SARS-CoV-2 Serologic Assays in Control and Unknown Populations Demonstrate the Necessity of Virus Neutralization Testing

Abstract

Background: To determine how serologic antibody testing outcome links with virus neutralization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we evaluated individuals for SARS-CoV-2 antibody level and viral neutralization.

Methods: We compared serum Ig levels across platforms of viral antigens and antibodies with 15 positive and 30 negative SARS-CoV-2 controls followed by viral neutralization assessment. We then applied these platforms to a clinically relevant cohort of 114 individuals with unknown histories of SARS-CoV-2 infection.

Results: In controls, the best-performing virus-specific antibody detection platforms were SARS-CoV-2 receptor binding domain (RBD) IgG (sensitivity 87%, specificity 100%, positive predictive value [PPV] 100%, negative predictive value [NPV] 94%), spike IgG3 (sensitivity 93%, specificity 97%, PPV 93%, NPV 97%), and nucleocapsid protein (NP) IgG (sensitivity 93%, specificity 97%, PPV 93%, NPV 97%). Neutralization of positive and negative control sera showed 100% agreement. Twenty individuals with unknown history had detectable SARS-CoV-2 antibodies with 16 demonstrating virus neutralization. Spike IgG3 provided the highest accuracy for predicting serologically positive individuals with virus neutralization activity (misidentified 1/20 unknowns compared to 2/20 for RBD and NP IgG).

Conclusions: The coupling of virus neutralization analysis to a spike IgG3 antibody test is optimal to categorize patients for correlates of SARS-CoV-2 immune protection status.

Keywords: SARS-CoV-2; antibody; immunity; neutralization; serologic; two-tiered testing.

Figure 1.

Comparison of ELISA IgG platforms. A, Positive and negative control serum sample total IgG antibody recognition of SARS-CoV-2 RBD, spike protein, and UV-inactivated SARS-CoV-2. B, Unknown serum samples total IgG RBD and spike assays. Mean ± SD, Student t tests for comparisons of mean of groups. Dotted lines represent cutoff of 3 SD from the mean of the negative control samples to designate “positive” antibody samples. Abbreviations: ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; RBD, receptor-binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 2.

Comparison of ELISA antibody isotype performance. A, IgM antibody recognition of SARS-CoV-2 RBD and spike proteins of all serum samples. B, IgG1, IgG3, and IgA antibody recognition of spike protein of all samples. Mean ± SD, Student t tests for comparisons of mean of groups. Dotted line represents cutoff of 3SD from the mean of the negative control samples to designate “positive” antibody samples. C, IgG SARS-CoV-2 Abbott NP chemiluminescent microparticle immunoassay results, assay quantitative measurement output, > 1.39 = positive result. Abbreviations: ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; NP, nucleocapsid protein; RBD, receptor-binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 3.

PRNT results. A, Linear regression analyses correlating magnitude of neutralization PRNT₈₀ to magnitude of ELISA serum: spike IgG, RBG IgG, NP IgG, and spike IgG3. B, PRNT₈₀ results for all samples and for those meeting neutralization criteria. Mean ± SD, Student t tests for comparisons of mean of 2 groups and ANOVA analyses for difference among means of 3 groups. Abbreviations: ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; NP, nucleocapsid protein; PRNT₈₀, 80% plaque reduction neutralization test; RBD, receptor-binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 4.

A summary of ELISA and PRNT testing performance across all platforms in the setting of unknown SARS-CoV-2 exposure individuals (n = 20). A, All of the ELISA platforms are compared based on ELISA assay detection and correlation with detectable neutralization via PRNT. B, Comprehensive comparison of the 3 assays best at detecting antibodies with neutralizing activity. Light color denotes positive ELISA group B subject; dark color denotes positive ELISA, SARS-CoV-2 group A subject; PRNT% agreement denotes number of ELISA positives in agreement with PRNT positives. MI denotes misidentified outcomes in which incongruent ELISA results with neutralization detection are not encompassed by the %PRNT agreement calculation. Number ranges within parentheses indicate 95% confidence intervals for the adjacent values. Abbreviations: ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; NP, nucleocapsid protein; PRNT, plaque reduction neutralization test; RBD, receptor-binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; N/A, not applicable.

Figure 5.

Assessment of coupled, sequential antibody tests to increase performance. Testing performance was assessed for 4 possible combinations of ELISA platforms to increase the ability to detect and predict neutralizing antibodies. All the coupled combinations were compared to spike IgG3 as the best performing single ELISA platform. Light color denotes positive ELISA group B subject; dark color denotes positive ELISA, SARS-CoV-2 group A subject; PRNT% agreement denotes number of ELISA positives in agreement with PRNT positives. MI denotes misidentified outcomes in which incongruent ELISA results with neutralization detection were not encompassed by the %PRNT agreement calculation. Abbreviations: ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; NP, nucleocapsid protein; PRNT, plaque reduction neutralization test; RBD, receptor-binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 6.

Study subject description and serologic prevalence estimates. A, A map of Seattle with circles denoting the zip codes of the subjects involved in the study. Underlying map colors represent the population density for the areas shown. B, Number of subjects, exposure status, and reported symptoms for positive subjects. Group A and B unknown subjects had never been diagnosed with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Group A subjects had close contact with a known infected SARS-CoV-2 individual. Group B subjects had no known exposure to SARS-CoV-2 infected individuals. *Subjects reported symptoms within 5 days of exposure to SARS-CoV-2–positive individuals. **Subjects reported symptoms over the possible exposure window in the Seattle area (21 January to 15 April 2020).