Several studies reported the presence of antibodies reacting with SARS-CoV-2 spike and nucleocapsid proteins in serum sampled before the pandemic and in HCoVs patients [20,21]. In a few previous reports of the described AWB [2224], a recombinant protein was used as the antigen whereas we used purified virus antigen directly produced in the biosafety level 3 laboratory [13]. compared ELISA (Cohens Kappa=0.64). Automated Western immunoblotting may be used as a second line test to monitor exposure of people to HCoVs including SARS-CoV-2. == Supplementary Info == The online Pseudoginsenoside-F11 version consists of supplementary material available at 10.1007/s10096-021-04203-8. Pseudoginsenoside-F11 Keywords:SARS-CoV-2, COVID-19, Cross-reactivity, Serology, Automated Western immunoblotting == Intro == To day, seven coronaviruses have been reported as human being pathogens, including four seasonal coronaviruses (Alphacoronavirus229E and NL63 andBetacoronavirusHKU1 and OC43) here referred to as HCoVs, which are associated with causing mild-to-severe top and lower respiratory tract infections [1]. Two additional betacoronaviruses that caused severe acute respiratory syndrome in 2002 in China (SARS-CoV) and the Middle East Respiratory Syndrome in 2012 in Saudi Arabia (MERS-CoV) [2] and theBetacoronavirusSARS-CoV-2 that is the agent of the COVID-19 pandemic have been demonstrated to infect a variety of animals and humans [3]. The second option is phylogenetically closely related to HCoV-HKU1 and presents a high sequence homology with SARS-CoV [2]. Serological assays used to explore exposure to seasonal HCoVs have previously indicated cross-immunity between all coronaviruses [46]. SARS-CoV-2 exhibits several antigens eliciting a Pseudoginsenoside-F11 serological response in COVID-19 individuals, including spike glycoprotein, its N-terminal (S1), and C-terminal (S2) subunits as well as nucleocapsid [7]. Most of regularly used serological COVID-19 assays integrated only one recombinant protein [810]. Second generation assays are combining two antigens to increase sensitivity and mostly specificity [7,11]. Pseudoginsenoside-F11 We developed an automated Western immunoblotting (AWB) assay in order to characterize serological reactions to SARS-CoV-2 and the potential cross-reactivity with HCoVs. == Individuals and methods == == Serum sample collections == A first set of 27 serum samples from 27 different individuals with RT-PCR-documented COVID-19 [12], collected between March and April 2020, at least RHPN1 2 weeks after the onset of symptoms were incorporated like a positive Pseudoginsenoside-F11 control group. All of them presented with an IgG titer 1:100 using in-house indirect immunofluorescence assay (IFA) [13]. Of these, 16 serum samples were utilized for standard immunoblotting including 3 samples exhibiting low (1:200), moderate (1:800), and high (1:3200) IgG titers using IFA that were used to fix optimal conditions to be used for AWB (antigen, serum, and secondary antibodies concentrations). One serum collected in 2018, before the onset of COVID-19 (having a negative RT-PCR for HCoVs on homologous respiratory specimen), was included as bad control. As for AWB, 223 serum samples (including the 27 serum samples described above) collected from 223 different RT-PCR-confirmed COVID-19 individuals between March and September 2020 were integrated like a positive control group. Twenty-seven of these sera were tested for antibodies to the recombinant S1 protein by EUROIMMUN SARS-CoV-2 IgG ELISA (Euroimmun, Bussy Saint-Martin, France) performed using the Elispeed DUO system (Euroimmun) according to the manufacturers recommendations. The percentage (AUC sample/AUC calibrator) was interpreted as follows: <0.8 negative; 0.8 to <1.0 undetermined; 1.1 positive. We regarded as undetermined results as bad for statistical analyses. A negative control group (37 serum samples) consisted of (i) 10 serum samples obtained less than 5 days after the onset of symptoms in individuals showing high viral loads of SARS-CoV-2 (Ct ideals < 20) collected in March and April 2020; (ii) 14 sera from asymptomatic healthcare workers largely exposed to the disease but exhibiting bad results for RT-PCR and serology by IFA.
