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Open AccessArticle

A Simple and High-Throughput ELISA-Based Neutralization Assay for the Determination of Anti-Flavivirus Neutralizing Antibodies

1
Graduate School of Biomedical Sciences, Nagasaki University, Sakamoto 1-12-4, Nagasaki 852-8523, Japan
2
Department of Virology, Institute of Tropical Medicine, Nagasaki University, Sakamoto 1-12-4, Nagasaki 852-8523, Japan
3
Program for Nurturing Global Leaders in Tropical and Emerging Communicable Diseases, Nagasaki University, Sakamoto 1-12-4, Nagasaki 852-8523, Japan
4
Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-murayama-shi, Tokyo 208-0011, Japan
5
Viet Nam Research Station, Center for Infectious Disease Research in Asia and Africa, Institute of Tropical Medicine, Nagasaki University, Sakamoto 1-12-4, Nagasaki 852-8523, Japan
*
Author to whom correspondence should be addressed.
Vaccines 2020, 8(2), 297; https://doi.org/10.3390/vaccines8020297
Received: 14 May 2020 / Revised: 3 June 2020 / Accepted: 5 June 2020 / Published: 10 June 2020
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. View Full-Text
Keywords: ELISA; microneutralization test; flavivirus; neutralizing antibodies; dengue; Zika ELISA; microneutralization test; flavivirus; neutralizing antibodies; dengue; Zika
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MDPI and ACS Style

Balingit, J.C.; Phu Ly, M.H.; Matsuda, M.; Suzuki, R.; Hasebe, F.; Morita, K.; Moi, M.L. A Simple and High-Throughput ELISA-Based Neutralization Assay for the Determination of Anti-Flavivirus Neutralizing Antibodies. Vaccines 2020, 8, 297.

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