2.1 Patients and serum samples
A total of 154 serum samples from 13 patients with COVID-19 confirmed by RT-qPCR were collected at about three-day intervals during hospitalization. All of the enrolled patients were Chengdu residents and they were treated in Public Health Clinical Centre of Chengdu, one of the biggest designated hospitals for COVID-19 in Sichuan province. The study was approved by the Ethics Committee of Public Health Clinical Centre of Chengdu (ref. No. PJ-K2020-23-01). Due to the retrospective nature of the study, written informed consent was waived. All methods were carried out in accordance with relevant guidelines and regulations of Declaration of Helsinki.
2.2 RT-qPCR assay
RT-qPCR was applied for viral RNA detection every 2–3 days after patients’ admission. Briefly, nasopharyngeal samples were collected from suspected patients and placed into collection tubes with virus preservation solution. Viral RNA samples were extracted on the Automatic Nucleic Acid Extraction System (NP968C, Tianlong Science & Technology Co., Ltd, Xi’an, China) using the Virus DNA/RNA Isolation Kit (Tianlong Science & Technology Co., Ltd, Xi’an, China) in line with manufacturer’s instructions. A commercial RT-qPCR kit, 2019 Novel Coronavirus (2019-nCoV) RNA detection kit (Daan Gene Co., Ltd. of Sun Yat-Sen University, Guangzhou, China, registration no. 20203400063) was used for the detection of SARS-CoV-2 open reading frame 1 ab (ORF1ab) and nucleocapsid protein (N) genes [11-13]. Patients with one positive RT-qPCR result were recognized as SARS-CoV-2 positive. After symptom resolution, patients with two consecutive negative RT-qPCR results with a sampling time interval of at least 24 hours were defined as SARS-CoV-2 negative [3, 14].
2.3 SARS-CoV-2-specific antibodies and antigen detection
The chemiluminescent microparticle immunoassays (CMIA) were applied for the virus-specific TAb, IgG, IgM, IgA, and Ag detection following the manufacturer’s instructions (Innodx Biotech Co. Ltd., Xiamen, China, Chinese national medical devices registration approval numbers 20203400198 (TAb)).
For TAb detection, the double antigen sandwich CMIA strategy was applied. In the first step, each sample was mixed with the magnetic microparticles coated with recombinant SARS-CoV-2 antigen (M_Ag1). The specific TAb in the sample interacted with the recombinant antigen coated on the magnetic microparticles, and the ingredients not bound to the magnetic microparticles were washed and removed. In the second step, recombinant antigen-labeled acridinium ester (Ag2_AE) was added to form a "M_Ag1–TAb–Ag2_AE" complex. After washing, the substrate solution was added. The chemiluminescence reaction signal was measured and expressed as relative luminescence unit (RLU). RLU positively correlated with the specific antibodies or antigen levels in the serum sample.
For IgG detection, the indirect CMIA strategy was applied. In the first step, each sample was mixed with the magnetic microparticles coated with recombinant SARS-CoV-2 antigen (M_Ag1). The specific IgG in the sample interacted with the recombinant antigen coated on the magnetic microparticles, and the ingredients not bound to the magnetic microparticles were washed and removed. In the second step, anti-human IgG labeled acridinium ester (antiIgG_AE) was added to form a "M_Ag1–IgG–antiIgG_AE" complex. After washing, the substrate solution was added. The chemiluminescence reaction signal was measured and expressed as RLU.
For IgM detection, the capture CMIA strategy was applied. In the first step, each sample was mixed with the magnetic microparticles coated with anti-human IgM monoclonal antibody (M_antiIgM), where the specific IgM in the sample interacted with the anti-human IgM monoclonal antibody coated on the magnetic microparticles, and the ingredients not bound to the magnetic microparticles were washed and removed. In the second step, recombinant SARS-CoV-2 antigen-labeled acridinium ester (Ag2_AE) was added to form a "M_antiIgM–IgM–Ag2_AE" complex. After washing, the substrate solution was added. The chemiluminescence reaction signal was measured and expressed as RLU.
For IgA detection, the indirect CMIA strategy was applied. In the first step, each sample was mixed with the magnetic microparticles coated with recombinant SARS-CoV-2 antigen (M_Ag1), where the specific IgA in the sample interacted with the recombinant antigen coated on the magnetic microparticles, and the ingredients not bound to the magnetic microparticles were washed and removed. In the second step, anti-human IgA labeled acridinium ester (antiIgA_AE) was added to form a "M_Ag1–IgA–antiIgA_AE" complex. After washing, the substrate solution was added. The chemiluminescence reaction signal was measured and expressed as RLU.
For Ag detection, the direct CMIA strategy was applied. In the first step, each sample was mixed with lysate and the magnetic microparticles coated with SARS-CoV-2-specific antibody (M_Ab1). The virus particles in the sample were lysed to expose the internal virus nucleocapsid protein antigen (NP). The NP antigen interacted with the specific antibody coated on the magnetic microparticles. After incubation, SARS-CoV-2-specific antibody-labeled acridinium ester (Ab2_AE) was added to form a "M_Ab1–Ag–Ab2_AE" complex. The ingredients not bound to the magnetic microparticles were washed and removed. Then, the substrate solution was added. The chemiluminescence reaction signal was measured and expressed as RLU.
All the tests were performed under rigorous biosafety conditions using an automated magnetic chemiluminescence analyzer (WANTAI BioPharm Caris200, Innodx Biotech Co. Ltd) in line with the manufacturer’s guidelines. The titer of antibodies or antigen was tested once per serum sample. The signal-to-cutoff (S/CO) ratio was calculated with S/CO < 1.0 being recognized as negative and S/CO ≥ 1.0 being recognized as positive. Seroconversion was interpreted as the SARS-CoV-2-specific TAb, IgG, IgM, IgA, or Ag detection results changing from negative to positive in sequential samples.
2.4 Statistical analyses
Continuous variables were presented as the median (interquartile range, IQR) and analyzed with Wilcoxon rank-sum test. Categorical variables were shown as counts (%) and analyzed using Fisher’s exact probability test. Correlation analysis was conducted using Spearman’s test. A P value <0.05 was recognized as statistically significant. Data were analyzed with Rstudio (version 3.6.1) and Statistical Product and Service Solutions (SPSS Inc. IBM Corp., Chicago, IL, version 25).