High-throughput, multiplexed IgG subclassing of antigen-specific antibodies from clinical samples

In vivo, the activity of antibodies relies critically on properties of both the variable domain, responsible for antigen recognition, and the constant domain, responsible for innate immune recognition. Here, we describe a flexible, microsphere-based array format for capturing information about both functional ends of disease-specific antibodies from complex, polyclonal clinical serum samples. Using minimal serum, we demonstrate IgG subclass profiling of multiple antibody specificities. We further capture and determine the subclass of epitope-specific antibodies. The data generated in this array provides a profile of the humoral immune response with multi-dimensional metrics regarding properties of both variable and constant IgG domains. Significantly, these properties are assessed simultaneously, and therefore information about the relationship between variable and constant domain characteristics is captured, and can be used to predict functions such as antibody effector activity. [Display omitted] ► We describe the use of Luminex bead arrays to characterize antibodies in clinical samples. ► Using this technology, up to 500 antigen specificities can be assessed at once. ► Simultaneous readouts of antibody subclass can be performed. ► This data provides a comprehensive profile of the antibody response. ► Array signatures can predict antibody functions.