Development of an enzyme-linked immunosorbent assay test system for quantifying Chikungunya virus E2 protein and calculating the mass of whole-virion antigen in culture fluid samples

INTRODUCTION. In recent years, Chikungunya virus (CHIKV) has spread in many parts of the world and has caused large-scale outbreaks with serious economic and social consequences. To improve the effectiveness of CHIKV control measures, it is necessary to develop and optimise diagnostic methods applicable not only to patient serum samples but also to mosquito samples (to identify and eliminate the foci of infection). In addition, it is important to determine antigens in culture fluid samples taken at various stages in the development and production of CHIKV vaccines.

AIM. This study aimed to develop a quantitative one-step sandwich enzyme-linked immunosorbent assay (ELISA) test system for detecting CHIKV E2 protein and a procedure for calculating the mass of whole-virion antigen in culture fluid samples.

MATERIALS AND METHODS. The study focused on two mouse monoclonal antibodies, purified CHIKV (Nika21 strain, GenBank ID: PQ673601), and recombinant CHIKV E2 protein. The sensitivity of ELISA was compared with that of real-time quantitative reverse transcription polymerase chain reaction (real-time RT-qPCR). The comparison used culture fluid samples collected at different time points after infection of Vero cells with CHIKV (18, 24, 46, and 72 h). The main analytical and technical characteristics of the ELISA test system developed were determined in accordance with GOST 51352-2013.

RESULTS. The sensitivity of the assay was not less than 0.625 ng/mL, and its coefficient of variation was not more than 3.56%. The recovery of the assay was 100%. The assay demonstrated an acceptable linearity of 90–110% in the concentration range of 1.5–16 ng/mL. The specificity of the assay was 100%, as no cross-reactivity was observed with samples containing dengue, yellow fever, Sindbis, rubella, and West Nile viruses. The ELISA test system developed in this study and real-time RT-qPCR showed similar results (1.06 and 1.09 μg/mL, respectively) in calculating the mass of whole-virion antigen in culture fluid samples with the use of a conversion factor.

CONCLUSIONS. A simple, specific, and sensitive ELISA test system was developed for the quantitative determination of CHIKV E2 protein in culture fluid samples (and for rapid testing of mosquito samples). The authors offered a method for calculating the mass of whole-virion antigen from the amount of E2 protein (ELISA) and the quantity of genomic equivalents (real-time RT-qPCR). The study demonstrated a strong negative correlation between optical density values obtained using ELISA and cycle threshold values derived from real-time RT-qPCR.

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