α-Synuclein is the major protein associated with Lewy body dementia, Parkinson's 
disease and multiple system atrophy. Since α-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 α-synuclein, synthetic peptides containing different amino 
acid sequences were used for immunization of mice. After generation of 
α-synuclein aggregates, ELISA and immunoblotting were used to test the 
specificity of antibodies. Tissue microarray sections originating from different 
human α-synucleinopathies were used to compare immunostaining with other, 
commercially available antibodies. Immunization of mice with the peptide 
TKEGVVHGVATVAE (amino acid 44-57 of α-synuclein) resulted in the generation of a 
monoclonal antibody (5G4), which was able to bind aggregated α-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 α-synuclein pathology. Immunoblotting of 
human brain tissue revealed an additional band seen in dementia with Lewy 
bodies, whereas the band representing monomeric α-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 α-synuclein in body fluids.