SARS-CoV-2 Serum Neutralization Assay: A Traditional Tool for a Brand-New Virus

Abstract

SARS-CoV-2 serum neutralization assay represents the gold standard for assessing antibody-mediated protection in naturally infected and vaccinated individuals. In the present study, 662 serum samples collected from February 2020 to January 2021 from acute and convalescent COVID-19 patients were tested to determine neutralizing antibody (NAb) titers using a microneutralization test (MNT) for live SARS-CoV-2. Moreover, anti-SARS-CoV-2 IgG, IgA, and IgM directed against different viral antigens were measured by high-throughput automated platforms. We observed higher levels of NAbs in elderly (>60 years old) individuals and in patients presenting acute respiratory distress syndrome. SARS-CoV-2 NAbs develop as soon as five days from symptom onset and, despite a decline after the second month, persist for over 11 months, showing variable dynamics. Through correlation and receiver operating characteristic (ROC) curve analysis, we set up a testing algorithm, suitable for the laboratory workload, by establishing an optimal cutoff value of anti-SARS-CoV-2 IgG for convalescent plasma donors to exclude from MNT samples foreseen to have low/negative NAb titers and ineligible for plasma donation. Overall, MNT, although cumbersome and not suitable for routine testing of large sample sizes, remains the reference tool for the assessment of antibody-mediated immunity after SARS-CoV-2 infection. Smart testing algorithms may optimize the laboratory workflow to monitor antibody-mediated protection in COVID-19 patients, plasma donors, and vaccinated individuals.

Keywords: SARS-CoV-2; neutralizing antibodies; protective immunity; serology.

Figure 1

Neutralizing antibody titers in different ages (A) and COVID-19 severity (B) groups of symptomatic COVID-19 patients. P/F = arterial pO2 divided by the fraction of inspired oxygen (FiO2) used as correlate of diseases severity. Dot line indicates the limit of neutralizing antibody (NAb) quantification, n = number of patients tested. Statistical analysis was performed by Mann–Whitney test p values < 0.05, < 0.01, < 0.001 are indicated as *, **, and *** respectively.

Figure 2

Neutralizing antibody titers in serum samples of SARS-CoV-2-infected patients collected at different time from symptom onset. Dot line indicates limit of NAb quantification. fso = from symptom onset, n = number of patients tested. Statistical analysis was performed by Mann–Whitney test; p values < 0.05 is indicated as *.

Figure 3

Patterns of NAb persistence. NAb titers in longitudinally collected samples from patients with at least 6 months follow-up post-infection are shown in the panels. Data are grouped according to evolution of NAb levels over time: stable (upper panel), increasing (central panel), or decreasing (lower panel). Each dot represents one serum sample and each line indicates one patient. Dot line indicates limit of NAb quantification. fso = from symptom onset.

Figure 4

Anti-SARS-CoV2 IgG correlation to NAb titers. Total anti-SARS-CoV-2 (left panel), anti-Spike (anti-S) (central panel), and anti-Nucleoprotein (anti-N) (right panel) IgG levels were compared to NAb titers. Horizontal dot line indicates limit of NAb level quantification, vertical dot line indicates the limit of IgG detection. Spearman’s test was used for correlation analysis.

Figure 5

ROC curve analysis performed for anti-S IgG to detect high NAb titer (≥1:160) serum samples.