A high-throughput test that can predict neutralizing activities is a critical step toward understanding herd immunity to covid-19.

By Lily Li, MD, PhD

The 2020 pandemic put the entire scientific and medical community to test with the urgent need to understand this novel virus (SARS-CoV-2) and the disease that it causes (covid-19).1,2 While the diagnostic industry responded to the call in record speed and gained substantial information, an enduring focus is to understand whether patients recovered from covid-19 will develop protection or immunity.3,4 Knowledge from the past year about acquired immunity to covid-19 not only provides valuable insights for the development of vaccines and therapeutics but also enables the implementation of suitable policies and strategies for effective pandemic control.5,6  

What Do We Mean by Immunity? And What Are Neutralizing Antibodies? 

Immunity is the ability to resist a disease or an infection. Immunity to viral infection is a combined outcome of both cellular and humoral (antibody) immune responses. While cellular immune response is often difficult to measure, antibody production serves as a hallmark of humoral immune response.6

Neutralizing antibodies, a subset of antibodies, can inhibit viral replication and represent a major mechanism of humoral immunity against viral infection.7 The protective function of neutralizing antibodies is mainly mediated by the blocking of the interaction between the virus and its host cells, resulting in the inhibition of viral entry to cells, thus preventing infection.7 Because of the mechanism of action, it is not surprising that most neutralizing antibodies are against viral surface proteins.7

Neutralizing Antibodies to SARS-CoV-2

The virus that causes covid-19, SARS-CoV-2, uses the spike protein (S) to bind to the receptor on host cells to trigger cell entry and infection. S protein consists of S1 and S2 subunits, and S1 interacts with the host cells via the receptor binding domain (RBD).8 Monoclonal antibodies to S1 protein that exhibit neutralizing activities are being developed as potential therapeutics for covid-19.9,10 Neutralizing antibodies to the S protein are the key active ingredients of convalescent plasma used to treat severe covid-19 patients.11 In addition, almost all the covid-19 vaccines, both approved and currently under development, target the S protein with the goal to induce neutralizing anti-S antibodies.12 Earlier vaccine studies showed a correlation between neutralizing antibody titers and protective efficacy in animal models.13

All these observations suggest that S protein is a primary target for neutralizing antibodies against SARS-CoV-2, and anti-S neutralizing antibodies play a key role in covid-19 immunity.

Humoral immune response and antibody production after SARS-CoV-2 infection have not been fully understood in various covid-19 patient populations. Early studies clearly showed that not all covid-19 patients produced detectable antibodies and certainly not all patients developed neutralizing antibodies.14,15 Even among convalescent plasma donors, around 10% of samples did not contain detectable neutralizing activities, putting the therapeutic efficacy of the convalescent plasma in question.16 Therefore, measuring neutralizing antibody activities is important to ensure qualified convalescent plasmas are used to achieve desirable therapeutic outcomes as well as to support the development of covid-19 vaccines.16 In addition, given the heterogeneity of the immune responses among covid-19 patients, it is important to assess neutralizing antibody activities for individual patients as well as in populations to help to address the key questions about covid-19 immunity:

1. What is the neutralizing antibody level sufficient to provide complete protection?

2. How long does the protection last?

Measuring Neutralizing Antibodies

Neutralizing antibody activity is typically measured by biological assays mimicking viral infection in cultured cells. The tests are time-consuming, labor-intensive, and low-throughput. The operational complexities of the tests make them unfeasible for scaled up routine testing in large populations.16,17 

A 2019 publication reported a modified neutralization assay with increased throughput. However, the test system still requires viral particles and live cells, which can be difficult to implement in a typical clinical laboratory.18 Therefore, there is a clear need to develop automated, high-throughput, and easy-to-operate serological tests as potential surrogate tests to evaluate neutralizing antibody activities in a large patient population.

How Can Neutralizing Antibodies Be Assessed More Easily? 

In responding to this need, a group of scientists and physicians at New York Blood Center evaluated six different serological tests for their correlation with neutralization assays.16 The tests included in this study represent a variety of technology platforms (lateral flow assay, enzyme-linked immunosorbent assay [ELISA], and automated chemiluminescent tests) with different viral targets (S1, nucleocapsid [N], and RBD). The Vitros Anti-SARS-CoV-2 Total assay targeting the S1 protein of SARS-CoV-2 was among the six tests evaluated. 

The study tested 370 convalescent plasmas on the serological tests, and the results were correlated with neutralizing titers generated from two different neutralization assays.16 In general, ELISA and chemiluminescent assays showed better correlation with neutralizing titers than the lateral flow assay, and assays targeting the S1 protein exhibited better correlation than assays targeting the N protein. Among all six tests evaluated, the Vitros Anti-SARS-CoV-2 Total test demonstrated the strongest correlation with neutralizing titers and produced the highest predictive value for neutralization assays. 

The authors concluded that tests such as the Vitros Anti-SARS-CoV-2 Total test may thus serve to predict antiviral activity against SARS-CoV-2.16 S protein is a primary target for neutralizing antibodies against SARS-CoV-2, and anti-S neutralizing antibodies play a key role in covid-19 immunity. 

It is expected that the Vitros Anti-SARS-CoV-2 Total test that targets the S1 protein demonstrates the strongest correlation with neutralization assays because most, if not all, neutralizing antibodies bind to S1 protein and should be detected by the Vitros assay. Assays targeting the N protein, on the other hand, rely mainly on indirect correlations. 

In the New York Blood Center study,16 14% of the convalescent plasma samples contained only anti-N but no detectable anti-S antibodies, which would make it hard to use results from an anti-N assay to predict neutralizing titers in these samples.

In addition, the high sensitivity, semi-quantitative capability, and the wide dynamic measuring range of the Vitros Anti-SARS-CoV-2 Total test are all crucial design features enabling more accurate prediction of a wide range of neutralizing titers observed among convalescent plasma donors in this study.16

Lily Li, MD, PhD, Ortho Clinical Diagnostics.

The validation of suitable serological tests as surrogates for SARS-CoV-2 neutralization assays is a substantial advancement in antibody testing. It makes the assessment of neutralizing activities in a large patient population possible to gain better understandings of community-based immunity or herd immunity. As it is still early in the covid-19 pandemic, understandably there are still major knowledge gaps of immunity to SARS-CoV-2.

We do not yet know the minimum neutralizing activity required for protection from reinfection or the sustainability of the protection. However, the possibility of predicting neutralizing activities at both individual and population level using an automated high-throughput, high-performance serological test will help us better understand covid-19 immunity. l

Lily Li, MD, PhD, is the medical safety officer at Ortho Clinical Diagnostics responsible for evaluating potential medical risks and guiding risk mitigation of all marketed products. She also serves as a director at medical and scientific affairs, providing evidence-based support to Ortho’s current and future products. Li obtained her medical degree from Peking University, China, and her PhD in immunology from the University of Alberta, Canada. She completed her postdoctoral training at the Scripps Research Institutes, San Diego. Li is the author of more than 40 scientific articles and has filed 17 patent applications. 


1. World Health Organization. Coronavirus disease (covid-19) pandemic. Available at: www.who.int/emergencies/diseases/novel-coronavirus-2019. Accessed January 7, 2021. 

2. Centers for Disease Control and Prevention. Covid-19. Available at: www.cdc.gov/coronavirus/2019-ncov/index.html. Accessed January 7, 2021.

3. Sethuraman N, Jeremiah SS, Ryo A. Interpreting diagnostic tests for SARS-CoV-2. JAMA 2020;323(22):2249-2251. doi: 10.1001/jama.2020.8259.

4. Melgacoa JG, Azamor T, Ano Bom APD. Protective immunity after covid-19 has been questioned: what can we do without SARS-CoV-2-IgG detection? Cell Immunol. 2020;353:104114. doi: 10.1016/j.cellimm.2020.104114.

5. World Health Organization. Scientific Brief: “Immunity passports” in the context of covid-19. April 24, 2020. Available at www.who.int/news-room/commentaries/detail/immunity-passports-in-the-context-of-covid-19. Accessed January 7, 2021.

6. Manners C, Bautista EL, Muacevic A, Adler JR. Protective adaptive immunity against severe acute respiratory syndrome coronaviruses 2 (SARS-CoV-2) and implications for vaccines. Cureus. 2020; 12(6):e8399. Doi: 10.7759/cureus.8399.

7. Murin CD, Wilson IA, Ward AB. Antibody responses to viral infections: a structural perspective across three different enveloped viruses. Nat Microbiol. 2019;4(5):734–747. doi:10.1038/s41564-019-0392-y.

8. Zhou G, Zhao Q. Perspectives on therapeutic neutralizing antibodies against the novel coronavirus SARS-CoV-2. Int J Biol Sci. 2020;16(10):1718-1723. doi: 10.7150/ijbs.45123.

9. Wang C, Li W, Drabek D, et al. A human monoclonal antibody blocking SARS-CoV-2 infection. Nat Commun.2020;11(1):2251. doi: 10.1038/s41467-020-16256-y.

10. Cao Y, Su B, Guo X, et al. Potent neutralizing antibodies against SARS-CoV-2 identified by high-throughput single-cell sequencing of convalescent patients’ B cells. Cell. 2020;182(1):73-84. doi: 10.1016/j.cell.2020.05.025.

11. Rojas M, Rodriguez Y, Monsalve DM, et al. Convalescent plasma in covid-19: Possible mechanisms of action. Autoimmun Rev. 2020;19(7);102554. doi: 10.1016/j.autrev.2020.102554.

12. Mullard A. COVID-19 vaccine development pipeline gears up. Lancet. 2020;395(10239):1751-1752. doi: 10.1016/S0140-6736(20)31252-6.

13. Yu J, Tostanoski LH, Peter L, et al. DNA vaccine protection against SARS-CoV-2 in rhesus macaques. Science.2020;369(6505):806-811. doi: 10.1126/science.abc6284.

14. Brochot E, Demey B, Touze A, et al. Anti-spike, anti-nucleocapsid and neutralizing antibodies in 1 SARS-CoV-2 hospitalized patients and asymptomatic carriers. Front Microbiol. 2020;11:2468. doi: 10.1101/2020.05.12.20098236.

15.Liu T, Wu S, Tao H, et al. Prevalence of IgG antibodies to SARS-CoV-2 in Wuhan – implications for the ability to produce long-lasting protective antibodies against SARS-CoV-2. medRxiv. Epub. June 6, 2020 doi: 10.1101/2020.06.13.20130252.

16. Luchsinger LL, Ransegnola B, Jin D, et al. Serological analysis of New York City covid-19 convalescent plasma donors. medRxiv. Epub. June 9, 2020. doi: 10.1101/2020.06.08.20124792.

17. Crawford KHD, Eguia R, Dingens AS, et al. Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 spike protein for neutralization assays. Viruses. 2020;12(5):513. doi: 10.3390/v12050513.

18. Vandergaast R, Carey T, Reiter S, et al. Development and validation of IMMUNO-COVTM: a high-throughput clinical assay for detecting antibodies that neutralize SARS-CoV-2. bioRxiv. Epub. May 27, 2020. doi: 10.1101/2020.05.26.117549.