α-Synuclein purification significantly impacts seed amplification assay performance and consistency

Area of Science:

• Neuroscience
• Biochemistry
• Clinical Diagnostics

Background:

• Seed amplification assays (SAAs) show promise for diagnosing synucleinopathies like Parkinson's disease.
• Reproducibility challenges in SAAs stem from α-synuclein substrate de novo aggregation and batch variability.
• Standardization of α-synuclein substrate purification methods is lacking.

Purpose of the Study:

• To compare the utility of recombinant α-synuclein purified by four different methods as SAA substrates.
• To evaluate substrate performance across two independent laboratories.
• To identify the optimal purification method for maximizing SAA reproducibility.

Main Methods:

• Recombinant α-synuclein was purified using four distinct methods.
• Purified α-synuclein substrates were tested in SAAs using α-synuclein preformed fibrils and patient brain homogenates.
• Assay performance was evaluated for de novo aggregation propensity and inter-batch reproducibility across two labs.

Main Results:

• Osmotic shock-purified α-synuclein monomer exhibited the lowest de novo aggregation.
• This substrate demonstrated superior performance in SAAs seeded with preformed fibrils or patient homogenates.
• Osmotic shock purification yielded the highest inter-batch reproducibility compared to other methods.

Conclusions:

• The purification method significantly impacts α-synuclein substrate quality for SAAs.
• Osmotic shock purification is the preferred method for producing α-synuclein monomer substrates.
• Adopting osmotic shock α-synuclein monomer as a universal substrate will enhance SAA diagnostic accuracy and reproducibility.