A Serological Assay To Detect Sars-Cov-2 Seroconversion in Humans
A Serological Assay To Detect Sars-Cov-2 Seroconversion in Humans
A Serological Assay To Detect Sars-Cov-2 Seroconversion in Humans
https://doi.org/10.1038/s41591-020-0913-5
Here, we describe a serological enzyme-linked immunosor- can help inform studies that aim to identify antibody responses that
bent assay for the screening and identification of human correlate with protection from SARS-CoV-2.
SARS-CoV-2 seroconverters. This assay does not require the Sarbecoviruses express a large (approximately 140 kDa) glycopro-
handling of infectious virus, can be adjusted to detect differ- tein termed spike protein (S, a homotrimer), which mediates bind-
ent antibody types in serum and plasma and is amenable to ing to host cells via interactions with the human receptor angiotensin
scaling. Serological assays are of critical importance to help converting enzyme 2 (ACE2)1–3. The S protein is highly immuno-
define previous exposure to SARS-CoV-2 in populations, iden- genic with the receptor-binding domain (RBD) being the target of
tify highly reactive human donors for convalescent plasma many neutralizing antibodies4. Individuals infected with coronavi-
therapy and investigate correlates of protection. ruses typically mount neutralizing antibodies5 and a neutralizing
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)— response has been demonstrated for SARS-CoV-2 in an individual
a member of the subgenus Sarbecovirus—has spread globally, case from day 9 onwards6. For human coronaviruses these responses
causing a pandemic with, so far, 3.6 million infections and 250,000 have been linked to protection for a period of time and future stud-
fatalities (as of 5 May 2020). ies will show if there is a correlation between neutralizing antibodies
Nucleic acid tests that detect the SARS-CoV-2 RNA genome and protection from SARS-CoV-2 infection as well5. Serum neutral-
are now widely employed to diagnose coronavirus disease 2019 ization can be measured using replication competent virus but the
(COVID-19). However, there remains a great need for assays process requires several days and must be conducted in a biosafety
that measure antibody responses and determine seroconversion. level 3 laboratory for containment of SARS-CoV-2. Potentially,
While such serological assays are not well suited to detect acute pseudotyped viral particle based entry assays using lentiviruses or
infections, they support a number of highly relevant applications. vesicular stomatitis virus could be used but these reagents are not
First, serological assays allow us to study the immune response(s) trivial to produce. A simple solution is the use of a binding assay,
to SARS-CoV-2 in a qualitative and quantitative manner. Second, e.g. an enzyme-linked immunosorbent assays (ELISA), with recom-
serosurveys are needed to determine the precise rate of infection in binant antigen as substrate, especially if ELISA results correlate with
an affected area, which is an essential variable to accurately deter- neutralization assay results. Here we report the development of
mine the infection fatality rate. Third, serological assays will allow such an assay and provide a protocol for both recombinant antigen
for the identification of individuals who mounted strong antibody production as well as the ELISA methodology7.
responses and who could serve as donors for the generation of We generated two different versions of the SARS-CoV-2 spike
convalescent serum/plasma therapeutics. Lastly, serological assays protein, based on the genomic sequence of the first virus isolate,
1
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 2Graduate School of Biomedical Sciences, Icahn School
of Medicine at Mount Sinai, New York, NY, USA. 3Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
4
Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria,
Australia. 5Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 6Global Health and Emerging
Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 7Division of Infectious Diseases, Department of Medicine, Icahn School
of Medicine at Mount Sinai, New York, NY, USA. 8Division of Pulmonary, Critical Care, and Sleep Medicine, Icahn School of Medicine at Mount Sinai,
New York, NY, USA. 9Department of Medicine, Mount Sinai Queens, Astoria, NY, USA. 10Division of Hospital Medicine, Mount Sinai Health System,
New York, NY, USA. 11Division of Clinical Immunology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
12
Department of Pediatrics, the Icahn School of Medicine at Mount Sinai, NY, USA. 13Department of Physiology and Biophysics, University of California,
Irvine, Irvine, CA, USA. 14Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 15Travel Medicine Program, Division of
Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 16School of Public Health and Preventive Medicine, Monash University
and Infection Prevention and Healthcare Epidemiology Unit Alfred Health, Melbourne, Victoria, Australia. 17Department of Virology, Medicum, University
of Helsinki, Helsinki, Finland. 18Veterinary Biosciences, Veterinary Faculty, University of Helsinki, Helsinki, Finland. 19Department of Virology and
Immunology, Helsinki University Hospital (HUSLAB), Helsinki, Finland. 20Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zürich, Zürich,
Switzerland. ✉e-mail: [email protected]
a 2.5
b 2.5
c 4
d 4
Post-NL63
2.0 2.0
3 3 Other negative control samples
1.5 1.5
OD490
OD490
OD490
OD490
SARS-CoV-2 samples
2 2
1.0 1.0
1 1
0.5 0.5
0 0 0 0
100 1,000 10,000 100 1,000 10,000 100 1,000 10,000 100 1,000 10,000
Reciprocal dilution Reciprocal dilution Reciprocal dilution Reciprocal dilution
e f g h
10,000 10,000 10,000 10,000
P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001
AUC
AUC
AUC
10 10 10 10
1 1 1 1
te 2
es
te 2
es
te 2
es
te 2
er V-
er V-
er V-
er V-
pl
pl
pl
pl
rs
rs
rs
rs
nv Co
nv Co
nv Co
nv Co
am
am
am
am
co -
co -
co -
co -
ro RS
ro RS
ro RS
ro RS
ls
ls
ls
ls
tro
tro
tro
tro
SA
SA
SA
se SA
on
on
on
on
C
C
se
se
se
i j k
4 2.0 10,000
Microneutralization IC50
Pearson's r = 0.9279
1,000 P < 0.0001
3 1.5
100
OD490
OD490
2 1.0
10
1 0.5
1 Negative controls
0 0 0.1
100 1,000 10,000 100 1,000 10,000 10 100 1,000 10,000 100,000
Reciprocal dilution Reciprocal dilution mSpike ELISA end-point titer
Fig. 1 | Reactivity of control and SARS-CoV-2 convalescent sera to different spike antigens. a–d, Reactivity to iRBD (a), mRBD (b), iSpike (c) and mSpike (d).
Red, green and black data points/lines show the results for sera from SARS-CoV-2-infected individuals, a convalescent serum sample post-NL63 infection and
other negative control samples, respectively. e–h, Data from the same experiment as in a–d, respectively, but plotted as AUCs to obtain a better quantitative
impression (control samples: n = 50 for iRDB, iSpike and mSpike; n = 59 for mRBD; convalescent samples: n = 4 for iRBD and iSpike; n = 16 for mRBD and
mSpike). Statistical analyses were performed using an unpaired two-tailed Student’s t-test in GraphPad Prism. Horizontal lines represent mean values.
i,j, Reactivity of the 50 negative control samples from a–h against spike protein from human coronaviruses 229E (i) and NL63 (j). k, Correlation between
ELISA titers and microneutralization titers (n = 12; the three samples from negative control sera overlap and are displayed as a single point). Statistical analysis
was performed using Pearson’s rank test in GraphPad Prism. The experiments were performed once. IC50, half-maximum inhibitory concentration.
Wuhan-Hu-1 (ref. 8). The first construct encodes a full-length tri- (iRBD) running slightly lower than the mammalian cell-derived
meric and stabilized version of the spike protein, whereas the second protein (mRBD) (Extended Data Fig. 1). The size difference prob-
produces only the much smaller RBD. Sequences were codon opti- ably reflects differences in glycan sizes between insect cells and
mized for mammalian cell expression. The full-length spike protein mammalian cells. The full-length S protein was also expressed in
sequence was modified to remove the polybasic cleavage site, which both systems with higher yields in mammalian cells (mSpike) than
is recognized by furin, and to add a pair of stabilizing mutations in insect cells (iSpike) (~5 versus ~0.5 mg l−1 of culture). Reducing
(Extended Data Fig. 1)2,9,10. These two modifications were included SDS-PAGE showed the full-length protein as a prominent band
to enhance the stability of the protein based on published litera- between 135 and 190 kDa, followed by a faint second band slightly
ture2,9. At amino acid P1213, the sequence was fused to a thrombin below, which may be a cleavage product.
cleavage site, a T4 foldon sequence for proper trimerization and a ELISAs were performed by serial dilution of the individual
carboxy (C)-terminal hexahistidine tag for purification (Extended serum samples. Values from the dilution curves were used to deter-
Data Fig. 1). The sequence was cloned into a pCAGGS vector mine the area under the curve (AUC), which was plotted on a graph.
for expression in mammalian cells and into a modified pFastBac Initially, we tested a panel of 50 (59 for mRBD) banked human
Dual vector for the generation of baculoviruses and expression in serum samples collected from study participants with and with-
insect cells. For expression of the RBD, the natural amino-terminal out confirmed previous viral infections (but otherwise healthy), to
signal peptide of S was fused to the RBD sequence (amino acids establish an ELISA with our proteins. These human sera were used
319–541) and joined with a C-terminal hexahistidine tag11. The to test the background reactivity to the SARS-CoV-2 spike in sam-
same vectors as for the full-length S protein were used to express ples representative of the general US population from individuals
the RBD. In mammalian cells (Expi293F), the RBD domain gave ranging from 20 to ≥65 years. An initial set of four plasma/serum
high yields (approximately 25–50 mg l−1 of culture), but expression samples from three COVID-19 survivors were used to determine
was lower in insect cells (approximately 1.5 mg l−1 of culture). Clear the reactivity of SARS-CoV-2-infected individuals to the RBD and
single bands were visible when the recombinant RBD proteins were the full-length spike (Fig. 1).
analyzed by reducing sodium dodecyl sulfate–polyacrylamide gel All COVID-19 plasma/serum samples reacted strongly to
electrophoresis (SDS-PAGE), with the insect cell-derived protein both RBD and full-length spike protein, whereas reactivity of the
AUC
AUC
difference between control sera and convalescent samples (initial
n = 4) was larger when the mRBD was used compared with the 100 100
iRBD. The same was true for the full-length spike protein. We tested
an additional 12 serum samples from patients with acute COVID-
19 disease, as well as convalescent participants, for reactivity to 10 10
mRBD and mSpike (Fig. 1). All 12 samples reacted with both RBD
in
in
d
d
te
te
m
m
iva
iva
and spike protein. Thus, our assay distinguished sera from par-
0
r6
r6
t
t
ac
ac
fo
fo
-in
-in
ticipants diagnosed with COVID-19 from serum samples collected
°C
°C
on
on
56
56
N
N
before the pandemic (for example, collected in the autumn of 2019).
Our initial set of negative controls included convalescent serum
c d 10,000
from a participant with a confirmed NL63 infection. Importantly, 10,000 P = 0.0311
this sample did not produce a signal against the SARS-CoV-2 RBD
or spike. Since human coronaviruses OC43, 229E, NL63 and/or
1,000 1,000
HKU1 are responsible for a large proportion of common colds every
AUC
year, cross-reactivity between SARS-CoV-2 and these seasonal coro-
AUC
naviruses is of particular importance and warrants further investi- 100 100
gation. To test how common antibodies to human coronaviruses
other than SARS-CoV-2 are in our pre-pandemic serum panel, P = 0.4448
we performed ELISAs coated with spike protein of coronaviruses 10 10
229E and NL63. While none of the negative control sera reacted to
a
m
sm
ru
ru
SARS-CoV-2 RBD and spike, the majority of samples yielded strong
as
Se
Se
a
Pl
Pl
signals to the spike proteins of these two human coronaviruses
(Fig. 1i,j). In addition, we tested 21 different batches (27 vials) of Fig. 2 | Effect of heat treatment and serum versus plasma on assay
pools of different products of normal human immune globulin performance. a,b, Reactivity of paired non-treated serum and heat-treated
(NHIG) that were intended for intravenous use and derived from serum samples to mRBD (a) and mSpike (b) of SARS-CoV-2 (n = 5).
>1,000 donors each. None of the NHIG preparations reacted with c,d, Reactivity of paired serum and plasma samples to mRBD (c) and
SARS-CoV-2 RBD or spike protein and the signal obtained was sim- mSpike (d) of SARS-CoV-2 (n = 7). Statistical analyses were performed
ilar to that of the three irrelevant human monoclonal antibodies. In using a paired two-tailed Student’s t-test in GraphPad Prism. The experiments
contrast, the RBD-binding monoclonal antibody CR3022 produced were performed once.
a strong signal in the ELISA (Extended Data Fig. 2a,b)12–14. Lastly,
we tested a panel of 50 plasma samples collected from patients posi-
tive for human immunodeficiency virus and banked from 2008 and and quick in its execution and can be performed at biosafety level 2
2011. Again, none of the samples reacted with the SARS-CoV-2 as it does not involve live virus. We have tested this method using
RBD or spike (Extended Data Fig. 2c,d). banked serum samples and NHIG preparations obtained from
For the plasma/sera of patients with COVID-19 from our ini- individuals before SARS-CoV-2 started to widely circulate in the
tial panel, we performed an isotyping and subtyping ELISA using United States. These serum samples produced low, close-to-baseline
the mammalian cell-expressed S proteins. Strong reactivity was signals in our ELISAs. The age of the participants ranged from 20
found for all samples for immunoglobulin G3 (IgG3), IgM and to ≥65 years of age and it is likely that most of these individuals
IgA (Extended Data Fig. 3a). An IgG1 signal was detected for the had experienced infections with human coronaviruses, including
majority of samples, in addition to low reactivity for IgG2 (in five of the alphacoronaviruses NL63 and 229E, as well as the betacoronavi-
the COVID-19 samples) and IgG4 (in four samples). Furthermore, ruses OC43 and HKU1 (ref. 5). In fact, the majority of our negative
we correlated the ELISA reactivity with the neutralizing activity control subjects had strong reactivity to the spike protein of NL63
of sera against the USA-WA1/2020 isolate. ELISA titers and micro- and 229E, but showed no cross-reactivity to SARS-CoV-2 RBD and
neutralization titers correlated significantly (Fig. 1k and Extended spike. We also included a convalescent serum sample from a partici-
Data Fig. 3b), with a Spearman’s r of 0.9279 (P < 0.0001). pant with a laboratory-confirmed coronavirus NL63 infection. Our
One complexity with measuring antibodies in the bodily fluids data show that there is no or only negligible cross-reactivity from
of patients with COVID-19 is that infectious virus could be pres- human coronaviruses to SARS-CoV-2 in these individuals. Similar
ent in the biospecimen. To limit this risk, serum or plasma is heat findings were reported in a recent study where sera from negative
inactivated for 1 h at 56 °C. To test whether such a heat treatment control subjects reacted well with spike proteins from human coro-
has an effect on detecting antibodies to the SARS-CoV-2 RBD navirus but not with SARS-CoV-2 (ref. 15). This is notable because
and spike, we compared the reactivity of matched non-treated and it suggests that humans are serologically naive to SARS-CoV-2,
heat-treated serum samples from patients with COVID-19. While which may explain the relatively high basic reproduction num-
slight differences were observed, they were minimal, suggesting that ber (or R0) of SARS-CoV-2 compared with that of other respira-
heat treatment may have no negative impact on assay performance tory viruses, such as influenza virus16. As a caveat, the reactivity of
(Fig. 2a,b). Similarly, we tested matched serum and plasma samples samples from SARS-CoV-1- or Middle East respiratory syndrome
from patients with COVID-19 and found negligible differences, coronavirus-infected individuals was not tested, and cross-reaction
suggesting that both types of specimens can be used in the assay might occur in this assay. Another caveat is of course the relatively
interchangeably (Fig. 2c,d). small number of samples tested.
Here, we describe a serological method to detect seroconver- Our data show strong seroconversion with ELISA AUC values
sion upon SARS-CoV-2 infection. The method is based on reactiv- in the 1:1,000 range after natural infection with SARS-CoV-2.
ity to the immunogenic S protein of the virus, is relatively simple The results from our assay suggest that antibodies mounted upon
infection target the full-length S protein as well as the RBD, which code availability are available at https://doi.org/10.1038/s41591-
is the major target for neutralizing antibodies for related corona- 020-0913-5.
viruses4. In fact, one of the SARS-CoV-2 samples was previously
tested in another study in neutralization assays and showed a neu- Received: 19 March 2020; Accepted: 28 April 2020;
tralizing titer of 1:160 (ref. 6). In addition, we performed microneu- Published: xx xx xxxx
tralization assays with a subset of our samples and found excellent
correlation between our ELISA titers against the spike protein and References
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Extended Data Fig. 1 | Constructs for recombinant protein expression. a, Visualization of the trimeric spike protein of SARS-CoV-2 based on PBD # 6VXX
using Pymol3. One monomer is colored in dark blue while the remaining two monomers are held in light blue. The receptor binding domain (RBD) of the
dark blue trimer is highlighted in red. b, Schematic of the wild type full length spike protein with signal peptide, ectodomain, receptor binding domain, furin
cleavage site, S1, S2, and transmembrane and endodomain domain indicated. c, Schematic of the soluble trimeric spike. The polybasic/furin cleavage site
(RRAR) was replaced by a single A. The transmembrane and endodomain were replaced by a furin cleavage site, a T4 foldon tetramerization domain and
a hexahistidine tag. Introduction of K986P and V987P has been shown to stabilize the trimer in the pre-fusion conformation. d, Schematic of the soluble
receptor binding domain construct. All constructs are to scale. e Reducing SDS PAGE of insect cell and mammalian cell derived soluble trimerized spike
protein (iSpike and mSpike). f Reducing SDS PAGE of insect cell derived and mammalian cell derived recombinant receptor binding domain (iRBD and
mRBD). Experiments were performed six times with the same result.
Extended Data Fig. 2 | Human normal immunoglobulin preparations and historic sera from HIV + patients do not react with the SAR-CoV-2 spike.
a, b, Reactivity of 21 different pools of human normal immunoglobulin (HNIG) preparations (27 different vials) to mRBD and mSpike of SARS-CoV-2.
MAb CR3022 was used as positive control, three different irrelevant human mAbs were used as negative control. c, d shows reactivity of historic
samples from 50 HIV + individuals to mRBD and mSpike of SARS-CoV-2. Both HNIG and serum samples from HIV + donors were collected before the
SARS-CoV-2 pandemic. Experiments were performed once. MAb CR3022 was used as positive control at a starting concentration of 100 ug/ml. Of note,
the experiments in A and C as well as B and D were done at the same time and their positive controls are shared and displayed in both panels. Experiments
were performed once.
Extended Data Fig. 3 | Isotypes and subtypes of antibodies from COVID19 patients to the soluble spike protein and microneutralization titers.
a, Mammalian cell derived spike protein was used to study isotype/subclass distribution of antibodies (n = 13 positive samples). Lines represent the
geometric mean. b, Microneutralization assay (n = 12) performed with authentic SARS-CoV-2. Lines represent curves fitted using an inhibitor (log) versus
response variable slope with four parameters function in Graphpad Prism. Experiments were performed once.
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative.
Sample size Sample size was determined/limited by available number of samples. The maximum number of samples available was analyzed. Positive and
negative control samples for assay development showed a clear difference in reactivity that could already be detected with an n of 4 positive
samples.
Replication Assays were repeated with 4 different substrates. ELISAs for each substrate were run once each. All attempts at replication were successful.
Randomization Randomization was not performed since the purpose of this work was assay development.
Blinding Blinding was not performed since the purpose of this work was assays development. Performance tests of this assay setup in our clinical
laboratory have been conducted using blinded operators.
Antibodies
Antibodies used mAb CR3022 is a published antibody with known reactivity to the RBD of SARS-CoV-1 and 2. 1C7 is an unpublished in-house mAb
with reactivity to the N protein of SARS-CoV-1 and 2.
Validation Both mAbs were validated by binding studies to cells infected with SARS-CoV-2.
Authentication No authentication was performed. All expression constructs were Sanger sequenced.
Mycoplasma contamination The cell lines were not tested for mycoplasma.
Population characteristics Only de-identified samples were used. This is considered non-human subject research. 16 samples were from COVID19 survivors,
109 negative control samples were from a non-COVID19 infected cohort age 20 to 65+.
Ethics oversight Alfred Hospital (ID #280/14) and University of Melbourne (ID #1442952.1, 1955465.2) Human Research Ethics Committees,
under research permit for project TYH2018322 of Helsinki University Hospital Laboratory and by the IRB of the Icahn School of
Medicine at Mount Sinai, NY
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Note that full information on the approval of the study protocol must also be provided in the manuscript.