Indian J Med Res 151, May 2020, pp 444-449 Quick Response Code:

DOI: 10.4103/ijmr.IJMR_2232_20
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Development of indigenous IgG ELISA for the detection of

anti-SARS-CoV-2 IgG

Gajanan Sapkal1,#, Anita Shete-Aich2,#, Rajlaxmi Jain2, Pragya D. Yadav2, Prasad Sarkale2, Rajen Lakra2,
imK/icbKUHM2OIeJcBKnJRfjC8MfTw== on 06/17/2024

Srikant Baradkar2, Gururaj Rao Deshpande1, Deepak Mali2, Bipin N. Tilekar1, Triparna Majumdar2,
Himanshu Kaushal3, Yogesh Gurav4, Nivedita Gupta8, Sreelekshmy Mohandas2, Ketki Deshpande1,
Ojas Kaduskar1, Malvika Salve2, Savita Patil2, Shivshankar Gaikwad1, A.P. Sugunan7, M. Ashok6,
Sidhartha Giri8, Jayanthi Shastri5, Priya Abraham†, Raman R. Gangakhedkar8 & COVID Support Team$

1
Diagnostic Virology Group, 2Maximum Containment Laboratory, 3Human Influenza Group, 4Epidemiology
Group, †ICMR-National Institute of Virology, Pune, 5Department of Microbiology, Kasturba Hospital for
Infectious Diseases, Mumbai, Maharashtra, 6ICMR-National Institute of Virology, Bangalore Unit, Bengaluru,
Karnataka, 7ICMR-National Institute of Virology, Kerala Unit, Alappuzha, Kerala & 8Division of Epidemiology
& Communicable Diseases, Indian Council of Medical Research, New Delhi, India

Background & objectives: Since the beginning of the year 2020, the pandemic caused by severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2) impacted humankind adversely in almost all spheres
of life. The virus belongs to the genus Betacoronavirus of the family Coronaviridae. SARS-CoV-2 causes
the disease known as coronavirus disease 2019 (COVID-19) with mild-to-severe respiratory illness. The
currently available diagnostic tools for the diagnosis of COVID-19 are mainly based on molecular assays.
Real-time reverse transcription-polymerase chain reaction is the only diagnostic method currently
recommended by the World Health Organization for COVID-19. With the rapid spread of SARS-CoV-2,
it is necessary to utilize other tests, which would determine the burden of the disease as well as the spread
of the outbreak. Considering the need for the development of such a screening test, an attempt was made
to develop and evaluate an IgG-based ELISA for COVID-19.
Methods: A total of 513 blood samples (131 positive, 382 negative for SARS-CoV-2) were collected and
tested by microneutralization test (MNT). Antigen stock of SARS-CoV-2 was prepared by propagating
the virus in Vero CCL-81 cells. An IgG capture ELISA was developed for serological detection of anti-
SARS-CoV-2 IgG in serum samples. The end point cut-off values were determined by using receiver
operating characteristic (ROC) curve. Inter-assay variability was determined.
Results: The developed ELISA was found to be 92.37 per cent sensitive, 97.9 per cent specific, robust and
reproducible. The positive and negative predictive values were 94.44 and 98.14 per cent, respectively.
Interpretation & conclusions: This indigenously developed IgG ELISA was found to be sensitive and
specific for the detection of anti-SARS-CoV-2 IgG in human serum samples. This assay may be used for
determining seroprevalence of SARS-CoV-2 in a population exposed to the virus.

Key words COVID-19 - diagnosis - ELISA - human - IgG antibodies - microneutralization test - SARS-CoV-2 - standardization

Equal contribution
#

© 2020 Indian Journal of Medical Research, published by Wolters Kluwer - Medknow for Director-General, Indian Council of Medical Research
444
 SAPKAL et al: IgG ELISA FOR SARS-CoV-2 445

COVID Support Team: Pawar S., Patshute S., Salve V., Natarajan V., Marwah V., Malhotra B., Jain A., Sathe P., More R.,
$

Khedekar R., Suryawanshi D., Kalele K., Kumar A., Upadhyaya C.P., Waghmare A., Gawande P., Gopale S., Acharya M., Holeppanavar M.,
Thorat S., Chopade G., Vidhate S., Khutwad K., Phagiwala D., Bhanarkar S., Rakhe A., Salvi V., Dhaigude S., Ciyona
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Coronavirus disease 2019 (COVID-19), caused actual burden of infection and its spread can be
by severe acute respiratory syndrome coronavirus 2 determined using detection of IgM and IgG antibodies
(SARS-CoV-2), was first reported from China during against SARS-CoV-2 by serological assays such as
December 20191. Within a short period of time, the ELISA10-14. Haveri et al15 reported the presence of
virus has spread across different continents affecting serum IgM and IgG antibodies against SARS-CoV-2
almost 215 countries and territories. Till date, SARS- using immunofluorescence assays. Commercial and
CoV-2 has infected about 5.4 million people and led non-commercial IgM/IgG ELISA for COVID-19
to the death of approximately 0.34 million people (n=61) have been certified for commercial purpose/
imK/icbKUHM2OIeJcBKnJRfjC8MfTw== on 06/17/2024

across the globe2. Coronaviruses are a large family research by different certifying agencies16. About 16
of viruses within the family Coronaviridae, and the ELISA kits are still in the stage of development. These
order Nidovirales3. They cause diseases in a variety assays would be easy to use and be cost-effective
of domestic and wild animals as well as in humans. tools for the detection and surveillance of COVID-19
Human coronoviruses (HCoVs)-229E and OC43 were in resource-limited settings14,15. Here, we report the
the first HCoVs discovered in the 1960s4. Although standardization of an indigenous IgG-ELISA for the
these viruses caused self-limiting respiratory illnesses, detection of anti-COVID-19 IgG antibodies at the
the first HCoV that caused SARS epidemic emerged Indian Council of Medical Research-National Institute
in China during 20035. A second outbreak of severe of Virology (ICMR-NIV), Pune, India.
respiratory illness called the Middle East respiratory
syndrome (MERS) occurred in Saudi Arabia Material & Methods
during 20126. Human clinical specimens: A total of 513 blood
The COVID-19 infection is known to spread samples were included for assay validation. Among
from one person to others via droplets produced while them, 150 serum samples were from real-time reverse
coughing and sneezing. The available data suggest transcription-polymerase chain reaction (real-time
an incubation period between 2 and 14 days, with RT-PCR)-confirmed COVID-19 patients after the
an average of five days. Approximately 80 per cent second week of the onset of the disease. Those
of the infected cases had mild illness; 14-15 per cent who were real-time RT-PCR positive for influenza
experienced severe illness with pneumonia and five A (H1N1) pdm09 (n=13), influenza A (H3N2)
per cent were seriously ill, mainly older individuals (n=5), human coronavirus OC43 (n=5), rhinovirus
and people with other medical conditions such as (n=2), respiratory syncytial virus (n=2), influenza B
asthma, hypertension, diabetes or heart disease7. (n=3), parainfluenza type 4 (n=3), hepatitis B virus
However, some COVID-positive individuals found to (n=5), hepatitis C virus (n=5) and dengue IgG and
be asymptomatic could act as carriers of the infection8. chikungunya IgG positive samples (n=10 each) were
One of the main challenges in containing the spread used as a panel to test cross-reactivity. Two hundred
of SARS-CoV-2 is the diagnosis of a large number of and seventy nine serum samples were COVID-19
infected individuals within a population. Majority of real-time RT-PCR negative from healthy blood
the asymptomatic cases may remain undetected due to donors collected prior to the SARS-COV-2 outbreak.
the limited resources for diagnosis. The remaining 21 serum samples were real-time RT-
As approved by the World Health Organization PCR negative for COVID-19, which were collected
(WHO), molecular assays are currently the mainstay during the current pandemic. All these serum
of diagnosis of COVID-199. Serological assays have samples had been tested using microneutralization
also been used as a screening tool for the surveillance test (MNT) for SARS-CoV-217. All samples were
of the emerging and re-emerging diseases. However, stored at −20°C until further use. Institutional
serological assays are not currently recommended Human Ethics Committee approved this study. A
for case detection and are not included in the WHO written informed consent was obtained from all the
laboratory testing guidelines for COVID-19. The participants.
 446 INDIAN J MED RES, MAY 2020

Cell and antigen preparation highest dilution resulting in an infection reduction of

>50 per cent. A cut-off of neutralization titre >20 was
Virus propagation and titration: SARS-CoV-2 considered as positive.
was isolated from throat/nasal swab specimen
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Anti-SARS-CoV-2 human IgG ELISA: An IgG

(NIV-2020-770) in Vero CCL-81 cells at the
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capture ELISA was developed for serological

containment facility of ICMR-NIV, Pune18.
SARS-CoV-2 virus stock was prepared by propagating diagnosis of SARS-CoV-2 infection from patients’
the virus in Vero CCL-81 cells. The cytopathic effect serum. Briefly, 96-well polystyrene microtitre ELISA
started at the post-infection day (PID) 2, and the culture plates (Nunc, Thermo Fisher Scientific, USA) were
supernatant was harvested at the PID 3. Virus titrations coated with inactivated SARS-CoV-2 antigen (1:10
were performed in Vero CCL-81 cells using tissue diluted, 100 µl/well) in 1x phosphate-buffered saline
culture infectious dose 50% (TCID50) assay. Virus (PBS) (pH 7.2, 1 M). The antigen-coated plates were
kept overnight at 4°C. These wells were blocked
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titre (TCID50/ml) was calculated by the Reed-Muench

method19 and found to be 106.5 TCID50/ml. with a Liquid Plate Sealer (CANDOR Bioscience
GmbH, Germany) for two hours at room temperature
Gamma inactivation of the virus: Gamma irradiation (25-30°C). The plates were washed three times with
of the virus stock was performed using Co-60 10 mM PBS, pH 7.4 with 0.1 per cent Tween-20
source (24 kGy) of GC-5000 Gamma chamber (PBST) (Sigma-Aldrich, USA). To the coated and
(BRIT, Mumbai). This irradiated stock was again blocked wells, 100 µl of 1:100 diluted human serum
inoculated in Vero CCL-81 twice for five days to samples were added and incubated at 37°C for one
confirm the inactivation of the virus. hour. These wells were washed five times using 1x
Concentration of gamma-inactivated antigen: Gamma- PBST. Following this, anti-human IgG horseradish
irradiated SARS-CoV-2-infected tissue culture fluid peroxidase (HRP) was diluted in StabilZyme
was concentrated using 30 kDa filters (Millipore, (SurModics, Inc., USA) and added 100 µl/well; and
Germany) and further passed through 0.2 µm filters, plates were incubated for one hour at 37°C. After
aliquoted and stored at −80°C. Concentrated viral incubation, the plates were washed as described above.
antigen was also aliquoted in 1 and 2 ml volumes Further, 100 µl of 3,3’,5,5’-tetramethylbenzidine
in frosted glass bottles and further lyophilized. The (TMB) substrate was added and incubated for 10
lyophilized vials were stored at −20°C to be used as min. The reaction was stopped by 1 N sulphuric acid
antigen in ELISA tests. (H2SO4), and the absorbance values were measured at
450 nm using an ELISA reader. Positive and negative
Microneutralization test (MNT): All serum samples controls were prepared as described below and were
were tested for neutralization against SARS-CoV-2 included in the respective wells in the test.
(ID NIV2020-770) in the Biosafety Level 3 (BSL-3)
laboratory of ICMR-NIV, Pune. The assay was Using virus neutralization test as the gold standard,
performed as described by Manenti et al17 with a few the sensitivity and specificity of IgG ELISA was
modifications. Briefly, two-fold (1:10 to 1:1280), determined.
serially diluted heat-inactivated serum samples (50 µl) Serum controls for anti- SARS-CoV-2 human IgG
in minimal essential medium (Gibco, Thermo Fisher ELISA: Human serum samples tested and confirmed
Scientific, USA) supplemented with two per cent foetal by real-time RT-PCR and MNT were selected for the
bovine serum (Gibco, Thermo Fisher Scientific, USA) preparation of positive and negative controls. High
were prepared. In the next step, 50 µl virus suspension positive control serum was prepared by pooling 15
of 2 log tissue culture infective dose of previously
serum samples collected from COVID-19 survivors
titered virus stock was added to each well of ELISA
infected during the current SARS-COV-2 pandemic.
plate and incubated at 37°C for one hour. The mixture
Negative control serum was prepared by pooling
was then added on to the preformed monolayers of
samples from 10 healthy individuals, whose samples
Vero CCL-81 cells in 96-well plates (Nunc, Thermo
were collected and stored before this pandemic.
Fisher Scientific, USA) and incubated at 37°C with
five per cent CO2. After incubation for 72 h, the Determination of cut-off values and assay validation:
medium was decanted and washed with saline and A total of 513 serum samples, including the 131
the plates were stained with one per cent amido black. positive and 382 negative samples as confirmed by the
The serum neutralization titre is the reciprocal of the MNT, were tested by ELISA. The end point cut-off
 SAPKAL et al: IgG ELISA FOR SARS-CoV-2 447

was determined by the analysis of a receiver operating Of the 131 MNT-positive serum samples tested
characteristic (ROC) curve based on positive divided by the in-house IgG ELISA, four were found to be
by negative (P/N) values. The panels of serum samples negative and 127 were positive. However, one serum
described above were selected to validate cross- sample which was IgG positive by the in-house ELISA
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reactivity. The inter-assay variability was assessed was negative by MNT. The inter-assay CV was nine
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by testing two negative and two positive control per cent for the positive control serum and 9.4 per cent
serum samples in 15 different work sessions, and four for the negative control. The intra-assay CV of four
replicates within each plate were used to determine the replicates within each plate for positive control was
coefficient of variation (CV). 3.5 per cent and for negative control it was 7.2 per cent.
Results Inter-laboratory evaluation of the assay:
Inter-laboratory comparison of indigenously developed
Among the 150 real-time RT-PCR-confirmed anti-SARS human IgG ELISA was performed at
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COVID-19 serum samples, 131 were positive by MNT two centres. A panel of 150 coded (75 positive and
and 19 were negative. In addition, among 21 serum 75 negative) human serum samples (including the
samples collected during this pandemic which were panel to test cross-reactivity) were provided along
negative by real-time RT-PCR, remained negative with four ready-to-use SARS-COV-2 IgG ELISA
when tested by MNT. kits. The concordance obtained was 99.33 and 100
Determination of cut-off value and assay per cent at both the centres when compared with the
performance: After the ROC analysis, the area under results of ICMR-NIV, Pune. A correlation analysis was
the curve (AUC) was found to be 0.986. The cut-off performed to analyze the inter-laboratory performance
threshold for the assay was set at 0.200, which gave between the different laboratories. The results indicated
a sensitivity of 92.37 per cent and a specificity of a positive correlation between the reference laboratory
97.9 per cent (Fig. 1). The calculated threshold cut- and the other laboratories (Lab 1: r=0.9153, P<0.001
off for the ELISA at optical density (OD) at 450 nm with r2 value 0.8387; Lab 2: r=0.9523, P<0.001 with r2
was more than the average OD of negative control value of 0.9068) (Fig. 2).
+0.2 and the sample to negative control a ratio >1.5,
Correlation between anti-SARS-CoV-2 human IgG
differentiating between the presence and absence of
ELISA and MNT: The correlation between the in-house
IgG antibodies in the samples. The assay was found to
IgG ELISA against the neutralization titres indicated a
have a negative predictive value of 98.14 per cent and
strong positive correlation (r=0.7836, P<0.001) (Fig. 3).
a positive predictive value of 94.44 per cent.
Discussion
The U.S. Food and Drug Administration (FDA)
has granted 31 molecular diagnostic kits (real-time RT-

Fig. 1. Receiver operating characteristic (ROC) analysis showing Fig. 2. Inter-laboratory correlation analysis: A panel of 150 serum
sensitivity versus specificity for discrimination of positive and samples tested at two external laboratories and compared with the
negative serum samples, which were derived for in-house IgG ELISA ICMR-National Institute of Virology reference laboratory indicated
compared to microneutralization test results (ROC area under the a positive correlation between the reference laboratory and the other
curve: 0.986). laboratories.
 448 INDIAN J MED RES, MAY 2020

antibodies among individuals who have been exposed

to SARS-CoV-2. The ELISA results correlated strongly
with virus neutralizing antibodies. This assay may
be used for ascertaining the seroprevalence against
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SARS-CoV-2 in a population and for epidemiological

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studies.
Financial support & sponsorship: None.

Conflicts of Interest: None.

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For correspondence: Dr Pragya D. Yadav, Maximum Containment Laboratory, Maximum Containment Facility,
ICMR-National Institute of Virology, Sus Road, Pashan, Pune 411 021, Maharashtra, India
e-mail: [email protected]