Detection of Synthetic Peptides As Target Antigens Using Yeast Cell Engineering

The detection of phosphorylated tau (p-tau) levels in clinical samples is of extreme importance for the detection of Alzheimer’s Disease (AD) as well as other neurodegenerative diseases. Recent reports show that detecting low levels of p-tau in plasma is a reliable biomarker for 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 (PTM) protein. However, the need for reliable and specific antibodies for PTM proteins persists due to lack of antibody validation and non-specific binding of popular antibodies.

Yeast surface display has proven to be an effective tool in cell panning applications being used to find binders against surface antigens of endothelial cells, glioblastoma stem-like cells, and mammalian cells. An advantage of yeast biopanning is the yeast cell’s ability to display many copies of antibody fragments which enhances the avidity effect and strengthens binding interactions for low affinity binders. However, due to the limited accessibility of antibody fragments on yeast surface, screening antibodies with low affinity or against low density surface antigens is limited.

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 PTM sites, particularly for targeting intrinsically disordered proteins such as the human tau protein. We show that our platform can specifically and robustly detect p-tau target peptide when compared against controls. 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 will also present optimization parameters towards biopanning antibody libraries against new PTM specific antibodies.