Quantitative ELISA Measurement of α-Synuclein, Aβ, and Tau

Frozen human frontal cortex primary gray matter was weighed and placed on dry ice. Freshly prepared, ice-cold 5 M Guanidine Hydrochloride in Tris-buffered saline (20 mM Tris-HCl, 150 mM NaCl, pH 7.4 TBS) containing 1:100 Halt protease inhibitor cocktail (Thermo Scientific) and 1:100 Phosphatase inhibitor cocktail 2 & 3 (Sigma, St. Louis, MO) was added to the brain tissue at 5:1 ratio and homogenized with Qiagen Tissue Lyser LT (Qiagen Inc., Germantown, MD) at 50 Hz for 5 minutes. The homogenate was then shaken (regular rocker) overnight at room temperature. The lysate was diluted with 1% Blocker A (Meso Scale Discovery (MSD) #R93BA-4, Rockville, MD) in wash buffer in accordance with the specific ELISA assays: 1:4000 for α-synuclein (MSD #K151TGD-2), 1:300 for total tau and tau phosphorylated at threonine 231 (pTau231; MSD #K15121D-2), and 1:4000 for β-amyloids 1-40 and 1-42 (MSD #K15200E-2). Samples were subsequently spun down at 17,000 g and 4°C for 15 minutes, and the supernatant was applied to the ELISA assays. A standard sandwich ELISA was used to measure tau phosphorylated at threonine 181 (pTau181), with the capturing antibody as AT270 against pTau181 (Thermo Scientific #MN1050) and a well-characterized T46 antibody against total tau as the detecting antibody (Thermo Scientific). Sulfo-tag conjugated anti-mouse secondary antibody (MSD) was used for signal detection by the MSD platform, and an MSD SECTOR S 600 Imager was used to measure analyte levels.