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Comparative Study
. 2012 Feb;50(2):372-7.
doi: 10.1128/JCM.01319-11. Epub 2011 Nov 30.

Preparation and evaluation of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for detection of total antibodies in human and animal sera by double-antigen sandwich enzyme-linked immunosorbent assay

Yongjun Jiao  1 , Xiaoyan Zeng, Xiling Guo, Xian Qi, Xiao Zhang, Zhiyang Shi, Minghao Zhou, Changjun Bao, Wenshuai Zhang, Yan Xu, Hua Wang
Affiliations
Comparative Study

Preparation and evaluation of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for detection of total antibodies in human and animal sera by double-antigen sandwich enzyme-linked immunosorbent assay

Yongjun Jiao et al. J Clin Microbiol. 2012 Feb.

Abstract

The recent emergence of the human infection confirmed to be caused by severe fever with thrombocytopenia syndrome virus (SFTSV) in China is of global concern. Safe diagnostic immunoreagents for determination of human and animal seroprevalence in epidemiological investigations are urgently needed. This paper describes the cloning and expression of the nucleocapsid (N) protein of SFTSV. An N-protein-based double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) system was set up to detect the total antibodies in human and animal sera. We reasoned that as the double-antigen sandwich ELISA detected total antibodies with a higher sensitivity than traditional indirect ELISA, it could be used to detect SFTSV-specific antibodies from different animal species. The serum neutralization test was used to validate the performance of this ELISA system. All human and animal sera that tested positive in the neutralization test were also positive in the sandwich ELISA, and there was a high correlation between serum neutralizing titers and ELISA readings. Cross-reactivity was evaluated, and the system was found to be highly specific to SFTSV; all hantavirus- and dengue virus-confirmed patient samples were negative. SFTSV-confirmed human and animal sera from both Anhui and Hubei Provinces in China reacted with N protein in this ELISA, suggesting no major antigenic variation between geographically disparate virus isolates and the suitability of this assay in nationwide application. ELISA results showed that 3.6% of the human serum samples and 47.7% of the animal field serum samples were positive for SFTSV antibodies, indicating that SFTSV has circulated widely in China. This assay, which is simple to operate, poses no biohazard risk, does not require sophisticated equipment, and can be used in disease surveillance programs, particularly in the screening of large numbers of samples from various animal species.

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Figures

Fig 1
Fig 1
SFTSV nucleocapsid protein gene cloning and expression. (A) N-protein-encoding gene was amplified with a pair of specific primers by one-step RT-PCR. Lanes: 1, amplified N-protein gene with a size of 738 bp; 2, DNA ladder. (B) SDS-PAGE of rN protein. Lanes: 1, protein marker; 2, lysate of pET-28(a)-NP-transformed bacterial cell after overnight induction; the prominent band at 26 kDa constitutes rN protein; 3, His tag-purified rN protein. (C) Western blot of rN protein using anti-histidine tag MAb-HRP conjugate. Lanes: 1, lysate of bacterial cell; 2, purified rN protein.
Fig 2
Fig 2
Mean ± SD of OD values of positive-control (pc) and negative-control (nc) sera in rN-protein-based double-antigen sandwich ELISA on 8 plates during routine runs of the assay over a period of 2 months. Each internal control was tested in quadruplicate for every plate.
Fig 3
Fig 3
Distribution of ELISA PP values (■) and serum neutralization titers (♢) in all 654 serum samples. (A) Results for 250 human field serum samples, including 150 from Jiangsu and 100 from Anhui; (B) results for 150 goat field serum samples, including 100 from Jiangsu and 50 from Anhui; (C) results for 154 animal field serum samples from 50 cattle, 50 pigs, 50 chickens, and 4 hedgehogs, all from Jiangsu; (D) results for 35 SFTSV-confirmed patient convalescent-phase serum samples, including 18 from Jiangsu, 15 from Anhui, and 2 from Hubei, and 30 hantavirus- and 35 dengue virus-confirmed human serum samples from Jiangsu. Two specimens from Jiangsu from a human (specimen 118) and a pig (specimen 33) (indicated with arrows in panels A and C) showed inconsistency in ELISA and serum neutralization tests. The horizontal lines represent the cutoff for serum neutralization test (—; l g10 [i.e., log1010 with a value of 1] equivalent to a serum dilution of ≥1:10), the cutoff for ELISA based on mean + 2× SD of PP values in negative-control sera (– – –; 1.29, equivalent to 19.29 PP of positive-control sera), and the cutoff for ELISA based on mean + 3× SD of PP values in negative-control sera (- - -; 1.33, equivalent to 21.56 PP).
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