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A multiplex ELISA-based protein array for screening diagnostic antigens and diagnosis of Flaviviridae infection

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European Journal of Clinical Microbiology & Infectious Diseases Aims and scope Submit manuscript

Abstract

Assays with the ability to detect multiple antibodies in parallel have a wide range of potential applications in epidemiologic research. Here, a multiplex enzyme-linked immunosorbent assay-based protein array (ELISA-array) was developed to simultaneously detect five Flaviviridae infections. The platform was based on an indirect ELISA and 15 antigens were constructed for specific antibody detection against five Flaviviridae viruses (Japanese B, tick-borne encephalitis, West Nile, dengue, and yellow fever viruses) and four serotypes of dengue virus. The specificity was evaluated by calculating the signal value cross-reacting with serum immunized with other viruses, and the sensitivity of antigens was compared with conventional ELISAs using immunized rabbit polyclonal antisera. IgG and IgM calibration curves were constructed to evaluate the reproducibility of the platform. Finally, 24 dengue fever (DF) infection and 15 tick-borne encephalitis (TBE) infection clinical sera were used to compare the advantage of ELISA-array to ELISA. After initial screening, 9 out of 15 antigens were chosen for ELISA-array printing. By using different virus-immunized rabbit antiserum, 7 out of 9 antigens showed good specificity in the ELISA-array. Eight out of 9 antigens showed four-fold greater sensitivity in ELISA-array compared to that in conventional ELISAs. The coefficients of determination (r 2) close to 1 showed high reproducibility, and clinical sera test showed that ELISA-array had higher specificity and sensitivity than traditional ELISA. ELISA-array was a good platform for antigen screening and this multiplexed assay might be a useful and convenient tool for multiple immunological detection of infectious viral antibodies.

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Acknowledgments

The authors would like to thank Dr. Wei Zhang for his help with the ELISA-array printing. We are grateful to Qiubo Huo and Licheng Liu for their assistance in collecting the serum samples. We thank Binyin Si and Yu Zhang for their help in the preparation of animal polyclonal antisera. This study was funded by the National Key Research Special Foundation of China (No.2013ZX10004101-003 and No.2011ZX10004-001).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics statement

All the experiments in the paper were conducted under the supervision of the Institutional Review Board of the Academy of Military Medical Sciences. Ethical approval to obtain samples and relevant clinical and personal details were provided by the hospitals. Each patient was informed about the study purpose and signed a consent form prior to sample and personal information acquisition.

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    Authors and Affiliations

    1. State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, 100071, China

      D. Wang, Y. Zheng, X. Kang, X. Zhang, L. Li, Q. Yuan, Y. Yang, Y. Jiang & H. Jiang

    2. CAS Key Laboratory of Pathogenic Microbiology and Immunity, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China

      H. Hao

    3. Genomics Institute in Shenzhen, Beishan Industry Beishan Road, Shenzhen, 518083, China

      W. Chen, L. Liu & X. Li

    4. College of Food Technology, Huazhong Agricultural University, Wuhan, China

      F. Chen

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    1. D. Wang
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    2. Y. Zheng
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    Correspondence to Y. Yang, Y. Jiang or H. Jiang.

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    D. Wang and Y. Zheng contributed equally to this work.

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    Wang, D., Zheng, Y., Kang, X. et al. A multiplex ELISA-based protein array for screening diagnostic antigens and diagnosis of Flaviviridae infection. Eur J Clin Microbiol Infect Dis 34, 1327–1336 (2015). https://doi.org/10.1007/s10096-015-2353-6

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    • DOI: https://doi.org/10.1007/s10096-015-2353-6

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