SARS-CoV-2 S1 and N-based serological assays reveal rapid seroconversion and induction of specific antibody response in COVID-19 patients

Abstract

As the Coronavirus Disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2, continues to spread rapidly around the world, there is a need for well validated serological assays that allow the detection of viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used multiple indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological assays to study the antibody response in COVID-19 patients. In order to validate the assays we determined the cut off values, sensitivity and specificity of the assays using sera collected from pre-pandemic healthy controls, COVID-19 patients at different time points after disease-onset, and seropositive sera to other human coronaviruses (CoVs). The developed SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N)-based ELISAs not only showed high specificity and sensitivity but also did not show any cross-reactivity with other CoVs. We also show that all RT-PCR confirmed COVID-19 patients tested in our study developed both virus specific IgM and IgG antibodies as early as week one after disease onset. Our data also suggest that the inclusion of both S1 and N in serological testing would capture as many potential SARS-CoV-2 positive cases as possible than using any of them alone. This is specifically important for tracing contacts and cases and conducting large-scale epidemiological studies to understand the true extent of virus spread in populations.

Figure 1

SARS-CoV-2 recombinant proteins and cut-off values for the developed ELISAs. Recombinant SARS-CoV-2 (a) S1 or (b) N proteins were detected by Western blot using anti-His tag antibodies, known seropositive COVID-19 human samples, or known seronegative COVID-19 human samples. All experiments showed protein bands with expected sizes (~ 110 kDa and ~ 46 kDa for S1 and N, respectively). A 100 serum samples from healthy controls collected before the COVID-19 pandemic were used to determine the cut-off values for (c) S1 IgG-ELISA, (d) rS1 IgM-ELISA, (e) N IgG-ELISA and (f) N IgM ELISA. Values were calculated as mean + 3SD. The square is a serologically positive sample from COVID-19 patient. The dotted lines represent the cut-off of each assay.

Figure 2

The specificity of the developed ELISAs. (a) Sequence homology analysis of SARS-CoV-2 N protein and S1 subunit compared to other human coronaviruses. (b) Developed ELISAs were tested for their specificity using sera known to be seronegative for SARS-CoV-2 and MERS-CoV (HC; n = 8), seropositive sera for MERS-CoV (MERS; n = 2) or seropositive sera for SARS-CoV-2 (COVID-19; n = 3). These serum samples were also tested for their reactivity in IgG and IgM ELISAs developed for MERS-CoV S1 and N proteins, as well as full S protein from hCoV-OC43, hCoV-NL63, hCoV-229E, and hCoV-HKU1 viruses. The dotted lines represent the cut-off of each assay. The cut-off values for hCoV-OC43, hCoV-NL63, hCoV-229E, and hCoV-HKU1 ELISAs were set at arbitrary value = blank mean + 3SD.

Figure 3

Humoral immune response to COVID-19. Serum samples from healthy controls (n = 125) or COVID-19 patients collected during the 1st week (n = 10), 2nd week (n = 23), 3rd week (n = 14), or 4th week (n = 5) of symptoms-onset were tested for IgG and IgM against SARS-CoV-2 S1 (a,b) and N (c,d) proteins using the developed ELISA. The dotted lines represent the cut-off of each assay. Correlation of S1 IgG (e), S1 IgM (f), N IgG (g) and N IgM (h) with days after symptom onset. Comparison of IgM and IgG for each patient based on the time of collection for S1 antibodies (i) and N antibodies (j).

Figure 4

Receiver operating characteristics (ROC) analysis. ROC analysis was applied to positive vs. negative SARS-CoV-2 samples as identified by RT-PCR assay for (a) S1 IgG-ELISA, (b) S1 IgM-ELISA, (c) N IgG-ELISA and (d) N IgM ELISA. Serum samples from healthy controls (n = 125) or COVID-19 patients collected during the 1st week (n = 10), 2nd week (n = 23), 3rd week (n = 14), or 4th week (n = 5) of symptoms-onset as well as all COVID-19 samples (n = 52). Correlation of (e) S1 and N IgG antibodies and (f) S1 and N IgM antibodies.