A throughput serological Western blot system using whole virus lysate for the concomitant detection of antibodies against SARS-CoV-2 and human endemic Coronaviridae

Abstract

BACKGROUND: Seroreactivity against human endemic coronaviruses has been linked to disease severity after SARS-CoV-2 infection. Assays that are capable of concomitantly detecting antibodies against endemic coronaviridae such as OC43, 229E, NL63, and SARS-CoV-2 may help to elucidate this question. We set up a platform for serum-screening and developed a bead-based Western blot system, namely DigiWest, capable of running hundreds of assays using microgram amounts of protein prepared directly from different viruses. METHODS: The parallelized and miniaturised DigiWest assay was adapted for detecting antibodies using whole protein extract prepared from isolated SARS-CoV-2 virus particles. After characterisation and optimization of the newly established test, whole virus lysates of OC43, 229E, and NL63 were integrated into the system. RESULTS: The DigiWest-based immunoassay system for detection of SARS-CoV-2 specific antibodies shows a sensitivity of 87.2 % and diagnostic specificity of 100 %. Concordance analysis with the SARS-CoV-2 immunoassays available by Roche, Siemens, and Euroimmun indicates a comparable assay performance (Cohen's Kappa ranging from 0.8799-0.9429). In the multiplexed assay, antibodies against the endemic coronaviruses OC43, 229E, and NL63 were detected, displaying a high incidence of seroreactivity against these coronaviruses. CONCLUSION: The DigiWest-based immunoassay, which uses authentic antigens from isolated virus particles, is capable of detecting individual serum responses against SARS-CoV-2 with high specificity and sensitivity in one multiplexed assay. It shows high concordance with other commercially available serologic assays. The DigiWest approach enables a concomitant detection of antibodies against different endemic coronaviruses and will help to elucidate the role of these possibly cross-reactive antibodies.

Antibody Response against SARS-CoV-2 and Seasonal Coronaviruses in Nonhospitalized COVID-19 Patients

“…IgG enzyme-linked immunosorbent assays (ELISAs) against the S1 protein (Euroimmun) and S-protein RBD (Mediagnost) and IgA and IgM against S-RBD (Mediagnost) as well as an electrochemiluminescence immunoassay (ECLIA) detecting IgG against the viral nucleocapsid (NC; Roche). Furthermore, we applied a throughput Western blot system (DigiWest) allowing detection of SARS-CoV-2 and seasonal coronavirus antibodies ( 16 ).…”

“…It is a matter of ongoing debate if antibodies against seasonal coronaviruses might confer cross-protection against SARS-CoV-2. To gain insight into this question, we employed a quantitative and high-throughput Western blot-based detection system identifying the bulk of IgGs against a specific coronavirus ( 16 ). As expected and in line with our previous data ( Fig.…”

“…Until now, it was unclear if antibodies against seasonal coronaviruses that are highly prevalent within the human population play a role in SARS-CoV-2 neutralization. We employed an innovative throughput Western blot system to concomitantly detect antibodies specific against SARS-CoV-2 as well as the seasonal coronaviruses 229E, OC43, and NL63 ( 16 ). In fact, 100% of individuals included in our study had antibodies against the three seasonal coronaviruses with high diversity in relative numbers (see Table S1 in the supplemental material).…”

“…Whole-viral-protein lysates from 229E, OC43, and NL63 (ZeptoMetrix Corp.) and from SARS-CoV-2 were used for DigiWest as described previously ( 16 ). Viral protein lysates were used for denaturing gel electrophoresis and Western blotting using the NuPAGE system.…”

“…The DigiWest analysis tool was used to assess serum reactivity against the viral proteins ( 16 ). Virus protein-specific peaks were identified, and average fluorescence intensity (AFI) values were calculated by integration of peak areas.…”