Validation and Establishment of a SARS-CoV-2 Lentivirus Surrogate Neutralization Assay as a pre-screening tool for the Plaque Reduction Neutralization Test

Merluza, J.; Ung, J.; Makowski, K.; Robinson, A.; Manguiat, K. J.; Mueller, N.; Audet, J.; Chen, J. C.-y.; Strong, J. E.; Wood, H.; Bello, A.

doi:10.1101/2022.09.13.507876

bioRxiv · 10.1101/2022.09.13.507876

Validation and Establishment of a SARS-CoV-2 Lentivirus Surrogate Neutralization Assay as a pre-screening tool for the Plaque Reduction Neutralization Test

Merluza, J.·Ung, J.·Makowski, K.·Robinson, A.·Manguiat, K. J.·Mueller, N.·Audet, J.·Chen, J. C.-y.·Strong, J. E.·Wood, H.·Bello, A.

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Abstract

Neutralization assays are important in understanding and quantifying neutralizing antibody responses towards SARS-CoV-2. The SARS-CoV-2 Lentivirus Surrogate Neutralization Assay (SCLSNA) can be used in biosafety level 2 (BSL-2) laboratories and has been shown to be a reliable, alternative approach to the plaque reduction neutralization test (PRNT). In this study, we optimized and validated the SCLSNA to assess its ability as a comparator and pre-screening method to support the PRNT. Comparability between the PRNT and SCLSNA was determined through clinical sensitivity and specificity evaluations. Clinical sensitivity and specificity produced acceptable results with 100% (95% CI: 94-100) specificity and 100% (95% CI: 94-100) sensitivity against ancestral Wuhan spike pseudotyped lentivirus. The sensitivity and specificity against B.1.1.7 spike pseudotyped lentivirus resulted in 88.3% (95% CI: 77.8 to 94.2) and 100% (95% CI: 94-100), respectively. Assay precision measuring intra-assay variability produced acceptable results for High (1:[≥] 640 PRNT50), Mid (1:160 PRNT50) and Low (1:40 PRNT50) antibody titer concentration ranges based on the PRNT50, with %CV of 14.21, 12.47, and 13.28 respectively. Intermediate precision indicated acceptable ranges for the High and Mid concentrations, with %CV of 15.52 and 16.09, respectively. However, the Low concentration did not meet the acceptance criteria with a %CV of 26.42. Acceptable ranges were found in the robustness evaluation for both intra-assay and inter-assay variability. In summary, the validation parameters tested met the acceptance criteria, making the SCLSNA method fit for its intended purpose, which can be used to support the PRNT.

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Merluza, J., Ung, J., Makowski, K., Robinson, A., Manguiat, K. J., Mueller, N., Audet, J., Chen, J. C.-y., Strong, J. E., Wood, H., Bello, A.. 2022-09-14. Validation and Establishment of a SARS-CoV-2 Lentivirus Surrogate Neutralization Assay as a pre-screening tool for the Plaque Reduction Neutralization Test. https://doi.org/10.1101/2022.09.13.507876

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