ELISA for Alpha-Synuclein Quantification

ELISA analysis of α-syn was performed as previously described with minor modifications (52 (link)). Costar 96 half-well plates were incubated with Syn211 antibody (Santa Cruz Biotechnology, Inc.) diluted 1:200 in carbonate coating buffer and incubated at 4 °C overnight. Plates were washed five times with PBS/Tween 20 (0.05%) (PBS-T) and blocked with 2% BSA/PBS for 2 h at 37 °C. After five more washes with PBS-T, samples were diluted in PBS-T (with 0.6% SDS) and protease inhibitor (Sigma), and then incubated overnight at 4 °C with rocking. Standard curve samples were prepared in equivalent concentrations of SDS from recombinant α-syn monomer. All standards and samples were run in duplicate. After 5 washes of PBS-T, polyclonal capture antibody FL-140 (Santa Cruz Biotechnology, Inc.) was diluted 1:200 in 0.05% BSA in PBS-T and added to each well. Samples were incubated for 2 h at 37 °C. After 5 washes in PBS-T, biotinylated anti-rabbit secondary horseradish peroxidase antibody (Jackson ImmunoResearch) was diluted 1:800 in 1% BSA/PBS-T and incubated in the dark for 1.5 h at room temperature. After 5 washes, the plate was developed with super slow 3,3′5,5′-tetramethylbenzidine (Sigma) and absorbance was determined at 650 nm on a Bio-Tek Synergy 2 plate reader after 45 s of shaking.