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.
Similar content being viewed by others
References
Mendoza LG, McQuary P, Mongan A, Gangadharan R, Brignac S, Eggers M (1999) High-throughput microarray-based enzyme-linked immunosorbent assay (ELISA). Biotechniques 27(4):778–780, 782–776, 788
Nielsen UB, Geierstanger BH (2004) Multiplexed sandwich assays in microarray format. J Immunol Methods 290(1–2):107–120. doi:10.1016/j.jim.2004.04.012
Kingsmore SF (2006) Multiplexed protein measurement: technologies and applications of protein and antibody arrays. Nat Rev Drug Discov 5(4):310–320. doi:10.1038/nrd2006
Lash GE, Scaife PJ, Innes BA, Otun HA, Robson SC, Searle RF, Bulmer JN (2006) Comparison of three multiplex cytokine analysis systems: Luminex, SearchLight and FAST Quant. J Immunol Methods 309(1–2):205–208. doi:10.1016/j.jim.2005.12.007
Liew M, Groll MC, Thompson JE, Call SL, Moser JE, Hoopes JD, Voelkerding K, Wittwer C, Spendlove RS (2007) Validating a custom multiplex ELISA against individual commercial immunoassays using clinical samples. Biotechniques 42(3):327–328, 330–333. doi:10.2144/000112332
Cunningham AA (2005) A walk on the wild side—emerging wildlife diseases. BMJ 331(7527):1214–1215. doi:10.1136/bmj.331.7527.1214
Blancou J, Chomel BB, Belotto A, Meslin FX (2005) Emerging or re-emerging bacterial zoonoses: factors of emergence, surveillance and control. Vet Res 36(3):507–522. doi:10.1051/vetres:2005008
Gao X, Nasci R, Liang G (2010) The neglected arboviral infections in mainland China. PLoS Negl Trop Dis 4(4):e624. doi:10.1371/journal.pntd.0000624
Lu Z, Liu H, Fu S, Lu X, Dong Q, Zhang S, Tong S, Li M, Li W, Tang Q, Liang G (2011) Liao ning virus in China. Virol J 8:282. doi:10.1186/1743-422X-8-282
Liu H, Gao X, Liang G (2011) Newly recognized mosquito-associated viruses in mainland China, in the last two decades. Virol J 8:68. doi:10.1186/1743-422X-8-68
Deneke Y, Sabarinath T, Gogia N, Lalsiamthara J, Viswas KN, Chaudhuri P (2014) Evaluation of recombinant LigB antigen-based indirect ELISA and latex agglutination test for the serodiagnosis of bovine leptospirosis in India. Mol Cell Probes 28(4):141–146. doi:10.1016/j.mcp.2014.01.001
Holbrook MR, Shope RE, Barrett AD (2004) Use of recombinant E protein domain III-based enzyme-linked immunosorbent assays for differentiation of tick-borne encephalitis serocomplex flaviviruses from mosquito-borne flaviviruses. J Clin Microbiol 42(9):4101–4110
Gritsun DJ, Jones IM, Gould EA, Gritsun TS (2014) Molecular archaeology of Flaviviridae untranslated regions: duplicated RNA structures in the replication enhancer of flaviviruses and pestiviruses emerged via convergent evolution. PLoS One 9(3):e92056. doi:10.1371/journal.pone.0092056
Bhardwaj S, Holbrook M, Shope RE, Barrett AD, Watowich SJ (2001) Biophysical characterization and vector-specific antagonist activity of domain III of the tick-borne flavivirus envelope protein. J Virol 75(8):4002–4007. doi:10.1128/JVI.75.8.4002-4007.2001
Crill WD, Roehrig JT (2001) Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. J Virol 75(16):7769–7773
Kuhn RJ, Zhang W, Rossmann MG, Pletnev SV, Corver J, Lenches E, Jones CT, Mukhopadhyay S, Chipman PR, Strauss EG, Baker TS, Strauss JH (2002) Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell 108(5):717–725
Oliphant T, Nybakken GE, Engle M, Xu Q, Nelson CA, Sukupolvi-Petty S, Marri A, Lachmi BE, Olshevsky U, Fremont DH, Pierson TC, Diamond MS (2006) Antibody recognition and neutralization determinants on domains I and II of West Nile Virus envelope protein. J Virol 80(24):12149–12159. doi:10.1128/JVI.01732-06
Crill WD, Roehrig JT (2001) Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. J Virol 75(16):7769–7773. doi:10.1128/jvi.75.16.7769-7773.2001
Heinz FX, Berger R, Tuma W, Kunz C (1983) Location of immunodominant antigenic determinants on fragments of the tick-borne encephalitis virus glycoprotein: evidence for two different mechanisms by which antibodies mediate neutralization and hemagglutination inhibition. Virology 130(2):485–501
Mandl CW, Guirakhoo F, Holzmann H, Heinz FX, Kunz C (1989) Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-borne encephalitis virus as a model. J Virol 63(2):564–571
Fonseca BA, Khoshnood K, Shope RE, Mason PW (1991) Flavivirus type-specific antigens produced from fusions of a portion of the E protein gene with the Escherichia coli trpE gene. Am J Trop Med Hyg 44(5):500–508
Holzmann H, Mandl CW, Guirakhoo F, Heinz FX, Kunz C (1989) Characterization of antigenic variants of tick-borne encephalitis virus selected with neutralizing monoclonal antibodies. J Gen Virol 70(Pt 1):219–222
Gromowski GD, Barrett AD (2007) Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virus. Virology 366(2):349–360. doi:10.1016/j.virol.2007.05.042
Chávez JH, Silva JR, Amarilla AA, Moraes Figueiredo LT (2010) Domain III peptides from flavivirus envelope protein are useful antigens for serologic diagnosis and targets for immunization. Biologicals 38(6):613–618. doi:10.1016/j.biologicals.2010.07.004
Holzmann H (2003) Diagnosis of tick-borne encephalitis. Vaccine 21(Suppl 1):S36–S40. doi:10.1016/S0264-410X(02)00819-8
Ludolfs D, Reinholz M, Schmitz H (2009) Highly specific detection of antibodies to tick-borne encephalitis (TBE) virus in humans using a domain III antigen and a sensitive immune complex (IC) ELISA. J Clin Virol 45(2):125–128. doi:10.1016/j.jcv.2009.03.016
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.
Author information
Authors and Affiliations
Corresponding authors
Additional information
D. Wang and Y. Zheng contributed equally to this work.
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10096-015-2353-6