Quantification of SARS-CoV-2 neutralizing antibody by wild-type plaque reduction neutralization, microneutralization and pseudotyped virus neutralization assays
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Virus neutralization assays measure neutralizing antibodies in serum and plasma, and the plaque reduction neutralization test (PRNT) is considered the gold standard for measuring levels of these antibodies for many viral diseases. We have developed procedures for the standard PRNT, microneutralization assay (MNA) and pseudotyped virus neutralization assay (PNA) for severe acute respiratory syndrome coronavirus 2. The MNA offers advantages over the PRNT by reducing assay time, allowing increased throughput and reducing operator workload while remaining dependent upon the use of wild-type virus. This ensures that all severe acute respiratory syndrome coronavirus 2 antigens are present, but Biosafety Level 3 facilities are required. In addition to the advantages of MNA, PNA can be performed with lower biocontainment (Biosafety Level 2 facilities) and allows for further increases in throughput. For each new vaccine, it is critical to ensure good correlation of the neutralizing activity measured using PNA against the PRNT or MNA. These assays have been used in the development and licensure of the ChAdOx1 nCoV-19 (AstraZeneca; Oxford University) and Ad26.COV2.S (Janssen) coronavirus disease 2019 vaccines and are critical for demonstrating bioequivalence of future vaccines.
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Live virus neutralization assays are considered the gold standard for assessing immune responses.
“Live virus neutralization assays are considered to be the gold standard for assessing immune responses25.”
From An Omicron-specific, self-amplifying mRNA booster vaccine for COVID-19: a phase 2/3 randomized trial (2024) · cites this paper as [25]
The Development of the protocol section calls the live-virus plaque reduction neutralization test (PRNT) the gold standard for assessing humoral correlates of protection for many viral diseases. The citing sentence broadens this to live-virus neutralization assays generally and to immune responses beyond the humoral response. (LLM, conf=0.88)
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Thiruvengadam, R., Awasthi, A., Medigeshi, G., Bhattacharya, S., Mani, S., Sivasubbu, S., Shrivastava, T., Samal, S., Rathna Murugesan, D., Koundinya Desiraju, B., et al. (2022) · The Lancet Infectious Diseases · doi:10.1016/s1473-3099(21)00680-0 · Cited by 96 · see citing works
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“Live virus neutralization assays are considered to be the gold standard for assessing immune responses 25 .”
- Authors
- Bewley, K. R., Coombes, N. S., Gagnon, L., McInroy, L., Baker, N., Shaik, I., St-Jean, J. R., St-Amant, N., Buttigieg, K. R., Humphries, H. E., Godwin, K. J., Brunt, E., Allen, L., Leung, S., Brown, P. J., Penn, E. J., Thomas, K., Kulnis, G., Hallis, B., Carroll, M., Funnell, S., & Charlton, S.
- Affiliations
- Public Health England (GB)
- Corresponding author
- Bewley, Kevin R.
- ORCID
- Venue
- Nature Protocols
- Published
- 2021-04-23
- Volume
- 16
- Issue
- 6
- Pages
- 3114-3140
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- OpenAlex
- W3155000349
- Cited by
- 293
- References
- 33
- Citations by year
- 2026: 21 · 2025: 38 · 2024: 58 · 2023: 66 · 2022: 84 · 2021: 25 · 2013: 1
- FWCI
- 18.51
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- Funders
- The PNA elements of this work were supported by the Global Health Vaccine Accelerator Platforms (GH-VAP) and the Coronavirus Immunotherapy Consortium (CoVIC) funded by BMGF
- Topics
- Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
- Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 detection and testing
- Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Animal Virus Infections Studies
- Keywords
- SARS-CoV-2, neutralizing antibodies, plaque reduction neutralization test, microneutralization, antibody quantification, ChAdOx1 nCoV-19 vaccine, Ad26.COV2-S
- MeSH terms
- COVID-19, SARS-CoV-2, COVID-19 Vaccines, Ad26COVS1, ChAdOx1 nCoV-19, Antibodies, Viral, Humans, Neutralization Tests, Time Factors, Antibodies, Neutralizing
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