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.

Claims

1. A method for detecting the presence of alpha-synuclein aggregation in a human cerebrospinal fluid (“CSF”) sample, the method comprising the steps: (i) providing a human CSF sample; (ii) providing a reaction sample comprising: (a) a population of beads, wherein the population of beads have a mean diameter of the beads of 0.1 mm; (b) thioflavin T (“ThT”); and (c) human recombinant full-length (1-140 aa) alpha-synuclein; (iii) combining the human CSF sample and the reaction sample to form a reaction mixture; (iv) incubating the reaction mixture with intermittent agitation cycles; (v) illuminating the reaction mixture with a wavelength of light that excites the ThT; and (vi) determining the level of fluorescence of the reaction mixture during incubation, wherein 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 human CSF sample.

2. The method according to claim 1, wherein the reaction sample is a buffered reaction sample, and wherein the reaction sample is buffered to maintain the pH of the reaction mixture at pH 8.2.

3. The method according to claim 1, wherein the reaction sample further comprises a phosphate buffer.

4. The method according to claim 3, wherein the phosphate buffer is present in a concentration of 100 mM.

5. The method according to claim 1, wherein the ThT is present in a concentration of 10 μM.

6. The method according to claim 1, wherein the reaction sample comprises 0.1 mg/mL of the human recombinant full-length (1-140 aa) alpha-synuclein to act as an aggregation substrate.

7. The method according to claim 1, wherein the beads of the population of beads of the reaction sample comprise zirconia/silica.

8. The method according to claim 1, wherein the reaction sample comprises from 20 mg to 30 mg of beads per 100 μL of reaction mixture.

9. The method according to claim 1, wherein the intermittent agitation cycles comprise agitation by double orbital shaking.

10. The method according to claim 1, wherein the intermittent agitation cycles comprise agitation by double orbital shaking at a rate of 200 rotations per minute.

11. The method according to claim 1, wherein the incubating the reaction mixture with intermittent agitation cycles comprises one minute of double orbital shaking at a rate of 200 rotations per minute followed by 14 minutes of incubation.

12. The method according to claim 1, wherein the method is carried out at a temperature of from 32° C. to 34° C.

13. The method according to claim 1, wherein the human CSF sample is provided in an amount of 5 μL.

14. The method according to claim 1, wherein the total volume of the reaction mixture is 100 μL.

15. The method according to claim 1, wherein the illuminating the reaction mixture with a wavelength of light that excites the ThT comprises illuminating with 450 nm excitation and 480 nm emission.

16. A method for detecting the presence of alpha-synuclein aggregation in a biological sample, the method comprising the steps: (i) providing a biological sample; (ii) providing a reaction sample comprising: (a) a population of beads, wherein the population of beads have a mean diameter of the beads from 1 mm to 0.1 mm; (b) thioflavin T (ThT); and (c) alpha-synuclein or a variant; (iii) combining the biological sample and the reaction sample to form a reaction mixture; (iv) incubating the reaction mixture with intermittent agitation cycles; (v) illuminating the reaction mixture with a wavelength of light that excites the ThT; and (vi) determining the level of fluorescence of the reaction mixture during incubation, wherein 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.

17. The method according to claim 16, wherein the reaction sample is a buffered reaction sample, and wherein the reaction sample is buffered to maintain the pH of the reaction mixture at pH 8.0.

18. The method according to claim 16, wherein the reaction sample further comprises a phosphate buffer.

19. The method according to claim 16, wherein the ThT is present in a concentration of 10 μM.

20. The method according to claim 16, wherein the reaction sample comprises 0.1 mg/mL of the alpha-synuclein or a variant to act as an aggregation substrate.

21. The method according to claim 16, wherein the beads of the population of beads of the reaction sample comprise silica, glass, or a combination thereof.

22. The method according to claim 16, wherein the intermittent agitation cycles comprise agitation by double orbital shaking.

23. The method according to claim 16, wherein the intermittent agitation cycles comprise agitation by double orbital shaking at a rate of at least 300 rotations per minute.

24. The method according to claim 16, wherein the incubating the reaction mixture with intermittent agitation cycles comprises one minute of double orbital shaking at a rate of at least 300 rotations per minute followed by incubation.

25. The method according to claim 16, wherein the method is carried out at a temperature of from 25° C. to 45° C.

26. The method according to claim 16, wherein the total volume of the reaction mixture is 100 μL.

27. The method according to claim 16, wherein the illuminating the reaction mixture with a wavelength of light that excites the ThT comprises illuminating with 450 nm excitation and 480 nm emission.

28. The method of claim 16, wherein the biological sample is a bodily fluid sample.

29. The method of claim 28, wherein the bodily fluid is selected from the group consisting of cerebrospinal fluid, blood, blood fractions, nasal fluid, nasal tissue, urine, faeces, and lymph.

30. The method of claim 28, wherein the biological sample is a cell-based tissue sample.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the accompanying drawings.

(2) FIG. 1: A schematic diagram showing the process of standard RT-QuIC;

(3) FIG. 2: RT-QuIC traces for brain homogenate (BH) samples from patients with Dementia with Lewy Bodies (DLB), Alzheimer's disease (AD) and sporadic Creutzfeldt-Jakob disease (sCJD) with patients with no neuropathological evidence of disease who died suddenly (SD) as controls;

(4) FIG. 3: RT-QuIC traces for BH samples from patients with mixed pathologies;

(5) FIG. 4: RT-QuIC traces for cerebrospinal fluid (CSF) samples from confirmed DLB and sCJD patients;

(6) FIG. 5: RT-QuIC traces for control CSF samples and one BH sample from a patient with DLB;

(7) FIG. 6: RT-QuIC traces for CSF samples from patients with AD and one BH sample from a patient with DLB;

(8) FIG. 7: RT-QuIC traces for CSF samples from patients with DLB and one BH sample from a patient with DLB;

(9) FIG. 8: RT-QuIC traces for CSF samples from patients with DLB and AD and one BH sample from a patient with DLB;

(10) FIG. 9: RT-QuIC traces for CSF samples from patients with Parkinson's Disease (PD) and CSF samples from control subjects;

(11) FIG. 10: RT-QuIC traces for SD BH samples A) with beads and B) without beads;

(12) FIG. 11: RT-QuIC traces for DLB BH samples A) with beads and B) without beads;

(13) FIG. 12: RT-QuIC traces for Parkinson's Disease cerebrospinal fluid (CSF) samples A) with beads and B) without beads;

(14) FIG. 13: RT-QuIC traces for Dementia with Lewy Bodies CSF samples A) with beads and B) without beads;

(15) FIG. 14: RT-QuIC traces for Dementia with Lewy Bodies BH samples with A) no beads B) 18.7 mg beads per well and C) 37.5 mg beads per well;

(16) FIG. 15: RT-QuIC traces for BH from control subjects (SDBH), Lewy body disease patients (LBDBH), Alzheimer's Disease (ADBH) or sporadic Creutzfeldt-Jakob disease (sCJDBH) or unseeded for (A) 0.1 mm zirconium/silica beads, (B) 0.5 mm zirconium/silica beads and (C) 2.3 mm zirconium/silica beads;

(17) FIG. 16: RT-QuIC traces for CSF samples from patients with Lewy Body disease (LB) or from control subjects with the addition of 37 mg of (A) 0.1 mm zirconium/silica beads, (B) 0.5 mm zirconium/silica beads and (C) 2.3 mm zirconium/silica beads;

(18) FIG. 17: RT-QuIC traces for BH samples from control subjects (SDBH), Lewy body disease patients (LBDBH) and Alzheimer's Disease (ADBH) and compared to those reactions left unseeded using (A) 0.5 mm steel beads or (B) 0.5 mm glass beads;

(19) FIG. 18: RT-QuIC traces for CSF samples from control subjects, and Lewy body disease patients (LBDBH) and compared to those reactions left unseeded using (A) 0.5 mm steel beads or (B) 0.5 mm glass beads; and

(20) FIG. 19: RT-QuIC traces for platelet samples isolated form EDTA anti-coagulated blood samples from PD and control subjects.

DETAILED DESCRIPTION

(21) While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

(22) To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

(23) General Materials and Methods

(24) Real-Time Quaking Induced Aggregation for Alpha-Synuclein

(25) The RT-QuIC reaction buffer (RB) was composed of 100 mM phosphate buffer (pH 8.2), 10 μM Thioflavin T (ThT) and 0.1 mg/mL human recombinant full-length (1-140aa) alpha-synuclein (Stratech, Cambridge, UK). Each well of a black 96-well plate with a clear bottom (Nalgene Nunc International, Fisher Scientific Ltd, UK) contained 98 μL, 90 μL or 85 μL RB (depending on volume of seed added) and 37±3 mg of 0.5 mm zirconium/silica beads (Thistle Scientific Ltd, Glasgow, UK). Reactions were seeded with 2 μL of working strength brain homogenate (BH), 5 μl, 10 μl or 15 μl of undiluted CSF to a final reaction volume of 100 μl. The plates were sealed with a plate sealer film (Fisher Scientific Ltd, UK) and incubated in a BMG OPTIMA FluoSTAR plate reader at 30° C. for 120 h with intermittent shaking cycles: double orbital with 1 minute shake (200 rpm), 14 minute rest. ThT fluorescence measurements (450 nm excitation and 480 nm emission) were taken every 15 minutes. Each sample was run in duplicate, allowing 2 negative control samples (reactions seeded with SD and AD BH), 1 positive control (reaction seeded with DLB BH), an unseeded reaction and 44 CSF samples to be tested on one plate.

(26) Patient Groups

(27) Initial phase of CSF RT-QuIC development was carried out on 99 CSF samples obtained from the OPTIMA cohort (Oxford Project to Investigate Memory and Ageing) with clinically and neuropathologically confirmed diagnosis of pure DLB (n=12), PD (n=2), progressive supranuclear palsy (PSP) (n=2), corticobasal degeneration (n=3), DLB with AD pathology (n=17), AD with incidental LBs (n=13), pure AD (n=30) and controls (n=20). OPTIMA initiated in 1988, is a prospective, longitudinal clinico-pathological study of dementia and aging including CSF collection at multiple time points during clinical follow-up. All clinical and pathological protocols have been described in detail.sup.13 and were approved by the local ethics committee and participants provided informed consent prior to enrolment.

(28) The validation phase of RT-QuIC was carried out on CSF samples (20 PD, 15 controls and 3 at-risk) obtained from the Oxford Discovery cohort, which is one of the largest, clinically best-characterized longitudinal PD cohorts to date. Full clinical details of this cohort have been described previously..sup.14 In brief, patients with idiopathic PD diagnosed within 3.5 years according to UK PD Society Brain Bank diagnostic criteria.sup.15 were recruited between September 2010 and September 2014 from a 2.9 million population (ethics study 10/H0505/71). Mean disease duration among 20 PD patients was 1.6±1.1 years (range 0.1-3.2 years) and Hoehn and Yahr stage 1.9±0.4 (range 1-3, maximum possible score 5). The control population were recruited from spouses and friends of patients taking part in the study, as well as the general public. The at-risk group comprised patients with REM sleep behaviour confirmed on overnight polysomnography,.sup.16 80% of which have shown to develop Lewy body disorder over time..sup.17 Demographic information is given in Table 1.

(29) TABLE-US-00002 TABLE 1 Patient demographic information for the Optima and Discovery patients investigated Age at death Mean ± SD (range) F/M OPTIMA patients (n) Pure LBD (12) 80.8 ± 6.5 (71-92)  4/8 Parkinson's disease (2) 77.5 ± 7.8 (72-83)  0/2 Mixed LBD/AD (17) 80.1 ± 6.4 (69-90) 10/7 AD with incidental LB (13) 79.8 ± 7.8 (67-91)  9/4 Pure AD (30) 77.7 ± 8.6 (61-93) 17/13 Progressive supranuclear palsy 69.5 ± 3.5 (67-72)  2/0 (PSP) (2) Corticobasal degeneration (CBD) (3) 64.0 ± 10.6 (52-72)  1/2 Controls (20) 82.9 ± 6.9 (68-93) 10/10 Discovery patients (n) Parkinson's disease (20) 65.1 ± 9.1 (42-80)  6/14 At-risk RBD patients (3) 67.6 ± 7.7 (59-74)  0/3 Controls (15) 65.8 ± 7.4 (55-83)  8/7
Brain Homogenates

(30) Brain tissue was provided the MRC Brain Bank in the NCJDRSU (ethical license 11/ES/0022). All tissue was frozen at −80° C. within 2 hours of being sampled and stored at −80° C. prior to analysis. Brain tissues had been stored between 2-18 years prior to use.

(31) Frontal cortex tissue was taken from patients with Alzheimer's disease (AD); sporadic Creutzfeldt-Jakob disease (sCJD); and Diffuse Lewy body dementia (DLB). In addition frontal cortex was obtained from individuals without neurodegenerative disease from the MRC Sudden Death Brain and Tissue Bank (Sudden Death (SD) controls). Both frontal cortex and substantia nigra tissue was obtained from patients with mixed AD/DLB; mixed sCJD/DLB and mixed AD/PD. All cases used had been examined histologically and the diagnosis reached using internationally accepted criteria..sup.18 Initial 10% w/v brain homogenates (BH) were prepared using phosphate buffered saline (PBS) containing 1 mM EDTA, 150 mM NaCl, 0.5% Triton X and complete protease inhibitor cocktail from Roche. Subsequent working strength BHs were prepared by diluting the above 1:20,000 with PBS.

(32) Cerebrospinal Fluid Samples

(33) CSF samples were stored in 0.5 mL aliquots in polypropylene tubes at −80° C. prior to analysis. 99 CSF samples from the OPTIMA cohort and 38 CSF samples from the Oxford Parkinson's Disease Centre (OPDC) Discovery study were received from the Nuffield Department of Clinical Neurosciences, University of Oxford. All CSF samples were transported from Oxford to Edinburgh on dry-ice and stored at −80° C. on arrival. In addition, CSF samples from patients with neuropathologically confirmed sCJD or DLB from the NCJDRSU CSF Bank were analysed. Ethical approval for the use of CSF samples from the NCJDRSU CSF Bank was covered by Multi-centre Research Ethics Committee for Scotland 05/MRE00/67. All CSF were spun and stored at −80° C. prior to analysis.

(34) Results

(35) The development of RT-QuIC was undertaken using frontal cortex BH from patients with a clinico-pathological diagnosis of DLB, Alzheimer's disease (AD) and sCJD. Patients with no neuropathological evidence of neurological disease who died suddenly and were part of the MRC Sudden Death brain bank were used as controls (SD) (FIG. 2). The RT-QuIC reactions seeded with BH from DLB had a lag-phase of 50 hours, after which an increasing thioflavine T fluorescent signal was seen that became maximal at 70 hours. None of the reactions seeded with BH from patients with other protein misfolding disorders (AD or sCJD) or the SD controls, gave a positive response even after 120 hours.

(36) Many disease pathologies commonly co-exist, especially AD-related and a-syn pathology.sup.19. To investigate whether the presence of an alternative protein-misfolding disorder can interfere with the a-syn aggregation induced by either DLB or PD, BHs from the frontal cortex of patients with mixed pathologies were examined (FIG. 3). The presence of a second protein-misfolding disorders such as AD or sCJD does not inhibit the RT-QuIC reaction induced by OLE or PD BHs (FIGS. 2 and 3). To investigate whether the RT-QuIC method developed was sensitive enough to detect a-syn in CSF, two CSF samples from patients with neuropathologically confirmed OLE and 1 CSF from a neuropathologically confirmed case of sCJD were analysed (FIG. 4). Both CSF samples from the OLE patients gave positive responses with a lag-phase between 60-100 hours.

(37) An exploratory set of 99 in vivo CSF samples obtained as part of the OPTIMA study from patients with subsequent neuropathologically confirmed disease were analysed at three different volumes (i.e. 5, 10 and 153) to investigate the sensitivity and specificity of the RT-QuIC and to calculate the optimal CSF volume for the analysis (Table 2). Using a volume of 15 μl a sensitivity of 92% was obtained for CSF samples from OLE (FIGS. 7 and 8) and a sensitivity of 65% was obtained for CSF samples from patients with mixed DLE/AD pathology. None of the CSF samples from the control subjects (FIG. 5) or patients with pure AO (FIG. 6), CEO or PSP were positive. Using this exploratory set of CSF samples a positive response was defined as a relative fluorescence unit (rfu) value of >2SD above the mean of the negative controls at 120 hours of at least one of the CSF duplicates.

(38) TABLE-US-00003 TABLE 2 Positive RT-QuIC reactions seeded with CSF samples from patients with neuropathologically confirmed DLB, mixed DLB/AD, AD with incidental LB, AD, PD and healthy controls (Exploratory Group) and patients with clinically diagnosed PD, at risk- PD, neuropathologically confirmed corticobasal degeneration and supranuclear palsy and PD controls (Confirmatory Group). A positive RT-QuIC response was classified as a relative fluorescence unit (rfu) value of >2SD above the mean of the negative controls at 120 hours of at least one of the CSF duplicates. Number of Number of Number of Positive RT- Positive RT- Positive RT- Exploratory QuIC (%) QuIC (%) QuIC (%) Patient Group (n) using 5 μl using 10 μl using 15 μl AD with incidental LB  2 (15%)  4 (31%)  2 (15%) (13) Healthy Controls (20)  0 (0%)  0 (0%)  0 (0%) Mixed DLB/AD (17)  9 (53%)  11 (65%)  11 (65%) Parkinson's disease (2)  2 (100%)  2 (100%)  2 (100%) Progressive Supranuclear  0 (0%)  0 (0%)  0 (0%) Corticobasal degeneration  0 (0%)  0 (0%)  0 (0%) (3) Pure AD (30)  2 (7%)  1 (3%)  0 (0%) Pure DLB (12)  10 (83%)  11 (92%)  11 (92%) Sensitivity (DLB)  83%  92%  92% Specificity (vs controls) 100% 100% 100% Specificity (vs AD)  93%  97% 100% Specificity (vs Controls +  96%  98% 100% AD) Number of Positive RT- Confirmatory QuiC (%) Patient Group (n) using 15 μl Parkinson disease (20) / /  19 (95%) At-risk PD patients (3) / /  3 (100%) Parkinson's disease / /  0 (0%) controls Sensitivity (PD) / /  95% Specificity / / 100%

(39) The second phase of the study was to apply these analytical conditions and cut-off criteria to a set of confirmatory in vivo CSF samples from 20 patients with clinically diagnosed PD, 15 control patients and 3 patients with REM sleep behaviour disorder (RBD) recognised to be at high risk of developing future alpha-synucleinopathies,.sup.20 obtained from the large prospective, OPDC Discovery cohort.sup.20. These CSF samples were coded, analysed and reported without prior knowledge of the final diagnosis. After the samples were de-coded the results showed that 19 of the 20 PD patients had a positive RT-QuIC response (FIG. 9) and none of the 15 controls were found to be positive. This resulted in a RT-QuIC sensitivity and specificity for PD of 95% and 100% respectively (Table 2). Interestingly, all three patients at-risk of developing PD had a positive RT-QuIC response. These patients had RBD, of whom 80% have been shown to progress to develop a Lewy body disorder..sup.20

(40) Discussion

(41) The early diagnosis of both DLB and PD is hampered by the lack of sensitive and reliable clinical diagnostic tests. Both conditions are underpinned by the neuropathological deposition of an aggregated form of a-syn which is released into the CSF. We have exploited the ability of the aggregated a-syn to induce further aggregation of non-aggregated a-syn in a cyclical manner to develop a technique that can detect abnormal CSF a-syn in DLB and PD with a sensitivity of 92% and 95% respectively with 100% specificity. Uniquely, we found that 3 RBD patients at high future risk of developing a Lewy body disorder gave a positive RT-QuIC response, suggesting that this test could be used as an early diagnostic test for prodromal PD. Future work focusing on test validation in a larger cohort of RBD patients, followed by ongoing longitudinal assessment, will test the assay's utility in risk-stratifying those prodromal individuals most at risk of early PD conversion in whom neuroprotective therapies might be trialed. We also found that CSF samples from CBD and PSP patients do not give positive RT-QuIC responses. These are movement disorders associated with abnormalities in tau protein rather than a-syn which may be mistaken for PD in the early stages. Therefore, RT-QuIC offers a new approach to the detection of abnormal a-syn and one which has the potential to improve the early clinical diagnosis of PD and DLB in addition to other alpha-synucleinopathies such as MSA.

(42) The Role of Beads in a Modified RT-QuiC Assay for the Detection of Alpha-Synuclein

(43) With reference to FIGS. 10-13, to show the role being played by the beads used in the above assays, parallel assays were carried out for Sudden Death (SD) Brain Homogenate (BH) samples A) with beads and B) without beads, Dementia with Lewy Bodies BH samples A) with beads and B) without beads, Parkinson's Disease cerebrospinal fluid (CSF) samples A) with beads and B) without beads, and Dementia with Lewy Bodies CSF samples A) with beads and B) without beads. These assays were carried out using the same general method described above. As can be seen from FIG. 10, no reading was seen for the SD samples. In addition, those samples with Lewy Body Disease and Parkinson's Disease without beads (FIG. 11B, FIG. 12B or FIG. 13B) showed no reading. However, readings are clearly seen for those samples with either Lewy Body Disease or Parkinson's Disease with the beads. Therefore, the presence of the beads is vital to the ability to detect the presence of a-syn aggregates using RT-QuiC.

(44) With reference to FIG. 14, the effect of bead concentration was investigated using Dementia with Lewy Bodies brain homogenate samples. Samples with no beads present in the reaction mixture showed a constant fluorescent signal, indicated a negative RT-QuiC result. In contrast, samples with 18.7 mg beads per well and 37.5 mg beads per well gave a clear increase in fluorescence and the higher concentration of beads giving a clearer and earlier signal.

(45) Therefore it is clear the presence of the beads in the reaction mixture is crucial for a signal for the detection of alpha-synuclein aggregates to be obtained.

(46) 1. A-Syn RT-QuIC Reactions Using 0.1 mm, 0.5 mm and 2.3 mm Zirconium/Silica Beads (Approx. 37±3 mg) and Seeded with BHs

(47) RT-QuIC reactions with the addition of 37 mg of 0.1 mm, 0.5 mm or 2.3 mm beads were seeded with 5 μL of 1:200,000 dilution of identical BH from control subjects (SDBH), Lewy body disease patients (LBDBH), Alzheimer's Disease (ADBH) or sporadic Creutzfeldt-Jakob disease (sCJDBH) or unseeded. The a-syn RT-QuIC responses obtained with 0.1 mm zirconium/silica beads are shown in FIG. 15A, those with 0.5 mm zirconium/silica beads are shown in FIG. 15B and those with 2.3 mm zirconium/silica beads are shown in FIG. 15C.

(48) It can be seen from FIGS. 15A,B,C that increasing the size of the zirconium/silica beads results in an increase in the time taken to illicit a positive a-syn RT-QuIC response with LBDBH seeded reactions. However despite having a quicker response time using 0.1 mm zirconium/silica beads, the a-syn RT-QuIC gave positive reactions with ADBH seeded reactions.

(49) 2. A-Syn RT-QuIC Reactions Using 0.1 mm, 0.5 mm and 2.3 mm Zirconium/Silica Beads (Approx. 37±3 mg) and Seeded with CSF Samples

(50) RT-QuIC reactions with the addition of 37 mg of 0.1 mm, 0.5 mm or 2.3 mm beads were seeded with 15 μL CSF samples from patients with Lewy Body disease (LB) or from control subjects. The a-syn RT-QuIC responses obtained with 0.1 mm zirconium/silica beads are shown in FIG. 16A, those with 0.5 mm zirconium/silica beads are shown in FIG. 16B and those with 2.3 mm zirconium/silica beads are shown in FIG. 16C.

(51) The use of 0.1 mm and 0.5 mm zirconium/silica beads resulted in positive a-syn RT-QuIC reactions seeded with LB CSF samples but not with control CSF samples. In contrast the use of 2.3 mm zirconium/silica beads did not support a-syn RT-QuIC reactions seeded with CSF samples from LB patients. Identical CSF samples were used in the experiments illustrated in FIGS. 16A, B and C.

(52) The results from the experiments illustrated in FIGS. 15 and 16 demonstrate that the use of 2.3 mm zirconium/silica does not accelerate the a-syn RT-QuIC reaction seeded by either BH or CSF from LB patients. The results a-syn RT-QuIC reactions using either the 0.1 mm or the 0.5 mm zirconium/silica beads were similar, although a-syn RT-QuIC reactions using the 0.1 mm zirconium/silica beads resulted in ADBH inducing a positive reaction. Handling 0.1 mm zirconium/silica beads was difficult as the very small size meant they were more prone to static than the larger 0.5 mm zirconium/silica. Therefore all further investigations into the composition of the beads were undertaken beads of 0.5 mm in diameter.

(53) 3. A-Syn RT-QuIC Reactions Using 0.5 mm Steel and 0.5 mm Glass Beads Seeded with BHs

(54) RT-QuIC reactions with the addition of 37 mg of 0.5 mm steel or glass beads were seeded with 5 μL of 1:200,000 dilution of identical BH from control subjects (SDBH), Lewy body disease patients (LBDBH) and Alzheimer's Disease (ADBH) and compared to those reactions left unseeded. The a-syn RT-QuIC reactions using 0.5 m steel beads are shown in FIG. 17A and those for glass beads are shown in FIG. 17B.

(55) The use of 0.5 mm steel beads does not support a-syn RT-QuIC reactions seeded with LBDBH. In contrast the use of 0.5 mm glass beads does result in positive a-syn RT-QuIC reactions seeded with LBDBH, however the unseeded and ADBH seeded reactions show a gradual increase in fluorescence resulting in a lack of a steady baseline.

(56) 4. A-Syn RT-QuIC Reactions Using 0.5 mm Steel and 0.5 mm Glass Beads Seeded with CSF Samples

(57) RT-QuIC reactions with the addition of 37 mg of 0.5 mm steel or glass beads were seeded with 15 μL CSF samples from patients with Lewy Body disease (LB) or from control subjects. The a-syn RT-QuIC reactions seeded with CSF using 0.5 m steel beads are shown in FIG. 18A and those for glass beads are shown in FIG. 18B. The use of steel beads inhibits the ability of CSF samples from LB patients to seed the a-syn RT-QuIC, whilst the use of glass beads results in non-specific increase in fluorescence and also does not support the a-syn RT-QuIC reaction.

(58) The overall conclusion from the above experiments is that the addition of 37 mg/well of 0.5 mm zirconium/silica beads is the best promoter of a-syn RT-QuIC reactions seeded with either BH or CSF samples from patients with LBD.

(59) Investigation of the Ability of Blood Components from Parkinson's Disease (PD) Patients to Seed the Alpha-Synuclein (a-Syn) Real-Time Quaking Induced Conversion (RT-QuIC)

(60) Platelets were isolated from EDTA anti-coagulated blood samples from two PD patients and a control subject. By increasing the shaking speed from 200 rpm to 600 rpm and the temperature of the reaction to 42° C. it was possible to seed the a-syn RT-QuIC reaction using 15 μL of platelets from these 2 patients with PD (FIG. 19). In contrast the platelets from the control subject failed to seed the reaction. Therefore, this demonstrated that the method of the invention is effective at determining whether a blood sample comprises aggregates of a-syn, and therefore to potentially diagnose whether the subject from whom the blood sample originated has an alpha-synucleinopathy such as Parkinson's Disease or Dementia with Lewy Bodies.

REFERENCES

(61) 1. Spillantini M G, Divane A, and Goedert M. Assignment of human alpha-synuclein (SNCA) and beta-synuclein (SNCB) geners to chromosome 4q21 and 5q35. Genomics. 1995; 27:379-381. 2. Goedert M, Spillantini M G, Del Tredici K, et al. 100 years of Lewy pathology. Nat Rev Neurol. 2013; 9:13-24. 3. Martf M J, Tolsa E, and Campdelacreu J. Clinical Overview of the Synucleinopathies.

(62) Movement disorders. 2003; 18:S21-S27. 4. Mollenhauer M, Cullen V, Kahn I, et al. Direct quantification of CSF alpha-synuclein by ELISA and first cross-sectional study in patients with neurodegeneration. Exp Neurol. 2008; 213:315-325. 5. Williams S M, Schult P, and Sierks M R. Oligomeric a-synuclein an B-amyloid variants as potential biomarkers for Parkinson's and Alzheimer's diseases. Eur J Neurosci. 2015. 6. Shi M, Bradner J, and Hancock A M. Cerebrospinal fluid biomarkers for Parkinson's disease diagnosis and progression. Annals of Neurology. 2011; 69:570-580. 7. Ohrfelt A, Grognet P, Andreasen N, et al. Cerebrospinal fluid alpha-synuclein in neurodegenerative disorders—a marker of synapse loss? Neuroscience Letts. 2009; 450:332-335. 8. Hong Z, Shi M, Chung K A, et al. DJ-1 and alpha-synucelin in human cerebrospinal fluid as biomarkers of Parkinson's disease. Brain. 2010; 133:713-726. 9. Kruse N, Persson S, Alcolea D, et al. Validation of a quantitative cerebrospinal fluid alpha-synuclein assay in a European-wide interlaboratory study. Neurobiol aging. 2015; 36:2587-2596. 10. Bernis M E, Babila J T, Breid S, et al. Prion-lie propagation of human brain-derived alpha-synuclein in transgenic mice expressing human wild-type alpha-synuclein. Acta Neuropathol Commun. 2015; 3. 11. Brandel J P, Corbille A G, Derkinderen P, et al. Is Parkinson's disease a prion disease? Rev Neurol (Paris). 2015; 171:812-824. 12. L McGuire, A Peden, C Orru, et al. Prion seeding activity in cerebrospinal fluid from patients with sporadic Creutzfeldt-Jakob disease patients using real-time QuIC analysis: a potential new clinical diagnostic test with high sensitivity and specificity. Annals of Neurology 72 (2), 278-285. 13. Clarke R, Smith A D, Jobst K A, et al. Folate, Vitamin B12 and serum total homocysteine levels n confirmed Alzheimer's disease. Archives of Neurology. 1998; 55:1449-1455. 14. Szewwczyk-Krolikowski K, Tomlinson P, Nithi K, et al. The influence of age and gender on motor and non-motor features of early parkinson's disease: initial findings form the Oxford Parkinson Disease Center (OPDC) discovery cohort. Parkinsonism Relat Disord. 2014; 20:99-105. 15. Hughes A J, Daniel S E, Kilford L, et al. Accuracy of clinical diagnosis of idiopathic Parkinson's disease—a clinicopathological study of 100 cases. J Neurol Neurosurg Psychiatry. 1992; 55:181-184. 16. Rolinski M, Zokaei N, Baig F, et al. Visual short-term memory deficits in REM sleep behaviour disordermirror those in parkinson's disease. Brain. 2016; 139:47-53. 17. Iranzo A, Tolosa E, Gelpi E, et al. Neurodegnerative disease status and post-mortem pathology in idiopathic rapid-eye-movement sleep behaviour disorder: an observational cohort study. Lancet. 2013; 12:443-453. 18. Thomas J Montine, Creighton H Phelps, Thomas G Beach, et al. National Institute on Aging-Alzheimer's Association guidleines for the neuropathological assessment of Alzheimer's disease: a practical approach. Acta Neuropathol. 2012; 123:1-11. 19. Kovacs G G, Alafuzoff I, A-SS, Arzberger T, et al. Mixed brain pathologies in dementia: the BrainNet Europe consortium experience. Dement Geriatr Cogn Disord. 2008; 26:343-350. 20. Iranzo A, Tolosa E, Gelpi E, et al. Neurodegnerative disease status and post-mortem pathology in idiopathic rapid-eye-movement sleep behaviour disorder: an observational cohort study. Lancet. 2013; 12:443-453.