Ct-αSyn complex vaccine induces antibodies to recognize αSyn.

Adult mice (n = 7) were intraperitoneally injected with Ct-αSyn complex vaccine four times at 2-week intervals. Sera were collected from tail veins before vaccination (pre-Vac) and after the fourth vaccination (post-Vac). Pooled sera and an αSyn-specific mouse monoclonal antibody (Syn211) were used as primary antibodies (1’ Ab) to probe αSyn in HEK293 cells transfected with or without pAAV-αSyn plasmids (encoding human αSyn) by (A) immunofluorescence staining and (B) Western blot analyses. In the immunofluorescence staining, Alexa488-conjugated polyclonal antibodies were used as secondary antibodies (green); cellular nuclei were labeled with DAPI (blue); scale bar = 20 μm. In the Western blot analysis, HRP-conjugated anti-mouse IgG antibodies were used as secondary antibodies (2’ Ab); the migration molecular weights of αSyn (14 kDa) and β-actin (41 kDa) were indicated. (C) All serum samples were 2-fold serially diluted for antibody titration by enzyme-linked immunosorbent assay conducted in microplates coated with αSyn110-130 peptide. The antibody titer was defined as the reciprocal of the highest serum dilution that gives an OD450 value 3-fold higher than the background value. Data are expressed as mean values ± SEM. Significant differences between groups are indicated (**p t-test).