Protein A/G agarose beads (Abcam) were pretreated with 1% BSA, dissolved in phosphate-buffered saline (PBS) buffer (20 mM phosphate, 150 mM NaCl, pH-7.4), to block any unspecific interactions. A large volume of CSF (50 mL) was pulled down from healthy controls, and endogenous immunoglobulins were depleted from the sample during 2-h incubation with Protein A/G agarose beads, followed by elimination of beads via centrifugation. The antibody recognizing αSN, used for similar approaches previously (25 (link)–27 (link, link)), was added to the obtained solution and incubated overnight in 4 °C. Thereafter, the sample was incubated for 6 h with Protein A/G agarose beads at room temperature (RT) and spun. The beads were washed, and protein elution was conducted by 0.2 M glycine pH 2.6. The sample was dialyzed, lyophilized, and stored at −20 °C for further analysis. In parallel, separate positive and negative control samples were prepared. Recombinant αSN served as a positive control. The negative control was a CSF sample without addition of the primary anti-αSN antibody.