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An improved respiratory syncytial virus neutralization assay based on the detection of green fluorescent protein expression and automated plaque counting

Yvonne van Remmerden1, Fang Xu1, Mandy van Eldik1, Jacco GM Heldens23, Willem Huisman23 and Myra N Widjojoatmodjo14*

Author Affiliations

1 Department of Vaccine Research, Vaccinology, National Institute for Public Health and the Environment, Bilthoven, The Netherlands

2 Nobilon International BV, Boxmeer, The Netherlands

3 Present address: Merck/MSD Animal Health, Boxmeer, The Netherlands

4 Present address: Crucell, Leiden, The Netherlands

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Virology Journal 2012, 9:253  doi:10.1186/1743-422X-9-253

Published: 31 October 2012



Virus neutralizing antibodies against respiratory syncytial virus (RSV) are considered important correlates of protection for vaccine evaluation. The established plaque reduction assay is time consuming, labor intensive and highly variable.


Here, a neutralization assay based on a modified RSV strain expressing the green fluorescent protein in combination with automated detection and quantification of plaques is described.


The fluorescence plaque reduction assay in microplate format requires only two days to complete and is simple and reproducible. A good correlation between visual and automated counting methods to determine RSV neutralizing serum antibody titers was observed.


The developed virus neutralization assay is suitable for high-throughput testing and can be used for both animal studies and (large scale) vaccine clinical trials.

RSV; Virus neutralization; EGFP