A synthetic nanobody library was screened against the distinct to select for oligomer-binding nanobodies. To enrich for high-affinity oligomer binders, yeast surface display was performed followed by two rounds of magnetic-activated cell sorting (MACS), and two rounds of fluorescence-activated cell sorting (FACS) against the amyloid-β oligomer. After each MACS and FACS run, flow cytometry was run for all samples to measure the population of oligomer-binding yeast cells. An increase in the population of yeast cells binding oligomers was observed.
The primed yeast cell colonies were isolated to determine their corresponding nanobody-encoding sequences, then colonies were grown, extracted, and amplified. Resulting nanobody sequences were analyzed with multiple sequence alignment to identify and eliminate redundant clones. Flow cytometry was used to determine the binding capabilities of the selected nanobody sequences and their respective colonies. Due to high expression and binding to amyloid-β oligomer, AbNb4-Fc was selected to test for specific binding against the amyloid-β monomer, oligomer, and fibril.
Dot blot was used to analyze the purified nanobodies binding affinity to amyloid-β monomers, oligomers, and fibrils. The oligomer specific nanobodies will then be identified after negative sorting of the synthetic nanobody library. This research will greatly contribute to future research concerning amyloid-β oligomer specific nanobodies. Identifying nanobodies with selective binding to amyloid-β oligomers represents an important step toward developing targeted therapeutics for Alzheimer’s disease.