Alpha-synuclein detection using beads
A method is provided for determining the presence of soluble, misfolded α-synuclein protein in a biological sample. The method comprises contacting the biological sample with a pre-incubation mixture, the pre-incubation mixture comprising: a monomeric α-synuclein protein; a buffer composition; a salt; and an indicator, to form an incubation mixture. An incubation cycle is conducted on the incubation mixture in the presence of either a silicon nitride bead or a borosilicate glass bead having a diameter of from about 1 mm to about 5 mm. The method further comprises determining if a detectable amount of misfolded α-synuclein aggregate is present in the biological sample.
1. A method for detecting the presence of α-synuclein (α-syn) aggregate in a biological sample, the method comprising:
(A) providing a biological sample;
(B) providing a pre-incubation mixture, the pre-incubation mixture comprising:
(1) a monomeric α-syn protein;
(2) a buffer composition;
(3) a salt;
(4) a fluorescent protein aggregation indicator; and
(5) a Si 3 N 4 bead comprising a coating of bovine serum albumin;
(C) combining the biological sample and the pre-incubation mixture to form an incubation mixture;
(D) incubating the incubation mixture with intermittent agitation cycles to form an incubated mixture;
(E) illuminating the incubated mixture with a wavelength of light that excites the fluorescent protein aggregation indicator; and
(F) determining a level of fluorescence during incubation,
wherein an increase in the level of fluorescence during incubation indicates the presence of α-syn aggregate 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 greater than 2.3 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 5 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 piperazine-N,N′-bis(2-ethanesulfonic acid) (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 fluorescent protein aggregation indicator comprises thioflavin T (ThT).
13. The method of claim 1 , wherein the intermittent agitation cycles comprise shaking.
14. The method of claim 1 , wherein the pre-incubation mixture further comprises sarkosyl.
15. A method for detecting the presence of α-synuclein (α-syn) aggregate in human cerebrospinal fluid (CSF), the method comprising:
(A) providing human CSF;
(B) providing 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;
(4) thioflavin T (ThT); and
(5) a Si 3 N 4 bead comprising a coating of bovine serum albumin (BSA) and having a diameter greater than 2.3 mm;
(C) combining the human CSF and the pre-incubation mixture to form an incubation mixture;
(D) incubating the incubation mixture with intermittent agitation cycles to form an incubated mixture;
(E) illuminating the incubated mixture with a wavelength of light that excites the ThT; and
(F) determining a level of ThT fluorescence during incubation,
wherein an increase in the level of ThT fluorescence during incubation indicates the presence of α-syn aggregate in the human CSF.
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 detecting the presence of α-synuclein (α-syn) aggregate in a biological sample, the method comprising:
(A) providing a biological sample;
(B) providing 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) a fluorescent protein aggregation indicator; and
(5) a borosilicate glass bead having a diameter greater than 2.3 mm;
(C) combining the biological sample and the pre-incubation mixture to form an incubation mixture;
(D) incubating the incubation mixture with intermittent agitation cycles to form an incubated mixture;
(E) illuminating the incubated mixture with a wavelength of light that excites the protein aggregation indicator; and
(F) determining a level of fluorescence during incubation,
wherein an increase in the level of fluorescence during incubation indicates the presence of α-syn aggregate in the biological sample.
19. The method of claim 18 , wherein the biological sample is human cerebrospinal fluid (CSF).
20. The method of claim 18 , wherein:
(i) the monomeric α-syn protein is present in a concentration of from about 5 μM to about 30 μM;
(ii) the buffer composition comprises piperazine-N,N′-bis(2-ethanesulfonic acid) (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; and
(v) the fluorescent protein aggregation indicator comprises thioflavin T (ThT) in a concentration of from about 5 μM to about 10 μM.