The Quest for Anti-α-Synuclein Antibody Specificity-Lessons Learnt From Flow Cytometry Analysis.

The accumulation of alpha-synuclein (aSyn) is the hallmark of a group of neurodegenerative conditions termed synucleopathies. Physiological functions of aSyn, including those outside of the CNS, remain elusive. However, a reliable and reproducible evaluation of aSyn protein expression in different cell types and especially in low-expressing cells is impeded by the existence of a huge variety of poorly characterized anti-aSyn antibodies and a lack of a routinely used sensitive detection methods. Here, we developed a robust flow cytometry-based workflow for aSyn detection and antibody validation. We test our workflow using three commercially available antibodies (MJFR1, LB509, and 2A7) in a variety of human cell types, including induced pluripotent stem cells, T lymphocytes, and fibroblasts, and provide a cell- and antibody-specific map for aSyn expression. Strikingly, we demonstrate a previously unobserved unspecificity of the LB509 antibody, while the MJFR1 clone revealed specific aSyn binding however with low sensitivity. On the other hand, we identified an aSyn-specific antibody clone 2A7 with an optimal sensitivity for detecting aSyn in a range of cell types, including those with low aSyn expression. We further utilize our workflow to demonstrate the ability of the 2A7 antibody to distinguish between physiological differences in aSyn expression in neuronal and non-neuronal cells from the cortical organoids, and in neural progenitors and midbrain dopaminergic neurons from healthy controls and in patients with Parkinson's disease who have aSyn gene locus duplication. Our results provide a proof of principle for the use of high-throughput flow cytometry-based analysis of aSyn and highlight the necessity of rigorous aSyn antibody validation to facilitate the research of aSyn physiology and pathology.