Yeast biopanning against site-specific phosphorylations in tau.

Antibodies that bind to protein phosphorylation sites are a critical tool for detecting the post-translational modification. The detection of site-specific phosphorylation in the microtubule-associated protein tau is emerging as a means to diagnose and monitor the progression of Alzheimer's Disease (AD) as well as other neurodegenerative diseases. 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 for yeast biopanning using synthetic peptides containing site-specific phosphorylations as antigens. 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. Using yeast cells displaying a previously validated phospho-tau (p-tau) single-chain variable region fragment (scFv), we show that our platform can discriminate yeast cell binding based on the presence of a single phosphate modification on the target peptide. By improving biopanning parameters, we enabled phospho-specific capture of yeast cells displaying scFvs against p-tau with a wide range of affinities (KD = 0.2 to 60 nM). Finally, we demonstrate the capability to screen large yeast libraries against p-tau by performing biopanning in 6-well plates. These results show that yeast biopanning can robustly capture yeast cells based on phospho-site specific antibody binding, opening doors for the facile identification of high-quality monoclonal antibodies.