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.

Early Humoral Response Correlates with Disease Severity and Outcomes in COVID-19 Patients

“…Recombinant SARS-CoV-2 S1 subunit (amino acids 1-685) was purchased commercially (Sino Biological, Beijing, China). Recombinant SARS-CoV-2 N protein was expressed and purified in-house as previously described [18]. Indirect S1-based or N-based ELISA were performed for the detection of specific IgG and IgM as previously described [18].…”

“…Recombinant SARS-CoV-2 N protein was expressed and purified in-house as previously described [18]. Indirect S1-based or N-based ELISA were performed for the detection of specific IgG and IgM as previously described [18]. Briefly, Recombinant S1 and N proteins were used to coat 96-well high binding ELISA plates (Greiner Bio One, Monroe, NC, USA) at 1 and 4 µg/mL in phosphate-buffered saline (PBS) with 50 µL per well, respectively.…”

“…The reaction was stopped by 100 µL per well of 0.16 M sulfuric acid, and absorbance was measured at 450 nm using the ELx808™ Absorbance Microplate Reader (BioTek, Winooski, VT, USA). Cutoff values for these ELISA were previously determined as 0.17 for IgG S1-ELISA, 0.3 for IgM S1-ELISA, 0.4 for IgG N-ELISA, and 0.55 for IgM N-ELISA [18].…”

“…Another important viral protein capable of inducing strong immune response is the abundantly expressed nucleocapsid (N) protein [17,18]. However, antibodies elicited towards the N protein are not neutralizing and might not provide protection against infection as shown in SARS-CoV infection model [19].…”

“…In the past few months, several studies have focused on studying the humoral response against SARS-CoV-2 in COVID-19 patients, with antibodies against S1 and N antigens found to emerge as early as one week following disease onset and persist for at least three month after infection [18,[20][21][22][23][24]. Here, we studied the kinetics of SARS-CoV-2 specific antibodies to S1 and N viral proteins in blood samples collected between 4 to 70 days post-symptoms onset from a cohort of 87 COVID-19 patients with different disease presentations (i.e., mild, moderate or severe) or outcomes (i.e., survival vs death).…”