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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.

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.

SUBMITTER: Langenhorst RJ

PROVIDER: S-EPMC3272925 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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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.

Langenhorst Robert J RJ Lawson Steven S Kittawornrat Apisit A Zimmerman Jeffrey J JJ Sun Zhi Z Li Yanhua Y Christopher-Hennings Jane J Nelson Eric A EA Fang Ying Y

Clinical and vaccine immunology : CVI 20111207 2

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 cov ...[more]

PMID: 22155765

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Project description:BackgroundA novel porcine deltacoronavirus (PDCoV), also known as porcine coronavirus HKU15, was reported in China in 2012 and identified in the U.S. in early 2014. Since then, PDCoV has been identified in a number of U.S. states and linked with clinical disease including acute diarrhea and vomiting in the absence of other identifiable pathogens. Since PDCoV was just recently linked with clinical disease, few specific antibody-based reagents were available to assist in diagnosis of PDCoV and limited serological capabilities were available to detect an antibody response to this virus. Therefore, the overall objective of this project was to develop and validate selected diagnostic reagents and assays for PDCoV antigen and antibody detection.ResultsThe nucleoprotein of PDCoV was expressed as a recombinant protein and purified for use as an antigen to immunize mice for polyclonal, hyperimmune sera and monoclonal antibody (mAb) production. The resulting mAbs were evaluated for use in fluorescent antibody staining methods to detect PDCoV infected cells following virus isolation attempts and for immunohistochemistry staining of intestinal tissues of infected pigs. The same antigen was used to develop serological tests to detect the antibody response to PDCoV in pigs following infection. Serum samples from swine herds with recent documentation of PDCoV infection and samples from expected naïve herds were used for initial assay optimization. The tests were optimized in a checkerboard fashion to reduce signal to noise ratios using samples of known status. Statistical analysis was performed to establish assay cutoff values and assess diagnostic sensitivities and specificities. At least 629 known negative serum samples and 311 known positive samples were evaluated for each assay. The enzyme linked immunosorbent assay (ELISA) showed diagnostic sensitivity (DSe) of 96.1% and diagnostic specificity (DSp) of 96.2%. The fluorescent microsphere immunoassay (FMIA) showed a DSe of 95.8% and DSp of 98.1%. Both ELISA and FMIA detected seroconversion of challenged pigs between 8-14 days post-infection (DPI). An indirect fluorescent antibody (IFA) test was also developed using cell culture adapted PDCoV for comparative purposes.ConclusionThese new, specific reagents and serological assays will allow for improved diagnosis of PDCoV. Since many aspects of PDCoV infection and transmission are still not fully understood, the reagents and assays developed in this project should provide valuable tools to help understand this disease and to aid in the control and surveillance of porcine deltacoronavirus outbreaks.

Project description:Surveillance of highly pathogenic viruses circulating in both human and animal populations is crucial to unveil endemic infections and potential zoonotic reservoirs. Monitoring the burden of disease by serological assay could be used as an early warning system for imminent outbreaks as an increased seroprevalance often precedes larger outbreaks. However, the multitude of highly pathogenic viruses necessitates the need to identify specific antibodies against several targets from both humans as well as from potential reservoir animals such as bats. In order to address this, we have developed a broadly reactive multiplex microsphere immunoassay (MMIA) for the detection of antibodies against several highly pathogenic viruses from both humans and animals. To this aim, nucleoproteins (NP) of Ebola virus (EBOV), Marburg virus (MARV) and nucleocapsid proteins (NP) of Crimean-Congo haemorrhagic fever virus, Rift Valley fever virus and Dobrava-Belgrade hantavirus were employed in a 5-plex assay for IgG detection. After optimisation, specific binding to each respective NP was shown by testing sera from humans and non-human primates with known infection status. The usefulness of our assay for serosurveillance was shown by determining the immune response against the NP antigens in a panel of 129 human serum samples collected in Guinea between 2011 and 2012 in comparison to a panel of 88 sera from the German blood bank. We found good agreement between our MMIA and commercial or in-house reference methods by ELISA or IIFT with statistically significant higher binding to both EBOV NP and MARV NP coupled microspheres in the Guinea panel. Finally, the MMIA was successfully adapted to detect antibodies from bats that had been inoculated with EBOV- and MARV- virus-like particles, highlighting the versatility of this technique and potentially enabling the monitoring of wildlife as well as human populations with this assay. We were thus able to develop and validate a sensitive and broadly reactive high-throughput serological assay which could be used as a screening tool to detect antibodies against several highly pathogenic viruses.

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Dataset Information

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.

Publications

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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