Quantification of Antibody Specificity Using Yeast Surface Display

The human microtubule associated protein tau is expressed in healthy neurons and is essential to normal function. It is known that tau undergoes phosphorylation, which is a chemical modification at threonine, serine, and tyrosine residues. Hyperphosphorylation of tau – phosphorylation at abnormally high numbers of residues - is involved in a number of diseases, including Alzheimer’s disease and chronic traumatic encephalopathy, collectively labeled tauopathies. Finding antibodies that can bind specifically to disease-associated phosphorylated residues in tau is essential to further research and, eventually, to the determination of a clinical solution to these diseases. The human tau protein is very long and contains many phosphorylation sites, and while there are many antibodies that bind to tau their specificity to phosphorylated tau is not well characterized and is challenging to quantify. To address this problem, we displayed the tau protein on the surface of yeast cells. Using flow cytometry, the binding of commercially available antibodies was quantified. This allows for the testing of large quantities of individual yeast cells expressing the protein, and expression level for each cell can be quantified using an epitope tag inserted at the C-terminus of tau. Specific serine and threonine residues were mutated with overlap extension polymerase chain reaction (OE-PCR) to alanine, glutamic acid, or aspartic acid in order to mimic a dephosphorylated or phosphorylated state. These phosphomimetic mutations were used to ensure pseudo-dephosphorylation or pseudo-phosphorylation at specific sites, and flow cytometry binding data for each was compared to determine epitope and phospho-specificity. This platform can be used to determine the binding and specificity of commercial antibodies, as well as newly created and affinity matured antibodies.