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. 2005 Aug;12(8):922-9.
doi: 10.1128/CDLI.12.8.922-929.2005.

Noninfectious Virus-Like Particle Antigen for Detection of Swine Vesicular Disease Virus Antibodies in Pigs by Enzyme-Linked Immunosorbent Assay

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Noninfectious Virus-Like Particle Antigen for Detection of Swine Vesicular Disease Virus Antibodies in Pigs by Enzyme-Linked Immunosorbent Assay

Young-Joon Ko et al. Clin Diagn Lab Immunol. .
Free PMC article

Abstract

An inactivated SVDV antigen is used in current enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies to swine vesicular disease virus (SVDV). To develop a noninfectious recombinant alternative, we produced SVDV-like particles (VLPs) morphologically and antigenically resembling authentic SVDV particles by using a dual baculovirus recombinant, which expresses simultaneously the P1 and 3CD protein genes of SVDV under different promoters. Antigenic differences between recombinant VLPs and SVDV particles were not statistically significant in results obtained with a 5B7-ELISA kit, indicating that the VLPs could be used in the place of SVDV antigen in ELISA kits. We developed a blocking ELISA using the VLPs and SVDV-specific neutralizing monoclonal antibody 3H10 (VLP-ELISA) for detection of SVDV serum antibodies in pigs. The VLP-ELISA showed a high specificity of 99.9% when tested with pig sera that are negative for SVDV neutralization (n=1,041). When tested using sera (n=186) collected periodically from pigs (n=19) with experimental infection with each of three different strains of SVDV, the VLP-ELISA detected SVDV serum antibodies as early as 3 days postinfection and continued to detect the antibodies from all infected pigs until termination of the experiments (up to 121 days postinfection). This test performance was similar to that of the gold standard virus neutralization test and indicates that the VLP-ELISA is a highly specific and sensitive method for the detection of SVDV serum antibodies in pigs. This is the first report of the production and diagnostic application of recombinant VLPs of SVDV. Further potential uses of the VLPs are discussed.

Figures

FIG. 1.
FIG. 1.
(A) Amplification of P1 and 3CD protein genes of SVDV by using reverse transcription-PCR. (B) Construction of a recombinant dual expression vector containing P1 and 3CD protein genes of SVDV. (C) A schematic strategy for production of virus-like particles induced by recombinant baculovirus in insect cells.
FIG. 2.
FIG. 2.
Characterization of recombinant SVDV-like particles. (A) Antigenic reactivity of each fraction from CsCl gradients. The reactivity of each fraction was measured by an indirect ELISA using 5B7 antibody. (B) Electron microscopy of SVDV-like particles from antigenic materials made by a dual baculovirus recombinant. (C) Western immunoblot analyses of SVDV-like particles. Proteins from SVDV-like particles (lane 1 and 2), SVDV (lane 3), and mock-infected cells (lane 4) were electrophoretically separated, transferred onto nitrocellulose membranes, and detected using guinea pig sera specific for SVDV (lanes 1, 3, and 4) and VP1 peptide (lane 2).
FIG. 3.
FIG. 3.
Frequency distribution of PI values obtained from VLP-ELISA using known SVDV-negative pig sera (n = 1,041). The cutoff value of the ELISA was set at a PI value of 50 (↓), based on the mean PI value of (62.4) of the most weakly positive serum (EU SVD RS4) minus 3× the standard deviation (3 × 4.2).
FIG. 4.
FIG. 4.
Kinetics of early antibody development as determined by VLP-ELISA and virus neutralization (VN) test in pigs (n = 19) with induced SVDV infections. Pigs of the four groups A, B, C, and D received inoculations with different SVDV strains of ITL/9/93, ITL/11/98, UKG/27/72, and ITL/9/93 (42 days postvaccination with SVDV P1), respectively. The dotted lines represent the cutoff PI value. Arrows represent the time of SVDV challenge (A to D) or P1 vaccination (D). Results obtained from VN test were presented as number positive/number tested.

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