A high-throughput multiplex array for antigen-specific serology with automated analysis

Abstract High-throughput customizable CBA automated script-based analysis The utility of high-throughput systems to evaluate antigen-specific antibody (Ab) has been highlighted by the SARS-CoV-2 pandemic. Pathogen-specific Ab levels are often used to assess protection following vaccination and, in the case of novel pathogens, an indication of prior exposure. Several platforms exist to visualize antigen-specific Ab, however most are not quantitative and are difficult to scale for population level studies. Additionally, the sensitivity across platforms differs making direct comparisons between studies difficult. Cytometric bead arrays (CBA) are an attractive platform for antigen-specific Ab measurements as they can be used to assess Ab reactivity against several antigens and of several isotypes to be performed simultaneously. Additionally, CBAs exhibit high sensitivity and can be designed to provide quantitative measurements. Using commercially available particles, a biotin-Streptavidin bead loading strategy, and the inclusion of indirect standards, we describe a flexible system that can be modified to include a variety of antigens. Here we describe two arrays, focused on antigens derived from either β-coronaviruses or influenza virus. To support the high-throughput capacity of this system, we developed a suit of automated software tools, the CBA Toolbox, to process raw data into antigen-reactive IgM, IgA, and IgG concentrations. We describe quality control requirements, assay performance, and normalizations to accurately quantitate antigen-specific Ig.