An Assessment of Serological Assays for SARS-CoV-2 as Surrogates for Authentic Virus Neutralization

doi:10.1128/Spectrum.01059-21

. 2021 Oct 31;9(2):e0105921.

doi: 10.1128/Spectrum.01059-21. Epub 2021 Oct 27.

An Assessment of Serological Assays for SARS-CoV-2 as Surrogates for Authentic Virus Neutralization

Nicholas Wohlgemuth¹, Kendall Whitt², Sean Cherry¹, Ericka Kirkpatrick Roubidoux¹, Chun-Yang Lin³, Kim J Allison¹, Ashleigh Gowen¹, Pamela Freiden¹, E Kaitlynn Allen³; St. Jude Investigative Team,; Aditya H Gaur¹, Jeremie H Estepp^{4

5}, Li Tang⁶, Tomi Mori⁶, Diego R Hijano¹, Hana Hakim¹, Maureen A McGargill³, Florian Krammer⁷, Michael A Whitt², Joshua Wolf¹, Paul G Thomas³, Stacey Schultz-Cherry¹

Affiliations

¹ Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
² Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Centergrid.267301.1, Memphis, Tennessee, USA.
³ Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
⁴ Department of Hematology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
⁵ Department of Global Pediatric Medicine, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
⁶ Department of Biostatistics, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
⁷ Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

PMID: 34704832
PMCID: PMC8549747
DOI: 10.1128/Spectrum.01059-21

An Assessment of Serological Assays for SARS-CoV-2 as Surrogates for Authentic Virus Neutralization

Nicholas Wohlgemuth et al. Microbiol Spectr. 2021.

. 2021 Oct 31;9(2):e0105921.

doi: 10.1128/Spectrum.01059-21. Epub 2021 Oct 27.

Authors

Affiliations

¹ Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
² Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Centergrid.267301.1, Memphis, Tennessee, USA.
³ Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
⁴ Department of Hematology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
⁵ Department of Global Pediatric Medicine, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
⁶ Department of Biostatistics, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
⁷ Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

PMID: 34704832
PMCID: PMC8549747
DOI: 10.1128/Spectrum.01059-21

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and has since caused a global pandemic resulting in millions of cases and deaths. Diagnostic tools and serological assays are critical for controlling the outbreak, especially assays designed to quantitate neutralizing antibody levels, considered the best correlate of protection. As vaccines become increasingly available, it is important to identify reliable methods for measuring neutralizing antibody responses that correlate with authentic virus neutralization but can be performed outside biosafety level 3 (BSL3) laboratories. While many neutralizing assays using pseudotyped virus have been developed, there have been few studies comparing the different assays to each other as surrogates for authentic virus neutralization. Here, we characterized three enzyme-linked immunosorbent assays (ELISAs) and three pseudotyped vesicular stomatitis virus (VSV) neutralization assays and assessed their concordance with authentic virus neutralization. The most accurate assays for predicting authentic virus neutralization were luciferase- and secreted embryonic alkaline phosphatase (SEAP)-expressing pseudotyped virus neutralizations, followed by green fluorescent protein (GFP)-expressing pseudotyped virus neutralization, and then the ELISAs. IMPORTANCE The ongoing COVID-19 pandemic is caused by infection with severe acute respiratory syndrome virus 2 (SARS-CoV-2). Prior infection or vaccination can be detected by the presence of antibodies in the blood. Antibodies in the blood are also considered to be protective against future infections from the same virus. The "gold standard" assay for detecting protective antibodies against SARS-CoV-2 is neutralization of authentic SARS-CoV-2 virus. However, this assay can only be performed under highly restrictive biocontainment conditions. We therefore characterized six antibody-detecting assays for their correlation with authentic virus neutralization. The significance of our research is in outlining the advantages and disadvantages of the different assays and identifying the optimal surrogate assay for authentic virus neutralization. This will allow for more accurate assessments of protective immunity against SARS-CoV-2 following infection and vaccination.

Keywords: SARS-CoV-2; immunoassays; neutralizing antibodies.

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Figures

**FIG 1**
Comparison of neutralization assays by sample groups. Shown are area under the curve (AUC) (A) and 50% neutralization dilution (ND₅₀) (B) calculations by neutralization assay type. AUC and ND₅₀ values were calculated and used to compare authentic virus neutralization (black), GFP pseudotype neutralization (pink), luciferase pseudotype (teal), and SEAP pseudotype (purple). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (mixed-effects model with the Geisser-Greenhouse correction and Tukey multiple-comparison posttest and P value adjustment). n = 34 samples run on each assay.

**FIG 2**
Bland-Altman analysis of SARS-CoV-2 pseudotyped virus neutralization assays. Bland-Altman analysis was performed between the log ND₅₀ values of authentic virus neutralization against each pseudotyped virus platform. The difference between the two assays for each sample is on the y axis, and the average of the two assays is on the x axis.

**FIG 3**
Correlation of SARS-CoV-2 serologic assays. (A) SARS-CoV-2-specific ELISAs and (B) VSV pseudotyped virus neutralization assays were compared by simple linear regression. The Pearson’s r values (a metric of correlation) and P values corresponding to each graph are to the lower left of each set of graphs. The background shading corresponds to the degree of correlation between the two assays. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

**FIG 4**
Principal-component analysis (PCA) of SARS-CoV-2 serological assays. Principal-component analysis was performed using all three ELISAs (spike, RBD, and nucleocapsid) and pseudotyped virus neutralization platforms (GFP, luciferase, and SEAP). The authentic virus ND₅₀ is indicated by the color of the data point. PCA loadings generated during the analysis are shown on the right.

**FIG 5**
Comparison of high-positive, low-positive, and negative ELISA groups across neutralization assays. (A) RBD-, (B) spike (S)-, and (C) nucleocapsid (N)-positive samples were divided into high and low positives by finding cutoff values using histograms (RBD ratio, 15; N ratio, 10, and spike value, 6). Log ND₅₀ values for the corresponding samples were then graphed and compared by Kruskal-Wallis test with Dunn's multiple-comparison tests. Significance thresholds: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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References

1. Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, Bleicker T, Brünink S, Schneider J, Schmidt ML, Mulders DG, Haagmans BL, van der Veer B, van den Brink S, Wijsman L, Goderski G, Romette J-L, Ellis J, Zambon M, Peiris M, Goossens H, Reusken C, Koopmans MP, Drosten C. 2020. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill 25:2000045. doi:10.2807/1560-7917.ES.2020.25.3.2000045. - DOI - PMC - PubMed
1. Wang W, Tang J, Wei F. 2020. Updated understanding of the outbreak of 2019 novel coronavirus (2019-nCoV) in Wuhan, China. J Med Virol 92:441–447. doi:10.1002/jmv.25689. - DOI - PMC - PubMed
1. Wu F, Zhao S, Yu B, Chen Y-M, Wang W, Song Z-G, Hu Y, Tao Z-W, Tian J-H, Pei Y-Y, Yuan M-L, Zhang Y-L, Dai F-H, Liu Y, Wang Q-M, Zheng J-J, Xu L, Holmes EC, Zhang Y-Z. 2020. A new coronavirus associated with human respiratory disease in China. Nature 579:265–269. doi:10.1038/s41586-020-2008-3. - DOI - PMC - PubMed
1. Zhou P, Yang X-L, Wang X-G, Hu B, Zhang L, Zhang W, Si H-R, Zhu Y, Li B, Huang C-L, Chen H-D, Chen J, Luo Y, Guo H, Jiang R-D, Liu M-Q, Chen Y, Shen X-R, Wang X, Zheng X-S, Zhao K, Chen Q-J, Deng F, Liu L-L, Yan B, Zhan F-X, Wang Y-Y, Xiao G-F, Shi Z-L. 2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579:270–273. doi:10.1038/s41586-020-2012-7. - DOI - PMC - PubMed
1. Grzelak L, Temmam S, Planchais C, Demeret C, Tondeur L, Huon C, Guivel-Benhassine F, Staropoli I, Chazal M, Dufloo J, Planas D, Buchrieser J, Rajah MM, Robinot R, Porrot F, Albert M, Chen K-Y, Crescenzo-Chaigne B, Donati F, Anna F, Souque P, Gransagne M, Bellalou J, Nowakowski M, Backovic M, Bouadma L, Le Fevre L, Le Hingrat Q, Descamps D, Pourbaix A, Laouénan C, Ghosn J, Yazdanpanah Y, Besombes C, Jolly N, Pellerin-Fernandes S, Cheny O, Ungeheuer M-N, Mellon G, Morel P, Rolland S, Rey FA, Behillil S, Enouf V, Lemaitre A, Créach M-A, Petres S, Escriou N, Charneau P, Fontanet A, et al. . 2020. A comparison of four serological assays for detecting anti-SARS-CoV-2 antibodies in human serum samples from different populations. Sci Transl Med 12:eabc3103. doi:10.1126/scitranslmed.abc3103. - DOI - PMC - PubMed

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[1] Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, Bleicker T, Brünink S, Schneider J, Schmidt ML, Mulders DG, Haagmans BL, van der Veer B, van den Brink S, Wijsman L, Goderski G, Romette J-L, Ellis J, Zambon M, Peiris M, Goossens H, Reusken C, Koopmans MP, Drosten C. 2020. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill 25:2000045. doi:10.2807/1560-7917.ES.2020.25.3.2000045. - DOI - PMC - PubMed

[2] Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, Bleicker T, Brünink S, Schneider J, Schmidt ML, Mulders DG, Haagmans BL, van der Veer B, van den Brink S, Wijsman L, Goderski G, Romette J-L, Ellis J, Zambon M, Peiris M, Goossens H, Reusken C, Koopmans MP, Drosten C. 2020. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill 25:2000045. doi:10.2807/1560-7917.ES.2020.25.3.2000045. - DOI - PMC - PubMed

[3] Wang W, Tang J, Wei F. 2020. Updated understanding of the outbreak of 2019 novel coronavirus (2019-nCoV) in Wuhan, China. J Med Virol 92:441–447. doi:10.1002/jmv.25689. - DOI - PMC - PubMed

[4] Wang W, Tang J, Wei F. 2020. Updated understanding of the outbreak of 2019 novel coronavirus (2019-nCoV) in Wuhan, China. J Med Virol 92:441–447. doi:10.1002/jmv.25689. - DOI - PMC - PubMed

[5] Wu F, Zhao S, Yu B, Chen Y-M, Wang W, Song Z-G, Hu Y, Tao Z-W, Tian J-H, Pei Y-Y, Yuan M-L, Zhang Y-L, Dai F-H, Liu Y, Wang Q-M, Zheng J-J, Xu L, Holmes EC, Zhang Y-Z. 2020. A new coronavirus associated with human respiratory disease in China. Nature 579:265–269. doi:10.1038/s41586-020-2008-3. - DOI - PMC - PubMed

[6] Wu F, Zhao S, Yu B, Chen Y-M, Wang W, Song Z-G, Hu Y, Tao Z-W, Tian J-H, Pei Y-Y, Yuan M-L, Zhang Y-L, Dai F-H, Liu Y, Wang Q-M, Zheng J-J, Xu L, Holmes EC, Zhang Y-Z. 2020. A new coronavirus associated with human respiratory disease in China. Nature 579:265–269. doi:10.1038/s41586-020-2008-3. - DOI - PMC - PubMed

[7] Zhou P, Yang X-L, Wang X-G, Hu B, Zhang L, Zhang W, Si H-R, Zhu Y, Li B, Huang C-L, Chen H-D, Chen J, Luo Y, Guo H, Jiang R-D, Liu M-Q, Chen Y, Shen X-R, Wang X, Zheng X-S, Zhao K, Chen Q-J, Deng F, Liu L-L, Yan B, Zhan F-X, Wang Y-Y, Xiao G-F, Shi Z-L. 2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579:270–273. doi:10.1038/s41586-020-2012-7. - DOI - PMC - PubMed

[8] Zhou P, Yang X-L, Wang X-G, Hu B, Zhang L, Zhang W, Si H-R, Zhu Y, Li B, Huang C-L, Chen H-D, Chen J, Luo Y, Guo H, Jiang R-D, Liu M-Q, Chen Y, Shen X-R, Wang X, Zheng X-S, Zhao K, Chen Q-J, Deng F, Liu L-L, Yan B, Zhan F-X, Wang Y-Y, Xiao G-F, Shi Z-L. 2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579:270–273. doi:10.1038/s41586-020-2012-7. - DOI - PMC - PubMed

[9] Grzelak L, Temmam S, Planchais C, Demeret C, Tondeur L, Huon C, Guivel-Benhassine F, Staropoli I, Chazal M, Dufloo J, Planas D, Buchrieser J, Rajah MM, Robinot R, Porrot F, Albert M, Chen K-Y, Crescenzo-Chaigne B, Donati F, Anna F, Souque P, Gransagne M, Bellalou J, Nowakowski M, Backovic M, Bouadma L, Le Fevre L, Le Hingrat Q, Descamps D, Pourbaix A, Laouénan C, Ghosn J, Yazdanpanah Y, Besombes C, Jolly N, Pellerin-Fernandes S, Cheny O, Ungeheuer M-N, Mellon G, Morel P, Rolland S, Rey FA, Behillil S, Enouf V, Lemaitre A, Créach M-A, Petres S, Escriou N, Charneau P, Fontanet A, et al. . 2020. A comparison of four serological assays for detecting anti-SARS-CoV-2 antibodies in human serum samples from different populations. Sci Transl Med 12:eabc3103. doi:10.1126/scitranslmed.abc3103. - DOI - PMC - PubMed

[10] Grzelak L, Temmam S, Planchais C, Demeret C, Tondeur L, Huon C, Guivel-Benhassine F, Staropoli I, Chazal M, Dufloo J, Planas D, Buchrieser J, Rajah MM, Robinot R, Porrot F, Albert M, Chen K-Y, Crescenzo-Chaigne B, Donati F, Anna F, Souque P, Gransagne M, Bellalou J, Nowakowski M, Backovic M, Bouadma L, Le Fevre L, Le Hingrat Q, Descamps D, Pourbaix A, Laouénan C, Ghosn J, Yazdanpanah Y, Besombes C, Jolly N, Pellerin-Fernandes S, Cheny O, Ungeheuer M-N, Mellon G, Morel P, Rolland S, Rey FA, Behillil S, Enouf V, Lemaitre A, Créach M-A, Petres S, Escriou N, Charneau P, Fontanet A, et al. . 2020. A comparison of four serological assays for detecting anti-SARS-CoV-2 antibodies in human serum samples from different populations. Sci Transl Med 12:eabc3103. doi:10.1126/scitranslmed.abc3103. - DOI - PMC - PubMed

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An Assessment of Serological Assays for SARS-CoV-2 as Surrogates for Authentic Virus Neutralization

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An Assessment of Serological Assays for SARS-CoV-2 as Surrogates for Authentic Virus Neutralization

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