ALPHA-SYNUCLEIN SEED AMPLIFICATION ASSAY FOR PERIPHERAL MATRICES
Methods and kits are provided for reproducible detection of misfolded aS aggregates in biological fluids and tissue that are less-invasively or non-invasively obtained compared to cerebrospinal fluid, such as skin, olfactory mucosa, saliva, and blood or blood parts.
1 . An in vitro method for detecting the presence of alpha-synuclein (αS) aggregate in a skin sample, the method comprising:
(A) providing a skin sample;
(B) providing a pre-incubation mixture, the pre-incubation mixture comprising:
(1) a monomeric αS substrate;
(2) a buffer composition;
(3) a salt solution;
(4) an indicator comprising a fluorophore;
(5) sarkosyl; and
(6) a bead,
(C) combining the skin sample and the pre-incubation mixture to form a reaction mixture;
(D) incubating the reaction mixture with an intermittent agitation cycle to form an incubated reaction mixture;
(E) illuminating the incubated reaction mixture with a wavelength of light that excites the fluorophore; and
(F) determining a level of fluorescence during incubation, wherein an increase in the level of fluorescence indicates the presence of αS aggregate in the skin sample.
2 . The method of claim 1 , wherein the monomeric αS substrate comprises SEQ ID NO: 2.
3 . The method of claim 1 , wherein the monomeric αS substrate comprises SEQ ID NO: 2 and is present in a concentration of 0.3 mg/ml±10%.
4 . The method of claim 1 , wherein the buffer composition comprises PIPES.
5 . The method of claim 1 , wherein the buffer composition comprises about 100 mM PIPES having a pH=6.5±10%.
6 . The method of claim 1 , wherein the salt solution comprises NaCl.
7 . The method of claim 1 , wherein the salt solution comprises about 500 mM NaCl.
8 . The method of claim 1 , wherein the indicator comprising a fluorophore comprises thioflavin T (ThT).
9 . The method of claim 1 , wherein the indicator comprising a fluorophore comprises thioflavin T (ThT) having a concentration of about 5 to about 10 μM.
10 . The method of claim 1 , wherein the sarkosyl is present in a concentration of about 0.1% w/v.
11 . The method of claim 1 , wherein the bead comprises silicone.
12 . The method of claim 1 , wherein the bead is a ceramic bead.
13 . The method of claim 1 , wherein the bead comprises glass.
14 . The method of claim 1 , wherein the bead comprises Si 3 N 4 .
15 . The method of claim 1 , wherein the bead comprises two Si 3 N 4 beads, each having a diameter of 3.175 mm±10%.
16 . The method of claim 1 , wherein the increase in the level of fluorescence comprises an increase of greater than two times a standard deviation of a maximum fluorescence compared to the fluorescence at any point during a lag phase.
17 . An in vitro method for detecting the presence of αS aggregate in an olfactory mucosa sample, the method comprising:
(A) providing an olfactory mucosa sample;
(B) providing a pre-incubation mixture, the pre-incubation mixture comprising:
(1) a monomeric αS substrate;
(2) a buffer composition;
(3) a salt solution;
(4) an indicator comprising a fluorophore;
(5) sarkosyl; and
(6) a bead,
(C) combining the diluted olfactory mucosa homogenate and the pre-incubation mixture to form a reaction mixture;
(D) incubating the reaction mixture with at least one agitation cycle to form an incubated reaction mixture;
(E) illuminating the incubated reaction mixture with a wavelength of light that excites the fluorophore; and
(F) determining a level of fluorescence during incubation, wherein an increase in the level of fluorescence indicates the presence of αS aggregate in the olfactory mucosa sample.
18 . The method of claim 17 , wherein the monomeric αS substrate comprises SEQ ID NO: 2.
19 . The method of claim 17 , wherein the monomeric αS substrate comprises SEQ ID NO: 2 and is present in a concentration of 0.3 mg/ml±10%.
20 . The method of claim 17 , wherein the buffer composition comprises PIPES.
21 . The method of claim 17 , wherein the buffer composition comprises about 100 mM PIPES having a pH=6.5±10%.
22 . The method of claim 17 , wherein the salt solution comprises NaCl.
23 . The method of claim 17 , wherein the salt solution comprises about 500 mM NaCl.
24 . The method of claim 17 , wherein the indicator comprising a fluorophore comprises thioflavin T (ThT).
25 . The method of claim 17 , wherein the indicator comprising a fluorophore comprises thioflavin T (ThT) having a concentration of about 5 to about 10 μM.
26 . The method of claim 17 , wherein the sarkosyl is present in a concentration of about 0.1% w/v.
27 . The method of claim 17 , wherein the bead comprises silicone.
28 . The method of claim 17 , wherein the bead is a ceramic bead.
29 . The method of claim 17 , wherein the bead comprises glass.
30 . The method of claim 17 , wherein the bead comprises Si 3 N 4 .
31 . The method of claim 17 , wherein the bead comprises two Si 3 N 4 beads, each having a diameter of 3.175 mm±10%.
32 . The method of claim 17 , wherein the increase in the level of fluorescence comprises an increase of greater than two times a standard deviation of a maximum fluorescence compared to the fluorescence at any point during a lag phase.