Objective: To develop a method for capturing α-synuclein (syn) seeds from cerebrospinal fluid (CSF) and other matrices with high affinity by means of thioflavin T (ThT) coated magnetic beads (ThT-MB).

Background: The dye ThT has been widely utilized to identify protein aggregates with cross-beta structure, often found in brain samples from people with clinical manifestations of neurodegenerative diseases. The biomarker test syn seed amplification assay (synSAA) multiplies and detects syn-seeds associated to underlying syn neuropathology present in synucleinopathies like Parkinson’s disease and multiple system atrophy.

Method: 9 biotinylated anti-syn antibodies (6 aggregate-specific, 3 nonspecific) were used individually to capture syn-seeds from CSF (2 synSAA+ CSF, 1 synSAA- CSF). Streptavidin-conjugated magnetic beads (Ab-MB) were used to bind the antibodies, and the captured syn-seeds were eluted and neutralized. The immunoprecipitated CSF, neutralized eluate, and post-elution Ab-MB were analyzed with synSAA. Similarly, ThT-MB were used to capture syn-seeds from CSF (13 synSAA+ CSF, 2 synSAA- CSF); the ThT-MB and syn-depleted CSF were analyzed with synSAA. synSAA+ and synSAA- CSF were spiked into skin homogenate (SH), saliva, and serum collected from healthy individuals; ThT-MB were used to capture syn-seeds, and the products were analyzed with synSAA.

Results: Immunoprecipitation of syn-seeds from CSF was ineffective; both synSAA+ CSF samples remained strongly positive in synSAA after immunoprecipitation with each of the 9 antibodies, while the neutralized eluates and post-elution Ab-MB were mostly negative, suggesting the syn-seeds remained overwhelmingly in the immunoprecipitated CSF. Conversely, ThT-MB removed syn-seeds from all synSAA+ CSFs with apparent full affinity; all synSAA+ CSFs became negative in synSAA after treatment with ThT-MB, while the ThT-MB showed strong positive signal. Skin homogenate, saliva, and serum did not interfere with the efficiency of ThT-MB syn-seed capture from spiked synSAA+ CSF. ThT-MB were negative in synSAA after interaction with synSAA- CSFs in all conditions.

Conclusion: ThT-MBs capture syn-seeds from CSF with full affinity using only one round of extraction. This method will be further investigated to isolate and enrich syn-seeds to better understand their nature and will be investigated as a potential tool for diagnostic purposes.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/isolation-of-seeding-competent-material-by-means-of-tht-extraction/