Development of a method for detection of specific antibodies to E protein of yellow fever virus (Flaviviridae: Flavivirus) by enzyme immunoassay

Abstract

Introduction. Yellow fever (YF) remains one of the most common natural focal infectious diseases in the world. In connection with the increasing tourist flow to countries endemic for YF, the discovery of stable populations of Aedes aegypti and Ae. albopictus which are the main vectors of the yellow fever virus (YFV), in the southern regions of Russia, and the fact that in medical institutions in our country it is possible to obtain a live attenuated vaccine against YF, but there is no way to evaluate the effectiveness of vaccination, the question arises of the development and implementation of diagnostic kits for detecting antibodies (AB) to the pathogen by enzyme immunoassay (ELISA). The aim of this study was to develop a method for detecting specific IgG antibodies to the E protein of YFV by ELISA and assessing its diagnostic characteristics. Materials and methods. A specific cDNA was synthesized by reverse transcription on an RNA template of YFV isolated on a cell culture of Aedes albopictus clone C6/36, and a fragment of the genome coding the YFV E protein was amplified and subsequently cloned into the plasmid pET160 (Thermo Fisher Scientific, USA). The resulting gene fragment was used as a DNA template to obtain a recombinant analog of the third domain of the YFV E protein in Escherichia coli cells (BL-21(DE3)). Next, the immunogenicity of the obtained antigen was evaluated and the analysis conditions were optimized. Results. The optimal conditions for the production of the obtained recombinant E protein of YFV were determined, its specificity was confirmed by immunological methods (Western blot and ELISA), sorption buffers and blocking solutions were selected, and sensitivity and specificity of detection of antibodies to YFV using the recombinant antigen were assessed. Conclusion. A method for the detection of specific IgG antibodies to the YFV E protein by ELISA was developed. This diagnostic kit can be used both to study the protective properties of the YF vaccine and to detect imported cases of infection in non-endemic areas.

Keywords

• yellow fever virus
• specific antibodies
• yellow fever diagnostics
• flavivirus e protein
• recombinant proteins
• enzyme immunoassay