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

Abstract

The utility of high-throughput systems to evaluate antigen-specific Ab has been highlighted by the SARS-CoV-2 pandemic. Pathogen specific antibody 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 difficult to scale for population levels studies. Additionally, the sensitivity across platforms differs making direct comparisons between studies difficult. Cytometric Bead Arrays are an attractive platform for antigen-specific Ab measurements as they allow antibodies reactive against several antigens and of several isotypes to be performed simultaneously. Additionally, cytometric arrays exhibit a high sensitivity and can be designed to provide quantitative measurements. Using commercially available particles, a biotin-Streptavidin loading strategy, and the inclusion of indirect standards, we describe here a flexible system that can be modified to include a variety of antigens. We generated two arrays, one focused on b-Coronavirus antigens and one focused on Influenza. To support the high throughput capacity of this system, we developed a suit of automated tools to process raw data into antigen-reactive IgM, IgA, and IgG. We describe quality control requirements, assay performance, and normalizations to accurately quantitate antigen specific Ig.