Characterizing antibody specificity to different protein morphologies by AFM

Protein misfolding and aggregation can lead to several neurodegenerative diseases including Alzheimer's Disease (AD), Parkinson's Disease (PD) and Huntington's Disease (HD). While the respective proteins involved in each disease differ in their pathological effects and amino acid sequences, the aggregated forms all share a common cross β-sheet conformation. Substantial controversy exists over the roles of the different aggregate morphologies in disease onset and progression, and analytical tools such as morphology specific antibodies are needed to distinguish between the different protein morphologies in situ. Here we utilize atomic force microscopy (AFM) to characterize the binding of three single chain antibody fragments (scFvs) to different morphologies of α-synuclein (αS). From the topographic images generated using the AFM, we were able to show that one scFv bound all morphologies of αS, a second bound only oligomeric αS, and a third bound only fibrillar αS by comparing the height distribution of the different αS morphologies with and without addition of the different scFvs. These results demonstrate the versatility of the AFM- based technique as an easy tool to characterize specific antigen-antibody binding and the potential applications of scFvs as promising immunodiagnostics for protein misfolding diseases.