Multiple indirect ELISAs for serological detection of SARS-CoV-2 antibodies

46 As the coronavirus disease 2019 (COVID-19), which is caused by the novel coronavirus SARS- 47 CoV-2, continues to spread rapidly around the world, there is an urgent need for validated 48 serological assays to evaluate viral specific antibody responses in COVID-19 patients or recovered 49 individuals. In this study, we established and used indirect Enzyme Linked Immunosorbent Assay 50 (ELISA)-based serological tests to study the antibody response in COVID-19 patients. In order to 51 validate the assays, we determined the cut-off values, sensitivity and specificity of the developed 52 assays using sera collected from COVID-19 patients in Saudi Arabia at different time points after 53 disease onset, as well as sera that are seropositive to other human CoVs; namely MERS-CoV, 54 hCoV-OC43, hCoV-NL63, hCoV-229E, and hCoV-HKU1. The SARS-CoV-2 S1 subunit of the 55 spike glycoprotein and nucleocapsid (N) ELISAs that we developed here not only showed high 56 specificity and sensitivity, but also did not show any cross-reactivity with other CoVs. We also 57 showed that all RT-PCR confirmed COVID-19 patients included in our study developed both virus 58 specific IgM and IgG as early as one week after the onset of disease. The availability of these 59 validated assays will enable us to determine the nature and duration of the antibody response 60 mounted in response to SARS-CoV-2 infection. It will also allow conducting large-scale 61 epidemiological studies to determine evidence of previous exposure to the virus and assess the true 62 extent of virus spread within communities. 63 In the current study, we report the development and validation of an ELISA-based serological assays for the detection of SARS-CoV-2 specific IgG and IgM antibodies in COVID-19 serum specimens. We showed that our ELISAs can specifically detect SARS-CoV-2 specific IgM and IgM antibodies in sera from COVID-19 patients, but not from sera derived from healthy human donors. Our data also show that our SARS-CoV-2 S1 and N ELISAs do not cross-react with sera that are seropositive to other human CoVs; including human CoVs that belong to the beta-CoV genus such as MERS-CoV, hCoV-OC43 and hCoV-HKU1, as well as those from alpha-CoVs such as hCoV-NL63 and the hCoV-229. Furthermore, using the developed ELISAs, we evaluated the production of SARS-CoV-2 specific IgG and IgM antibodies in a cohort of hospitalized COVID- 19 patients (n = 52), including samples collected during the 1 st week (n = 10), 2 nd week (n = 23), 3 rd week (n = 14) or 4 th week (n = 5) of symptoms-onset. Our analysis showed that SARS-CoV-2 IgM or IgG specific antibodies for either SASR-CoV-2 S1 or N antigens can be virtually detected 250 in all RT-PCR confirmed COVID-19 patients in this study. We showed that both virus specific IgG and IgM can be detected as early as one week after disease-onset but significantly increased by week two and three, with IgG persisting through week four (last time point in our study) compared IgM peaked week 2 or 3 before declining. This increase in IgG over time


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In December 2019, a cluster of atypical pneumonia was reported in Wuhan City, the capital of The other third of the genome encodes the four main structural proteins (envelope (E), membrane 87 (M), spike (S), and nucleocapsid (N) proteins) as well as other accessory proteins [4,5].

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As SARS-CoV-2 continues to spread around the globe, it is important to understand the duration 90 and nature of immunity mounted in response to infection, which is currently not fully understood 91 and investigated. Furthermore, the actual extent of the current global COVID-19 pandemic is not 92 well known; therefore, serological assays are critically needed to shed light on all these 93 unanswered questions. Here, we report the development and validation of multiple indirect 94 ELISA-based serological assays that can be adapted and used by laboratories to determine the 95 immune status of individuals in surveillance and epidemiological studies, as we have previously 96 described for MERS-CoV [6,7]. Using sera derived from either confirmed COVID-19 patients or 97 known noninfected healthy controls, we validated our ELISAs and determined their cut off values, 98 sensitivity and specificity. We also showed that our assays had no cross-reactivity using sera with 99 known positivity to MERS-CoV and other common CoVs. Our study shows that SARS-CoV-2 column according to the manufacturer's protocol and as previously described [6]. Positive fractions 129 of N proteins were pooled, aliquoted and stored at −80°C until used. SARS-CoV-2 proteins were 130 confirmed by Western blot using anti-His tag antibodies as well as SARS-CoV-2 seropositive and 131 seronegative human serum samples as previously described [6].

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Expression and production of SARS-CoV-2 proteins 163 The S protein of SARS-CoV-2 is a major immunogen and is divided into two subunits; S1 which analysis showed that both S1 (~110 KDa, Figure 1a) and N (~46 KDa, Figure 1b) proteins were 173 detected using anti-His antibodies and shown to bind specifically to sera derived from COVID-19 174 patients but not to COVID-19 seronegative sera from normal human donors collected prior to the 175 pandemic. These data indicate that both S1 and N proteins are antigenically similar to native 176 proteins and able to strongly and specifically detect SARS-CoV-2 antibodies in serum samples.

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We developed four different types of indirect ELISAs for the testing of IgM and IgG antibodies 180 using purified SARS-CoV-2 S1 and N proteins as coating antigens. We initially optimized the 181 coating conditions for the ELISA using known SARS-CoV-2 seronegative and seropositive sera 182 and found that the optimal working concentrations of each antigen were 1 μg/mL and 4 μg/mL for recombinant S1 and N proteins, respectively (data not shown). Furthermore, optimal serum 184 dilution was determined using checkerboard titration where the highest OD ratio values of positive 185 to negative samples (P/N) were obtained. After optimization, we tested sera from 100 normal 186 human donors and one serum sample from an RT-PCR confirmed COVID-19 patient in the 187 developed ELISAs at a dilution of 1:100 to determine the cut-off values (mean + 3 SD). As shown 188 in Figure 2, the cut-off values were found to be 0.17 (mean = 0.09, SD = 0.3) for S1 IgG-ELISA,  199 or to other known human CoVs, we found that our developed SARS-CoV-2 S1 and N-based ELISAs. As shown in Table 1, the specificity of the assays ranged between 91.2%-97.6%. The 218 sensitivity, however, was dependent on the sampling time in relevance to disease onset. During 219 the first week post symptoms onset, the sensitivity of IgM and IgG ELISAs ranged between 20%-220 30% and 40%-60%, respectively (Table 1). Nonetheless, the sensitivity of the assays increased to 221 88.5%, 84.6%, 100% and 88.5% for S1 IgG-ELISA, S1 IgM-ELISA, N IgG-ELISA and N IgM-222 ELISA, respectively by week two. Importantly, while these sensitivity values were maintained at 223 100% for N IgG-ELISA or increased to 100% for both S1 IgG-ELISA and S1 IgM-ELISA during 224 week three and four post symptoms onset, N IgM-ELISA's sensitivity declined.

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Next, we conducted ROC analysis to examine the diagnostic power of each developed assay as 227 shown in Figure 5. Our analysis showed high accuracy of S1 IgG-ELISA, S1 IgM-ELISA and N Discussion 238 In the current study, we report the development and validation of an ELISA-based serological 239 assays for the detection of SARS-CoV-2 specific IgG and IgM antibodies in COVID-19 serum 240 specimens. We showed that our ELISAs can specifically detect SARS-CoV-2 specific IgM and 241 IgM antibodies in sera from COVID-19 patients, but not from sera derived from healthy human 242 donors. Our data also show that our SARS-CoV-2 S1 and N ELISAs do not cross-react with sera Another important finding in our study is that using both S1 and N in serology could lead to the 292 detection of as many potential seropositive specimens as possible than using any of them alone. testing algorithm is its relatively small size and lack of glycosylation sites, which makes it easy to 308 clone and produce in prokaryotic expression systems, especially in resource-limited settings [3]. 309 We believe that using both S1 and N ELISAs would capture as many potential SARS-CoV-2 310 positive cases as possible than using any of them alone. We believe that our assays are well validated, highly specific, sensitive and can be used for       Week 1 samples Week 2 samples Week 3 samples Week 4 samples SARS-CoV-2 S1 IgG ELISA SARS-CoV-2 S1 IgM ELISA SARS-CoV-2 N IgG ELISA SARS-CoV-2 N IgM ELISA a b c d