Yeast biopanning for detecting antibody binding to site-specific phosphorylations in tau

The detection of phosphorylated tau (p-tau) levels in clinical samples is of extreme importance for the detection of Alzheimers Disease (AD) as well as other neurodegenerative diseases. Recent reports show that detecting low levels of p-tau in plasma can be used as a reliable biomarker for detecting AD prior to the onset of memory loss. The ability to detect such low levels of p-tau is dependent on antibodies specific to the post translationally modified protein. However, the need for reliable phospho-site specific antibodies persists due to a lack of approaches for identifying monoclonal antibodies and characterizing non-specific binding. Here, we report a novel approach using the principles of yeast biopanning to create a robust platform that uses synthetic peptides as target antigens. Using peptides as antigens enables screening antibodies against defined post-translational modification sites, particularly for targeting intrinsically disordered proteins such as the human tau protein. To readily assess yeast binding and distinguish non-specific binding, we developed bi-directional expression vectors that allow antibody fragment surface display and intracellular fluorescent protein expression. We show that our platform can specifically and robustly detect a specific site within the p-tau target peptide when compared against non-phosphorylated controls. By improving biopanning parameters, we enabled phospho-specific capture of yeast cells displaying single-chain variable region fragments (scFvs) against p-tau with a wide range of affinities (KD = 0.2 to 60 nM). These results demonstrate that yeast biopanning can robustly capture yeast cells based on phospho-site specific antibody binding, opening doors for facile identification of high-quality monoclonal antibodies.