key: cord-0964398-g6z5ci2k
authors: Kulkarni, Ruta; Shrivastava, Shubham; Patil, Harshad P.; Kore, Pravin; Rane, Prajakta; Palkar, Sonali; Lalwani, Sanjay; Mishra, Akhilesh Chandra; Arankalle, Vidya A.
title: Performance assessment of SARS-CoV-2 IgM & IgG ELISAs in comparison with plaque reduction neutralization test
date: 2021
journal: Indian J Med Res
DOI: 10.4103/ijmr.ijmr_3806_20
sha: 7d171d9543c12b6550270fe126cdb591d7a6a132
doc_id: 964398
cord_uid: g6z5ci2k

BACKGROUND & OBJECTIVES: Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) continues to be a devastating pandemic. This study was aimed at performance assessment of SARS-CoV-2 IgM and IgG ELISAs, and investigation of their utility for patient diagnosis and sero-epidemiologic investigations. METHODS: Serum/plasma samples from COVID-19 patients or asymptomatic contacts (n=180) and healthy donors (n=90) were tested in parallel using two commercial IgM ELISAs (Erbalisa and Inbios), and four IgG ELISAs (Kavach, Euroimmun, Erbalisa and Inbios) along with an indigenous β-propiolactone inactivated virus-based ELISA (IRSHA-IgG-ELISA). Plaque reduction neutralization test (PRNT) was used as reference test. RESULTS: Among 180 COVID-19 patients, 125 tested positive by PRNT. Inbios-IgM-ELISA showed sensitivity (Se)/specificity (Sp)/positive predictive value (PPV)/negative predictive value (NPV) of 93.6/97.8/98.4/94.4 per cent in relation to PRNT, and performed better than Erbalisa-IgM-ELISA (Se: 48%, Sp: 95.6%, PPV: 95.2%, NPV: 65.2%). During the first week of disease, only 47.4 per cent of the COVID-19 patients tested IgM positive by Inbios-IgM-ELISA, detection improving at two weeks and beyond (~86-100%). Among IgG tests, Inbios-IgG-ELISA ranked first in terms of sensitivity (83.2%), followed by IRSHA (64.8%), Euroimmun (64%), Erbalisa (57.6%) and Kavach (56%) tests. For all IgG tests, sensitivity improved during the third (73.9-95.7%) and fourth week (100%) of illness. The specificity (96.7-100%) and PPV (96.2-100%) of all IgG tests were high; NPV ranged between 71.9 and 87.1 per cent with Inbios-IgG-ELISA scoring highest. INTERPRETATION & CONCLUSIONS: Our results show that IgM detection by the current, most sensitive ELISAs cannot replace molecular diagnosis, but may aid as a supplement test. The available IgG tests are suitable for serosurveys for the assessment of previous virus exposure.

Currently, COVID-19 diagnosis is carried out by testing respiratory tract samples for viral RNA using reverse transcription-polymerase chain reaction (RT-PCR) 2 . Although highly sensitive, this method has limitations due to dependence on sampling technique, sample type/quality and virus genetic variability 3 . Further, the performance of RT-PCR is affected by the timing of sample collection relative to the day of illness, as viral RNA is detectable for a limited period post-disease onset [4] [5] [6] . Thus, there is a need for sensitive and specific antibody detection tests to supplement molecular diagnosis, particularly if the patients seek medical advice late, when the RNA positivity is bound to be lower. In addition, for seroepidemiologic studies and vaccine immunogenicity testing, IgG tests are crucial.

Since the emergence of the COVID-19 pandemic, extensive efforts have been made for development of antibody detection immunoassays, and several enzymelinked immunosorbent assays (ELISAs) and lateral flow assays (LFAs) are now commercially available. While the LFAs offer the advantage of rapid results and point-of-care use, their lower sensitivity limits the application of these assays in comparison to ELISAs 7 . The plaque reduction neutralization test (PRNT) remains the gold standard for detection of neutralizing antibodies, however, the test is time-consuming and needs biosafety level 3 (BSL3) facility for handling the live SARS-CoV-2. ELISAs are more suitable for high throughput screening, and allow detection of nonneutralizing antibodies as well.

Performance comparison of SARS-CoV-2 IgM and IgG ELISAs is of special importance for the SARS-CoV-2, for which the antibody dynamics are not yet clearly understood. While initial studies have reported late appearance of antibodies and IgG preceding IgM 6,8-10 , there is a need to revisit this issue by using newer/better tests. The present study was aimed at the assessment of commercially available SARS-CoV-2 IgM and IgG ELISAs, and our indigenously developed IgG ELISA 11 in a clinical setting. In the absence of a reference ELISA recommended by international/national bodies, PRNT was used as the gold standard. 

ELISA: SARS-CoV-2 isolation, propagation and inactivation using 0.1 per cent β-propiolactone (Sigma-Aldrich., Inc., Saint Louis, MO, USA) was carried out in the BSL3 facility of IRSHA. The inactivated virus was used as the coating antigen for an indirect IgG ELISA, hereafter referred to as IRSHA IgG ELISA. The ELISA protocol described previously 11 was followed.

Plaque reduction neutralization test: SARS-CoV-2 PRNT was performed in IRSHA BSL3 laboratory using Vero CCL81 cells procured from ATCC and maintained in minimum essential medium (MEM; Gibco, Waltham, MA, USA) with 10 per cent foetal bovine serum (FBS; Gibco, Waltham, MA, USA) and antibiotics including penicillin-streptomycin (100 µg/ml; Thermo Fisher Scientific, Waltham, MA, USA) at 37°C in a humidified incubator with five per cent carbon dioxide. For the assay, cells were seeded at a density of 1×10 5 cells/well in a 24-well plate, one day prior to infection. Serum samples diluted 1:5 (v/v) in MEM containing two per cent FBS and antibiotics, were subjected to heat inactivation followed by 4-fold serial dilutions. Each dilution was mixed with equal volume of 20-40 pfu of SARS-CoV-2, followed by incubation at 37°C. After one hour, each virus-serum mixture was added in duplicate wells of the seeded 24-well plate, and incubated for one hour, followed by the addition of overlay medium containing MEM, one per cent carboxymethyl cellulose (Aquacide-II, Merck, Calbiochem-Merck, San Diego, CA, USA), two per cent FBS and antibiotics. At five days post-infection, cells were fixed using 3.7 per cent formaldehyde and stained using one per cent crystal violet (Sigma-Aldrich., Inc., Saint Louis, MO, USA). Plaques were counted and PRNT 50 titre was determined using Karber's formula 12 . Samples with PRNT 50 titre ≥20 were considered seropositive.

Statistical analysis: Sensitivity, specificity, positive and negative predictive values (PPV and NPV) of the IgM and IgG ELISAs were assessed against PRNT as the reference test. Uncertainty was expressed by 95 per cent confidence intervals (95% CI). Proportions were compared using Chi-square test. The analyses were conducted using RStudio version 3.4.1 (RStudio, Inc., Boston, MA, USA).

Among RT-PCR confirmed COVID-19 patients, neutralizing antibody positivity was 69.4 per cent (125/180). The antibody detection rate increased over the course of illness -Week 1: 38/76 (50%), Week 2: 54/63 (85.7%), Week 3: 23/23 (100%), Week 4: 5/5 (100%) ( Table I) . PRNT did not detect anti-SARS-CoV-2 antibodies in the serum/plasma samples of 90 healthy blood donors obtained prior to the emergence of COVID-19.

IgM ELISAs: Among the 125 PRNT-positive samples, 60 (48%) and 117 (93.6%) tested positive, respectively using the Erbalisa and Inbios IgM ELISAs, with the IgM positivity rising with increase in post-onset day (POD) of disease (Table I) . The Inbios ELISA detected IgM in eight PRNT-negative samples, while for the Erbalisa kit this number was higher (n=19). Of these, five IgMreactive samples were common to both ELISAs.

IgG ELISAs: For IgG detection, 81 (64.8%), 70 (56%), 80 (64%), 72 (57.6%) and 104 (83.2%) of the PRNTpositive samples tested positive, respectively using IRSHA, Kavach, Euroimmun, Erbalisa and Inbios IgG ELISAs, with the IgG detection improving steadily over the course of the illness (Table I) . Among the 55 PRNT-negative samples, 3-5 were positive for IgG using different ELISAs; the samples identified as IgG-reactive were different in these ELISAs. These IgG ELISA-positive, PRNT-negative samples could represent the presence of non-neutralizing antibodies not detected by PRNT, or ELISA false positivity.

To assess specificity and PPV of the different ELISAs, 90 blood donor serum/plasma samples obtained prior to the emergence of COVID-19, and tested negative by PRNT were used (Table II) (Table II) .

For IgG detection, the Inbios ELISA showed the highest sensitivity (83.2%, P<0.05), followed by IRSHA (64.8%), Euroimmun (64%), Erbalisa (57.6%) and Kavach (56%) ELISAs (Table II) . The NPV of the Inbios IgG ELISA (87.1%) was significantly higher than the other four tests (71.9-76%, P<0.05). On analysis in relation to POD, the test sensitivity ranged between 36.8 and 71.1 per cent for the IgG ELISAs during the first week of illness, improved steadily during the second (53.7-85.2%) and third week (73.9-95.7%). During the fourth week, only five samples were available, and all tested positive with each of the ELISAs evaluated. The Inbios ELISA detected IgG in higher proportion of the PRNT-positive samples during the first (71.1%) and second week of illness (85.2%), as compared to the other four tests, and was thus the most sensitive assay in early disease phase (P<0.05).

On comparison of the IgM (Inbios ELISA) and IgG (Inbios ELISA) markers for 

The present study reports comparative assessment of two commercial SARS-CoV-2 IgM and four IgG ELISAs along with one indigenous IgG ELISA using PRNT as the reference test. While PRNT detects only neutralizing antibodies, ELISAs detect both neutralizing and non-neutralizing (binding) antibodies.

Diagnostic utility of serologic testing in COVID-19 has been questionable because of the observations of delayed antibody response and IgG appearance before IgM 6, [8] [9] [10] . Our data revealed that Inbios IgM ELISA showed very good sensitivity in relation to PRNT. However, only 47.4 per cent (36/76) of the RT-PCR positives identified during the first week of disease were IgM positive, PRNT identifying 50 per cent. Thus, utility of Inbios IgM ELISA as a single diagnostic test would have limited application for diagnosis during the first week of disease, when viral RNA is likely to be detected. Blood collection and IgM/PRNT testing among the COVID-19 patients was done after confirmation of viral RNA positivity (1-4 days later), and hence possibility of RNA negativity at the time of blood collection cannot be ruled out. Despite this apparent lower detection rate in early disease phase, the Inbios IgM ELISA could still be considered for diagnosis in remote places or wherever RNA testing facilities are not available. During the second week of disease, when RNA positivity is bound to be lower, the Inbios IgM ELISA detection improved to about 86 per cent. Therefore, for patients seeking medical advice post-one week of clinical symptoms, IgM ELISA may be the preferred diagnostic test. Importantly, 91.1 per cent agreement was noted between IgM (binding antibodies in ELISA) and neutralizing (PRNT) antibodies. The performance of Erbalisa IgM 13 . Thus, to assess diagnostic utility of IgM testing, IgM detection and RNA testing should be done simultaneously.

During the current pandemic, efficient IgG detection is of paramount importance for identification of virus exposure among contacts of symptomatic patients, high risk groups as well as for populationbased serosurveys and vaccine evaluations. In the present study, the Inbios IgG ELISA ranked first in terms of sensitivity (83.2%, P<0.05), followed by IRSHA (64.8%), Euroimmun (64%), Erbalisa (57.6%) and Kavach (56%) tests. Importantly, the Inbios ELISA showed >70 per cent sensitivity in the first week of illness, indicating its potential for early IgG detection. The data reveal that if sensitive ELISAs are used, antibodies can be detected early during the disease. For all the ELISAs, sensitivities improved during the third week of illness (73.9-95.7%). In the fourth week, only five samples were available and all were IgG positive. The test sensitivities determined in relation to PRNT confirm earlier reports of superiority of cell-based neutralization tests over IgG ELISAs 14, 15 .

On comparison of IgG and IgM markers using Inbios ELISAs, the most sensitive tests identified during the present study, IgM positivity was higher than IgG during the first and second weeks of disease, suggestive of IgM as a useful marker for current/ recent SARS-CoV-2 infection. This is in contrast to earlier observations suggesting late appearance of anti-SARS-CoV-2 IgM, and thus limited utility for diagnosis 6, [8] [9] [10] 16, 17 . Our data thus suggest that when sensitive and specific tests are employed, antibody dynamics of SARS-CoV-2 appears to be similar to other viral infections, IgM appearing before IgG and serving as a marker of recent infection. Comparison of the ELISAs with PRNT allowed us to assess the ability of different ELISAs in identifying samples with neutralizing antibodies. Our data revealed low ELISA positivity among the PRNT-negative COVID-19 patient samples. Based on our data, it may be surmised that for comparing both IgM and IgG tests, samples collected during the first and second week post-infection should be used as almost 100 per cent positivity is achieved thereafter by all the tests.

Our study had certain limitations. First, RT-PCRbased diagnosis and blood collection for antibody testing was not done on the same day. The possibility of viral RNA negativity on the day of blood collection cannot be ruled out. Thus, the diagnostic utility of IgM cannot be truly determined. Second, cross-reactivity of the different ELISAs with closely related coronaviruses causing common cold such as HCoV-OC43, HCoV-NL63, HCoV-229E, HCoV-HKU1, could not be investigated due to the unavailability of known serum samples positive for antibodies against these viruses.

In conclusion, IgM detection by the currently available, most sensitive ELISAs cannot replace RT-PCR, but may prove useful as a supplement to molecular diagnosis. The available IgG tests should suffice the current need of assessment of previous exposure. In our study, Inbios IgM and IgG ELISAs provided optimum performance.

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Dr Vidya A. Arankalle, Department of Communicable Diseases, Interactive Research School for Health Affairs, Bharati Vidyapeeth (Deemed to be University)

Bhosale and Ms Anamika Solaskar for technical assistance and Shri Rahul Patil for statistical analysis. 

None.