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. 2012;7(12):e53026.
doi: 10.1371/journal.pone.0053026. Epub 2012 Dec 31.

Development of a blocking ELISA for detection of serum neutralizing antibodies against newly emerged duck Tembusu virus

Xuesong Li  1 , Guoxin Li, Qiaoyang Teng, Lei Yu, Xiaogang Wu, Zejun Li
Affiliations

Development of a blocking ELISA for detection of serum neutralizing antibodies against newly emerged duck Tembusu virus

Xuesong Li et al. PLoS One. 2012.

Abstract

Background: Since April 2010, domesticated ducks in China have been suffering from an emerging infectious disease characterized by retarded growth, high fever, loss of appetite, decline in egg production, and death. The causative agent was identified as a duck Tembusu virus (DTMUV), a member of the Ntaya virus (NTAV) group within the genus Flavivirus, family Flaviviridae. DTMUV is highly contagious and spreads rapidly in many species of ducks. More than 10 million shelducks have been infected and approximately 1 million died in 2010. The disease remains a constant threat to the duck industry; however, it is not known whether DTMUV can infect humans or other mammalians, despite the fact that the virus has spread widely in southeast China, one of the most densely populated areas in the world. The lack of reliable methods to detect the serum antibodies against DTMUV has limited our ability to conduct epidemiological investigations in various natural hosts and to evaluate the efficiency of vaccines to DTMUV.

Methodology/principal findings: A neutralizing monoclonal antibody (mAb) 1F5 binding specifically to the E protein was developed. Based on the mAb, a blocking enzyme-linked immunosorbent assay (ELISA) was developed for the detection of neutralizing antibodies against DTMUV. The average value of percent inhibition (PI) of 350 duck serum samples obtained from DTMUV-free farms was 1.0% ± 5.8% (mean ± SD). The selected cut-off PI values for negative and positive sera were 12.6% (mean +2SD) and 18.4% (mean +3SD), respectively. When compared with a serum neutralizing antibody test (SNT) using chicken embryonated eggs, the rate of coincidence was 70.6% between the blocking ELISA and SNT, based on the titration of 20 duck DTMUV-positive serum samples.

Conclusions/significance: The blocking ELISA based on a neutralizing mAb allowed rapid, sensitive, and specific detection of neutralization-related antibodies against DTMUV.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Characterization of monoclonal antibody 1F5 by immunofluorescence assay.
Monoclonal antibody 1F5 was used to perform indirect immunofluorescence assay on DF-1 cells infected with DTMUV FX2010 and 293T cells transfected with pCAGGS-E plasmids. A) DF-1 cells infected with DTMUV FX2010, B) control DF-1 cells, C) 293T cells transfected with recombinant plasmid pCAGGS-E, and D) control 293T cells fixed with 4% paraformaldehyde, and then incubated with mAb 1F5 and FITC-conjugated goat anti-mouse IgG, in turn. Cells were mounted with 10 mM p-phenylenediamine (PPD) in glycerol-PBS and observed under a fluorescent microscope.
Figure 2
Figure 2. Specificity of the b-ELISA to anti-DTMUV serum.
To test the abilities of mAb to bind specifically to domain III of E protein, western blot was conducted with purified fusion protein including both the domain III (12 kDa) of E protein and TF tag protein (52 kDa) (line 1), and purified TF tag protein (52 kDa) (line 2) expressed by pCold plasmids. The mAb 1F5 was able to bind specifically to the 64-kDa fusion protein, but could not bind to purified TF tag protein (Fig 2).
Figure 3
Figure 3. Specificity of the b-ELISA to anti-DTMUV serum.
Seven antisera against different viruses were investigated. The PI value of anti-DTMUV serum reached a maximum of 69.13%, while the other antisera against H5N1 AIV, H9N2 AIV, NDV, DHV-1, DPV, RV, and JEV ranged from −2.7% to 2.3%.
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Grants and funding

This study was supported the National Natural Science Foundation of China (no. 31172332), the Ministry of Agriculture’s Major Projects for Transgenic Research Program, China (no. 2009ZX08010-022B), the Special Fund for Agro-scientific Research in the Public Interest, China (no. 201003012), the Special Fund for International Communication and Cooperation (no. S2011ZR0429), and the National High Technology Research and Development Program of China (863 Program) (no. 2011AA10A200). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.