Expression and purification of recombinant SARS-CoV-2 nucleocapsid protein (N-protein)
A codon optimized synthetic gene expressing the full-length SARS-CoV-2 N-protein (Uniprot QHD43423.2) was obtained and cloned into pET28a by General Biosystems [6]. This plasmid was named pLHSarsCoV2-N and transformed into E. coli BL21 (λDE3) enabling the expression of N-terminal His-tagged N-protein after induction with IPTG. The cells were grown in 100 ml LB medium at 120 rpm at 37 °C to OD600nm of 0.4. The incubator temperature was lowered to 16 °C, after 30 min, IPTG was added to a final concentration 0.3 mM and the culture was kept at 120 rpm at 16 °C overnight. Cells were collected by centrifugation at 3000 × g for 5 min. The cell pellet was resuspended in 20 ml of buffer 1 (Tris–HCl pH 8, 50 mM, KCl 100 mM, glycerol 10%). Cells were disrupted by sonication on an ice bath. The soluble fraction was recovered after centrifugation at 20,000 × g for 20 min and loaded onto a 5 ml His-Trap column (Cytiva), which had been previously equilibrated with buffer 1. The column was washed with 10 ml of buffer 1, and bound proteins were eluted with buffer 1 containing increasing concentrations of imidazole. The His-tagged N-protein was eluted at 300 mM imidazole. The final protein preparation yield was ~ 8 mg and was homogeneous as judged by SDS-PAGE analysis. The purified protein was stored in aliquots at 4 °C for 1 week or at − 20 °C for up to 4 months.
Human samples
Samples were collected at the Hospital de Clínicas (CHC/UFPR), Hospital Erasto Gaertner, Hospital do Trabalhador, and Secretária Municipal de Saúde de Guaratuba. Samples for serological analysis comprised both serum and plasma-EDTA. COVID-19-positive cases were confirmed by the detection of SARS-CoV-2 RNA by RT-qPCR from nasopharyngeal sample swabs. The time point of sampling of serum ranged from 1 to 70 days after onset of symptoms. The cohort of 210 negative controls consisted of pre-pandemic samples. The Institutional Ethics Review Board of CHC/UFPR (n# 30,578,620.7.0000.0008), CEP/HEG (n# 31,592,620.4.1001.0098), CEP/HT (n# 31,650,020.5.0000.5225) and CEP/UFPR (n# 43946621.7.0000.0102) approved this study.
ELISA assays
The purified SARS-CoV-2 N-protein was diluted to 2 ng/µl in 50 mM Tris–HCl pH 8, 100 mM KCl, 10% (v/v) glycerol, and 0.1 ml aliquots were transferred to the wells of ELISA plates (Olen) and incubated overnight at 4 °C. Unbound excess protein was removed, and wells were washed twice with 0.2 ml of 1 × TBST buffer pH 7.6 (Tris 2.42 g.l−1, NaCl 8 g.l−1, and Tween 80 0.1% v/v). Wells were blocked with 0.2 ml 3% (w/v) skimmed milk in 1 × TBST overnight at 4 °C. The serum was diluted at 1:1000 in 1 × TBST containing 1% (w/v) skimmed milk, and 0.1 ml was loaded onto the ELISA duplicate wells following by incubation at room temperature for 1 h. The wells were washed three times with 0.2 ml of 1 × TBST followed by incubation with 0.1 ml of anti-human IgG-HPR from goat (Thermo Fisher—cat number 62–8420) diluted 1:3000 in 1 × TBST. Wells were washed three times with 0.2 ml of 1 × TBST. The HPR substrate TMB (Thermo Fisher—cat number 00–2023) was added to the wells (0.1 ml). The reaction was stopped after a 10-min incubation at room temperature by addition of 0.1 ml 1 M HCl. The plates were put on the top of a white light transilluminator device and photographed. Samples were run in duplicates and data were reported as the percent of a positive reference serum. The optical density was measured at 450 nm using a TECAN M Nano plate reader (TECAN) monochromator at bandwidth 9 nm and 25 flashes.
The commercial Euroimmun Anti-SARS-CoV-2 ELISA IgG test was used following the manufacturer’s instructions.
Data analysis
One COVID-19-positive serum was used as a positive control and reference throughout the study. All data were expressed as the percent of this positive control before applying receiver operating analysis using GraphPad Prism 7.0. Statistical analyses were performed using the t test.