CA-3029204-C - ALPHA-SYNUCLEIN DETECTION ASSAY AND METHOD FOR DIAGNOSING ALPHA-SYNUCLEINOPATHIES

Abstract

A method of detecting the presence of alpha-synuclein aggregation in a biological sample is provided whereby a biological sample is mixed with a reaction sample comprising a population of beads, a fluorophore adapted to bind to protein aggregates and to increase fluorescence when bound to protein aggregates, and alpha-synuclein or a fragment or variant thereof to form a reaction mixture, the reaction mixture is illuminated and at the same time incubated with intermittent agitation cycles, wherein a significant increase in the fluorescence of the reaction mixture during incubation is indicative of the presence of aggregates of alpha-synuclein in the biological sample. Method of diagnosing alpha-synucleinopathies such as Parkinson's disease or Dementia with Lewy Bodies.

Inventors

• Alison Green
• Graham Fairfoul

Assignees

• THE UNIVERSITY COURT OF THE UNIVERSITY OF EDINBURGH

Dates

Publication Date

20260505

Application Date

20170706

Priority Date

20160707

Claims (1)

1. CLAIMS 1. An in vitro method of detecting the presence of alpha-synuclein aggregation in a biological sample, the method comprising the steps: 5 (i) providing a reaction sample comprising: (a) a population of beads comprising zirconia and/or silica, wherein the population of beads have a mean diameter of from 1 mm to 0.1 mm ±10%; (b) thioflavin T; and (c) alpha-synuclein or a variant of alpha-synuclein having from 1 to 10 10 amino acid substitutions or insertions that do not substantially affect the ability of the peptide to aggregate in the reaction conditions of the method; (ii) combining the biological sample and the reaction sample to form a reaction mixture; (iii) incubating the reaction mixture with intermittent agitation cycles; 15 (iv) illuminating the reaction mixture with a wavelength of light that excites the thioflavin T; and (v) determining the level of fluorescence of the reaction mixture during incubation, wherein steps (iii) to (v) are carried out at the same time, and a significant 20 increase in the fluorescence of the reaction mixture during steps (iii) to (v) is indicative of the presence of aggregates of alpha-synuclein in the reaction mixture, and wherein the presence of aggregates of alpha-synuclein in the reaction mixture is indicative of the presence of aggregates of alpha-synuclein in the biological sample. 25 2. The method according to claim 1, wherein the biological sample is a bodily fluid sample. 3. The method according to claim 1, wherein the biological sample is selected from the group consisting of cerebrospinal fluid, blood, blood fractions, nasal fluid, nasal tissue, urine, 30 faeces, and lymph. 4. The method according to claim 1, wherein the reaction sample is a buffered reaction sample. 35 5. The method according to claim 4, wherein the reaction sample is buffered to maintain the pH of the reaction sample from about pH 6 to about pH 8.5. Date Rer;ue/Date Recieved 2024-06-04 6. The method according to claim 1, wherein the aggregates of alpha-synuclein comprise beta-sheet content. 7. The method according to claim 1, wherein the reaction sample comprises from 5 approximately 0.01 mg/ml alpha-synuclein to about 10 mg/ml alpha-synuclein to act as an aggregation substrate. 8. The method according to claim 1, wherein the alpha-synuclein of the reaction sample is full length alpha-synuclein. 9. The method according to claim 1, wherein the reaction sample comprises from approximately 1 mg to approximately 150 mg of beads per 100 μl of reaction mixture. 10. The method according to claim 1, wherein the presence of aggregates of alpha- 15 synuclein in the biological sample is indicative of a disease associated with abnormal aggregation of alpha-synuclein. 11. The method according to claim 1, wherein the reaction mixture is incubated for more than 40 hours. 12. The method according to claim 1, wherein the method is carried out at a temperature of from 25 °c to 45 °c. 13. An in vitro method of detecting the presence of alpha-synuclein aggregation in a 25 biological sample, the method comprising the steps: (i) providing a reaction sample comprising: (a) a population of beads comprising zirconia and/or silica, wherein the population of beads have a mean diameter of the beads from 1 mm to 0.1 mm ±10%; (b) thioflavin T; and (c) alpha-synuclein or a variant of alpha-synuclein having from 1 to 10 amino acid substitutions or insertions that do not substantially affect the ability of the peptide to aggregate in the reaction conditions of the method; (ii) combining the biological sample and the reaction sample to form a reaction mixture; 35 (iii) incubating the reaction mixture with intermittent agitation cycles; (iv) illuminating the reaction mixture with a wavelength of light that excites the thioflavin T; and 26 Date Rer;ue/Date Recieved 2024-06-04 (v) determining the level of fluorescence of the reaction mixture during incubation, wherein steps (iii) to (v) are carried out at the same time, and a significant increase in the fluorescence of the reaction mixture during steps (iii) to (v) is indicative of a 5 disease associated with abnormal aggregation of alpha-synuclein. 14. The method according to claim 13, wherein the disease is Parkinson's disease or Dementia with Lewy Bodies. 10 15. The method according to claim 13, wherein the biological sample is selected from the group consisting of cerebrospinal fluid, blood, blood fractions, nasal fluid, nasal tissue, urine, faeces, and lymph. 16. The method according to claim 13, wherein the reaction sample is a buffered reaction 15 sample, wherein the reaction sample is buffered to maintain the pH of the reaction sample from about pH 6 to about pH 8.5. 17. The method according to claim 13, wherein the reaction sample comprises from about 0.01 mg/ml alpha-synuclein to about 10 mg/ml alpha-synuclein to act as an 20 aggregation substrate. 18. The method according to claim 13, wherein the reaction sample comprises from 1 mg to 150 mg of beads per 100 μL of reaction mixture. 25 19. An in vitro method for detecting the presence of alpha-synuclein aggregation in human cerebrospinal fluid, the method comprising the steps: (i) providing a reaction sample comprising: (a) a population of beads comprising zirconia/silica, wherein the population of beads have a mean diameter of the beads of 0.1 mm ±10%; 30 (b) thioflavin T; and (c) human recombinant full-length (1-140 aa) alpha-synuclein; (ii) combining the human cerebrospinal fluid and the reaction sample to form a reaction mixture; (iii) incubating the reaction mixture with intermittent agitation cycles; 35 (iv) illuminating the reaction mixture with a wavelength of light that excites the thioflavin T; and (v) determining the level of fluorescence of the reaction mixture during 27 Date Rer;ue/Date Recieved 2024-06-04 incubation, wherein a significant increase in the fluorescence of the reaction mixture during steps (iii) to (v) is indicative of the presence of aggregates of alpha-synuclein in the reaction mixture, and wherein the presence of aggregates of alpha-synuclein in the 5 reaction mixture is indicative of the presence of aggregates of alpha-synuclein in the human cerebrospinal fluid. 20. The method according to claim 19, wherein the reaction sample is a buffered reaction sample, and wherein the reaction sample is buffered to maintain the pH of the reaction 10 mixture at about pH 8.2. 21. The method according to claim 19, wherein the reaction sample further comprises a phosphate buffer. 15 22. The method according to claim 21, wherein the phosphate buffer is present in a concentration of about 100 mM. 23. The method according to claim 19, wherein the ThT is present in a concentration of about 10 μM. 24. The method according to claim 19, wherein the reaction sample comprises about 0.1 mg/ml of the human recombinant full-length (1-140 aa) alpha-synuclein to act as an aggregation substrate. 25 25. The method according to claim 19, wherein the reaction sample comprises from about 20 mg to about 40 mg of beads per 100 μL of reaction mixture. 26. The method according to claim 19, wherein the intermittent agitation cycles comprise agitation by double orbital shaking. 27. The method according to claim 19, wherein the intermittent agitation cycles comprise agitation by double orbital shaking at a rate of about 200 rotations per minute. 28. The method according to claim 19, wherein the incubating the reaction mixture 35 with intermittent agitation cycles comprises one minute of double orbital shaking at a rate of about 200 rotations per minute followed by about 14 minutes of incubation. 28 Date Rer;ue/Date Recieved 2024-06-04 29. The method according to claim 19, wherein the method is carried out at a temperature of from about 30 °C to about 34 °c. 30. The method according to claim 19, wherein the total volume of the reaction mixture is 5 about 100 μL. 31. The method according to claim 19, wherein the illuminating the reaction mixture with a wavelength of light that excites the ThT comprises illuminating with about 450 nm excitation and about 480 nm emission. 32. An in vitro method for detecting the presence of alpha-synuclein aggregation in a biological sample, the method comprising the steps: (i) providing a reaction sample comprising: (a) a population of beads comprising zirconia and/or silica, wherein 15 the population of beads have a mean diameter of the beads of 0.8 mm ±10%; (b) thioflavin T; and (c) alpha-synuclein or a variant of alpha-synuclein having from 1 to 10 amino acid substitutions or insertions that do not substantially affect the ability of the peptide to aggregate in the reaction conditions of the method; 20 (ii) combining the biological sample and the reaction sample to form a reaction mixture; (iii) (iv) incubating the reaction mixture with intermittent agitation cycles; illuminating the reaction mixture with a wavelength of light that excites the thioflavin T; and 25 (v) determining the level of fluorescence of the reaction mixture during incubation, wherein a significant increase in the fluorescence of the reaction mixture during steps (iii) to (v) is indicative of the presence of aggregates of alpha-synuclein in the reaction mixture, and wherein the presence of aggregates of alpha-synuclein in the 30 reaction mixture is indicative of the presence of aggregates of alpha-synuclein in the biological sample. 33. The method according to claim 32, wherein the reaction sample is a buffered reaction sample, and wherein the reaction sample is buffered to maintain the pH of the reaction 35 mixture at about pH 8.0. 34. The method according to claim 32, wherein the reaction sample further comprises a 29 Date Rer;ue/Date Recieved 2024-06-04 phosphate buffer. 35. The method according to claim 32, wherein the thioflavin T is present in a concentration of about 10 μM. 36. The method according to claim 32, wherein the reaction sample comprises about 0.1 mg/ml of the alpha-synuclein or the variant to act as an aggregation substrate. 37. The method according to claim 32, wherein the intermittent agitation cycles 10 comprise agitation by double orbital shaking. 38. The method according to claim 32, wherein the intermittent agitation cycles comprise agitation by double orbital shaking at a rate of at least 200 rotations per minute. 39. The method according to claim 32, wherein the incubating the reaction mixture with intermittent agitation cycles comprises one minute of double orbital shaking at a rate of at least 200 rotations per minute followed by incubation. 20 40. The method according to claim 32, wherein the method is carried out at a temperature of from about 25 °C to about 45 °c. 41. The method according to claim 32, wherein the total volume of the reaction mixture is about 100 μL. 42. The method according to claim 32, wherein the illuminating the reaction mixture with a wavelength of light that excites the ThT comprises illuminating with about 450 nm excitation and about 480 nm emission. 30 43. The method of claim 32, wherein the biological sample is selected from the group consisting of cerebrospinal fluid, blood, blood fractions, nasal fluid, nasal tissue, urine, faeces, and lymph. 44. The method of claim 32, wherein the biological sample is a cell-based tissue. 45. An in vitro method for detecting the presence of alpha-synuclein aggregation in a Date Rer;ue/Date Recieved 2024-06-04 brain homogenate, the method comprising the steps: (i) providing a reaction sample comprising: (a) a population of beads comprising glass; (b) thioflavin T; and (c) alpha-synuclein or a variant of alpha-synuclein having from 1 to 1 O amino acid substitutions or insertions that do not substantially affect the ability of the peptide to aggregate in the reaction conditions of the method; (ii) combining the brain homogenate and the reaction sample to form a reaction mixture; (iii) (iv) incubating the reaction mixture with intermittent agitation cycles; illuminating the reaction mixture with a wavelength of light that excites the thioflavin T; and (v) determining the level of fluorescence of the reaction mixture during incubation, 15 wherein a significant increase in the fluorescence of the reaction mixture during steps (iii) to (v) is indicative of the presence of aggregates of alpha-synuclein in the reaction mixture, and wherein the presence of aggregates of alpha-synuclein in the reaction mixture is indicative of the presence of aggregates of alpha-synuclein in the brain homogenate. 31 Date Rer;ue/Date Recieved 2024-06-04

Description

Alpha-Synuclein Detection Assay and Method for Diagnosing Alpha- Synucleinopathies Field of the Invention The invention relates to an assay for the detection of aggregated proteins in a biological sample, more specifically to an assay for the detection of aggregated alpha-synuclein in a biological sample. 10 Background of the Invention Alpha-synuclein (a-syn) is a well conserved, small acidic protein of 140-amino acids and a molecular weight of 19kDa that is encoded by the SNCA gene located on chromosome 4.1 It is located in high concentration in the presynaptic nerve terminals within the central nervous 15 system where it plays a role in synaptic vesicle biology.2 The synucleinopathies are a set of neurodegenerative disorders associated with the deposition of fibrillary aggregates of a-syn within selective populations of neurones and glia. These deposits can be found within neuronal soma as Lewy bodies (LB) or in dystrophic neurites in diseases such as Parkinson’s disease (PD) or Dementia with Lewy bodies (DLB); or in glial cytoplasmic 20 inclusions in multiple system atrophy (MSA).3 The presence of a-syn has been detected in biological fluids such as cerebrospinal fluid (CSF)4 and serum. 5 The measurement of a-syn concentrations in CSF by ELISAs has been proposed as a biomarker for a-syn related disorders. However, despite many studies 25 showing a reduction in CSF a-syn levels in PD and in DLB4,6, overall results are not consistent.7 Even in those studies where a reduction in CSF a-syn has been demonstrated, the differences are small8 and there is considerable overlap within patient groups and between patient and control groups. In addition the standardisation of CSF a-syn measurement between laboratories has proven difficult.9 The aggregation properties of a-syn have recently been compared to prion protein, the aggregation of which causes transmissible spongiform encephalopathies (TSEs).10 Indeed, there is much discussion in current literature as to whether the alpha-synucleinopathies are actually prion-like diseases.11 A recently-described technique called real-time quaking 35 induced conversion (RT-QuIC), which exploits the ability of prion protein to induce selfaggregation, has been used to develop a diagnostic CSF test for sporadic Creutzfeldt-Jakob disease (sCJD), the most common human form of TSE.12 However, given the significant differences between prion proteins and a-syn, these techniques have failed to detect in any reliable way the presence of a-syn or a-syn aggregation in a biological sample. Therefore, there remains a need for a reliable assay to detect the presence of a-syn and a-syn aggregation in a biological sample. At least one aim of the present invention is to provide an improved assay to detect the presence of a-syn and/or the aggregation of a-syn in a biological sample. Summary of the Invention According to a first aspect of the invention there is provided a method of detecting the presence of alpha-synuclein aggregation in a biological sample, the method comprising the steps: (i) providing a biological sample; 15 (ii) providing a reaction sample comprising a population of beads, a fluorophore adapted to bind to protein aggregates and to increase fluorescence when bound to protein aggregates, and alpha-synuclein or a fragment or variant thereof; (iii) combining the biological sample and the reaction sample to form a reaction mixture; 20 (iv) incubating the reaction mixture with intermittent agitation cycles; (v) illuminating the sample with a wavelength of light that excites the fluorophore of the reaction sample; and (vi) determining the level of fluorescence of the reaction mixture during incubation, 25 wherein steps (iv) to (vi) are carried out at the same time, and a significant increase in the fluorescence of the reaction mixture during steps (iv) to (vi) is indicative of the presence of aggregates of alpha-synuclein in the reaction mixture, and wherein the presence of aggregates of alpha-synuclein in the reaction mixture is indicative of the presence of aggregates of alpha-synuclein in the biological sample. According to a second aspect of the invention there is provided an in vitro method of detecting the presence of alpha-synuclein aggregation in a biological sample, the method comprising the steps: (i) providing a reaction sample comprising: (a) a population of beads comprising zirconia and/or silica, or glass, wherein the population of beads have a mean diameter of from 1 mm to 0.1 mm ±10%; (b) thioflavin T; and 2 Date Re<;ue/Date Received 2023-07-26 (c) alpha-synuclein or a variant of alpha-synuclein having from 1 to 10 amino acid substitutions or insertions that do not substantially affect the ability of the peptide to aggregate in the reaction conditions of the method; (ii) combining the biological sample and the reaction sample to form a reaction 5 mixture; (iii) (iv) incubating the reaction mixture with intermittent agitation cycles; illuminating the reaction mixture