Multiplexed Serum Antibody Screening Platform Using Virus Extracts from Endemic Coronaviridae and SARS-CoV-2

Abstract

The presence of antibodies against endemic coronaviruses has been linked to disease severity after SARS-CoV-2 infection. Assays capable of concomitantly detecting antibodies against endemic coronaviridae such as OC43, 229E, NL63, and SARS-CoV-2 may help to elucidate this question. We developed a serum screening platform using a bead-based Western blot system called DigiWest, capable of running hundreds of assays using microgram amounts of protein prepared directly from different viruses. Characterization of the immunoassay for detection of SARS-CoV-2 specific antibodies revealed a sensitivity of 90.3% and a diagnostic specificity of 98.1%. Concordance analysis with the SARS-CoV-2 immunoassays available by Roche, Siemens, and Euroimmun indicates comparable assay performances (Cohen’s κ ranging from 0.8874 to 0.9508). Analogous assays for OC43, 229E, and NL63 were established and combined into one multiplex with the SARS-CoV-2 assay. Seroreactivity for different coronaviruses was detected with high incidence, and the multiplex assay was adapted for serum screening.

Correlation of patient serum IgG, IgA and IgM antigen binding with COVID-19 disease severity using multiplexed SARS-CoV-2 antigen microarray and maintained relative IgA and IgM antigen binding over time

“…Our multiplexed platform was sensitive, accurate, and reproducible without the need for a separate blocking step or inclusion of blocking agent in the applied sample that is required for the majority of other reported multiplexed platforms [26][27][28][29][30][31][32][33][34]. We observed differences in quantitative and relative binding pattern between purified serum IgG, IgA, and IgM compared to the binding of same isotypes in the presence of all others in serum dilutions detected by antiisotype secondary antibodies.…”

“…Several multiplexed SARS-CoV-2 antigen assay platforms have been developed recently, ranging from bead-based assays to multiplexed antigens conjugated to functional glass or gold surfaces and using fluorescence-or plasmon-based detection [26][27][28][29][30][31][32][33][34]. Most of these formats were employed to detect antigen binding of up to two antibody isotypes, IgG and IgM or IgG and IgA, and all used a dilution of serum or plasma as the serum antibody source with detection of isotype binding using a fluorescently labelled anti-isotype secondary antibody.…”

NeutrobodyPlex—monitoring SARS‐CoV‐2 neutralizing immune responses using nanobodies

“…Currently available serological assays provide data on the presence and distribution of antibody subtypes against different SARS‐CoV‐2 antigens within serum samples of infected and recovered SARS‐CoV‐2 patients (Amanat et al, 2020; preprint: Lassaunière et al, 2020; Robbiani et al, 2020; preprint: Roxhed et al, 2020; Stadlbauer et al, 2020; Becker et al, 2021; Fink et al, 2021). However, they do not differentiate between total and neutralizing RBD‐binding antibodies, which sterically inhibit viral entry via ACE2 (Ju et al, 2020; Rogers et al, 2020; Tang et al, 2020).…”

Serum antibody fingerprinting of SARS-CoV-2 variants in infected and vaccinated subjects by label-free microarray biosensor

“…The relevance of this approach in serodiagnostics is, however, still to be confirmed. In the context of SARS-CoV-2, a few multiplexed antigen assay platforms have been proposed, which include fluorescence protein microarray ( 20 , 21 ), as well as bead-based approaches ( 22 ). Despite the validity of these methodologies, the COVID-19 pandemic experience has highlighted the critical need for affordable new assay formats that offer highly sensitive, quantitative, multiplexed, and rapid immune protection profiling.…”