Fl 140

Total and oligomeric CSF α-syn were measured as previously reported (Tokuda et al., 2006 (link)). Briefly, for CSF t-α-syn anti-human α-syn monoclonal antibody (clone Syn211) (Santa Cruz Biotechnology, USA) was used for capturing while the anti-human α-syn polyclonal antibody FL-140 (Santa Cruz Biotechnology, USA) was used for antigen detection. The standard curve for the ELISA assay was constructed using recombinant human α-syn solution at different concentrations diluted in blocking buffer. For α-syn oligomers, the antibody clone Syn211 was used for capturing, while biotinylated Syn211 (Santa Cruz Biotechnology, USA) was used for antigen detection. The plate was incubated with 50 μ L/well of ExtrAvidin-Peroxidase (Sigma-Aldrich, UK) and with the enhanced chemiluminescent substrate. For both immunoassays, the samples were screened in blind fashion and were randomly tested. A series of internal controls were run to check for run-to-run variations.

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.

Three µg/mL of α-synuclein and TDP-43 diluted in PBS were coated on separate polystyrene plates (ThermoFisher, Waltham, MA, USA), and incubated overnight at 4 °C. The plates were then washed three times with PBST and blocked with 200 µL of 3% bovine serum albumin (BSA; Millipore, Burlington, MA, USA) diluted in PBS for 1 h at room temperature. After washing, 100 µL of serially diluted (50, 10, and 2 ng/mL) of recombinant TDP-43 and α-synuclein in PBST were applied on to α-synuclein and TDP-43 coated wells, respectively. The plates were then left to incubate for 1 h at 37 °C. After washing, 100 µL of 0.1 µg/mL of biotinylated TDP-43 antibodies (10782-2-AP and 12892-1-AP; Abcam, Cambridge, UK) diluted in PBST with 10% BlockAce, and anti-α-synuclein antibodies (FL-140; Santa Cruz Biotechnology, Dallas, TX, USA) were applied on wells treated with TDP-43 and α-synuclein, respectively, and left to incubate for 1 h at 37 °C. After washing, the wells were treated with 100 µL of HRP-conjugated streptavidin diluted 10,000 times in PBST with 10% BlockAce and left to incubate for 30 min at room temperature. After washing, chemiluminescent substrate (SuperSignal ELISA Pico, Thermo Scientific) was added to achieve greater sensitivity. The resulting luminescence was then measured using a Victor 3 multilabel reader.