Discriminating Parkinson's Disease from Multiple System Atrophy Using Alpha-Synuclein PMCA

1 - 14 . (canceled)

15 . A method for distinguishing a cerebrospinal fluid (CSF) sample as originating from a human subject having Parkinson's disease (PD) versus originating from a human subject having Multiple System Atrophy (MSA), the method comprising:

(A) contacting the CSF sample (i) obtained from the human subject and (ii) comprising soluble, misfolded alpha-synuclein (αS) protein, with a pre-incubation mixture, the pre-incubation mixture comprising:

(1) a monomeric αS substrate;

(2) a buffer composition comprising piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES);

(3) a salt comprising sodium chloride (NaCl);

(4) a fluorescent protein aggregation indicator comprising thioflavin-T (ThT);

(5) sarkosyl; and

(6) a bead comprising borosilicate glass, to form an incubation mixture;

(B) incubating and agitating the incubation mixture to form an incubated mixture comprising misfolded αS aggregates;

(C) illuminating the incubated mixture with a wavelength of light that excites the fluorescent protein aggregation indicator; and

(D) determining a level of fluorescence during incubation, including:

(1) determining a maximum fluorescence (Fmax); and

(2) determining a time when the fluorescence reaches the Fmax.

16 . The method of claim 15 , further comprising comparing the Fmax and the time to a positive control of a CSF sample originating from a human subject having neuronal synuclein disease and/or a positive control of a CSF sample originating from a human subject having MSA.

17 . The method of claim 15 , wherein the determining further includes:

(1) determining a maximum slope (Smax) of fluorescence versus time; and

(2) determining a time when the Smax occurs.

18 . The method of claim 17 , further comprising comparing the Smax and the time to a positive control of a CSF sample originating from a human subject having neuronal synuclein disease and/or a positive control of a CSF sample originating from a human subject having MSA.

19 . The method of claim 15 , wherein the monomeric αS substrate comprises SEQ ID NO: 2.

20 . The method of claim 15 , wherein the bead has a diameter of from about 2.3 mm to about 5 mm.

21 . The method of claim 20 , wherein the bead has a diameter of about 2.45 mm.

22 . The method of claim 15 , wherein the pre-incubation mixture comprises two beads, each having a diameter of about 2.45 mm.

23 . The method of claim 19 , wherein the monomeric αS substrate is present in a concentration of about 0.3 mg/mL.

24 . The method of claim 15 , wherein the buffer composition has a pH of about 6.5.

25 . The method of claim 15 , wherein the NaCl is present in a concentration of about 300 mM to about 500 mM.

26 . The method of claim 15 , wherein the ThT is present in a concentration of about 5 μM.

27 . The method of claim 15 , wherein the sarkosyl is present in a concentration of about 0.1%.

28 . The method of claim 15 , wherein the method is performed at a temperature of about 42° C.

29 . The method of claim 15 , wherein the fluorescence includes excitation and emission maxima of about 440 nm and about 490 nm, respectively.

30 . A method for distinguishing a cerebrospinal fluid (CSF) sample as originating from a human subject having Parkinson's disease (PD) versus originating from a human subject having Multiple System Atrophy (MSA), the method comprising:

(A) contacting a CSF sample (i) obtained from the human subject and (ii) comprising soluble, misfolded alpha-synuclein (αS) protein, with a pre-incubation mixture, the pre-incubation mixture comprising:

(1) a monomeric αS substrate comprising SEQ ID NO: 2 in a concentration of about 0.3 mg/mL;

(2) a buffer composition comprising about 100 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) at a pH of about 6.5;

(3) a salt comprising sodium chloride (NaCl) in a concentration of about 300 mM to about 500 mM;

(4) a fluorescent protein aggregation indicator comprising thioflavin-T (ThT) in a concentration of about 5 μM;

(5) sarkosyl in a concentration of about 0.1%; and

(6) two beads comprising borosilicate glass, each having a diameter of about 2.45 mm, to form an incubation mixture;

(B) incubating and agitating the incubation mixture to form an incubated mixture comprising misfolded αS aggregates;

(C) illuminating the incubated mixture with a wavelength of light that excites the fluorescent protein aggregation indicator; and

(D) determining a level of fluorescence during incubation, including:

(1) determining a maximum fluorescence (Fmax); and

(2) determining a time when the fluorescence reaches the Fmax.

31 . The method of claim 30 , further comprising comparing the Fmax and the time to a positive control of a CSF sample originating from a human subject having neuronal synuclein disease and/or a positive control of a CSF sample originating from a human subject having MSA.

32 . The method of claim 30 , wherein the determining further includes:

(3) determining a maximum slope (Smax) of fluorescence versus time; and

(4) determining a time when the Smax occurs.

33 . The method of claim 32 , further comprising comparing the Smax and the time to a positive control of a CSF sample originating from a human subject having neuronal synuclein disease and/or a positive control of a CSF sample originating from a human subject having MSA.

34 . The method of claim 30 , wherein the method is performed at a temperature of about 42° C.

35 . The method of claim 30 , wherein the fluorescence includes excitation and emission maxima of about 440 nm and about 490 nm, respectively.