/1
N2 - 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 [GP51-255], M1-162, and N 1-110), as well as partial sequences of these structural proteins (GP51-116, GP575-112, GP555-98, M 88-162, and N1-69) 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 GP555-98 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 GP555-98 MIA and VNT outcomes correlated significantly (r = 0.84; P < 0.0001). Although the GP555-98 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. Copyright © 2008, American Society for Microbiology. All Rights Reserved.
AB - 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 [GP51-255], M1-162, and N 1-110), as well as partial sequences of these structural proteins (GP51-116, GP575-112, GP555-98, M 88-162, and N1-69) 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 GP555-98 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 GP555-98 MIA and VNT outcomes correlated significantly (r = 0.84; P < 0.0001). Although the GP555-98 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. Copyright © 2008, American Society for Microbiology. All Rights Reserved.
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U2 - 10.1128/CVI.00388-07
DO - 10.1128/CVI.00388-07
M3 - RGC 21 - Publication in refereed journal
C2 - 18032597
SN - 1556-6811
VL - 15
SP - 76
EP - 87
JO - Clinical and Vaccine Immunology
JF - Clinical and Vaccine Immunology
IS - 1
ER -