Severe Acute Respiratory Syndrome Coronavirus 2-Specific Antibody Responses in Coronavirus Disease Patients

Abstract

A new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has recently emerged to cause a human pandemic. Although molecular diagnostic tests were rapidly developed, serologic assays are still lacking, yet urgently needed. Validated serologic assays are needed for contact tracing, identifying the viral reservoir, and epidemiologic studies. We developed serologic assays for detection of SARS-CoV-2 neutralizing, spike protein-specific, and nucleocapsid-specific antibodies. Using serum samples from patients with PCR-confirmed SARS-CoV-2 infections, other coronaviruses, or other respiratory pathogenic infections, we validated and tested various antigens in different in-house and commercial ELISAs. We demonstrated that most PCR-confirmed SARS-CoV-2-infected persons seroconverted by 2 weeks after disease onset. We found that commercial S1 IgG or IgA ELISAs were of lower specificity, and sensitivity varied between the 2 assays; the IgA ELISA showed higher sensitivity. Overall, the validated assays described can be instrumental for detection of SARS-CoV-2-specific antibodies for diagnostic, seroepidemiologic, and vaccine evaluation studies.

Keywords: COVID-19; ELISA; HCoV; RBD; SARS-CoV-2; Severe acute respiratory syndrome coronavirus 2; antibodies; coronavirus; coronavirus disease 2019; human coronavirus; neutralization; nucleocapsid protein; receptor-binding domain; respiratory infections; serologic analysis; spike protein; viruses; zoonoses.

Figure 1

Kinetics of antibody responses against SARS-CoV-2 after infection. We tested 1 patient who had severe coronavirus disease 2019 (red) and 2 patients who had mild coronavirus disease 2019 (green and black) for antibody responses against A) S protein, B) S protein S1 subunit, C) S N-terminal (S1^A) domain, D) RDB, and E) N protein by using ELISAs. F) Virus-neutralizing antibodies were tested by using a PRNT₅₀. G, H) Reactivities of serum samples from the 3 patients at different time points against whole S (G) and S1 (H) of SARS-CoV-2, SARS-CoV, and MERS-CoV were tested by ELISAs. Dotted horizontal lines indicate ELISA cutoff values. MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleocapsid; OD, optical density; PRNT₅₀, 50% plaque reduction neutralization test; RBD, receptor-binding domain; S, spike; SARS-CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 2

Correlations between ODs of ELISAs and PRNT results for PCR-confirmed COVID-19 patients. A) S; B) S1; C) S1^A; D) RBD; E) N. Ten serum samples were collected 6–27 days after diagnosis from 3 COVID-19 patients in France. Dots indicate patients. Dotted horizontal lines indicate ELISA cutoff values. COVID-19, coronavirus disease 2019; N, nucleocapsid; OD, optical density; PRNT₅₀, 50% plaque reduction neutralization test; RBD, receptor-binding domain; S, spike; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 3

Validation of use of S1 (A, B), RBD (C), and N protein (D) ELISAs for detection of SARS-CoV-2–specific antibodies infections. Gray dots indicate specificity cohorts A–C, including healthy blood donors (n = 45), non-CoV respiratory infections (n = 76), and HCoV infections (n = 75); blue dots indicate non-SARS-CoV-2 zoonotic coronavirus infections (i.e., MERS-CoV [n = 7] and SARS-CoV [n = 2]); red dots indicate patients with severe COVID-19; and green and black dots indicate patients with mild COVID-19. Dotted horizontal lines indicate ELISA cutoff values. CoV, coronavirus; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleocapsid; OD, optical density; RBD, receptor-binding domain; RFU, relative fluorescence unit; S, spike; SARS-CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2; severe acute respiratory syndrome coronavirus 2.

Figure 4

Validation of 2 commercial ELISAs for detection of SARS-CoV-2–specific IgG (A, C, E, G) and IgA (B, D, F, H). A, B) Validation of the specificity of the 2 ELISA platforms; C, D) kinetics of antibody responses in 3 COVID-19 patients; E, F) cross-reactivity of HCoV-OC43 serum samples in commercial platforms; G, H) correlation between antibody responses detected by the ELISAs and the plaque reduction neutralization assay. Gray dots indicate specificity cohorts A–C, including healthy blood donors (n = 45), non-CoV respiratory infections (n = 76), and HCoV infections (n = 75); blue dots indicate non-SARS-CoV-2 zoonotic coronavirus infections (i.e., MERS-CoV [n = 7] and SARS-CoV [n = 2]); red dots indicate patients with severe COVID-19; and green and black dots indicate patients with mild COVID-19. Dotted horizontal lines indicate ELISA cutoff values. CoV, coronavirus; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleocapsid; OD, optical density; PRNT₅₀, plaque reduction neutralization assay; RBD, receptor-binding domain; RFU, relative fluorescence unit; S, spike; SARS-CoV-2; severe acute respiratory syndrome coronavirus 2.

Figure 5

Sensitivity of 2 commercial ELISAs for detection of SARS-CoV-2 specific IgG (A, C, E) and IgA (B, D, F). A, B) Kinetics of antibody responses in 9 COVID-19 patients from Germany; C, D) correlation between antibody responses detected by the ELISAs and the plaque reduction neutralization assay; E, F) kits were tested for specificity by using 18 serum samples from patients infected with HCoV (4 from patients infected with HCoV-229E, 3 from patients infected with HCoV-HKU1, 4 from patients infected with HCoV-NL63, and 7 from patients infected with HCoV-OC43), MERS-CoV (n = 3), and SARS-CoV (n = 3). Dotted horizontal lines indicate ELISA cutoff values. COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleocapsid; OD, optical density; PRNT₅₀, plaque reduction neutralization assay; RBD, receptor-binding domain; RFU, relative fluorescence unit; S, spike; SARS-CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2; severe acute respiratory syndrome coronavirus 2.