# US 11,079,396 B2 · Alpha-synuclein detection using beads > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,079,396 B2 - Document ID: US11079396B2 - Status: Granted - Kind: B2 - Application number: 17/011,374 - Filed: 2020-09-03 - Published: 2021-03-04 - Granted: 2021-08-03 - Earliest claimed: 2019-09-04 - Assignees: Amprion, Inc. - Applicants: Amprion, Inc. - Inventors: Luis Concha; Carly Farris; Bret Holguin; Russell Lebovitz; Benedikt Vollrath; Frank Espin - Web page: https://patentgrep.com/patent/US11079396B2 ## CPC - G01N 33/6896: {Neurological disorders, e.g. Alzheimer's disease} - G01N 2800/2814: Dementia; Cognitive disorders - G01N 2800/2835: Movement disorders, e.g. Parkinson, Huntington, Tourette - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract 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. ## Claims ### Claim 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 Si3N4 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. ### Claim 2 (depends on 1) The method of claim 1, wherein the Si3N4 bead has a diameter of from about 1 mm to about 5 mm. ### Claim 3 (depends on 1) The method of claim 1, wherein the Si3N4 bead has a diameter of greater than 2.3 mm. ### Claim 4 (depends on 1) The method of claim 1, wherein the biological sample comprises human cerebrospinal fluid (CSF). ### Claim 5 (depends on 1) 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. ### Claim 6 (depends on 1) The method of claim 1, wherein the monomeric α-syn protein comprises SEQ ID NO. 2. ### Claim 7 (depends on 6) The method of claim 6, wherein the monomeric α-syn protein is present in a concentration of about 19.6 μM. ### Claim 8 (depends on 1) The method of claim 1, wherein the buffer composition has a pH of between about 6.2 to about 6.5. ### Claim 9 (depends on 1) The method of claim 1, wherein the buffer composition comprises piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES). ### Claim 10 (depends on 1) The method of claim 1, wherein the salt comprises NaCl. ### Claim 11 (depends on 1) The method of claim 1, wherein the salt comprises NaCl in a concentration between about 500 mM to about 700 mM. ### Claim 12 (depends on 1) The method of claim 1, wherein the fluorescent protein aggregation indicator comprises thioflavin T (ThT). ### Claim 13 (depends on 1) The method of claim 1, wherein the intermittent agitation cycles comprise shaking. ### Claim 14 (depends on 1) The method of claim 1, wherein the pre-incubation mixture further comprises sarkosyl. ### Claim 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 Si3N4 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. ### Claim 16 (depends on 15) The method of claim 15, wherein the monomeric α-syn protein comprises SEQ ID NO. 2. ### Claim 17 (depends on 16) The method of claim 16, wherein the monomeric α-syn protein is present in a concentration of about 19.6 μM. ### Claim 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. ### Claim 19 (depends on 18) The method of claim 18, wherein the biological sample is human cerebrospinal fluid (CSF). ### Claim 20 (depends on 18) 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.