Serum samples
Serum samples from 180 healthy volunteers collected before the SARS-CoV-2 infection outbreak at the Guizhou Provincial People’s Hospital. Additionally, 182 serial serum samples collected from 88 patients diagnosed with COVID-19 collected in Guizhou province. Of the COVID-19 patients, 42 were male and 46 were female. The age distribution of these patients ranged from 5 months to 84 years old. The patients had complications recorded including hypertension, diabetes, heart disease, surgery, chronic bronchitis, chronic gastritis, tuberculosis and hepatitis B virus infection.
According to diagnosis and treatment protocol for novel Coronavirus pneumonia (Trial Version 8) which was released by the National Health Commission & State Administration of Traditional Chinese Medicine, defined the clinical classification of asymptomatic COVID-19 patients as those who have no clinical symptoms, yet test positive for SARS-CoV-2 using respiratory tract and other specimens or have a positive IgM antibody test; mild cases were defined as those who had symptoms but no sign of pneumonia by chest X-ray imaging; moderate cases have fever and respiratory symptoms with radiological findings of pneumonia; severe cases and critical cases were typified by a severe respiratory response that require ICU care and failure of other organs .
Expression and purification of the recombinant SARS-CoV-2-N protein
SARS-CoV-2 cDNA was prepared in BCL3 laboratory at the Nagasaki University, Japan. Briefly, SARS-CoV-2 virus TY-WK-521/20202 strain (Genebank accession number: LC522975) was isolated from a Japanese patient and propagated in a Vero cell line maintained at 37 °C in Eagle’s minimum essential medium supplemented with 2% fetal calf serum and 0.2 mM of each nonessential amino acid for four days. The viral RNA was extracted from the infected culture media and cDNA synthesized using a cDNA synthesize kit with random primers (Takara, JP). The full length SARS-CoV-2 N gene was amplified by RT-PCR using primers 5′- CAAGGATCCATGTCTGATAATGGACCCCAA-3′ (Bam HI site underlined) and 5′- TGCGTCGACTTAGGCCTGAGTTGAGTC-3′ (Sal I site underlined). The PCR product was successfully cloned into the BamHI and Sall sites of the pET28a expression vector. The sequence and reading frame of the N gene within the recombinant plasmid was confirmed by DNA sequencing. The rSARS-CoV-2-N protein was produced by inserting the recombinant plasmid into E. coil (strain BL-21), which was cultured at 37 °C in Luria-Bertani (LB) medium containing 50 μg/ml of kanamycin. When the optical density at 600 nm (OD600) of the culture reached 0.5, the expression of the recombinant proteins was induced by the addition of 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 5 h. The cells were harvested by centrifugation, washed in phosphate-buffered saline (PBS) solution, and resuspended in 10 mM PBS PH 7.5 with 500 mM NaCl and frozen at − 80 °C. After freezing and thawing three times, the cell suspension was sonicated for 10 min with an interval of 3 s between pulses and centrifuged at 13,000 g for 30 min at 4 °C. The supernatant was passed through a Talon™ IMAC resin column (Clontech, US). After being washed with binding buffer (10 mM PBS with 500 mM NaCl containing 30 mM imidazole, PH 7.5), the purified protein was eluted using elution buffer (10 mM PBS with 500 mM NaCl containing 250 mM imidazole, PH 7.5). The protein solution was aliquoted and stored at a final concentration of 10% glycerol at − 80 °C until use. The protein concentrations were determined by the Bradford method using a Bio-Rad protein assay reagent kit (Bio-Rad, USA), and the purity of the protein was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).
Western blot analysis
A western blot analysis was done as mentioned [27]. Briefly, the proteins were separated in a 10% polyacrylamide gel were transferred to a polyvinylidene difluoride (PVDF) membrane (Sigma-; US) using a semidry electroblotter (Sartorius, DE). The membrane was blocked with PBS-T with 5% skimmed milk (BD, US) overnight at 4 °C to prevent nonspecific staining. After which, the membrane was exposed to reaction with rSARS-CoV-2-N-immunized mouse serum (1:800 dilution), or COVID-19 patient serum (1:400 dilution) for 1 h at 37 °C; and subsequently incubated with horseradish peroxidase conjugated-goat anti-mouse IgG, or anti-human IgG (1:1000 dilution) for 1 h at 37 °C. The reaction was visualized by dimethyl amino benzidine (DAB) staining.
Checkerboard titration of rSARS-CoV-2-N based IgG ELISA
To evaluate the usefulness for diagnosis of the rSARS-CoV-2-N protein, an indirect IgG ELISA was established for the laboratory diagnosis of SARS-CoV-2 infection in human’s serum sample. The rSARS-CoV-2-N protein was used as antigen. The optimal concentrations of recombinant nucleocapsid protein were determined by checkerboard titration with different dilutions of coating recombinant protein. Briefly, purified rSARS-CoV-2-N protein was diluted in a 2-fold serial dilution started from 200 ng/well/100 μl to 12.5 ng/well/100 μl in phosphate buffered saline (PBS) and coated to ELISA plate overnight at 4 °C respectively. After washing the plate, 2 positive and 2 negative serum samples were diluted in a 2-fold serial dilution started from 1:100 to 1:800 and 100 μl of the diluted sera was reacted with the coated rSARS-CoV-2-N protein wells at 37 °C for 1 h respectively. After washing the plate, 100 μl of 1:30,000 diluted horseradish peroxidase-conjugated goat anti-human IgG was added to each well and reacted at 37 °C for 1 h. After washing the plate, the color was developed by TMB substrate and the OD value of each well recorded.
ELISA procedures using the recombinant nucleocapsid protein
To evaluate the diagnostic utility of the rSARS-CoV-2-N protein as an antigen in an indirect IgG ELISA to detect SARS-CoV-2 infection in human serum sample. The optimal concentrations of recombinant nucleocapsid protein were determined by checkerboard titration with different dilutions of recombinant protein coating. Ninety-six-well Nunc immunoplates (Nest Biotechnology, CN) were coated with 50 ng recombinant nucleocapsid protein antigen per ELISA well in 100 μl phosphate buffered saline (PBS) overnight at 4 °C. After the immunoplates were washed three times with PBS-Tween 20, 100 μl of 1:400 human serum diluted in PBS-Tween 20 with 5% nonfat milk (BD, US) was add to each well and incubated for 1 h at 37 °C. The plates were washed six times with PBS-Tween 20 and incubated for 1 h at 37 °C with 100 μl 1:30,000 horseradish peroxidase-conjugated goat anti-human IgG (American Qualex, US). After washing six times with PBS-Tween 20, 100 μl of TMB single-component substrate solution (Beijing Solarbio Science & Technology, CN) was added to each well and incubated in the dark at room temperature for 30 min. The reaction was stopped by adding 100 μl HCI (1 mol/L) to each well. OD values at 450 nm were recorded on a continuous wavelength ELISA reading instrument (Epoch). Each serum sample was tested in duplicate, and the mean OD for each sample was calculated. Any OD more than twice the mean OD of the negative control serum was considered positive.
Serum samples collected at fifth week after the onset of the disease were used to compare the IgG antibody difference of the COVID-19 patients with different severity. ELISA titers for these sera were calculated from standardized reciprocal dilution values using Thermo-Labsystem’s Ascent photospectrometric data analysis software, version 2.6 according to an established protocol [20].
Detection of SARS-CoV-2 IgG antibody by colloidal gold kit
The detection of serum IgG antibodies for SARS-CoV-2 using the colloidal gold kit (INNOVITA, CN) which used colloid gold labeled SARS-CoV-2 N and S fusion protein as an indicator was conducted according to the manufacturer’s instructions. Briefly, 10 μl of serum was added to the sample hole, 2 drops of serum sample diluent was added immediately, the result was observed within 15 min. If a clear purple-red band appeared on the T line and C line of the interpretation window, it is judged as the IgG antibody positive. If only the C line has the purple-red band, it is judged as the IgG antibody negative.
Detection of SARS-CoV-2 IgG antibody by SARS-CoV-2 spike S1 protein ELISA kit
The detection of serum IgG antibodies for SARS-CoV-2 using the SARS-CoV-2 spike S1 protein IgG antibody ELISA Kit (ABclonal, CN) was done according to the manufacturer’s instructions. Briefly, 100 μl of control antibody and 1:100 serum samples diluted in dilution buffer was add to each well of the 96 well plate coated with SARS-CoV-2 spike S1 protein and incubated for 2 h at 37 °C, then the plates was washed three times with wash buffer, 100 μl of secondary antibody working solution was added to each well, and the plates were incubated at 37 °C for 1 h. After three times washing with wash buffer, 100 μl substrate solution was added to each well and incubated in the dark at 37 °C for 15–20 min. The reaction was then stopped by adding 50 μl of stop solution. Finally, the OD values at 450 nm were recorded on a continuous wavelength ELISA reading instrument (Epoch) within 5 min. OD value more than twice the negative control was considered positive.
Statistical analysis
The data was presented using the mean of the original data after logarithmic conversion. The significance of the differences between the groups was determined using a one-way ANOVA, the differences between the N-based IgG ELISA and colloid gold antibody detection kit, the differences between the N-based IgG ELISA and S1 protein IgG ELISA kit were determined by the McNemar square test, with SPSS version 22.0.
