Novel Multiplexed Assays for Antibody Isotypes against SARS-CoV-2 Using Flow Cytometry

Abstract

Abstract The ongoing COVID-19 pandemic has prompted intense research on the role of antibodies in characterizing the emergence and sustenance of the immune response. Antibody isotype information is important in understanding disease mechanism, seroconversion, and vaccine response. While several methods provide qualitative information on SARS-CoV-2 antibodies, there is a need for quick methods with high detection sensitivity that provide relative response levels against multiple SARS-CoV-2 antigens. In this study, we show results from the development of a high-sensitivity bead-based immunoassay for flow cytometry that enables detection of IgG, IgM, or IgA antibodies in serum or plasma samples against multiple SARS-CoV-2 antigens. Antigenic targets include SARS-CoV-2 nucleoprotein, S1-subunit of the spike protein, and receptor-binding domain of the spike protein. The assay allows for quick processing, displays clear visual shifts, and provides fluorescent intensity information allowing for high detection sensitivity. Application of the assay to 30 PCR positive SARS-CoV-2 samples and 30 pre-pandemic samples provides high sensitivity and specificity for IgG analysis. IgG detection was observed in SARS-CoV-2 samples ranging from 6–75 days post-onset of symptoms in the study. IgM and IgA antibodies were also detected in several samples. The assay demonstrates good precision, with low %CVs of <10% typically observed. Given that SARS-CoV-2 research encompasses both cellular and serological aspects, the ability to obtain antibody isotype information on flow cytometry platforms that enable cellular as well as bead-analysis can benefit many researchers. For Research Use Only. Not for use in diagnostic procedures.