ALPHA-SYNUCLEIN DETECTION USING BEADS

1 . A method for determining the presence of soluble, misfolded α-synuclein (α-syn) protein in a biological sample, comprising:

(A) contacting the biological sample with a pre-incubation mixture, the pre-incubation mixture comprising:

(1) a monomeric α-syn protein;

(2) a buffer composition;

(3) a salt; and

(4) an indicator,

to form an incubation mixture;

(B) conducting an incubation cycle on the incubation mixture, the incubation cycle being conducted:

(1) two or more times on the incubation mixture effective to form an amplified portion of misfolded α-syn protein from the monomeric α-syn protein, each incubation cycle comprising:

(i) incubating the incubation mixture effective to cause misfolding and/or aggregation of at least a portion of the monomeric α-syn protein in the presence of the soluble, misfolded α-syn protein; and

(ii) physically disrupting the incubation mixture;

(2) in the presence of a Si 3 N 4 bead comprising a coating of bovine serum albumin (BSA); and

(C) determining if a detectable amount of misfolded α-syn aggregate is present in the biological sample, wherein detection of misfolded α-syn aggregate indicates the presence of soluble, misfolded α-syn protein in the biological sample.

2 . The method of claim 1 , wherein the Si 3 N 4 bead has a diameter of from about 1 mm to about 5 mm.

3 . The method of claim 1 , wherein the Si 3 N 4 bead has a diameter of about 2.38 mm.

4 . The method of claim 1 , wherein the biological sample comprises human cerebrospinal fluid (CSF).

5 . The method of claim 1 , wherein the monomeric α-syn protein is present in a concentration of from about 10 μM to about 30 μM.

6 . The method of claim 1 , wherein the monomeric α-syn protein comprises SEQ ID NO. 2.

7 . The method of claim 6 , wherein the monomeric α-syn protein is present in a concentration of about 19.6 μM.

8 . The method of claim 1 , wherein the buffer composition has a pH of between about 6.2 to about 6.5.

9 . The method of claim 1 , wherein the buffer composition comprises PIPES.

10 . The method of claim 1 , wherein the salt comprises NaCl.

11 . The method of claim 1 , wherein the salt comprises NaCl in a concentration between about 500 mM to about 700 mM.

12 . The method of claim 1 , wherein the indicator comprises thioflavin T (ThT).

13 . The method of claim 12 , wherein the detection comprises measuring ThT fluorescence.

14 . The method of claim 1 , wherein the physically disrupting comprises shaking.

15 . A method for determining the presence of soluble, misfolded α-syn protein in human CSF, comprising:

(A) contacting the human CSF with a pre-incubation mixture, the pre-incubation mixture comprising:

(1) a monomeric α-syn protein in a concentration of from about 10 μM to about 30 μM;

(2) a buffer composition having a pH between about 6.2 to about 6.5;

(3) NaCl in a concentration between about 500 mM to about 700 mM; and

(4) ThT,

to form an incubation mixture;

(B) conducting an incubation cycle on the incubation mixture, the incubation cycle being conducted:

(1) two or more times on the incubation mixture effective to form an amplified portion of misfolded α-syn protein from the monomeric α-syn protein, each incubation cycle comprising:

(i) incubating the incubation mixture effective to cause misfolding and/or aggregation of at least a portion of the monomeric α-syn protein in the presence of the soluble, misfolded α-syn protein; and

(ii) shaking the incubation mixture;

(2) in the presence of a Si 3 N 4 bead comprising a coating of BVA and having a diameter greater than 2.3 mm; and

(C) determining if a detectable amount of misfolded α-syn aggregate is present in the biological sample, wherein detection of misfolded α-syn aggregate indicates the presence of soluble, misfolded α-syn protein in the biological sample, and wherein the detection comprises measuring ThT fluorescence.

16 . The method of claim 15 , wherein the monomeric α-syn protein comprises SEQ ID NO. 2.

17 . The method of claim 16 , wherein the monomeric α-syn protein is present in a concentration of about 19.6 μM.

18 . A method for determining the presence of soluble, misfolded α-syn protein in a biological sample, comprising:

(A) contacting the biological sample with a pre-incubation mixture, the pre-incubation mixture comprising:

(1) a monomeric α-syn protein comprising SEQ ID NO. 2;

(2) a buffer composition;

(3) a salt; and

(4) an indicator,

to form an incubation mixture;

(B) conducting an incubation cycle on the incubation mixture, the incubation cycle being conducted:

(1) two or more times on the incubation mixture effective to form an amplified portion of misfolded α-syn protein from the monomeric α-syn protein, each incubation cycle comprising:

(i) incubating the incubation mixture effective to cause misfolding and/or aggregation of at least a portion of the monomeric α-syn protein in the presence of the soluble, misfolded α-syn protein; and

(ii) physically disrupting the incubation mixture;

(2) in the presence of a borosilicate glass bead having a diameter greater than 2.3 mm; and

(C) determining if a detectable amount of misfolded α-syn aggregate is present in the biological sample, wherein detection of misfolded α-syn aggregate indicates the presence of soluble, misfolded α-syn protein in the biological sample.

19 . The method of claim 18 , wherein the biological sample is human CSF.

20 . The method of claim 18 , wherein:

(i) the monomeric α-S protein is present in a concentration of from about 10 μM to about 30 μM;

(ii) the buffer composition comprises PIPES and has a pH of from about 6.2 to about 6.5;

(iii) the salt comprises NaCl in a concentration of from about 500 mM to about 700 mM;

(iv) the borosilicate glass bead has a diameter of 2.45 mm;

(v) the indicator comprises ThT in a concentration of from about 5 μM to about 10 μM; and

(vi) the detection comprises measuring ThT fluorescence.