A Test System for Tick-Borne Encephalitis Virus Detection Based on Bioluminescent Immunoassay

The tick-borne encephalitis virus (TBEV) is the causative agent of one of the most severe human neuroinfections. The infection transmitted by ixodid ticks is spread throughout the forest and foreststeppe zones of the temperate climatic belt of the Eurasian continent, including the Siberian region of the Russian Federation. Despite the availability of commercial analytical systems for the detection of TBEV, the task of developing approaches to a quick and reliable analysis that can be performed routinely, particularly in environmental studies, remains topical. A solid-phase bioluminescent immunoassay for determining the tick-borne encephalitis virus (TBEV) in ticks was developed. The assay is based on the hybrid protein consisting of a modified thermostable version of Renilla muelleri luciferase and a single-chain mini-antibody to protein E. This unique protein had been obtained and investigated by the authors earlier. The current study describes the expression of the hybrid protein in two different strains of recombinant E. coli cells. The optimal conditions for obtaining a highly purified protein were found. The bioluminescent reaction of the luciferase domain was triggered with the help of the stable natural form of the substrate, a Ca2+-dependent coelenterazine-binding protein, the recombinant variant of which was obtained by the authors. The conditions for production and storage of the immunoassay components (the hybrid protein, the stable form of the luciferase substrate, and activated microplates) were determined. Using the developed test system, more than 900 tick samples were analyzed for TBEV. In terms of sensitivity (89.5%) and specificity (98.9%), the proposed method is not inferior to colorimetric detection and is much simpler and faster than the latter