Development of a Fluorescent Microsphere Immunoassay for Detection of Antibodies against Porcine Reproductive and Respiratory Syndrome Virus Using Oral Fluid Samples as an Alternative to Serum-Based Assays |
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Authors: | Robert J. Langenhorst Steven Lawson Apisit Kittawornrat Jeffrey J. Zimmerman Zhi Sun Yanhua Li Jane Christopher-Hennings Eric A. Nelson Ying Fang |
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Affiliation: | aDepartment of Veterinary and Biomedical Science, South Dakota State University, Brookings, South Dakota, USA;bDepartment of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, Iowa, USA |
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Abstract: | For effective disease surveillance, rapid and sensitive assays are needed to detect antibodies developed in response to porcine reproductive and respiratory syndrome virus (PRRSV) infection. In this study, we developed a multiplexed fluorescent microsphere immunoassay (FMIA) for detection of PRRSV-specific antibodies in oral fluid and serum samples. Recombinant nucleocapsid protein (N) and nonstructural protein 7 (nsp7) from both PRRSV genotypes (type I and type II) were used as antigens and covalently coupled to Luminex fluorescent microspheres. Based on an evaluation of 488 oral fluid samples with known serostatus, the oral fluid-based FMIAs achieved >92% sensitivity and 91% specificity. For serum samples (n = 1,639), the FMIAs reached >98% sensitivity and 95% specificity. The assay was further employed to investigate the kinetics of the antibody response in infected pigs. In oral fluid, the N protein was more sensitive for the detection of early infection (7 and 14 days postinfection), but nsp7 detected a higher level and longer duration of antibody response (28 days postinfection). In serum, the antibodies specific to nsp7 and N proteins were detected as early as 7 days postinfection, and the responses lasted more than 202 days. This study provides a framework from which a more robust assay could be developed to profile the immune response to multiple PRRSV antigens in a single test. The development of oral fluid-based diagnostic tests will change the way we survey diseases in swine herds and improve our ability to cheaply and efficiently track PRRSV infections in both populations and individual animals. |
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