Assay for the detection of alpha-synuclein seeding activity associated with synucleinopathies

A method of determining whether a subject has a synucleinopathy, comprising:

• A) performing an alpha synuclein (aSyn) seeding assay on a biological sample from the subject comprising:
• i) contacting the biological sample with a soluble mutated recombinant alpha synuclein (rαSyn) to form a reaction mixture, wherein the reaction mixture comprises about 0.1 mg/ml of the soluble mutated rαSyn, about 4 to 8 beads per 100 μl, and a pH of about 8.0, and wherein the soluble mutated rαSyn comprises an amino acid sequence comprising a) one to eight point mutations in SEQ ID NO:
• 1, wherein the point mutations are at residues K23, D2, K21, K45, T59, G67, V77, and/or A78 and/or b) a deletion of up to ten amino acids in amino acids 2-11 of SEQ ID NO:
• 1;
• ii) incubating the reaction mixture at a temperature of 35-50° C. to permit coaggregation of misfolded αSyn aggregates present in the biological sample with the mutated rαSyn;
• iii) maintaining incubation conditions that promote coaggregation of the mutated rαSyn with the misfolded αSyn aggregates to result in a conversion of the soluble mutated rαSyn to mutated rαSyn aggregates while inhibiting spontaneous aggregation of soluble mutant rαSyn;
• and
• iv) agitating mutated rαSyn aggregates formed during step iii), wherein the conditions comprise shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking;
• and
• B) detecting misfolded mutated rαSyn aggregates in the reaction mixture, and wherein detection of misfolded mutated rαSyn aggregates in the reaction mixture indicates that the subject has the synucleinopathy.