Abstract
Mosquito-borne flavivirus infections, including dengue virus and Zika virus, are major public health threats globally. While the plaque reduction neutralization test (PRNT) is considered the gold standard for determining neutralizing antibody levels to flaviviruses, the assay is time-consuming and laborious. This study, therefore, aimed to develop an enzyme-linked immunosorbent assay (ELISA)-based microneutralization test (EMNT) for the detection of neutralizing antibodies to mosquito-borne flaviviruses. The inhibition of viral growth due to neutralizing antibodies was determined colorimetrically by using EMNT. Given the significance of Fcγ-receptors (FcγR) in antibody-mediated neutralization and antibody-dependent enhancement (ADE) of flavivirus infection, non-FcγR and FcγR-expressing cell lines were used in the EMNT to allow the detection of the sum of neutralizing and immune-enhancing antibody activity as the neutralizing titer. Using anti-flavivirus monoclonal antibodies and clinical samples, the utility of EMNT was evaluated by comparing the end-point titers of the EMNT and the PRNT. The correlation between EMNT and PRNT titers was strong, indicating that EMNT was robust and reproducible. The new EMNT assay combines the biological functional assessment of virus neutralization activity and the technical advantages of ELISA and, is simple, reliable, practical, and could be automated for high-throughput implementation in flavivirus surveillance studies and vaccine trials.
Highlights
Mosquito-borne viruses of the genus Flavivirus in the family Flaviviridae, including dengue virus (DENV) and Zika virus (ZIKV), are major public health threats in approximately a third of the world population that lives in transmission areas [1,2]
To develop the ELISA-based microneutralization test (EMNT), several parameters were tested in order to optimize the assay for sensitivity, sensitivity, reproducibility and efficiency
enzyme-linked immunosorbent assay (ELISA) method [37], the results suggest that the EMNT was technically easy to perform as compared to the plaque reduction neutralization test (PRNT) and the results were comparable to that of the PRNT
Summary
Mosquito-borne viruses of the genus Flavivirus in the family Flaviviridae, including dengue virus (DENV) and Zika virus (ZIKV), are major public health threats in approximately a third of the world population that lives in transmission areas [1,2]. As conventional PRNT uses cell lines that do not express FcγR receptors (FcγR) [10], the conventional method exclusively detects neutralization activity of antibodies but not the infection-enhancement activity (ADE activity) of antibodies This limitation of the conventional PRNT has been highlighted in several studies, which hypothesized that the neutralizing titers as determined by FcγR-bearing cells better reflect the biological function of antibodies [11,12]. In this context, vaccine efficacy studies found that despite the induction of reasonable levels of neutralizing antibodies against four DENV serotypes, protection in some vaccinated participants was minimal [13,14,15]
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