High-titer antibody depletion enhances discovery of diverse serum antibody specificities

The human antibody repertoire is a unique repository of information regarding infection, inflammation, and autoimmunity of the past, present, and future. However, antibodies can span vast ranges of concentrations with varying affinities and the repertoire is often heavily polarized by a few species. These complexities lead to difficulties detecting and characterizing low abundance antibody species that may be relevant to disease. We therefore developed a method to selectively remove antibodies from a sample in proportion to the titer of the species prior to analysis, referred to as high-titer depletion (HTD). Peptides from a large random peptide display library were enriched towards binding high-titer antibody species and utilized as binding reagents to deplete the corresponding species from the specimen. HTD enabled the discovery of antibody binding specificities using random peptide library screening with reduced cross-reactivity and background signal and improved coverage of low abundance species. With HTD, three monoclonal antibody species were detected at concentrations at least an order of magnitude lower than without HTD. Additionally, 92 serum antibody specificities were readily discovered from an individual specimen using HTD compared to only 25 specificities without HTD. Parameters affecting the extent of depletion such as the concentration of depleted serum were also adjusted to reproducibly improve the coverage of antibody specificities. These results demonstrate that HTD could be employed for the discovery of rare specificities related to disease and enable extensive characterization of the antibody repertoire. Moreover, the strategy of depletion in proportion to titer could be extended to other applications with complex biological samples to improve discovery.