An antibody with high reactivity for disease-associated α-synuclein reveals extensive brain pathology

alpha-Synuclein is the major protein associated with Lewy body dementia, Parkinson's disease and multiple system atrophy. Since alpha-synuclein is present in the brain in physiological conditions as a presynaptic protein, it is crucial to characterize disease-associated modifications to develop an in vivo biomarker. With the aim to develop antibodies showing high specificity and sensitivity for disease-associated alpha-synuclein, synthetic peptides containing different amino acid sequences were used for immunization of mice. After generation of alpha-synuclein aggregates, ELISA and immunoblotting were used to test the specificity of antibodies. Tissue microarray sections originating from different human alpha-synucleinopathies were used to compare immunostaining with other, commercially available antibodies. Immunization of mice with the peptide TKEGVVHGVATVAE (amino acid 44-57 of alpha-synuclein) resulted in the generation of a monoclonal antibody (5G4), which was able to bind aggregated alpha-synuclein preparation in sandwich ELISA or coated on magnetic beads. 5G4 proved to be superior to other antibodies in comparative immunohistochemical studies by revealing more widespread and distinct alpha-synuclein pathology. Immunoblotting of human brain tissue revealed an additional band seen in dementia with Lewy bodies, whereas the band representing monomeric alpha-synuclein was very weak or lacking. In summary, the 5G4 antibody is most promising for re-evaluation of archival material and may offer new perspective for the development of in vivo diagnostic assays for detecting disease-associated alpha-synuclein in body fluids.