Detection of Structural Forms of Proteins

Abstract

The present invention relates to a method of detecting one or more tissue-derived aggregated proteins in a biological sample.

Claims

1. A method of detecting and/or analysing the structural forms of a protein in a biological sample comprising: i. binding some or all of the structural forms of the protein to a surface, ii. eluting at least a portion of the structural forms of the protein from the surface using conditions that reverse the binding interaction, iii. detecting one or more of the structural forms of the protein; and optionally iv. analysing the content of the different structural forms of the protein in the eluate.

2. The method of claim 1 wherein the structural forms analysed in step iv) include total protein; monomeric forms; oligomeric forms; multimeric forms and aggregated forms.

3. The method of claim 1 wherein the pH of the eluting conditions is less than 4 or greater than 10.

4. The method of claim 3 wherein the pH of the eluting conditions is between 1.5 and 4, or between 10 and 14; or between 2 to 4, or between 11 to 13; or between 2.8 or 11.5.

5. (canceled)

6. (canceled)

7. The method of claim 1 wherein the biological sample comprises whole blood, serum, plasma or the cellular fraction whererin the cellular fraction is the red cell fraction, the white cell fraction, the platelets or a combination thereof.

8. A method of detecting and/or analysing the structural forms of a protein in a biological sample comprising: i. contacting the sample with a binding agent capable of binding to the structural forms of a protein to form a binding agent protein complex, wherein the structural forms comprise any two or more of the following: monomer, oligomer; multimer and aggregates; ii. subjecting the binding agent-protein complex to conditions that are capable of eluting at least a portion of the bound structural protein forms and which maintain at least a portion of the oligomer; multimer and aggregate forms intact; and iii. detecting any two or more of the monomer, oligomer; multimer and aggregate forms of the protein in the eluate

9-12. (canceled)

13. The method of claim 1 wherein protein which has bound and then been eluted is neutralised by addition of alkali or acid.

14. The method of claim 1 wherein elution is achieved at a temperature of between 20° C. and 40° C.

15. The method of claim 1 wherein the detection method comprises the use of solid phase ELISAs, biosensor platforms, surface plasmon resonance, immunometric methods comprising synthetic ligands with binding specificity for aggregated proteins and/or mass spectrometry.

16. The method of claim 1 wherein the protein species to be detected comprises one or more of: beta-amyloid, alpha synuclein (ASN), tau, phosphorylated tau, prion protein, huntingtin and SOD.

17. (canceled)

18. The method of claim 1 for detecting the presence or absence of a neurodegenerative disease; and/or optionally, for detecting the presence or absence of any one or more neurodegenerative diseases in the group comprising: Alzheimer's Disease (AD), Parkinson's Disease (PD), Huntington's Disease, Dementia with Lewy Bodies, Multiple System Atrophy, Progressive Supranuclear Palsy, Amyotrophic Lateral Sclerosis (ALS) or Epilepsy.

19. (canceled)

20. The method of claim 1 wherein the binding agent comprises any suitable ligand capable of binding to structural forms of a protein; or optionally, wherein the ligand comprises any one or more of the following: an antibody and/or antigen-binding fragment thereof, an aptamer, a lectin, an affibody or a synthetic mimic of an antibody.

21. A kit of parts for use in the method of claim 1, said kit comprising: i) a binding agent capable of binding to the structural forms of a protein; ii) an eluting agent capable of eluting at least a portion of the bound protein forms and which maintain at least a portion of the oligomer; multimer and larger aggregate forms intact; and iii) a detecting agent for detecting two or more of total protein; monomeric forms; oligomeric forms; multimeric forms and aggregated forms.

22. The method of claim 1 wherein step i) comprises the use of a detergent.

23. The method of claim 22 wherein the detergent is SDS and/or TWEEN.

Description

[0084] FIG. 1 shows the fluorescence measurements from Example 1;

[0085] FIG. 2 shows the results from the capture, elution and detection experiment of Example 2;

[0086] FIG. 3a shows the results of Example 4 with the fresh set of blood samples from patents diagnosed with AD, control blood samples from spouses (age matched) and a further range of different dementias (MCI—mild cognitive impairment, PD—Parkinson Disease, FTD—fronto temporal dementia, DLB—dementia with lewy bodies, PSP-progressive supranuclear palsy);

[0087] FIG. 3b shows the same data in 3a presented as a scattergram;

[0088] FIG. 3c shows the ROC curve for the AD results vs. age matched spouse controls, this data is summarised in Table 2;

[0089] FIG. 4 shows the results of the aggregated Abeta assay carried out according to the method of Example 4.

[0090] FIG. 5 shows the results of the assays for aggregated Abeta in whole blood from younger subjects

[0091] FIG. 6 shows the results of the assay for total Abeta and the aggregated fraction in whole blood.

EXAMPLES

Example 1. Investigation into Protein Solubilising and Disrupting Reagents that Maximise Immunocapture of Abeta Spiked into Whole Blood

[0092] Beads derivatized with Protein A (GE Healthcare, magnetic Sepharose, diameter 50 μm to 100 μm) were coated with mouse monoclonal anti Abeta antibody (4G8) by incubating for 30 min at room temperature (r.t.) with a solution of 4G8 in PBS at 40 μg/ml, with gentle agitation. Beads were blocked with a 1% skimmed milk solution for 1 hour. Pooled whole blood from normal controls was spiked at 50 pg/ml with a synthetic Abeta 1-42 peptide which had been labelled with biotin; this solution had previously been shown to contain a proportion of peptide aggregates.

[0093] 100 μl volumes of blood and antibody-coated beads were mixed overnight at 4° C. and were then washed 3× with PBS buffer pH 7.5 containing 0.2% Tween 20. Then immunocaptured Abeta was detected directly on the beads by adding 100 μl of a streptavidin-europium conjugate (Pierce), incubating for 60 min, washing the beads in PBS Tween, displacing europium using the standard dissociation solution and reading the fluorescence signal in the supernatant in a time resolved fluorimeter. During the immunocapture step various protein solubilising and disrupting agents were added to the blood according to the list in Table 1.

TABLE-US-00001 TABLE 1 Sample Reagents added A None B SDS 0.2% C RI-OR2-TAT 2 nM D SDS 0.2%, RI-OR2-TAT 2 nM E SDS 0.2%, RI-OR2-TAT 0.2 nM F SDS 0.1%, RI-OR2-TAT 2 nM G Tween 0.05%, SDS 0.15% H Tween 0.1%, SDS 0.1% I Tween 0.15%, SDS 0.05% J None, Dynabeads

[0094] FIG. 1 shows the fluorescence measurements. These data indicate that the whole blood matrix was inhibitory to the immunocapture of spiked Abeta peptide (Sample A) whilst recovery was considerably improved by adding either detergents or the peptide displacing agent RI-OR2-TAT (supplied by Lancaster University, U. K.) that is known to disrupt Abeta interactions. The Tween/SDS mix in reaction I gave the best recovery. (The results for F are an anomaly). In reaction J the large beads from GE Healthcare were replaced by small (1 μl diameter) Protein A-coated magnetic Dynabeads supplied by Invitrogen Inc. These were clearly far less successful in extracting spiked Abeta from the whole blood matrix. There was no additive effect when SDS was combined with RI-OR2-TAT suggesting that they are acting on the same population of Abeta in the blood. Note that this experiment did not discriminate between the monomeric and aggregated forms of Abeta; it simply showed the effect of added reagents on the efficiency of immunocapture of all forms of the spiked protein in the blood matrix.

Example 2. Detection of Synthetic Aggregated Abeta 1-42 Peptide Spiked into Buffer and Whole Blood

[0095] Beads derivatized with Protein A (GE Healthcare, magnetic Sepharose) were coated with mouse monoclonal anti beta amyloid antibody (4G8) according to Example 1. Abeta 1-42 peptide which had been aggregated previously by incubating at r.t. for 16-24 hours was spiked into PBS or fresh whole blood (1 ml) at 100 pg/ml and 50 μl of 4G8-decorated beads were added. The detergents SDS and Tween 20 were added to a final concentration of 0.05% and 0.15% respectively. As demonstrated previously in Example 1 the addition of these detergents improved the recovery of spiked Abeta in the whole blood matrix. The mixture was agitated for 1 hour at r.t. to capture Abeta onto the beads, which were then captured with a magnet and washed six times with PBS buffer to remove the blood matrix. After aspiration of the supernatant 100 μl of glycine-HCl elution buffer pH 2.8 was added and the beads were incubated for 20 min at r.t. to dissociate Abeta from the 4G8 binding agent and most probably to release some or all of the 4G8 antibody from the covalently attached Protein A on the bead. The eluate was neutralised by addition of 200 μl 0.2M-NaOH and transferred to an Abeta aggregate ELISA detection kit supplied by Microsens Biotechnologies (London, UK). The kit has a microplate well coated with a polymeric ligand that is thought to bind all forms of aggregated Abeta and the manufacturers' protocol involves aggregate capture, binding with a different monoclonal antibody with specificity for Abeta (BAM10) and detection with a secondary rabbit anti mouse HRP conjugate. After incubating with TMB substrate for 30 min the reaction was stopped with 0.1M HCl and the absorbance in the wells was measured at 450 nm.

[0096] FIG. 2 shows the results from this capture, elution and detection experiment. In the Figure sample 1 is the positive control in the assay (Abeta solution as supplied by the manufacturer), sample 2 is the negative control (no Abeta), sample 3 is a bead control (no 4G8 antibody added), sample 4 is the Abeta sample at 100 pg/ml spiked into PBS and sample 5 is the Abeta sample at 100 pg/ml spiked into whole blood. It is estimated that, based on previous analytical studies by the manufacturer and described on their website that the aggregate content of this spiked sample was 1% or less, hence the assay was detecting<1 pg/ml aggregated beta amyloid. This small amount of aggregated material was clearly bound by the immobilised 4G8 antibody on the beads and eluted using a brief acid incubation. The matrix effect of whole blood on the assay i.e. inhibition of binding was also still apparent in that the amount of Abeta recovered was significantly reduced compared to PBS alone, but was still measurable. This Example shows that at least a proportion of the aggregated Abeta spiked into blood was carried through the entire procedure of immunoconcentration in the presence of detergents, extensive washing to remove blood components, acid elution from the beads and neutralisation such that sufficient material remained in the eluate to give a signal in the aggregated protein ELISA detection kit.

Example 3. Measurement of Aggregated Abeta in Whole Blood from Dementia Patients Using an Ultrasensitive Time Resolved Fluorescence Method

[0097] Beads derivatized with Protein A (GE Healthcare, magnetic Sepharose) were coated with mouse monoclonal anti beta amyloid antibody (4G8) according to Example 1 and 50 μl of 4G8-decorated beads were then added and incubated with 1 ml of whole blood taken from patients diagnosed as having a neurological disease, and also from healthy age-matched controls. The blood samples had been aliquotted and frozen at −80° C. immediately after drawing. Hence they had been subjected to a single freeze/thaw cycle prior to assay. The disrupting reagents SDS and Tween 20 were also added to the thawed blood at a final concentration of 0.05% and 0.15% respectively. The mixture was agitated for 1 hour at room temperature (r.t.) to capture Abeta onto the beads, which were then captured with a magnet and washed six times with 1 mL PBS buffer to remove the blood matrix. After aspiration of the supernatant 100 μl of glycine-HCl elution buffer pH 2.8 was added and the beads were incubated for 20 min at r.t. to dissociate Abeta from the 4G8 binding agent The eluate was neutralised by addition of 200 μl 0.2M-NaOH and transferred to an Abeta aggregate ELISA detection kit supplied by Microsens Biotechnologies (London, UK). The detection antibody in the kit was replaced with an alternative commercially available antibody (designation 6E10) which had been biotinylated. In the revised assay a wash step was used to remove excess biotinylated 6E10 and then a solution of streptavidin-europium conjugate in PBS buffer was added, incubated for 30 min, excess conjugate was removed by further washing and the signal in the microwell was detected after addition of enhancer/dissociation solution (Pierce) using the standard procedure of time resolved fluorescence (TRF) in a microplate fluorimeter. TRF is an analytical method with a much lower detection limit (1000×) for the europium label employed than the conventional enzyme label used in the ELISA described in Example 2.

[0098] FIG. 3a shows the results with the blood samples from patents diagnosed with AD, control blood samples from spouses (age matched) and a further range of different dementias (MCI— mild cognitive impairment, PD— Parkinson Disease, FTD— fronto temporal dementia, DLB—dementia with lewy bodies, PSP-progressive supranuclear palsy. The Student's t-test for spouse vs. Alzheimer's gave a p value of 0.00152 (a highly significant difference) whilst the t-test for Alzheimer's vs. other dementias was 0.264 (no significant difference).

[0099] FIG. 3b shows the same data in 3a presented as a scattergram.

[0100] FIG. 3c shows the ROC curve for the AD results vs. age matched spouse controls, this data is summarised in Table 2. The AUC of 0.736 represents a fair discrimination between age matched controls and AD patients.

TABLE-US-00002 TABLE 2 Area Under the Curve Std. Asymptotic Asymptotic 95% Confidence Interval Area Error.sup.a Sig..sup.b Lower Bound Upper Bound .736 .066 .003 .606 .866 .sup.aUnder the nonparametric assumption .sup.bNull hypothesis: true area = 0.5

Example 4. Determination of Optimal pH Elution Conditions to Release Abeta from Capture Beads Using an Ultrasensitive Time Resolved Fluorescence Method

[0101] The procedure described in Example 3 was carried out using a solution of aggregated Abeta at 100 pg/ml in PBS. A pH range from 1.5 to 4 was investigated to determine the optimal elution conditions for the Abeta peptide from the beads whilst maintaining at least a proportion of the aggregated forms intact.

[0102] FIG. 4 shows that the release of Abeta from the beads is more efficient at lower pH, and is also improved using a higher temperature during elution (40° C.). Surprsingly even at the very low pH of 1.5 aggregated Abeta is still detectable and so has not been completely dissociated and denatured under these harsh elution conditions.

Example 5. Measurement of Aggregated Abeta in Whole Blood from Dementia Patients Using a Double Antibody ELISA Method

[0103] The procedure described in Example 3 was repeated including the step of elution of captured Abeta from the beads. The subsequent detection procedure was revised however and a double antibody assay method was used instead of the polymeric ligand assay. In this revised assay method the same monoclonal antibody was used on both sides of the ELISA ‘sandwich’ i.e. the microplate well was coated with 6E10 as the capture antibody and the detection antibody conjugate was biotinylated 6E10. Final detection of bound biotinylated antibody in the microwell was carried out with a streptavidin-europium conjugate and measurements were undertaken as before in a time resolved microplate fluorimeter. This revised assay configuration theoretically detects only the oligomers, multimers and aggregates of Abeta present in blood since no signal would be obtained from the assay if a multi-epitopic version of Abeta was not present (due to epitope masking by the antibody).

[0104] Without the prior antibody-coated bead capture step; there was no significant difference in a T-test between age-matched spouse and dementia patients However, using the same blood samples with the antibody-coated bead capture step prior to the double antibody assay method gave a T-test on spouse vs. AD patients of 0.07 which is close to a significant result, thus indicating that the initial antibody-coated bead capture step for Abeta is critical to obtaining a useful assay result.

Example 6. Measurement of Aggregated Tau Spiked into Whole Blood

[0105] Tau protein is a microtubule-associated protein (MAP) that is highly soluble in the cytoplasm. Tauopathies are a class of neurodegenerative diseases associated with the pathological aggregation of tau protein in the brain.

[0106] To detect aggregated tau protein in whole blood beads were first coated with anti-tau monoclonal antibody (Ab64193, Abcam) using the method described in Example 1. Tau protein (rPeptide, 4241 Mars Hill Road, Bogart, Ga. 30622, USA) in PBS buffer was aggregated by the addition of 1.5 μM arachidonic acid (Chirita et al. J Biol Chem. 2003 Jul. 11;278(28):25644-50. Anionic micelles and vesicles induce tau fibrillization in vitro). The aggregated tau protein was spiked into a whole blood sample from a healthy volunteer (exhibiting no signs of dementia) at 20 pg/ml. The detergents sarcosyl (0.1% w/v) and Tween 20 (0.1% v/v) were added to the blood sample to enhance recovery of tau protein by the anti-tau antibody coated beads. After incubating for 60 min the beads were separated from the whole blood and washed 6× in PBS buffer. The bound tau protein was then eluted at pH 2.8 as described above for Abeta assays and then aggregated forms were detected in the ligand-based microplate assay described in earlier examples using a second anti-tau antibody (HT7, Thermo Scientific) and a streptavidin-europium conjugate. The results obtained are shown in Table 3.

TABLE-US-00003 TABLE 3 Detection of aggregated tau protein spiked into whole blood Relative fluorescence units (RFU) in TRF Sample spectrometer Negative control 5000 (no tau aggregate added) Positive control 22000 (20 pg/ml aggregated tau protein added)

Example 7. Measurement of Aggregated Alpha Synuclein (ASN) Spiked into Whole Blood

[0107] Alpha-synuclein is abundant in the human brain where it is found mainly in the presynaptic terminals. The protein is involved in maintaining a supply of synaptic vesicles in presynaptic terminals and in regulating the release of the neurotransmitter dopamine.

[0108] The assay protocol described in Example 6 was followed with the substitution of anti-ASN monoclonal antibody (sc 211, Santa Cruz) on the beads and biotinylated anti-ASN monoclonal antibody (LB 509, Abcam) as the detection antibody in the ligand-based microplate assay for aggregated proteins. ASN protein (rPeptide) in PBS was aggregated by shaking at 37° C. for 5 days, at a protein concentration of 50 μM (Foulds et al FASEB J. 2011 Dec.;25(12):4127-37. Phosphorylated α-synuclein can be detected in blood plasma and is potentially a useful biomarker for Parkinson's disease). The aggregated tau protein was spiked into a whole blood sample from a healthy volunteer (exhibiting no signs of dementia) at 20 pg/ml. The detergents sarcosyl (0.1% w/v) and Tween 20 (0.1% v/v) were added to the whole blood sample and the pH was lowered to 4 with citrate buffer to enhance recovery of ASN protein by the anti-ASN antibody coated beads. The results obtained in the aggregated protein assay are shown in Table 4.

TABLE-US-00004 TABLE 4 Detection of aggregated ASN protein spiked into whole blood Relative fluorescence units (RFU) in TRF Sample spectrometer Negative control 25000 (no ASN aggregate added) Positive control 29000 (20 pg/ml aggregated ASN protein added)

Example 8. Detection of Synthetic Aggregated Abeta 1-42 Peptide Spiked into PBS and Whole Blood Using High pH Elution

[0109] The procedure involving capture of aggregated Abeta from whole blood drawn from a normal subject, as described in Example 2, was repeated with a change in elution conditions to pH 11.5 using 0.1M CAPS buffer followed by neutralisation to pH 7 with 0.1M HCl. The results from the aggregated protein assay are shown in Table 5.

TABLE-US-00005 TABLE 5 Detection of aggregated Abeta protein spiked into whole blood and eluted from the capture beads at pH 11.5 Relative fluorescence units (RFU) in TRF spectrometer Sample (single 1 ml samples) Negative control 12000 (no Abeta aggregate added) Positive control 18000 (20 pg/ml aggregated Abeta protein added)

[0110] In a further experiment the elution pH was raised to 12.8 using 0.2M KCl/NaOHbuffer and compared to elution at pH 3 using glycine-HCl. Aggregated Abeta was spiked into both PBS buffer and whole blood taken from a normal subject. Table 6 shows the comparison of high pH and low pH elution which, under these conditions, proved to give very similar results. In this experiment 1 ml whole blood or PBS samples were tested in triplicate and the controls used capture beads which had not been coated with the 4G8 Abeta-specific antibody.

TABLE-US-00006 TABLE 6 Detection of aggregated Abeta protein spiked into whole blood or PBS and eluted from the capture beads at pH 3 and 12.8 Relative fluorescence units (RFU) in TRF spectrometer (mean and SD of Sample 1 ml triplicate samples) Assay blank  5469 SD = 898 PBS pH 3, control, no capture 15838 antibody on beads  SD = 4630 pH 3, with capture antibody 176582   SD = 38815 pH 12.8 control, no capture 16457 antibody on beads  SD = 5846 pH 12.8, with capture antibody 163453   SD = 21515 Blood pH 3, control, no capture 44956 antibody on beads  SD = 6832 pH 3, with capture antibody 120661   SD = 36020 pH 12.8 control, no capture 29545 antibody on beads SD = 819 pH 12.8, with capture antibody 114882   SD = 58265

Example 9. Levels of Aggregated Abeta in Young Normal Controls

[0111] Two pools of whole blood were prepared from normal volunteers, the first group was <40 years and the second>40 years, but<60. The results of the aggregated Abeta assay carried out according the method of Example 4 are presented in FIG. 5. The data indicate that the levels reported in these young normal individuals are in the range of 10,000 to 15,000 RFUs; this is significantly below the mean of age matched (>60 year) controls shown in FIG. 3b which indicates that aggregated Abeta levels in the blood increase with age.

Example 10. Measurement of the Content of Total Abeta and Total Aggregated Forms in Whole Blood Using an ELISA

[0112] The first stage of the assay procedure described in Example 3 was carried out using Abeta-specific antibody-coated beads, followed by acid elution at pH 2.8 and neutralisation. Then 100 μl of the neutralised eluate was added to duplicate wells of a commercially available total Abeta 1-42 ELISA kit (Life Technologies) and the manufacturer's recommended protocol was followed. This kit measures total Abeta 1-42 content rather than the aggregated form only. In a further variant of this protocol 200 μl of the neutralised eluate was added to the wells of the aggregate detection kit provided by Microsens Biotechnologies and incubated for 45 min to bind at least a proportion of the total aggregates present (including all the oligomeric forms) under the low ionic strength conditions employed; then the supernatant in the wells was combined and transferred to a single well of the total Abeta 1-42 ELISA kit and a total protein assay was carried out as above. In addition 100 μl of the original whole blood sample was added directly to the wells of the Abeta ELISA and the manufacturer's protocol was followed to determine whether this assay could be used to detect total Abeta in the unprocessed original whole blood sample.

[0113] FIG. 6 shows the data from the total Abeta measurements in the Life Technologies kit. The results obtained with whole blood added directly to the wells have been omitted as the background levels were very high and variable and no useful data was obtained. However, the results obtained with the neutralised eluate from the bead capture step showed that significant levels of total Abeta 1-42 could be detected in two patients diagnosed with Alzheimer's Disease (“AD 1 beads or AD2 beads”), whilst the age matched (spouse) control level was zero (“Spouse 1 beads”). The Figure shows the mean of duplicate assays in the ELISA and the standard variation. The results from the samples that had been treated with the aggregate-binding microwells prior to the total Abeta 1-42 ELISA showed a reduction of approx. 60% in signal in the ELISA from the AD patients (“AD1”, “AD2”). This result provided evidence that the neutralised eluate contained approx. 60% total aggregated and approx. 40% non-aggregated forms of Abeta 1-42 and indicated that determining the content of these forms of the protein could be used to improve the accuracy of a blood test for Alzhiemer's Disease.

[0114] A repeat of this experiment using capture conditions of high ionic strength in the aggregate detection kit (which ensures that only larger aggregates and not smaller oligomeric forms are bound) substantially reduced the signal from the AD patients indicating that the oligomeric forms predominated in the whole blood sample

[0115] This Example shows that the immunoconcentration step using protein specific antibody-coated capture beads is an efficient way of extracting all structural forms of proteins from whole blood and the neutralised eluate from the bead capture step could be used in a commercial ELISA for total protein (this Example) as well as in the aggregate-specific protein assays (previous Examples).

Example 11. Measurement of Aggregated Abeta in Whole Blood and Plasma from Alzheimer's Disease Patients

[0116] The assay procedure described in Example 3 was carried out to measure aggregated Abeta in 1 ml aliquots of whole blood or 5 ml aliquots of plasma taken from Alzheimer's Disease (AD) patients. Aggregated Abeta can be detected in both sample types, however the data also indicates that levels in plasma were at least 6×lower in plasma than in whole blood suggesting that the majority of the aggregated forms of this protein were associated with the cellular fraction of blood.

Example 12. Measurement of Aggregated Abeta, Aggregated Alpha Synuclein and Aggregated Tau in Whole Blood from Dementia and Parkinson's Disease Patients

[0117] The method described in Examples 3, 6 and 7 was used to determine the levels of aggregated Abeta, aggregated alpha synuclein and aggregated tau in separate 1 ml aliquots of whole blood taken from Alzheimer's Disease (AD), Dementia with Lewy bodies (DLB) and Parkinson's Disease (PD) patients (8 subjects in total). The assays were conducted in duplicate. The blood samples had previously been aliquotted and frozen at −80° C. immediately after drawing. Hence they had been subjected to a single freeze/thaw cycle prior to assay.

[0118] Table 6 is a summary of the levels of these three aggregated proteins determined to be present in the blood samples whilst Table 7 shows the expected results from the known levels of large aggregates of these proteins in brain tissue of patients with a specific neurodegenerative disease. It is clear that 100% of the AD patients tested had the expected levels of circulating aggregated proteins present in blood, whilst 66% of the DLB or PD patients had the expected levels of proteins.

TABLE-US-00007 TABLE 6 Summary of levels of aggregated proteins measured in whole blood. Disease β-amyloid α-synuclein tau Age matched Low Low Low control (spouse) AD 1 High Low High AD 2 High Low Above control DLB 1 High High High DLB 2 Low High High DLB 3 High High High PD 1 Low High Low PD 2 Low High ND PD 3 High High ND

TABLE-US-00008 TABLE 7 Expected values Disease β-amyloid α-synuclein tau AD High Low High DLB High High High PD Low High Low