COVID-19 Serological Tests: How Well Do They Actually Perform? - MDPI
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diagnostics
Review
COVID-19 Serological Tests: How Well Do They
Actually Perform?
Abdi Ghaffari 1,2, * , Robyn Meurant 3 and Ali Ardakani 1, *
1 Novateur Ventures Inc., Vancouver, BC V6E 3P3, Canada
2 Department of Pathology and Molecular Medicine, Queen’s University, Kingston, ON K7L 3N6, Canada
3 NSF Health Sciences, Kirkbymoorside, York YO62 6AF, UK; rmeurant@nsf.org
* Correspondence: ghaffari@queensu.ca (A.G.); ali@novateur.org (A.A.); Tel.: +1-844-200-6682 (A.A.)
Received: 14 June 2020; Accepted: 1 July 2020; Published: 4 July 2020
Abstract: In only a few months after initial discovery in Wuhan, China, SARS-CoV-2 and the associated
coronavirus disease 2019 (COVID-19) have become a global pandemic causing significant mortality
and morbidity and implementation of strict isolation measures. In the absence of vaccines and effective
therapeutics, reliable serological testing must be a key element of public health policy to control
further spread of the disease and gradually remove quarantine measures. Serological diagnostic tests
are being increasingly used to provide a broader understanding of COVID-19 incidence and to assess
immunity status in the population. However, there are discrepancies between claimed and actual
performance data for serological diagnostic tests on the market. In this study, we conducted a review of
independent studies evaluating the performance of SARS-CoV-2 serological tests. We found significant
variability in the accuracy of marketed tests and highlight several lab-based and point-of-care rapid
serological tests with high levels of performance. The findings of this review highlight the need for
ongoing independent evaluations of commercialized COVID-19 diagnostic tests.
Keywords: COVID-19; serological diagnostic test; SARS-CoV-2; performance
1. Background
Coronavirus disease 2019 (COVID-19) was first discovered in a cluster of patients with severe
respiratory symptoms in Hubei Province, China, in December 2019. The early nucleic acid analysis
of known pathogen panels led to negative results, suggesting the causative agent was of unknown
origin. By early January 2020, analysis of bronchoalveolar lavage (BAL) fluid from infected patients
revealed a pathogen, later named SARS-CoV-2, with 50%, 80%, and 96% genetic sequence overlap to
the genome of the Middle East respiratory syndrome virus (MERS-CoV), the severe acute respiratory
syndrome virus (SARS-CoV), and bat coronavirus RaTG13, respectively [1,2]. Like SARS-CoV and
MERS-CoV, SARS-CoV-2 is a single-stranded RNA virus belonging to the beta genus Coronavirus in the
Coronaviridae family [3].
As SARS-CoV-2 can be transmitted from human to human, the disease has spread swiftly to over
200 countries, infecting nearly 6 million people and resulting in at least 350,000 deaths worldwide (as
of 27 May 2020) [4]. An unprecedented and rapidly growing global effort is underway to develop
COVID-19 vaccines and therapeutics, but at the time of this review, there are no vaccines, and only one
antiviral drug (remdesivir) with modest clinical benefit has been approved under the U.S. Food and
Drug Administration (FDA) Emergency Use Authorization (EUA) [5,6]. Under these circumstances,
countries were forced to implement physical distancing measures to control the outbreak and, in the
process, place approximately 3 billion people under lockdown.
Diagnostics 2020, 10, 453; doi:10.3390/diagnostics10070453 www.mdpi.com/journal/diagnostics
Diagnostics 2020, 10, 453 2 of 14
2. COVID-19 Diagnostic Tests
In any infectious disease outbreak, accurate and accessible diagnostic testing must be one
Diagnostics 2020, 10, x FOR PEER REVIEW 2 of 13
of the pillars of control-measure policies to understand and minimize the spread of disease.
The epidemiological studies
In any infectious of theoutbreak,
disease outbreakaccurate
in China andestimated
accessiblethe proportion
diagnostic of undetected
testing must be oneCOVID-19
of the
cases pillars
to be as of high
control-measure
as 86% [7]. policies to understand
As asymptomatic and cases
or mild minimize theplay
could spread of disease.
a significant The
role in the
epidemiological
transmission and spreadstudies ofofthe
theSARS-CoV-2
outbreak in Chinavirusestimated the proportion
[7,8], symptoms aloneofareundetected COVID-19
not reliable diagnostic
casesThere
markers. to be are
as high as 86%types
two major [7]. As
ofasymptomatic or mild cases
diagnostic technologies could play
available a significant
to address this: role in the and
molecular
transmission and spread of the SARS-CoV-2 virus [7,8], symptoms alone are not reliable diagnostic
serological tests. Currently, much of the focus is on the SARS-CoV-2 molecular test, which can detect,
markers. There are two major types of diagnostic technologies available to address this: molecular
with high accuracy, the virus-specific RNA molecules circulating in the host body. The gold-standard
and serological tests. Currently, much of the focus is on the SARS-CoV-2 molecular test, which can
molecular
detect,test
withishigh
based on reverse
accuracy, transcriptase
the virus-specific RNApolymerase chain reaction
molecules circulating (RT-PCR)
in the host body. The technology.
gold-
However,
standard molecular test is based on reverse transcriptase polymerase chain reaction (RT-PCR)ones
the PCR test is not useful in distinguishing between highly infective viruses versus
that have been neutralized
technology. However, the byPCRthe host,
test isand
not it cannot
useful assess immunity
in distinguishing status
between against
highly SARS-CoV-2
infective viruses [9].
Serologically
versus ones basedthat antibody
have beentests can complement
neutralized by the host, molecularly
and it cannot based tests by providing
assess immunity status againsta more
SARS-CoV-2
accurate estimate [9]. Serologically incidence
of SARS-CoV-2 based antibody
and by tests can complement
potentially molecularly
detecting individuals based
withtests by
immunity
providing a more accurate estimate of SARS-CoV-2
against the disease, as these tests detect markers of the immune response. incidence and by potentially detecting
individuals with immunity against the disease, as these tests detect markers of the immune response.
3. Humoral Immune Response to SARS-COV-2
3. Humoral Immune Response to SARS-COV-2
In humoral immune response to infection, pathogen-specific antibodies, produced by B cells,
In humoral immune response to infection, pathogen-specific antibodies, produced by B cells,
neutralize and prevent further spread of the disease. The activation and differentiation of B cells into
neutralize and prevent further spread of the disease. The activation and differentiation of B cells into
antibody-secreting plasma B cells are triggered by a cascade of events involving virus digestion by
antibody-secreting plasma B cells are triggered by a cascade of events involving virus digestion by
antigen-presenting
antigen-presentingcells cells
(e.g., (e.g.,
dendritic cells, cells,
dendritic macrophages) and presentation
macrophages) and presentation of virus-specific antigens
of virus-specific
to helper T cells
antigens (FigureT 1).
to helper Antibodies
cells (Figure 1).protect the host
Antibodies by the
protect binding
host byto binding
specific toantigens
specific (proteins)
antigens on
the virus to neutralize
(proteins) its fusion
on the virus and entry
to neutralize into and
its fusion the host
entrycell
intoand facilitate
the host recognition
cell and and killing by
facilitate recognition
and killing by phagocytic immune cells [10]. In humans, three
phagocytic immune cells [10]. In humans, three types of antibodies or immunoglobulins have types of antibodies orbeen
immunoglobulins
the target of COVID-19 have been the target
serological tests: of COVID-19
IgM, IgG, andserological tests: IgM,
IgA. Although theIgG, and IgA.ofAlthough
dynamics the immune
the in
response dynamics
COVID-19 of the immune
are not response in COVID-19
fully understood, typically IgMare antibodies
not fully understood,
are produced typically
by host IgM
immune
antibodies are produced by host immune cells during the early stages of a viral infection. IgG is often
cells during the early stages of a viral infection. IgG is often the most abundant antibody in the blood
the most abundant antibody in the blood and plays a more prominent role in the later stages of
and plays a more prominent role in the later stages of infection and in establishing long-term immune
infection and in establishing long-term immune memory [11]. While IgM and IgG antibodies have
memory [11]. While IgM and IgG antibodies have been the leading candidates in COVID-19 serological
been the leading candidates in COVID-19 serological test development, recent studies show that IgA,
test development,
predominatelyrecent
present studies
in theshow
mucosalthattissue,
IgA, predominately
may also play a present in the
critical role mucosal
in the immune tissue, may also
response
play aandcritical role in the immune
disease progression [12]. response and disease progression [12].
Figure 1. The
Figure 1. human
The human antibody
antibodyresponse
responsetotoSARS-CoV-2 infection.(1)(1)The
SARS-CoV-2 infection. The SARS-CoV-2
SARS-CoV-2 virus
virus enters
enters
the host
the cell
hostvia
cellinteraction between
via interaction betweenviral
viralspike
spike(S)
(S) and hostangiotensin-converting
and host angiotensin-converting enzyme
enzyme 2 (ACE2)
2 (ACE2)
Diagnostics 2020, 10, 453 3 of 14
proteins. (2,3) Following replication and release from the host cells, a subset of viruses will be engulfed
and digested by antigen-presenting cells (APCs) like macrophages or dendritic cells. (4) Fragmented
SARS-CoV-2 antigen(s) will be presented to T helper cells, which in turn will interact and activate B cells.
(5) Activated B cells will proliferate and differentiate into plasma or memory B cells with high-affinity
binding receptors for the original SARS-CoV-2 antigen. Plasma cells secrete their SARS-CoV-2-specific
receptors in the form of IgM, IgG, or IgA antibodies. (6) Antibody-mediated neutralization occurs
when SARS-CoV-2-specific antibodies bind to viral antigen(s) and prevent virus interaction and entry
into host cells.
4. Serological Antibody Test
Serological, or antibody, tests detect immunoglobulins produced by the host’s plasma B cells
following exposure to foreign antigens. The SARS-CoV-2 genome encodes approximately 25 proteins
that are required for infection and replication, including four major structural proteins: spike (S),
envelope (E), membrane (M), and nucleocapsid (N) (Figure 1). The S protein plays a critical role in
fusion and entry into the host cell, and it comprises an N-terminal S1 receptor-binding domain (RBD),
N-terminal domain (NTD), and a C-terminal S2 subunits. The primary function of the SARS-CoV-2 N
protein (NP) is binding and packing of the viral RNA genome into a helical nucleocapsid structure
during viral replication [13,14]. Studies on the serum of recovered COVID-19 patients suggest that
host-neutralizing antibodies primarily work against S and N proteins [15,16]. Consequently, the
likelihood of predicting immunity status could increase in serological tests that target various regions
of S or N proteins. Therefore, the characterization of specific SARS-CoV-2 antigen domains targeted by
the humoral immune response becomes an integral part of the serological test development.
There are four major types of serological diagnostic tests: the rapid diagnostic test (RDT),
enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and
neutralization assay. The neutralization assay is a lab-based test that uses live virus and cell culture
methods to determine if patient antibodies can prevent viral infection in vitro. This test must be
performed in laboratories with designated biosafety certificates to culture SARS-CoV-2-infected cells
and has a time-to-result of 3–5 days. An RDT is a simple and rapid test based on lateral flow
immunoassay (LFIA) technology, commonly found in pregnancy test kits, for example. RDT can
potentially be administered as a point-of-care (POC) test or self-test. Typically, RDT test strips use a
drop of blood to detect the presence of patient antibodies (IgG, IgM, or IgA) produced against a specific
SARS-CoV-2 antigen (Figure 2). An RDT is simple to use with a time-to-result anywhere between 10 and
30 min. Therefore, it has the potential to be deployed in large-scale serological surveys. ELISA assay,
currently the most commonly used format of the serological test, is a lab-based test with an average
time-to-result of 2–5 h. ELISA typically uses a surface coated with specific viral antigen(s) to bind to
and detect the corresponding patient antibodies (IgG, IgM, IgA) in blood, plasma, or serum samples.
The bound antigen–antibody complex is then detected by using a second antibody and a substrate
that produces a color- or fluorescent-based signal. ELISA assays can be found in different formats
including direct, competitive, and, the most commonly used, sandwich or double-antigen-bridging
assay (DABA) (Figure 3). CLIA technology follows a similar concept to ELISA by taking advantage of
high binding affinity between the viral antigen(s) and host antibodies but uses chemical probes that
yield light emission through a chemical reaction to generate a positive signal. CLIA has an average
time-to-result of 1–2 h. CLIA and ELISA are both high-throughput laboratory-based assays with high
level of analytical agreement [17,18].
Diagnostics 2020, 10, 453 4 of 14
Diagnostics 2020, 10, x FOR PEER REVIEW 4 of 13
Figure 2. Overview of rapid diagnostic serological test. Rapid diagnostic tests (RDTs) are typically
Figure 2. Overview of rapid diagnostic serological test. Rapid diagnostic tests (RDTs) are typically
based on colorimetric lateral flow immunoassay, in which host antibodies migrate across an adhesive
based on colorimetric lateral flow immunoassay, in which host antibodies migrate across an adhesive
pad (e.g., nitrocellulose) and interact with bound virus-specific antigens and secondary antibodies
pad (e.g., nitrocellulose) and interact with bound virus-specific antigens and secondary antibodies
(antihuman IgM/G antibodies). Conjugated SARS-CoV-2-specific antigen(s) (labeled with gold here)
(antihuman IgM/G antibodies). Conjugated SARS-CoV-2-specific antigen(s) (labeled with gold here)
will bind with the corresponding host antibodies. As antibody–antigen complexes travel up the
will bind with the corresponding host antibodies. As antibody–antigen complexes travel up the
membrane, bound anti-SARS-CoV-2 IgM antibodies interact with fixed anti-IgM secondary antibodies
membrane, bound anti-SARS-CoV-2 IgM antibodies interact with fixed anti-IgM secondary
on the M line, and anti-SARS-CoV-2 IgG antibodies interact with anti-IgG antibodies on the G line.
antibodies on the M line, and anti-SARS-CoV-2 IgG antibodies interact with anti-IgG antibodies on
If the blood sample does not contain SARS-CoV-2-specific antibodies, the M or G lines do not appear in
the G line. If the blood sample does not contain SARS-CoV-2-specific antibodies, the M or G lines do
the final test results; only the control (C) line will be revealed.
not appear in the final test results; only the control (C) line will be revealed.
Diagnostics
Diagnostics 2020,
2020, 10,10,
453x FOR PEER REVIEW 55ofof1413
Figure 3. Overview of enzyme-linked immunosorbent assay (ELISA)-based diagnostic test. ELISA can
Figure 3. Overview of enzyme-linked immunosorbent assay (ELISA)-based diagnostic test. ELISA can
be presented in different formats based on differences in antigen immobilization and antibody labeling.
be presented in different formats based on differences in antigen immobilization and antibody
In direct ELISA, SARS-CoV-2 antigen(s) bound to a plastic solid phase is detected by the addition
labeling. In direct ELISA, SARS-CoV-2 antigen(s) bound to a plastic solid phase is detected by the
of a conjugated antibody. In sandwich ELISA, the capture antibody is attached to the plastic solid
addition of a conjugated antibody. In sandwich ELISA, the capture antibody is attached to the plastic
phase. Antigen(s) in the sample will bind to the capture antibody and then be detected by a second
solid phase. Antigen(s) in the sample will bind to the capture antibody and then be detected by a
enzyme-labeled antibody. In competitive ELISA, sample SARS-CoV-2 antigen is preincubated with
second enzyme-labeled antibody. In competitive ELISA, sample SARS-CoV-2 antigen is preincubated
the primary antibody and then added to a well coated with a secondary antibody along with an
with the primary antibody and then added to a well coated with a secondary antibody along with an
enzyme-conjugated antigen that competes with the sample antigen for binding with the primary
enzyme-conjugated antigen that competes with the sample antigen for binding with the primary
antibody. The more SARS-CoV-2 antigen in the sample, the less conjugated antigen will be bound and
antibody. The more SARS-CoV-2 antigen in the sample, the less conjugated antigen will be bound
the lower the signal will be.
and the lower the signal will be.
5. Time Kinetics of Antibody Response in COVID-19
Knowledge of virus and host immune response dynamics are essential in formulating diagnostic
testing and treatment strategies. Studies of COVID-19 suggest that seroconversion, when antibody
levels become detectable in the blood, may take place days after the viral load has peaked [19].
Therefore, serological tests would be less effective in the early stages of COVID-19. Wolfel and colleagues
further confirmed these findings by reporting IgM and IgG seroconversion in 50% of patients at 1 week
after the onset of symptoms [20]. The median time for the detection of IgM and IgG in COVID-19 patients
was reported to be 5 and 14 days, respectively [21]. Yu and colleagues detected the seroconversion of IgA
Diagnostics 2020, 10, 453 6 of 14
5. Time Kinetics of Antibody Response in COVID-19
Knowledge of virus and host immune response dynamics are essential in formulating diagnostic
testing and treatment strategies. Studies of COVID-19 suggest that seroconversion, when antibody
levels become detectable in the blood, may take place days after the viral load has peaked [19]. Therefore,
serological tests would be less effective in the early stages of COVID-19. Wolfel and colleagues further
confirmed these findings by reporting IgM and IgG seroconversion in 50% of patients at 1 week after
the onset of symptoms [20]. The median time for the detection of IgM and IgG in COVID-19 patients
was reported
Diagnostics to be
2020, 10, 5 and
x FOR PEER14 days, respectively [21]. Yu and colleagues detected the seroconversion
REVIEW 6 of 13
of IgA on day 2 and IgM/IgG on day 5 after onset of symptoms. Furthermore, the study reported
on day
that 100% 2 and IgM/IgG
of cases had on day 5 after
detectable onset
levels of of symptoms.
IgA, IgM, andFurthermore,
IgG on day 32 theafter
study reported
onset that 100%
of symptoms of
[12].
cases had
Their detectable
findings levels of IgM
also revealed IgA, and
IgM,IgG
andlevels
IgG on to day 32 after onsethigher
be significantly of symptoms
in severe[12]. Their findings
COVID-19 cases
also revealed
than IgMwith
in patients and IgG mildlevels to be significantly
or moderate disease higher in severe COVID-19
[12], suggesting cases than
that serological in require
tests patientshigh
with
mild or moderate
sensitivity to detectdisease [12], suggesting
lower levels of antibodiesthatinserological
mild cases.tests require
Studies highpersistence
on the sensitivityoftoantibodies
detect lowerin
levels of
blood antibodies
suggest that in mild
high cases.ofStudies
levels IgG areondetectable
the persistence
for atofleast
antibodies
49 daysinafter
bloodthesuggest
onsetthat high levels
of symptoms,
of IgGIgM
while are detectable
levels declinedfor at rapidly
least 49 days after
on day 35the onset of symptoms,
postinfection [22]. Thewhile
diagram IgMin levels declined
Figure rapidly
4 depicts the
on day 35 and
timelines postinfection
peak levels [22].
forThe diagram inviral
SARS-CoV-2 Figure
load4 depicts
relativethe timelines
to blood IgM,and peak
IgG, andlevels for SARS-
IgA antibodies.
CoV-2 viralunderstanding
Improved load relative toofblood IgM,antibody
humoral IgG, and IgA antibodies.
response Improved
time kinetics understanding
in COVID-19 of humoral
is crucial to the
antibody
correct response of
application time kinetics in
serological COVID-19 is crucial to the correct application of serological tests.
tests.
Figure
Figure 4.4. Time kinetics of antibody response in coronavirus
coronavirus disease
disease 2019
2019 (COVID-19).
(COVID-19). The illustration
illustration
demonstrates
demonstrates the relative levels of host immunoglobulins (IgM, IgG, IgA) and SARS-CoV-2 viral
the relative levels of host immunoglobulins (IgM, IgG, IgA) and SARS-CoV-2 viral load
load
at
at different stages of COVID-19. Antibody-specific seroconversion occurs when the antibody reaches
different stages of COVID-19. Antibody-specific seroconversion occurs when the antibody reaches
aa detectable
detectable level
level in
in blood.
blood. Disclaimer: This graphic
Disclaimer: This graphic is
is for
for illustrative
illustrative purposes
purposes only
only and
and does
does not
not
represent
represent actual
actual levels
levels of
of each
each antibody.
antibody.
6. Serological Test Performance
6. Serological Test Performance
The urgent need for the development of serological diagnostic tests in response to the COVID-19
The urgent need for the development of serological diagnostic tests in response to the COVID-
outbreak has compelled regulatory bodies to implement emergency use authorization programs
19 outbreak has compelled regulatory bodies to implement emergency use authorization programs
to expedite the commercialization process of these tests. In light of this, independent and
to expedite the commercialization process of these tests. In light of this, independent and robust post-
robust post-market evaluations of COVID-19 serological tests are needed to confirm manufacturers’
market evaluations of COVID-19 serological tests are needed to confirm manufacturers’ performance
performance claims. The basic measures of quantifying diagnostic test performance are sensitivity and
claims. The basic measures of quantifying diagnostic test performance are sensitivity and specificity.
specificity. Sensitivity is the ability of a test to detect the disease agent or the host’s response to the
Sensitivity is the ability of a test to detect the disease agent or the host’s response to the disease (i.e.,
disease (i.e., antibodies) when it is truly present, whereas specificity is the ability of a test to correctly
antibodies) when it is truly present, whereas specificity is the ability of a test to correctly return a
return a negative result when disease or host response is absent [23].
negative result when disease or host response is absent [23].
We conducted a systematic review of independent studies that assessed the performance of
currently available SARS-CoV-2 serological tests. We included studies that reported sensitivity and
specificity, stage of disease (early, intermediate, or late), the test format (CLIA, ELISA, RDT), and
antibody target (IgA, IgG, IgM, or IgG + IgM) [24,25]. If available, the SARS-CoV-2 antigens used for
antibody detection was recorded. The studies that did not specify the disease stage of test samples
were grouped under the “overall” category and assessed separately. In total, we reviewed
Diagnostics 2020, 10, 453 7 of 14
We conducted a systematic review of independent studies that assessed the performance of
currently available SARS-CoV-2 serological tests. We included studies that reported sensitivity and
specificity, stage of disease (early, intermediate, or late), the test format (CLIA, ELISA, RDT), and
antibody target (IgA, IgG, IgM, or IgG + IgM) [24,25]. If available, the SARS-CoV-2 antigens used for
antibody detection was recorded. The studies that did not specify the disease stage of test samples
were grouped under the “overall” category and assessed separately. In total, we reviewed performance
data on 5 serological CLIA tests, 15 serological ELISA tests, and 42 serological RDTs currently on the
market (see Supplementary Materials).
The distribution plot of the data shows a higher degree of variability in test sensitivity values
Diagnostics 2020,
compared 10, x FOR PEER
to specificity REVIEW
(Figure 7 of 13
5). This level of variability further emphasizes the need for independent
evaluations of serological tests on the market. The sensitivity/specificity plots highlight tests at various
effective in later stages of the disease when higher IgG and IgM levels are present in the blood (Figure
stages of COVID-19 and confirm the expectation that serological tests are more effective in later stages
6). The heatmap of tests in the “overall” category ranks the highest-performing test in each target
of the disease when higher IgG and IgM levels are present in the blood (Figure 6). The heatmap of
antibody category based on sensitivity, followed by specificity (Figure 7). Top-performing COVID-
tests in the “overall” category ranks the highest-performing test in each target antibody category based
19 serological tests (>95% sensitivity and specificity) from the XY plots and heatmap are summarized
on sensitivity, followed by specificity (Figure 7). Top-performing COVID-19 serological tests (>95%
in Table 1.
sensitivity and specificity) from the XY plots and heatmap are summarized in Table 1.
Figure 5. Overview of reported sensitivity and specificity of COVID-19 serological tests. Reported
Figure 5. Overview of reported sensitivity and specificity of COVID-19 serological tests. Reported
performance data from categories were plotted based on target antibody (IgG, IgM, IgG/IgM) to
performance data from categories were plotted based on target antibody (IgG, IgM, IgG/IgM) to
provide an overview of variability distribution in marketed diagnostic tests. Blue bars represent the
provide an overview of variability distribution in marketed diagnostic tests. Blue bars represent the
mean values.
mean values.
Diagnostics 2020, 10, 453 8 of 14
Diagnostics 2020, 10, x FOR PEER REVIEW 8 of 13
Figure 6. Independent evaluation of COVID-19 serological tests at various stages of the disease.
XYFigure
plots of reported sensitivity
6. Independent and of
evaluation specificity
COVID-19 in detection
serologicaloftests
IgM,atIgG, or IgG/IgM
various antibodies
stages of wereXY
the disease.
generated at different stages of the disease, including early (1–7 days after symptom onset),
plots of reported sensitivity and specificity in detection of IgM, IgG, or IgG/IgM antibodies were intermediate
(7–14 days after
generated symptomstages
at different onset),ofandthelate (>14 days
disease, after symptom
including early (1–7onset).
daysELISA: enzyme-linked
after symptom onset),
immunosorbent
intermediate (7–14assay;days
CLIA: chemiluminescence
after symptom onset), immunoassay;
and late (>14 daysRDT: after
rapidsymptom
diagnosticonset).
test (lateral
ELISA:
flow immunoassay).
enzyme-linked Dotted lines represent
immunosorbent assay; 90% sensitivity
CLIA: and specificity immunoassay;
chemiluminescence levels. Refer to Figure
RDT: 7rapid
for
test names and manufacturers associated with number shown here.
diagnostic test (lateral flow immunoassay). Dotted lines represent 90% sensitivity and specificity
levels. Refer to Figure 7 for test names and manufacturers associated with number shown here.
Diagnostics 2020, 10, 453 9 of 14
Diagnostics 2020, 10, x FOR PEER REVIEW 9 of 13
No. Test Name Manufacturer Ref No.
1 IgG/IgM ELISA kits Beijing Wantai Biological Pharmacy 1 No. Test Name Manufacturer Ref No.
2 Wantai SARS-CoV-2 Ab ELISA Beijing Wantai Biological Pharmacy 15 31 Anti-SARS-CoV-2 Rapid Test AutoBio Diagnostics 15
3 Wantai SARS-CoV-2 IgG ELISA Beijing Wantai Biological Pharmacy 9 32 Coronavirus Diseases 2019 (COVID-19) IgM/IgG Antibody Test Artron Laboratories 15
4 Wantai SARS-CoV-2 IgM ELISA Beijing Wantai Biological Pharmacy 9 33 NG-Test IgM-IgG COVID All-in-One lateral flow immunoassay NG Biotech 20
5 Wantai SARS-CoV-2 Ab Rapid Test Beijing Wantai Biological Pharmacy 9 34 Covid-19 IgG/IgM Antibody Test Biomerica 20
6 CMIA-IgM Xiamen InnoDx Biotech Co., Ltd. 9 35 Acro Biotech COVID-19 Rapid POC test Acro Biotech 20
7 IgG/IgM ELISA kit Zhuhai Livzon Diagnostics Inc 2 36 Rapid SARS-CoV-2 Antibody (IgM/IgG) InTec Products 20
8 2019-nCoV IgG/IgM Antibody Detection Kit Zhuhai Lizhu Reagent 3 37 DiagnoSure COVID-19 IgG/IgM rapid test cassette GritOverseas Pte. 20
9 SARS-CoV-2 IgM/IgG CLIA kits Shenzhen Yhlo Biotech 4 38 Diagnostic kit IgM/IgG of Novel Coronavirus COVID-19 Shanghai LiangRun, Biomedicine Tech 20
10 Chemiluminescence detection kit Shenzhen Yhlo Biotech 7 39 COVID-19 IgG/IgM Rapid Test Cassette (Whole Blood/Serum/Plasma) Orient Gene / Healgen 20
11 2019-nCoV IgG/IgM Rapid Test Cassette Zhejang Orient Gene Biotech 5 40 COVID-19 rapid test PharmaAct AG 16
12 STANDARD Q COVID-19 IgM/IgG Duo Test SD BIOSENSOR, Inc. 20 41 Rapid SARS-CoV-2 Antibody (IgM/IgG) AMEDA Labordiagnostik GmbH 20
13 MAGLUMI 2019-nCoV IgM (CLIA) Snibe Co. 20 42 SARS-CoV-2 IgM/IgG Ab Rapid Test Sure Bio-Tech (USA) 17
14 COVID-19 IgG/IgM Combo Rapid Test Device Liming Bio-Products 20 43 COVID-19 IgM-IgG Dual Antibody Rapid Test BioMedomics 17
15 AllTest COV-19 IgG / IgM kit AllTest Biotech 6 44 Novel Coronavirus
p g(2019-nCoV)
g Ab Test
g (Colloidal Gold) y Innovita Biological Technology 17
16 Cellex qSARS-CoV-2 IgGIgM Cassette Rapid Test Cellex 20 45 test Jiangsu Medomics Medical Technologies 11
17 SARS-CoV-2 IgM/IgG Ab Rapid Test Shanghai Kehua Bio-engineering 20 46 COVID-19 (SARS-CoV-2) IgG/IgM Antibody Test Kit (Colloidal Gold) DeepBlue Medical Technology 17
18 COVID-19 rapid test PRIMA Lab S.A. 20 47 Novel Coronavirus (SARSCoV-2) IgM/IgG Combo Rapid Test-Cassette Decombio Biotechnology 17
19 COVID-19 ELISA IgG DIA PRO Diagnostic BioProbes 20 48 Novel Coronavirus (2019-nCoV) Ab Test (Colloidal Gold) Innovita Biological Technology 17
20 COVID-19 ELISA IgG Epitope Diagnostics 20 49 COVID-19 IgG/IgM Rapid Test Cassette Premier Biotech 17
21 VivaDiag COVID-19 IgM/IgG Rapid Test VivaChek Biotech 8 50 Coronavirus IgG/IgM Antibody (COVID-19) Test Cassette UCP Biosciences 17
22 Coronavirus IgG/IgM antibody GICA kit Zhuhai Livzon Diagnostics 9 51 EDI? Novel Coronavirus COVID-19 IgM ELISA Kit Epitope Diagnostics 17
23 SARS-CoV-2 ELISA Darui Biotech 10 52 Coronavirus IgG/IgM Antibody (COVID-19) Test Cassette UCP Biosciences 17
24 Rapid SARS-CoV-2 Antibody (IgM/IgG) InTec Products 20 53 COVID-19 IgG ELISA Mologic Ltd 18
25 COVID-19 IgG/IgM Rapid Test Cassette (Whole Blood/Serum/Plasma) Orient Gene / Healgen 20 54 One Step Novel Coronavirus (COVID-19) IgM/IgG Antibody Test Artron Laboratories 19
26 2019-nCoV IgG/IgM Rapid Test Dynamiker Biotechnology 20 55 AllTest COV-19 IgG / IgM kit AllTest Biotech 6
27 Novel Coronavirus IgG/IgM antibody ELISA kit Zhuhai Livzon Diagnostics 12 56 SARS-COV-2 ELISA (IgG) EUROIMMUN 21
28 Novel Coronavirus (2019- nCoV) IgM/IgG Antibody Test Kit Zhuhai Livzon Diagnostics 14 57 COVID-19 IgG/IgM Rapid Test Cassette Healgen 21
21 VivaDiag COVID-19 IgM/IgG Rapid Test VivaChek Biotech 8 58 COVID-19 IgM-IgG Rapid Test Kit Biomedomics 21
29 Anti-SARS-CoV-2 ELISA (IgG) (manual, automated) EUROIMMUN AG 15 59 COVID-19 Rapid Test Phamatech 21
30 OnSite COVID-19 IgG/IgM Rapid Test CTK Biotech, Inc. 15 60 SARS-CoV-2 IgG/IgM Antibody Detection Kit Tianjin Beroni Biotechnology 21
Figure Figure 7. Independent
7. Independent evaluation
evaluation ofofSARS-CoV-2
SARS-CoV-2 serological
serological test
testoverall
overallperformance. Sensitivity
performance. and and
Sensitivity
specificity data from studies that did not specify the COVID-19 stage (”overall” group) are
specificity data from studies that did not specify the COVID-19 stage (“overall” group) are represented
represented in a heatmap. The heatmap is ordered according to the antibody target (IgG, IgM, and
in a heatmap. The heatmap is ordered according to the antibody target (IgG, IgM, and IgG/IgM)
IgG/IgM) followed by sensitivity and specificity values. All analyses were conducted using software
followed by sensitivity and specificity values. All analyses were conducted using software R version
R version 3.6.3. Heatmaps were generated using the gplots and RColorBrewer packages. ELISA:
3.6.3. enzyme-linked
Heatmaps wereimmunosorbent
generated using the gplots
assay; CLIA:and RColorBrewer packages.
chemiluminescence ELISA:RDT:
immunoassay; enzyme-linked
rapid
immunosorbent assay;
diagnostic test. CLIA: chemiluminescence immunoassay; RDT: rapid diagnostic test.
Table 1. Top-performing COVID-19 serological tests based on independent evaluations of
sensitivity/specificity.
Disease Sensitivity Specificity
No. Test Name [ref] Manufacturer Format Target Antibody Antigen
Stage (%) (%)
Beijing Wantai
Wantai SARS-CoV-2
2 Biological ELISA IgG/IgM/IgA Overall 95.4 100 RBD
Ab ELISA [26]
Pharmacy
2019-nCoV IgG/IgM
Zhuhai Lizhu Late (>14
8 Antibody Detection ELISA IgG/IgM 95 100 NP
Reagent days)
Kit [27]
Xiamen InnoDx
6 CMIA-Ab [28] CLIA IgG/IgM Overall 96.2 99.3 RBD
Biotech Co., Ltd.
Chemiluminescen. Shenzhen Yhlo IgM 100 98.5
10 CLIA Overall n/a
detection kit [29] Biotech IgG 100 99
MAGLUMI 2019-
13 Snibe Co. CLIA IgG Overall 98.8 95.1 n/a
nCoV IgG [25]
Architect SARS-CoV- Late (>14
61 Abbott CLIA IgG 97.2 100 NP
2 IgG Assay [30] days)
Beijing Wantai
Wantai SARS-CoV-2
5 Biological RDT IgG/IgM/IgA Overall 97.5 95.2 RBD
Ab Rapid Test [28]
Pharmacy
NG-Test IgM-IgG Late (>14
33 NG Biotech RDT IgG/IgM 97 100 n/a
COVID All-in-One days)Diagnostics 2020, 10, 453 10 of 14
Table 1. Top-performing COVID-19 serological tests based on independent evaluations of sensitivity/specificity.
Target Sensitivity Specificity
No. Test Name [ref] Manufacturer Format Disease Stage Antigen
Antibody (%) (%)
Beijing Wantai Biological
2 Wantai SARS-CoV-2 Ab ELISA [26] ELISA IgG/IgM/IgA Overall 95.4 100 RBD
Pharmacy
8 2019-nCoV IgG/IgM Antibody Detection Kit [27] Zhuhai Lizhu Reagent ELISA IgG/IgM Late (>14 days) 95 100 NP
Xiamen InnoDx Biotech
6 CMIA-Ab [28] CLIA IgG/IgM Overall 96.2 99.3 RBD
Co., Ltd.
IgM 100 98.5
10 Chemiluminescen. detection kit [29] Shenzhen Yhlo Biotech CLIA Overall n/a
IgG 100 99
13 MAGLUMI 2019-nCoV IgG [25] Snibe Co. CLIA IgG Overall 98.8 95.1 n/a
61 Architect SARS-CoV-2 IgG Assay [30] Abbott CLIA IgG Late (>14 days) 97.2 100 NP
Beijing Wantai Biological
5 Wantai SARS-CoV-2 Ab Rapid Test [28] RDT IgG/IgM/IgA Overall 97.5 95.2 RBD
Pharmacy
NG-Test IgM-IgG COVID All-in-One Lateral Flow
33 NG Biotech RDT IgG/IgM Late (>14 days) 97 100 n/a
Immunoassay [25]
Mid.
34 Covid-19 IgG/IgM Antibody Test [25] Biomerica RDT IgG 100 100 n/a
(8–14 days)
Mid.
35 Acro Biotech COVID-19 Rapid POC Test [25] Acro Biotech RDT IgG 100 100 n/a
(8–14 days)
IgG Mid. 100 100
37 DiagnoSure COVID-19 IgG/IgM Rapid Test Cassette [25] GritOverseas Pte. RDT n/a
IgM (8–14 days) 100 100
AMEDA Labordiagnostik
41 Rapid SARS-CoV-2 Antibody (IgM/IgG) [25] RDT IgG/IgM Late (>14 days) 95.7 97.4 n/a
GmbH
IgM 100 100
57 COVID-19 IgG/IgM Rapid Test Cassette [24] Healgen RDT Overall n/a
IgG 96.7 97.5Diagnostics 2020, 10, 453 11 of 14
7. Seroprevalence of SARS-COV-2-Specific Antibodies
In light of policies to ease the lockdown and reopen the economy, large-scale seroprevalence
studies to screen for immunity status are being implemented in several jurisdictions. Critics point
to gaps in our understanding of immune response to COVID-19 infection, including the ability of
serological tests to detect neutralizing antibodies and the capacity of the immune system to provide
long-term immunity against SARS-CoV-2. However, some argue that in the context of a global
viral outbreak with a relatively high mortality rate, inaction due to uncertainty can have negative
consequences compared to the harm caused by false-positive and false-negative serological test
results [31]. Several jurisdictions have initiated seroprevalence studies to provide a more accurate
estimate of cases with positive SARS-CoV-2-specific antibodies, irrespective of disease symptoms.
In Los Angeles County, the prevalence of SARS-CoV-2 antibodies in the community was estimated to
be 4.65%, equivalent to 367,000 adults, which was substantially greater than the 8430 confirmed cases
in the same county at the time of the study [32]. In New York City, 19.9% of the population has been
estimated to have SARS-CoV-2 antibodies, compared to 2.1% confirmed cases as of 2 May 2020 [33].
Similar studies from Germany, the U.K., Singapore, and China show significantly higher estimates of
positive SARS-CoV-2 antibody cases compared to symptomatic cases confirmed by molecular tests [34].
As undetected cases with mild or no symptoms can transmit the virus, it is not surprising that countries
(e.g., South Korea, Germany, and Singapore) with large-scale and well-organized testing programs,
combined with extensive isolation and contact tracing for infected individuals, have had some success
in minimizing COVID-19-related death in their populations [35].
8. Concluding Remarks
As serological tests are in high demand, in part due to an increase in large-scale seroprevalence
studies, it is imperative for national and regional governments to continue coordinated efforts
to independently validate serological test performance and partner with industry to scale up
manufacturing and production capacity. Existing emergency authorization programs, intended
to accelerate the manufacturing of diagnostic tests, must also be accompanied by clear and informed
guidelines on preferred and minimally acceptable profiles of COVID-19 serological tests designed for
specific indications.
Despite the unprecedented response to the outbreak, major gaps remain in our understanding of
the interaction between SARS-CoV-2 and the immune system, which can negatively impact serological
testing utilization. Coordinated research efforts are urgently needed to investigate some of the key
gaps in our knowledge, including:
1. Which serological tests can identify SARS-CoV-2-neutralizing antibodies?
2. Is there cross-reactivity between neutralizing antibodies and other coronaviruses?
3. Which SARS-CoV-2 antigens are optimal for the detection of neutralizing antibodies?
4. What is the correlation between SARS-CoV-2-specific antibodies and protective immunity status?
5. How long does protective immunity last in recovered patients? Are individuals susceptible to
reinfection with SARS-CoV-2?
6. Is humoral antibody response the best indicator for protective immunity, or are there other
immune-cell-based mechanisms?
In the context of the COVID-19 outbreak and the execution of return-to-work policies, failing
to take advantage of available diagnostic tools due to uncertainty can have profound consequences.
Medical professionals frequently rely on imperfect evidence with the possibility of false positives and
false negatives. It is, however, important to clearly understand the limits and potential of serological
tests to make informed decisions based on risk and benefit assessment in each specific situation.
In the words of Tedros Adhanom Ghebreyesus, Director-General of the World Health Organization,
“Countries cannot fight this pandemic blindfolded. Countries should know where the cases are.”Diagnostics 2020, 10, 453 12 of 14
Supplementary Materials: The following are available online at http://www.mdpi.com/2075-4418/10/7/453/s1.
Author Contributions: Conceptualization, A.G., R.M.; methodology, A.G.; formal analysis, A.G.; data curation,
A.G.; writing—original draft preparation, A.G.; writing—review and editing, A.G., R.M. and A.A.; project
administration, A.A.; funding acquisition, A.A. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by Novateur Ventures Inc.
Conflicts of Interest: The authors declare no conflict of interest. The funder had no role in the design of the study;
in the collection, analyses, or interpretation of data
Abbreviations
COVID-19 Coronavirus Disease 2019
SARS Severe Acute Respiratory Syndrome
SARS-CoV Severe Acute Respiratory Syndrome Coronavirus
SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2
MERS Middle East Respiratory Syndrome
BAL Bronchoalveolar Lavage
EUA Emergency Use Authorization
RT-PCR Reverse Transcriptase Polymerase Chain Reaction
S SARS-CoV-2 Spike Protein
E SARS-CoV-2 Envelope Protein
M SARS-CoV-2 Membrane Protein
NP SARS-CoV-2 Nucleocapsid Protein
RBD Receptor-Binding Domain
ELISA Enzyme-Linked Immunosorbent Assay
RDT Rapid Diagnostic Test
CLIA Chemiluminescence Immunoassay
LFIA Lateral Flow Immunoassay
PoC Point of Care
DABA Double-Antigen-Bridging Assay
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