key: cord-0969916-ld6fvnt9 authors: Deshpande, Ketki; PT, Ullas; Kaduskar, Ojas; Vijay, Neetu; Rakhe, Aparna; Vidhate, Shankar; Khutwad, Kirtee; Deshpande, Gururaj Rao; Tilekar, Bipin; Saka, Sanskruti; Gadekar, Kshitija; Patil, Roshni; Yadav, Pragya; Potdar, Varsha; Gurav, Yogesh; Gupta, Priyanka; Kaur, Harmanmeet; Narayan, Jitendra; Sapkal, Gajanan; Abraham, Priya title: Performance assessment of seven SARS‐CoV‐2 IgG enzyme‐linked immunosorbent assays date: 2021-08-10 journal: J Med Virol DOI: 10.1002/jmv.27251 sha: f07a7a31445bb7a271cdb1e30fb643715580ac30 doc_id: 969916 cord_uid: ld6fvnt9 The pandemic of COVID‐19 has caused enormous fatalities worldwide. Serological assays are important for detection of asymptomatic or mild cases of COVID‐19, and sero‐prevalence and vaccine efficacy studies. Here, we evaluated and compared the performance of seven commercially available enzyme‐linked immunosorbent assay (ELISA)s for detection of anti‐severe acute respiratory syndrome corona virus 2 (SARS‐CoV‐2) immunoglobulin G (IgG). The ELISAs were evaluated with a characterized panel of 100 serum samples from qRT‐PCR confirmed COVID‐19 patients, collected 14 days post onset disease, 100 SARS‐CoV‐2 negative samples and compared the results with that of neutralization assay. Results were analysed by creating the receiver operating characteristic curve of all the assays in reference to the neutralization assay. All kits, were found to be suitable for detection of IgG against SARS‐CoV‐2 with high accuracy. The DiaPro COVID‐19 IgG ELISA showed the highest sensitivity (98%) among the kits. The assays demonstrated high sensitivity and specificity in detecting the IgG antibodies against SARS‐CoV‐2. However, the presence of IgG antibodies does not always correspond to neutralizing antibodies. Due to their good accuracy indices, these assays can also aid in tracing mild infections, in cohort studies and in pre‐vaccine evaluations. The corona virus disease of 2019 believed to have been originated in Wuhan, Hubei province in China, is caused by severe acute respiratory syndrome corona virus 2 (SARS-CoV-2). 1, 2 The disease was declared a pandemic by the World Health Organization and has since infected more than 178 million people and caused over three million fatalities worldwide. 3 The SARS-CoV-2 belongs to the Nidovirales order of the Coronaviridae family, which includes SARS-CoV and MERS-CoV, which caused outbreaks in 2003 and 2012 respectively. The SARS-CoV-2 causes respiratory infections of varying severity. The most common symptoms include fever, dry cough, tiredness and the more severe symptoms include acute respiratory distress syndrome (ARDS), coagulation disorders, multiorgan dysfunction and central nervous system infection. 3 The diagnosis of SARS-CoV-2 is mainly dependent on the detection of viral RNA by real time reverse transcriptase polymerase chain reaction (qRT-PCR). The viral RNA can be detected from 72 h before onset of symptoms 4 to more than 80 days post first detection. 5, 6 In patients with mild and asymptomatic infection, low PCR positivity rate has been reported in samples collected 8 days after onset of symptoms. 7 As the recent trend indicates an increase in the number of asymptomatic cases, there is a pressing need for serodiagnosis of the SARS-CoV-2. IgM and IgG antibodies against the virus can be detected in the serum of the patients as early 4-7 days post onset of disease (POD) 4, 8, 9 and up to 95% of infected individuals may show seropositivity after 8 days POD. [10] [11] [12] The currently available enzyme immunoassays for the detection of exposure to SARS-CoV-2 are based on the detection of IgA, IgM, IgG, or total antibodies against the virus. [13] [14] [15] The SARS-CoV-2 contains four structural proteins-spike (S), nucleocapsid (N), envelope (E) and membrane (M). Of the four proteins, S and N proteins are most immunogenic. 15 While the N protein facilitates viral replication, assembly and release, 15,16 the S protein, mediates binding of the virus to the ACE-2 cellular receptors. The S protein comprises two sub units, S1 and S2, responsible for binding to host cell receptor (ACE-2) and fusion of cellular and viral membranes respectively. 17, 18 Majority of the serological assays have S or N proteins as their target antigens. The serodiagnosis of the SARS-CoV-2 is still being explored for accurate and reliable diagnosis. Several ELISAs and other antibody testing assays such as chemiluminescence based immunoassay and lateral flow (rapid diagnostic) assays are now available from different manufacturers. The detection accuracy of IgG ELISA may considerably vary among the test kits, highlighting the need of validation before using them in field settings. Here, we have evaluated the performance of seven commercially available anti-SARS-CoV-2 IgG ELISA kits, which can be used to address different requirements. We analysed their performance in correlation to neutralization assay, which is a gold standard in assessing immunity against SARS-CoV-2. All the samples collected were with informed consent from patients and the study was approved by ICMR-NIV Institutional Ethics Committee. The test kits were evaluated with a panel of 100 serum specimens intra-assay and inter-assay replicates (two positives and two negatives). The test characteristics are compared in Table 1 . The PRNTs were performed as described by Deshpande et al (2020). 9 Briefly, all the sera were heat inactivated and serially diluted 4-fold starting at a dilution of 1:10. Further these samples were mixed with an equal amount of virus suspension containing 50-60 plaqueforming units (PFU) in 0.1 ml. After incubating the mixtures at 37°C for 1 h, each virus-diluted serum sample (0.1 ml) was inoculated onto a 24-well tissue culture plate containing a confluent monolayer of Vero CCL-81 cells. After incubating the plate at 37°C for 60 min, an overlay medium consisting of 2% carboxymethyl cellulose with 2% fetal calf serum in 2× MEM was added to the cell monolayer and the plate was further incubated at 37°C in 5% CO 2 for 5 days. Plates were stained with 1% amido black for an hour. Antibody titers were determined as the highest serum dilution that resulted in >50 (PRNT50) reduction in the number of plaques. Inter and intra assay precision for each kit was analysed with 4 serum samples (two positive and two negatives) tested on each run of all the kit manufacturers. Each of these samples was tested in three different kit lots for inter-assay assessment and four replicates within each plate were taken for intra-assay precision. The %CV for all the replicates of one kit was calculated to assess the repeatability of the kit ( Table 3 ). The data indicated that, with an exception to two assays, all the assays showed low inter-and intra-assay %CV. While, the T A B L E 1 Detailed kit specifications for seven commercial ELISA kits The measures of diagnostic accuracy of all the kits were analysed using the panel of 200 specimens ( The Cohen's kappa (κ) analysis was performed to understand the inter rater agreement between the test kits and the standard assay. In The agreement between the kits was analysed by percent concordance of results for all the kits with each other (Figure 3 ). The results of individual samples were compared for evaluating con- The nucleic acid detection test is currently the gold standard method for the diagnosis of SARS-CoV-2 infection, but ELISAs, CLIA and rapid tests for detection of antibodies, might also be useful in for detecting exposure to the virus. In this study, we compared seven Ease of performance is a distinct advantage of ELISAs. A few manufacturers employed coloured reagents as a pipetting guide in the assay. All the kits detected IgG antibody to the virus in serum. The time required for the assay performance ranged from 80 min (Aspen ELISA) to 130 min (J. Mitra). All the assays allowed testing of more than 90 samples per assay. All the assays displayed sensitivity and specificity in range of 91%-98% and 95-98%, respectively. The variation in the sensitivity and specificity between the different assays may be attributed to the different assay formats and target antigen. The high negative predictive value of these assays also suggests their usefulness in detection of past infections. Studies suggest that serum antibody levels against N and S proteins in the COVID-19 patients tend to increase after 10-17 days post onset of symptoms. 12, 19 Liu W, et al. 20 reported comparable sensitivities of S and N antigen based ELISAs, which was also observed in our study. The inter kit agreement between the kits are also high suggesting usefulness of the kits in detection of the SARS-CoV-2 IgG. The findings of this study are consistent with similar recent studies which suggest overall good performance of the SARS-CoV-2 IgG assays. [21] [22] [23] [24] [25] [26] [27] [28] Detailed studies are necessary to evaluate the performance of these assays on samples collected during different phases of infection. Also, cross-reactivity of the assays should be determined using a larger number of samples positive for closely related viruses. The results of this study may guide the use of various commercial assays in the field studies and help in public health decisions related to COVID-19. We are also studying the antibody responses to different antigens and emergence of antibodies against different viral proteins, which will help in understanding the response curve as well as to formulate a serological testing algorithm for detection of antibodies against SARS-CoV-2. In conclusion, the antibody tests have an important role in diagnosis and add value to the molecular diagnosis. This study provides background for the utility of these commercial IgG assays in screening, contact tracing and sero-prevalence studies for the SARS-CoV-2. The assays evaluated were highly specific and sensitive in detecting the IgG antibodies against SARS-CoV-2. However, it is also to be kept in mind that the presence of IgG antibodies does not always correspond to neutralizing antibodies. Due to their high sensitivity and specificity, these assays can be readily used in tracing those who had asymptomatic/mild infections, in longitudinal studies and in vaccine studies. A pneumonia outbreak associated with a new coronavirus of probable bat origin Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID-19): the epidemic and the challenges World Health Organization. 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