Study subjects and diagnosis
The study was approved by the Ethics Committee of the Pulmonary Hospital of Lanzhou (Lan fei lun: 2020-01). Serum samples were collected from 97 patients with COVID-19 and 41 patients with suspected COVID-19, as diagnosed by SARS-CoV-2 RT-PCR. Patients were treated at the Pulmonary Hospital of Lanzhou (Lanzhou, China) and Nanjing Gulou Hospital (Nanjing, China); 88 patients had general fever or pulmonary infection (COVID-19-negative) in the outpatient service of 940 Hospital of Chinese People’s Liberation Army Joint Logistic Support, 61 had Mycoplasma pneumoniae infection, 125 patients with respiratory tract infection were treated at the 940 Hospital, 48 doctors and nurses worked in the fever outpatient service of the 940 hospital (COVID-19-negative), and 56 health professionals underwent a medical examination in the health examination center of Gansu Province Tumor Hospital (Gansu Province, China).
Establishment of recombinant plasmids encoding the N and S1 fragments of SARS-CoV-2
Full-length N and S1 proteins of SARS-CoV-2 were synthesized according to the published sequence (General Biosystems (Anhui) Co. Ltd., Anhui, China). The full-length N gene was ligated into the pET-28a vector, which was used to transform DH5α E. coli cells. Colonies were selected on Luria Bertani (LB) agar plates containing 30 µg/mL kanamycin. Recombinant plasmids were extracted using a Plasmid DNA Miniprep Kit (Qiagen, Hilden, Germany), and analyzed for the presence of the N gene by sequencing and the use of BamH I and Xho I restriction enzymes. The recombinant plasmid expressing the S1 gene was acquired and analyzed by the same procedure, except that the pET-30a vector and BamH I and Apa I restriction enzymes were used. To express the N protein and S1 protein, recombinant plasmids encoding the N protein (pET-28a-SARS-CoV-2-N) and S1 protein (pET-30a-SARS-CoV-2-S1) were transformed into E. coli BL21 (DE3) cells. Transformants were selected on kanamycin (30 µg/mL) plates. BL21 (DE3) cells containing recombinant plasmids were cultivated in 5 mL LB broth and the protein expression was induced by the addition of 1 mM IPTG, followed by shaking for 3 h at 37 °C. The bacterial pellet was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to analyze expression of the recombinant protein.
SDS-PAGE analysis
To analyze expression of the recombinant N protein, 100 mL of the induced cultures were centrifuged and re-suspended in 10 mL sample buffer (10 mM phosphate-buffered saline (PBS), pH 7.4) and sonicated 60 times with 5-min pulses at 5-min intervals. The supernatant and pellet were separated by centrifugation at 10,000 rpm at 4 °C for 20 min. The pellet was washed once using 2 M urea and centrifuged. Finally, the pellet was dissolved in 8 M urea, after which the supernatant, 2 M urea solution, and 8 M urea solution were analyzed by SDS-PAGE. To analyze expression of the recombinant S1 protein, 100 mL culture was sonicated in buffer (50 mM Tris, pH 8.5) 100 times with 5-min pulses at 5-min intervals. The supernatant and pellet were separated by centrifugation at 10,000 rpm at 4 °C for 20 min, and the pellet was washed with 2 M urea and dissolved in 8 M urea as above. Finally, the supernatant, 2 M urea solution, and 8 M urea solution were analyzed by SDS-PAGE.
Purification of recombinant N protein and S1 protein
To purify the N protein, after sonication, the supernatant was loaded onto the Ni-NTA column and eluted with 10 mM PBS (pH 7.4) containing 100 mM imidazole. The purified recombination N protein was analyzed by SDS-PAGE. To purify the S1 protein, the pellet was re-suspended in 10 mL of 2 M urea and centrifuged at 10,000 rpm at 4 °C for 20 min. The supernatant was discarded and the pellet was dissolved in 6 mL of 8 M urea. To obtain active proteins, the denatured recombination protein was re-folded in 50 mM Tris (pH 8.5) buffer containing 150 mM NaCl by decreasing the concentration of urea from 8 M to 3 M. The refolded protein was purified by gel filtration on the Sephacryl S-300 column (US Pharmacia International, Inc., Rockville, MD, USA) and analyzed by SDS-PAGE.
Preparation of SARS-CoV-2 antibody lateral flow assay with purified recombinant N protein
To prepare a recombinant N protein-colloidal gold conjugate, 1.2 mg/mL purified recombinant N protein was added to 100 mL of 0.1% (w/v) colloidal gold (pH 8.5, adjusted using 0.2 M potassium carbonate buffer), followed by 20 min of magnetic stirring and subsequent addition of 500 mg bovine serum albumin (BSA). After 10 min of magnetic stirring and 2 h incubation at 4 °C, the colloidal gold solution was centrifuged at 1000 rpm and 4 °C for 60 min. Then the supernatant was discarded, and the sediment (recombinant N protein-colloidal gold conjugate) was re-dissolved in 0.02 M (pH 7.4) Tri-HCI buffer containing 0.5% BSA (w/v) and 0.1% ProClin (w/v), and stored at 4 °C before use. The anti-human IgG monoclonal antibody (mAB)-colloidal gold conjugate was prepared using the same procedure as that used for the recombinant N protein-colloidal gold conjugate. The recombinant N protein-colloidal gold conjugate and anti-human IgG mAb-colloidal gold conjugate were mixed at a 1:1 ratio and sprayed on fiberglass (1 × 30 cm) by a special coating machine. The sprayed fiberglass was dried at 25 °C for more than 15 h and used as a conjugate pad. To prepare the reaction membrane, 1.2 mg/mL recombinant N protein and 1 mg/mL anti-mouse IgG (Shanghai JieNing Biotechnology Co. Ltd., Shanghai, China) were immobilized at the test line (T line) and control line (C line) on a nitrocellulose membrane (Vivid™ 170; Pall Life Sciences, Port Washington, NY, USA) using a special coating machine. The reaction membrane was solidified at 45 °C for 1 h. The lateral flow assay consisted of five parts: plastic backing, sample pad, conjugate pad, absorbent pad, and reaction membrane. The sample pad was 1.8 × 30 cm fiberglass (No. 8964; Hangzhou Hangan Biotechnology Co. Ltd., Zhejiang, China) pre-treated with 0.02 M (pH 7.4) containing 1% BSA (w/v) and 0.5% Tween-20 (v/v). The absorbent pad was 300 g general filter paper. The lateral flow assay is summarized in Fig. 1a. The individual assays (3 × 65 mm) were done in a special plastic box (Fig. 2a) and sealed in aluminum pouches with a silica desiccant pellet. The packages were stored at room temperature.
Preparation of the SARS-CoV-2 IgM antibody lateral flow assay with purified recombinant S1 protein
The recombinant S1 protein-colloidal gold conjugate was prepared using the same procedure as described above. The recombinant S1 protein−colloidal gold conjugate and anti-human IgG mAb−colloidal gold conjugate were also mixed at a 1:1 ratio and sprayed on a fiberglass (1 × 30 cm) to prepare the conjugate pad using the same procedure as above. The reaction membrane was prepared as above, except that 1 mg/mL anti-human IgM (Lanzhou Rujie Biotechnology Co. Ltd., Lanzhou, China) was immobilized at the T line. The structure and components of the SARS-CoV-2 IgM antibody lateral flow assay were the same as those for the SARS-CoV-2 antibody lateral flow assay (Fig. 1b).
Sample testing
The test began immediately after the pouch was opened. Briefly, 10 L serum or plasma samples were pipetted into the sample port of the test card followed by the addition of two drops (80−100 µL) of dilution buffer (0.01 M PBS, pH 7.2). The test result was acquired in 20 min. The sample was positive when the C line and T line both appeared red, and was negative when only the C line was red. If the C line did not appear red, the test was invalid, and the sample was tested using another test card (Fig. 2b).
Statistical analysis
The specificity and sensitivity of the lateral flow assay were calculated according to the following formulas: specificity (%) = [true negative numbers/(true negative numbers + false positive numbers)] × 100%: sensitivity (%) = [true positive numbers/ (true positive numbers + false negative numbers)] × 100%. Analysis was performed using SPSS 23.0, and the chi-square test was applied to compare two proportions. p < 0.05 was considered to be statistically significant.