Serology characteristics of SARS-CoV-2
infection after exposure and
post-symptom onset
Bin Lou
1,2,3,7
, Ting-Dong Li
4,5,7
, Shu-Fa Zheng
1,2,3,7
, Ying-Ying Su
4,5,7
,
Zhi-Yong Li
5
, Wei Liu
4,5
, Fei Yu
1,2,3
, Sheng-Xiang Ge
4,5,8
, Qian-Da Zou
1,2,3
,
Quan Yuan
4,5
, Sha Lin
1,2,3
, Cong-Ming Hong
4,5
, Xiang-Yang Yao
5
,
Xue-Jie Zhang
4,5
, Ding-Hui Wu
5
, Guo-Liang Zhou
4,5
, Wang-Heng Hou
4,5
,
Ting-Ting Li
4,5
, Ya-Li Zhang
4,5
, Shi-Yin Zhang
4,5
, Jian Fan
1,2,3,8
, Jun Zhang
4,5,8
,
Ning-Shao Xia
4,5
and Yu Chen
1,2,3,6,8
@ERSpublications
Antibody responses were induced after SARS-CoV-2 infection, and the complementary diagnostic
value of the antibody test to the RNA test was observed. Antibody tests are critical to the clinical
management and control of SARS-CoV-2 infection and COVID-19. https://bit.ly/3fQZwZp
Cite this article as: Lou B, Li T-D, Zheng S-F, et al. Serology characteristics of SARS-CoV-2 infection after
exposure and post-symptom onset. Eur Respir J 2020; 56: 2000763 [https://doi.org/10.1183/
13993003.00763-2020].
ABSTRACT
Background: Timely diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
infection is a prerequisite for treatment and prevention. The serology characteristics and complement
diagnosis value of the antibody test to RNA test need to be demonstrated.
Method: Serial sera of 80 patients with PCR-confirmed coronavirus disease 2019 (COVID-19) were collected at
the First Affiliated Hospital of Zhejiang University, Hangzhou, China. Total antibody (Ab), IgM and IgG
antibodies against SARS-CoV-2 were detected, and the antibody dynamics during the infection w er e described.
Results: The seroconversion r ates for Ab , Ig M an d I gG were 98.8%, 93.8% and 93.8%, respectively. The firs t
detectible serology marker was Ab, followed by IgM and IgG, with a median seroconversion time of 15, 18 and
20 day s post exposure (d.p.e.) or 9, 10 and 12 days post onset (d.p.o.), respectively . The antibody levels
increased rapidly beginning at 6 d.p.o. and were accompanied by a decline in vir al load. For patients in the early
stage of illness (0–7 d.p.o), Ab show ed the highest sensitivity (64.1%) compared with IgM and IgG (33.3% for
both; p<0.001). The sensitivities of Ab, IgM and IgG increased to 100%, 96.7% and 93.3%, r espectively, 2 weeks
later. When the same antibody type was detected, no significant difference was observed between enzyme-
linked immunosorbent assays and other forms of immunoassays.
Conclusions: A typical acute antibody response is induced during SARS-CoV-2 infection. Serology testing
provides an important complement to RNA testing in the later stages of illness for pathogenic-specific
diagnosis and helpful information to evaluate the adapted immunity status of patients.
This article has supplementary material available from erj.ersjournals.com
This article has an editorial commentary: https://doi.org/10.1183/13993003.02411-2020
Data availability: We will share individual participant data that underlie the results reported in this article after
de-identification (text, tables, figures and appendices). The data will be available beginning 6 months after the major
findings from the final analysis of the study were published, ending 2 years later. The data will be shared with
investigators whose proposed use of the data has been approved by an independent review committee identified for
individual participant data meta-analysis. Proposals should be directed to chenyuzy@zju.edu.cn. To gain access, data
requestors will need to sign a data access agreement.
Received: 19 March 2020 | Accepted after revision: 8 May 2020
Copyright ©ERS 2020. This version is distributed under the terms of the Creative Commons Attribution Non-
Commercial Licence 4.0.
https://doi.org/10.1183/13993003.00763-2020 Eur Respir J 2020; 56: 2000763
|
ORIGINAL ARTICLE
INFECTIOUS DISEASE
Introduction
In early December 2019, a novel coronavirus (severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2)) was first reported to cause lethal pneumonia in humans, and person-to-person
transmission was demonstrated soon in Wuhan, the capital city of Hubei Province, China [1]. The virus
rapidly spread through China and then many other countries globally. By 6 May 2020, the virus had
resulted in >3.5 million laboratory-confirmed cases of coronavirus disease 2019 (COVID-19) and >243000
deaths in 215 countries [2]. The World Health Organization (WHO) declared COVID-19 a public health
emergency of international concern and gave it a “very high” risk assessment on a global level [3]. A
recent report from China showed that the median (interquartile range (IQR)) incubation period of
COVID-19 infection was 4 (2–7) days [4]. Fever, cough and fatigue are the most common symptoms [1].
Severe cases could rapidly progress to acute respiratory distress syndrome and septic shock. Abnormalities
on chest computed tomography, particularly ground-glass opacity and bilateral patchy shadowing, were
found in >80% of patients [4]. >80% of patients had lymphopenia, and ∼60% of patients had elevated
C-reactive protein [5]. However, the clinical and laboratory findings of COVID-19 infection are not
distinguishable from pneumonia caused by infection of some common respiratory tract pathogens, such as
influenza virus, Streptococcus pneumoniae and Mycoplasma pneumoniae [6]. Hence, the timely diagnosis
of SARS-CoV-2 infection is important for providing appropriate medical support and preventing the
spread by quarantining.
Currently, the diagnosis of SARS-CoV-2 infection almost solely depends on the detection of viral RNA
using PCR-based techniques [7]. Unfortunately, the sensitivity of the RNA test in the real world is not
satisfactory, particularly when samples collected from the upper respiratory tract are used [6, 8–10]. In
Wuhan, the overall positive rate of RNA testing is estimated to be ∼30–50% in patients with COVID-19
when they come to the hospital [11]. Furthermore, the overall throughput of available RNA tests is highly
limited by the fact that they require high workload, need skilful operators for testing and sample collection,
and need costly instruments and special operation places [12]. As a result, convenient serological detection
is expected to be helpful. However, current knowledge of the antibody response to SAR-CoV-2 infection is
very limited. The diagnostic value of the antibody test remains to be clearly demonstrated. How many
patients would raise an antibody response, and how long will it take for the antibody to convert to positive
since the exposure? Are there any meaningful differences between patients with short and long incubation
periods? What are the sensitivities of antibody detection for patients in different illness stages? Is there any
temporal association between the antibody response and the decline in viral load? To answer some of these
questions, we investigated the characteristics of antibody responses in 80 patients with COVID-19 during
their hospitalisation periods by detecting total antibodies, IgM and IgG using immunoassays.
Methods
Study design and participants
A confirmed COVID-19 case was defined based on the New Coronavirus Pneumonia Prevention and
Control Program (6th edition), published by the National Health Commission of China [13]. Briefly, a
confirmed case should meet three criteria: 1) fever and/or respiratory symptoms; 2) abnormal lung
imaging findings; and 3) a positive result of the nucleic acid of SARS-CoV-2. The degree of severity of the
patient was categorised as critical if any of the following clinical findings appeared: 1) acute respiratory
distress syndrome or oxygen saturation <93% and requiring mechanical ventilation either invasively or
noninvasively; 2) shock; and 3) complications of organ functional failure and requiring intensive care unit
support. A COVID-19 patient who did not meet the above criteria was defined as a non-critical case.
The study enrolled a total of 80 COVID-19 cases, where all patients were admitted to the hospital between
19 January and 9 February 2020, and were willing to donate their blood samples. All enrolled cases were
Affiliations:
1
Dept of Laboratory Medicine, the First Affiliated Hospital, College of Medicine, Zhejiang
University, Hangzhou, China.
2
Key Laboratory of Clinical In Vitro Diagnostic Techniques of Zhejiang Province,
Hangzhou, China.
3
Institute of Laboratory Medicine, Zhejiang University, Hangzhou, China.
4
The State Key
Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine
Development in Infectious Diseases, Collaborative Innovation Center of Biologic Products, School of Public
Health and School of Life Science, Xiamen University, Xiamen, China.
5
School of Public Health, Xiamen
University, Xiamen, China.
6
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases,
Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital,
College of Medicine, Zhejiang University, Hangzhou, China.
7
Bin Lou, Ting-Dong Li, Shu-Fa Zheng and Ying-
Ying Su contributed equally to this article.
8
Yu Chen, Jian Fan, Sheng-Xiang Ge and Jun Zhang contributed
equally to this article as lead authors and jointly supervised the work.
Correspondence: Yu Chen, Dept of Laboratory Medicine, First Affiliated Hospital, College of Medicine,
Zhejiang University, Hangzhou, China. E-mail: chenyuzy@zju.edu.cn, 1200011@zju.edu.cn
https://doi.org/10.1183/13993003.00763-2020 2
INFECTIOUS DISEASE | BIN LOU ET AL.
confirmed to be infected by SARS-CoV-2 through quantitative real-time reverse transcriptase PCR
(qRT-PCR) testing. The date of illness onset, clinical classification, RNA testing results during the
hospitalisation period, and personal demographic information were obtained from the clinical records. A
total of 300 healthy people were enrolled from the local community during the circulation of the virus. All
the controls had not reported close contact with any confirmed COVID-19 patient. The study was
reviewed and approved by the Medical Ethics Committee of the First Affiliated Hospital of Zhejiang
University (Hanzhou, China; approval number 2020-IIT-47). Written informed consent was obtained
from each enrolled subject.
Laboratory confirmation of COVID-19 by reverse-transcription polymerase chain reactions
For each patient, the sputum specimen was collected preferentially. Where sputum was not produced,
saliva after deep cough was collected, as previously reported [14]. The sputum was first treated with an
equal volume of protease K solution (0.4 mg·mL
−1
) at room temperature for 20 min. 200 μL of treated
sputum or saliva was subjected to total nucleic acid extraction by MagNA Pure LC 2.0 (Roche, Basel,
Switzerland), and each sample obtained 50 μL elution. A commercial one-step real-time reverse
transcriptase polymerase chain reaction (RT-PCR) assay (Bio-Germ, Shanghai, China) targeting the
nucleocapsid gene and open reading frame lab gene was performed with 5 μL of total nucleic acid
according to the manufacturer’s instructions.
Antibody measurement
The total antibody (Ab), IgM antibody and IgG antibody against SARS-CoV-2 in plasma samples were
tested using three enzyme-linked immunosorbent assays (ELISA-Ab, ELISA-IgM and ELISA-IgG), three
colloidal-gold lateral-flow immunoassays (LFIA-Ab, LFIA-IgM and LFIA-IgG) and two
chemiluminescence microparticle immunoassays (CMIA-Ab and CMIA-IgM). The ELISA reagents and
LFIA reagents were supplied by Beijing Wantai Biological Pharmacy Enterprise Co., Ltd (Beijing, China),
and the CMIA reagents were supplied by Xiamen InnoDx Biotech Co., Ltd (Xiamen, China). Total Ab
detection was based on a double-antigen sandwich immunoassay, and IgM antibody detection was based
on a μ-chain capture immunoassay. Mammalian cell-expressed recombinant antigens containing the
receptor binding domain (RBD) of the SARS-CoV-2 spike protein were used to develop total Ab and IgM
antibody assays. The IgG antibody kits were indirect immunoassays, and a recombinant nucleoprotein of
SARS-CoV-2 expressed in Escherichia coli was used as the coating antigen. The antigens used in these
assays were in-house preparations by Beijing Wantai Biological Pharmacy Enterprise Co., Ltd, and the
same antigen or antigen pairs were used for ELISA, CMIA and LFIA assays.
The measurement processes of ELISA and CMIA were conducted with automatic ELISA analyser
HB-300E ( Jiaxing CRED Medical Equipment Co. Ltd, Zhejiang, China) and automatic CMIA analyser
Caris 200 (Xiamen UMIC Medical Instrument Co. Ltd, Xiamen, China) according to the manufacturers’
instructions. The details of the procedures are described in the supplementary material. When LFIA-Ab
and LFIA-IgM were performed, 10 μL of sample was pipetted onto the sample receiving zone, followed by
two drops of sample buffer. For LFIA-IgG, the sample was diluted 1000-fold with sample buffer, and then
80 μL of dilution was added onto the sample receiving area. 15 min after the sample was added, the LFIA
results were observed by eye and recorded. It takes 75 min, 29 min and 15 min to get the results back for
ELISA, CMIA and LFIA assays, respectively.
Statistical analysis
The incubation period was defined as the interval between the earliest date of SARS-CoV-2 exposure and
the earliest date of symptom onset. Non-normally distributed continuous data were described as the
median (IQR) and compared using the Wilcoxon test. Categorical data were summarised as counts and
percentages. The 95% confidence intervals of sensitivity and specificity were estimated using the binomial
exact test. Categorical data were compared using the Chi-squared test or Fisher’s exact test for unpaired
proportions and McNemar’s test for paired proportions. Cumulative seroconversion rates were calculated
using the Kaplan–Meier method. All statistical analyses were conducted using SAS 9.4 (SAS Institute,
Cary, NC, USA).
Patient and public involvement
No patients were involved in setting the research question or the outcome measures, nor were they
involved in the design and implementation of the study.
Dissemination declaration
Dissemination to these groups is not possible/applicable.
https://doi.org/10.1183/13993003.00763-2020 3
INFECTIOUS DISEASE | BIN LOU ET AL.
Results
Characteristics of enrolled patients with COVID-19
Of the 81 COVID-19 patients admitted to the hospital (before 9 February 2020), 80 (99%) were enrolled in the
study (table 1). The median (IQR) age of the patients was 55 (45–64) years, and 38.7% were females. Critical
patients were significantly older than non-critical patients (p<0.001). The time of SARS-CoV-2 exposure
before onset in 45 patients (15 critical cases) was determined according to unambiguous close contact with
confirmed patients with COVID-19 through epidemiological inspection upon admission to the hospital. The
incubation period ranged 0–23 days with a median (IQR) of 5 (2–10) days. By 15 February 2020, a total of 32
(40%) patients (all non-critical cases) were recovered and discharged from hospital; none died.
The performance of SARS-CoV-2 antibody detection kits
A total of 80 patients with COVID-19 and 100–300 healthy people were tested for antibodies against
SARS-CoV-2 using different immunoassays. The seroconversion rates for Ab, IgM and IgG in patients
were 98.8% (79 out of 80), 93.8% (75 out of 80) and 93.8% (75 out of 80), respectively (table 2 shows the
combined sensitivity of three methods). The last blood sample from the only patient who had not
seroconverted was collected at 7 days post onset (d.p.o.). For the Ab, IgM and IgG tests, ELISAs seemed to
perform the best, although the differences were generally not significant. Therefore, the following
serological analyses were all based on the results of ELISAs, unless specifically noted.
Seroconversion sequentially appeared for Ab, IgM and IgG, with median onset times of 9, 10 and 12 days,
respectively (figure 1a). No significant difference of the time of seroconversion was observed between
critical and non-critical patients (supplementary figure E1). The seroconversion of Ab was significantly
quicker than that of IgM and IgG (p<0.001). The cumulative seroconversion curve showed that the rate
for Ab and IgM reached 100% and IgG reached 97.1% on days 16, 21 and 29 post symptom onset,
respectively. The antibody levels increased rapidly starting at 6 d.p.o. (figure 1b). The decline in viral load
co-occurred with increasing antibody levels. For patients in the early stage of illness (0–7 d.p.o.),
ELISA-Ab showed the highest sensitivity (64.1%) compared with ELISA-IgM and ELISA-IgG (33.3% for
both; p<0.001) (table 3). The sensitivities of Ab, IgM and IgG detection increased significantly when the
patient entered the later stage and reached 100%, 96.7% and 93.3%, respectively, 2 weeks later (p<0.05).
The antibody dynamics after exposure to SARS-CoV-2
Because of the difficulty of determining the exact infection date for patients with prolonged exposure, the
antibody dynamics after the earliest date of exposure instead of those after infection were investigated in
45 patients whose exposure times had been determined (figure 2). Seroconversion appeared sequentially
for Ab, IgM and IgG, and the levels increased rapidly, with median days post exposure (d.p.e.) of 15, 18
TABLE 1 Demographics and clinical characteristics of enrolled patients with coronavirus
disease 2019 (COVID-19)
Total Non-critical Critical p-value
Subjects n 80 54 26
Sex
Female 31 (38.7) 24 (44.4) 7 (26.9) 0.132
Male 49 (61.3) 30 (55.6) 19 (73.1)
Age years 55 (45–64) 51 (38–39) 65 (52–74) <0.001
Clinical outcome
Recovery 26 (32.5) 26 (48.1) 0 (0.0) <0.001
Still in hospital 54 (67.5) 28 (51.9) 26 (100.0)
Incubation period
#
5(2–10) 4 (2–12) 7 (1–10) 1.000
Day of first positive SARS-CoV-2 RNA finding 4(1–6) 4 (2–6) 3 (1–6) 0.179
Day when first antibody testing sample was collected 8(6–10) 7 (5–10) 8 (7–10) 0.215
Samples tested for antibodies for each case 4(3–5) 4 (3–4) 4 (3–5) 0.888
Total plasma samples n 305 205 100
Data are presented as n (%) or median (interquartile range), unless otherwise stated. SARS-CoV-2: severe
acute respiratory syndrome coronavirus 2.
#
: for patients who had no travel history to Wuhan, China, but
had close contact with a confirmed COVID-19 patient (index patient) within 14 days before symptom onset,
the exposure time was defined as the first day of close contact with the index patient. The time of
SARS-CoV-2 exposure before onset in 45 patients was determined through epidemiological inspection;
among them, 15 were critical cases.
https://doi.org/10.1183/13993003.00763-2020 4
INFECTIOUS DISEASE | BIN LOU ET AL.
and 20, respectively (figure 3). The median seroconverting times post exposure in critical patients were not
different from those in non-critical patients (supplementary figure E2). The decline in viral load
co-occurred with increasing antibody levels. The cumulative positive rates for Ab, IgM and IgG separately
reached 100%, 94.2% and 96.7% at 37 d.p.e. Patients who reported symptoms within 5 days since exposure
were assigned to the short incubation period group (0–5 days), and the remaining patients were assigned
TABLE 2 Sensitivity and specificity of different immunoassays to detect antibodies against
severe acute respiratory syndrome coronavirus 2
Type of
immunoassay
#
Sensitivity Specificity
¶
Patients
n
Positive
n
% (95% CI) Uninfected
n
Negative
n
% (95% CI)
Ab
ELISA-Ab 80 78 97.5 (91.3–99.7) 300 300 100.0 (98.8–100.0)
CMIA-Ab 80 77 96.3 (89.4–99.2) 300 298 99.3 (97.6–99.9)
LFIA-Ab 80 78 97.5 (91.3–99.7) 209 199 95.2 (91.4–97.7)
Combined 80 79 98.8 (93.2–100.0) 209 197 94.3 (90.2–97.0)
IgM
ELISA-IgM 80 74 92.5 (84.4–97.2) 300 300 100 (98.8–100)
CMIA-IgM 80 69 86.3 (76.7–92.9) 300 298 99.3 (97.6–99.9)
LFIA-IgM 80 71 88.8 (79.7–94.7) 209 205 98.1 (95.2–99.5)
Combined 80 75 93.8 (86.0–97.9) 209 203 97.1 (93.9–98.9)
IgG
ELISA-IgG 80 71 88.8 (79.7–94.7) 100 100 100.0 (96.4–100.0)
LFIA-IgG 80 69 86.3 (76.7–92.9) 209 208 99.5 (97.4–100.0)
Combined 80 75 93.8 (86.0–97.9) 100 99 99.0 (94.6–100)
Ab: total antibody; CMIA: chemiluminescence microparticle immunoassay; LFIA: lateral flow immunoassay.
#
: the combined sensitivities were calculated based on positive findings by any of the assays; the combined
specificities were calculated based on negative findings for all assays.
¶
: none of the controls reported
close contact with any confirmed coronavirus disease 2019 patients, and individuals who were positive for
any of the antibody tests were tested to be PCR negative with sputum or saliva samples collected later.
With the very limited local community spread of the virus during the period in mind, and for more rigorous
evaluation of an assay’s specificity, the community controls with positive results in antibody assays were
considered false-positive.
100
a)
80
60
40
20
0
Cumulative seroconversion rate %
Days after onset
Seronegative n
5
0 1015202530
75
76
78
80
80
80
Ab
IgM
IgG
39
56
59
2
9
16
0
1
3
0
20
ELISA-Ab
ELISA-IgM
ELISA-IgG
30 20
25
30
35
40
45
b)
25
15
20
10
5
0
Relative antibody binding S/CO
RNA rRT-PCR Ct (value)
Days after onset
5
01015202530
ELISA-Ab
Cut-off
ELISA-IgM
ELISA-IgG
RNA
FIGURE 1 Cumulative seroconversion rates and the dynamics of antibody levels since the onset of illness in 80 patients with coronavirus disease
2019. a) Curves of the cumulative seroconversion rates for total antibody (Ab), IgM and IgG detected by ELISAs plotted according to Kaplan–Meier
methods. The serological status of patients was assigned to be negative before the time that the first sample was collected. b) The antibody levels
were surrogated and expressed using the relative binding signals compared with the cut-off value of each assay (signal to cut-off (S/CO)). Four
parametric logistic fitting curves (solid lines) were used to mimic the trends of antibody levels. rRT-PCR: real-time reverse transcriptase PCR;
Ct: cycle thresholds
https://doi.org/10.1183/13993003.00763-2020 5
INFECTIOUS DISEASE | BIN LOU ET AL.
