Validation of a d34 recombinant antigen‐based ELISA for monitoring protective humoral immunity to dengue virus in MVA‐d34‐immunized mice

Abstract

To validate an enzyme‐linked immunosorbent assay (ELISA) test system for the semi‐quantitative detection of specific IgG antibodies against domain III of the E protein of dengue virus (DENV) serotypes 1–4 in mouse serum. Commercial reagent kits (JSC BTK Bioservice, Moscow, Russia) were utilised. The recombinant d34 protein expressed in a baculovirus system served as the immunosorbent. Analytical performance was evaluated using a panel of 100 serum samples from BALB/c mice (11 groups). Virusneutralising antibodies against DENV serotypes D1–D4 were determined by a virus neutralisation test in Vero cell culture.The specificity and sensitivity of the test system developed both reached 100 % (95 % CI: 91.2–100.0 %), with a critical optical density (ODcrit) of 0.16 and a coefficient of variation (CV) not exceeding 8.2 %. Stability testing at 37°C for 14 days revealed a 4.7 % signal decrease (95 % CI: –11.3 % to +1.9 %), indicating that the immunosorbent is expected to retain its functional properties for up to 12 months when stored at 2–8°C. The systematically higher ELISA values compared to neutralisation test titers reflect differences in the detected antibody pools and support the combined use of both methods for a comprehensive evaluation of the humoral immune response.The validated ELISA test system exhibits high diagnostic performance and is suitable for the semi‐quantitative determination of specific IgG antibodies against domain III of the DENV E protein in laboratory mouse serum. Its application provides a reliable tool for preclinical monitoring of vaccine candidate immunogenicity and establishes a methodological basis for standardising the quality control of biologics targeting serotypespecific epitopes.