This retrospective cohort study involved 404 serum specimens obtained from 172 COVID-19 patients who were hospitalized in Wuhan during the epidemic peak period and discharged. These serum specimens were collected at onset, hospitalization and rehabilitation period. All the participants were recorded with clinical information such as epidemiology history, exposure history, signs and symptoms, laboratory findings, typical CT image, positive with RT-PCR for SARS-CoV-2. The proportions of age, sex and disease severity of the cohort patients are shown in Table 1.
Number Proportion of the total (%) Age (years old) < 65 117 68.0 ≥ 65 55 32.0 Sex Male 91 52.9 Female 81 47.1 Severity Mild 107 62.2 Severe 58 33.7 Critical severe 7 4.1
Table 1. Constitution of the COVID-19 patient cohort in this study.
In order to analyze the dynamic and distribution of positive rate and antibody titer, the samples were divided into two categories, diseased and convalescent period. The samples of the diseased period were collected in the first 6 weeks with the onset of symptoms to be the initial time point. While the samples collected in the period when the patients discharged from the hospital with two consecutive negative RT-PCR results, were categorized as convalescent period, the duration of this period was mainly from the 12th to the 26th week.
All serum samples were inactivated at 56 ℃ for 30 min and stored at − 20 ℃ before testing. The antibodies against SARS-CoV-2 were measured using SARS-CoV-2 chemiluminescent immunoassay (CLIA) kits supplied by Shenzhen Mindray Bio-Medical Electronics Co., Ltd. with a chemiluminescence immunoassays analyzer (CL-900i, DE/CA05/IvD-238321–0297-00). The SARS-CoV-2 chemiluminescent immunoassay kits used in this study were SARS-CoV-2 IgM kit, SARS-CoV-2 IgG kit, SARS-CoV-2 N-IgM kit, SARS-CoV-2 RBD- IgM kit, SARS-CoV-2 N-IgG kit, SARS-CoV-2 RBD-IgG kit, and SARS-CoV-2 Total Antibody kit. All kits were validated with the standard protocol. To simplify the description, the antibodies against SARS-CoV-2 detected in this study are referred to as IgM, N-IgM, RBD-IgM, IgG, N-IgG, RBD-IgG, and S1-total antibody.
According to the manufacturer's instructions, IgG and IgM detections were developed based on an indirect method. The recombinant antigens containing the nucleoprotein (N protein) or/and receptor-binding domain (RBD) of the spike protein (S protein) of SARS-CoV-2 were coated on paramagnetic microparticles used to capture corresponding antibodies. The anti-human IgG/IgM antibody-conjugated alkaline phosphatase was used respectively as the detection antibody. IgG and IgM detections consist of two steps. In the first step, antigen-coated paramagnetic microparticles captured specific antibodies in the sample. After a wash step to remove unbound substances, anti-human IgG/IgM antibody-conjugated alkaline phosphatase was added to bind to antibodies captured by paramagnetic microparticles. After the second wash step to remove unbound substances, 3-(2′-spiroadamantyl)-4-methoxy-4-(3′'-phosphoryloxy)-phenyl-1,2-dioxetane (AMPPD) was added and catalyzed by alkaline phosphatase to emit light at 540 nm. The resulting chemiluminescent reaction was measured as Relative Light Units (RLUs) by a photomultiplier in the instrument.
The total antibody kit was developed based on a double-antigen sandwich immunoassay. The recombinant antigens containing S1 protein were coated on paramagnetic microparticles and conjugated alkaline phosphatase. The detection is a one-step method. Antigen-coated paramagnetic microparticles and antigen-conjugated alkaline phosphatase bound the specific antibodies in the sample. After a wash step to remove unbound substances, AMPPD was added and catalyzed by alkaline phosphatase to emit light at 540 nm. The resulting chemiluminescent reaction was measured as RLUs.
IgG antibodies and S1-total antibodies were calculated as AU/mL and the Cutoff value is 10 AU/mL. IgM antibodies were presented as the measured Relative Light Units (RLUs) divided by the cutoff value (cutoff index, COI): COI ≥ 1 was defined as positive and COI < 1 as negative. The cutoff value of the positive antibody titer was determined according to the manufacturer's instructions.
Based on the antibody level changed over time from the first to the 26th week (with exclusive of 6th to 12th week), dynamics of antibodies were described by locally weighted regression and smoothing scatterplots (LOESS) model. Pearson correlation coefficient analysis (Pearson correlation) was applied to analyze the correlation of antibody dynamics. The similarity of antibody dynamic in different groups was quantitatively analyzed by the Kolmogorov–Smirnov test, including antibody level (distance) and distribution pattern (P value). Unless otherwise stated, statistical analysis was conducted by the software R (The R Foundation, http://www.r-project.org, version 4.0.0) and GraphPad Prism 8.0.