阅读说明：本技术 帕金森氏病的确定 (Determination of Parkinson's disease ) 是由 C.切赫 S.齐亚德克 L.佩斯蒂克德拉戈维奇 L.唐 T.克雷默 W.M.扎戈 T-S 于 2019-08-08 设计创作，主要内容包括：本发明公开了用于精确鉴定帕金森氏病的方法和组合物。更特别地,本公开内容涉及在死前组织样品中对帕金森氏病的确定。(Methods and compositions for accurately identifying Parkinson's disease are disclosed. More particularly, the present disclosure relates to the determination of parkinson's disease in pre-mortem tissue samples.)

1. A method for determining whether a subject has Parkinson's Disease (PD), the method comprising:

(a) contacting a biological sample comprising at least one neurological characteristic from the subject with a primary antibody capable of binding phosphorylated alpha-synuclein;

(b) detecting whether said primary antibody capable of binding phosphorylated alpha-synuclein is located within a neurological signature of said biological sample; and

(c) determining that the subject has PD when the primary antibody capable of binding phosphorylated alpha-synuclein is located within the neural signature.

2. The method of claim 1, wherein the sample comprises a tissue sample.

3. The method of claim 1, wherein the neural feature comprises a neural cell.

4. The method of claim 1, wherein the neural feature comprises a precursor neural cell.

5. The method of claim 1, wherein the neural feature is adjacent to a neural cell.

6. The method of claim 1, wherein the sample is contacted with at least one protease prior to contacting the primary antibody capable of binding phosphorylated a-synuclein.

7. The method of claim 6, further comprising contacting the sample with at least one phosphatase.

8. The method of claim 1, further comprising contacting the biological sample from the subject with a primary antibody capable of binding the neurological feature.

9. The method of claim 8, wherein the primary antibody capable of binding to the neural feature is selected from an antibody capable of binding to a protein selected from the group consisting of: ubiquitin C-terminal hydrolase L1(PGP9.5, UCHL1, NDGOA; PARK 5; PGP 95; SPG 79; Uch-L1; HEL-117; PGP 9.5; HEL-S-53), RNA-bound FOX-1 homolog 3(RBFOX3, FOX3, NEUN, FOX-3, HRNBP3), microtubule-associated protein 2(MAP2, MAP2A, MAP2B, MAP2C), 160kDa neurofilament medium chain (NEFM, NFM, NEF3, NF-M), 200kDa neurofilament heavy chain (NEFH, NFH, CMT2CC), synapsin (SYP, MRX96, MRYP) and Discs lager MAGGUK scaffold protein 4(DLG 29, DLGH4, PSD 3995, PSD 4642, SAP-90, SAP A).

10. The method of claim 2, wherein the detecting comprises histochemical analysis.

11. The method of claim 1, wherein the primary antibody capable of binding phosphorylated a-synuclein detects a-synuclein phosphorylated at residue S129.

12. The method of claim 1, wherein the sample is fixed.

13. The method of claim 12, wherein the sample is a formalin-fixed, paraffin-embedded (FFPE) sample.

14. The method of claim 1, wherein the sample is a frozen sample.

15. The method of claim 1, wherein the sample comprises a slice of the neural feature.

16. The method of claim 1, wherein the sample is selected from the group consisting of: skin tissue, colon tissue and submandibular glands.

17. The method of claim 8, wherein the primary antibody capable of binding phosphorylated a-synuclein and the primary antibody capable of binding to the neurological feature are from the same host species, wherein the host species is mouse or rabbit.

18. The method of claim 1, wherein step (a) further comprises contacting the sample with a first secondary antibody having a first label conjugated thereto, wherein the first secondary antibody is immunoreactive with the primary antibody capable of binding phosphorylated a-synuclein.

19. The method of claim 18, comprising contacting the sample with a set of reagents reactive with a first label of the first secondary antibody to generate a first detectable signal in proximity to phosphorylated a-synuclein in the sample.

20. The method of claim 8, wherein prior to contacting the sample with the primary antibody capable of binding to the neural feature, the method comprises denaturing the immune complexes in the sample by incubating the sample at 100 ℃ for at least 15 minutes.

21. The method of claim 20, wherein the method further comprises contacting the sample with a second secondary antibody having a second label conjugated thereto, wherein the second secondary antibody is immunoreactive with the primary antibody capable of binding the neurological feature.

22. The method of claim 21, comprising contacting the sample with a set of reagents reactive with a second label of the second secondary antibody to generate a second detectable signal in the sample proximal to the neural feature.

23. The method of claim 22, wherein the first detectable signal and the second detectable signal are different.

24. The method of claim 23, wherein the first detectable signal is silver stain and the second detectable signal is QM-Dabsyl or fast red.

25. The method of claim 1, wherein the subject is suspected of having PD.

26. A kit, comprising:

(a) a primary antibody capable of binding phosphorylated alpha-synuclein; and

(b) primary antibodies capable of binding neural features.

27. The kit of claim 26, further comprising:

(a) a first secondary antibody having a first label conjugated thereto, wherein the first secondary antibody is immunoreactive with the primary antibody capable of binding phosphorylated a-synuclein;

(b) a set of reagents that generates a first detectable signal when reacted with a first label of the primary secondary antibody;

(c) a second secondary antibody having a second label conjugated thereto, wherein the second secondary antibody is immunoreactive with the primary antibody capable of binding to the neural characteristic;

(d) a set of reagents that generates a second detectable signal when reacted with a second label of the second secondary antibody;

wherein the first detectable signal and the second detectable signal are different.

28. The kit of claim 27, wherein the first detectable signal is silver stain and the second detectable signal is QM-Dabsyl or fast red.

29. The kit of claim 26, wherein the primary antibody capable of binding to the neurological feature is selected from an antibody capable of binding to a protein selected from the group consisting of: ubiquitin C-terminal hydrolase L1(PGP9.5, UCHL1, NDGOA; PARK 5; PGP 95; SPG 79; Uch-L1; HEL-117; PGP 9.5; HEL-S-53), RNA-bound FOX-1 homolog 3(RBFOX3, FOX3, NEUN, FOX-3, HRNBP3), microtubule-associated protein 2(MAP2, MAP2A, MAP2B, MAP2C), 160kDa neurofilament medium chain (NEFM, NFM, NEF3, NF-M), 200kDa neurofilament heavy chain (NEFH, NFH, CMT2CC), synapsin (SYP, MRX96, MRYP) and Discs lager MAGGUK scaffold protein 4(DLG 29, DLGH4, PSD 3995, PSD 4642, SAP-90, SAP A).

30. A method for determining whether a subject has Parkinson's Disease (PD), the method comprising:

(a) preparing a fixed or frozen section of a biological sample from a subject suspected of having PD;

(b) detecting whether the phosphorylated a-synuclein is within a neural signature of the slice; and

(c) diagnosing the subject as having PD when the phosphorylated alpha-synuclein is located within a neural feature.

31. The method of claim 30, wherein step (c) further comprises:

contacting the section with a primary antibody capable of binding phosphorylated alpha-synuclein and a primary antibody capable of binding the neural feature.

32. The method of claim 31, wherein the primary antibody capable of binding to the neural characteristic is selected from an antibody capable of binding to a protein selected from the group consisting of: ubiquitin C-terminal hydrolase L1(PGP9.5, UCHL1, NDGOA; PARK 5; PGP 95; SPG 79; Uch-L1; HEL-117; PGP 9.5; HEL-S-53), RNA-bound FOX-1 homolog 3(RBFOX3, FOX3, NEUN, FOX-3, HRNBP3), microtubule-associated protein 2(MAP2, MAP2A, MAP2B, MAP2C), 160kDa neurofilament medium chain (NEFM, NFM, NEF3, NF-M), 200kDa neurofilament heavy chain (NEFH, NFH, CMT2CC), synapsin (SYP, MRX96, MRYP) and Discs lager MAGGUK scaffold protein 4(DLG 29, DLGH4, PSD 3995, PSD 4642, SAP-90, SAP A).

33. The method of claim 31, further comprising:

i. contacting the section with a first secondary antibody having a first label conjugated thereto, wherein the first secondary antibody is immunoreactive with the primary antibody capable of binding phosphorylated alpha-synuclein, and

contacting the section with a second secondary antibody having a second label conjugated thereto, wherein the second secondary antibody is immunoreactive with the primary antibody capable of binding to the neurological feature.

34. The method of claim 33, further comprising:

i. contacting the section with a set of reagents reactive with a first label of the first secondary antibody to generate a first detectable signal in proximity to phosphorylated a-synuclein in the sample;

contacting the section with a set of reagents reactive with a second label of the second secondary antibody to generate a second detectable signal in the sample proximal to the neural feature.

35. The method of claim 34, further comprising denaturing the immune complexes in the sample after contacting the section with the primary antibody capable of binding phosphorylated a-synuclein and before contacting the section with the primary antibody capable of binding the neurological feature.

36. The method of claim 30, wherein the section is contacted with at least one protease prior to contacting with the primary antibody.

37. The method of claim 36, further comprising contacting the section with at least one phosphatase.

38. The method of claim 31, wherein the primary antibody capable of binding phosphorylated a-synuclein and the primary antibody capable of binding the neurological feature are from the same host species.

39. The method of claim 34, wherein the first detectable signal and the second detectable signal are different.

40. The method of claim 39, wherein the first detectable signal is silver stain and the second detectable signal is QM-Dabsyl or fast Red.

41. A method of diagnosing PD in a subject, the method comprising:

(a) obtaining a biological sample comprising at least one neurological feature from a subject suspected of having PD;

(b) detecting whether the phosphorylated alpha-synuclein is within the neural signature of the sample by contacting the sample with an anti-pgp.5 antibody and determining co-localization between the phosphorylated alpha-synuclein and PGP 9.5; and

(c) diagnosing the subject with PD when the presence of co-localization between phosphorylated alpha-synuclein and PGP9.5 in the neurological signature is positively determined.

42. A method of diagnosing and treating PD in a subject, the method comprising:

(a) obtaining a biological sample comprising at least one neurological feature from a subject suspected of having PD;

(b) detecting whether the phosphorylated alpha-synuclein is within the neural signature of the sample by contacting the sample with an anti-pgp.5 antibody and determining co-localization between the phosphorylated alpha-synuclein and PGP 9.5;

(c) diagnosing the subject with PD when the presence of co-localization between phosphorylated a-synuclein and PGP9.5 in the neurological feature is positively determined; and

(d) administering a therapy to treat PD in a subject diagnosed with PD.

43. A method of treating a subject diagnosed with PD, comprising administering to the subject an effective regime of an antibody to alpha-synuclein, wherein the antibody capable of binding phosphorylated alpha-synuclein and the antibody capable of binding a neurological feature have been shown to co-localize in a neurological feature in a skin sample from the subject.

44. A method of treating a subject determined to have PD comprising administering to the subject an effective regime of an a-synuclein antibody, wherein the subject is determined to have PD by the method of any one of claims 1-25 or 30-41.

45. The method of claim 43 or claim 44, wherein the effective regime of a-synuclein antibodies comprises a-synuclein antibodies selected from the group consisting of: monoclonal antibodies that bind to residues 1-20 of alpha-synuclein, residues 1-10 of alpha-synuclein, residues 4-15 of alpha-synuclein, residues 91-99 of alpha-synuclein, residue 117-123 of alpha-synuclein, residue 118-126 of alpha-synuclein, prasuzuzumab (PRX002), humanized antibodies having the CDRs of antibody clone 1H7(ATCC accession No. PTA-8220), humanized antibodies having the CDRs of antibody clone 9E4(ATCC accession No. PTA-8221), the CDRs of antibody clone NI-202.21D11 and the CDRs of antibody clone NI-202.12F4, e.g., the alpha-synuclein antibodies disclosed in U.S. patent nos. 8,092,801, 8,609,820, 8,790,644, 8,940,276, 9,580,493.

46. The method of any one of claims 1-25 or 30-31, or kit of any one of claims 26-29, wherein the antibody capable of binding phosphorylated a-synuclein is one of: monoclonal antibody clone 7E2 from Roche, monoclonal antibody clone 3G2 from Roche, monoclonal antibody clone MJF-R13(8-8) from Abcam (P/N ab168381), monoclonal antibody P-syn/81A from Abcam (P/N ab184674), monoclonal antibody clone pSyn #64 from WAKO (P/N015-25191), monoclonal antibody clone from AbcamAntibody clone LB509(P/N ab27766), monoclonal antibody clone 5C12 (from Prothena)No. PTA-9197) and monoclonal antibody clone 11A5 (from Prothena: (TM) ((TM))No.PTA-8222)。

47. The method of claims 1-25 or 30-31, or kit of any one of claims 26-29, wherein the primary antibody capable of binding the neural characteristic is one of: the monoclonal antibody clone EPR4118(P/N ab108986) from Abcam, the monoclonal antibody clone 13C/I3C4(P/N ab8189) from Abcam or from Cell Marque^TMThe polyclonal antibody with RTD P/N760-4434.

48. A method for detecting phosphorylated a-synuclein, the method comprising:

(a) contacting the biological sample with a primary antibody capable of binding phosphorylated alpha-synuclein; and

(b) detecting the primary antibody capable of binding phosphorylated alpha-synuclein.

49. The method of claim 48, wherein the sample is contacted with at least one protease prior to contacting the primary antibody capable of binding phosphorylated a-synuclein.

50. The method of claim 49, further comprising contacting the sample with at least one phosphatase.

51. The method of claim 48, wherein said detecting comprises histochemical analysis.

52. The method of claim 48, wherein the primary antibody capable of binding phosphorylated a-synuclein detects a-synuclein phosphorylated at residue S129.

53. The method of claim 52, wherein the primary antibody capable of binding phosphorylated a-synuclein is the 7E2 antibody clone or the 3G2 antibody clone.

54. The method of claim 48, wherein the sample is fixed.

55. The method of claim 54, wherein the sample is a formalin-fixed, paraffin-embedded (FFPE) sample.

56. The method of claim 48, wherein the sample is a frozen sample.

57. The method of claim 48, wherein step (a) further comprises contacting the sample with a first secondary antibody having a first label conjugated thereto, wherein the first secondary antibody is immunoreactive with the primary antibody capable of binding phosphorylated a-synuclein.

58. The method of claim 57, further comprising contacting the sample with a set of reagents reactive with a first label of the first secondary antibody to generate a first detectable signal in proximity to phosphorylated a-synuclein in the sample.

Technical Field

The present disclosure relates to methods and compositions for accurately identifying parkinson's disease. More particularly, the present disclosure relates to the determination of parkinson's disease in pre-mortem tissue samples.

Background

Currently, Parkinson's Disease (PD) is assessed by clinical examination of the subject's symptoms and imaging of dopamine transporter function in the brain (dasscan). The development of therapeutic agents for parkinson's disease has been hampered by the lack of diagnostic tests that accurately identify subjects as having PD. A clear diagnosis of PD is the presence of aggregated alpha-synuclein (aSyn) in neurons and loss of dopaminergic neurons in the substantia nigra region of the brain and can only be performed after necropsy.

Disclosure of Invention

The inventors have found that there is a need in the art to unambiguously and accurately identify parkinson's disease in a living subject.

Provided herein are methods for determining whether a subject has Parkinson's Disease (PD), the method comprising: (a) contacting a biological sample comprising at least one neurological characteristic from a subject with a primary antibody capable of binding phosphorylated alpha-synuclein; (b) detecting whether a primary antibody capable of binding phosphorylated alpha-synuclein is located within a neurological signature of the biological sample; and (c) determining that the subject has PD when a primary antibody capable of binding phosphorylated alpha-synuclein is located within the neurological signature.

In some of the methods, the sample comprises a tissue sample. In some of the methods, the neural feature comprises a neural cell. In some of the methods, the neural feature comprises a precursor neural cell. In some of the methods, the neural feature is adjacent to a neural cell.

In some of the methods, the sample is contacted with at least one protease prior to contacting with the primary antibody capable of binding phosphorylated alpha-synuclein. In some embodiments, the method further comprises contacting the sample with at least one phosphatase.

In some of the methods, the method further comprises contacting the biological sample from the subject with a primary antibody capable of binding a neurological feature. In some embodiments, the primary antibody capable of binding a neurological feature is selected from an antibody capable of binding a protein selected from the group consisting of: ubiquitin C-terminal hydrolase L1(PGP9.5, UCHL1, NDGOA; PARK 5; PGP 95; SPG 79; Uch-L1; HEL-117; PGP 9.5; HEL-S-53), RNA-bound FOX-1 homolog 3(RBFOX3, FOX3, NEUN, FOX-3, HRNBP3), microtubule-associated protein 2(MAP2, MAP2A, MAP2B, MAP2C), 160kDa neurofilament medium chain (NEFM, NFM, NEF3, NF-M), 200kDa neurofilament heavy chain (NEFH, NFH, CMT2CC), synapsin (SYP, MRX96, MRYP) and Discs lager MAGGUK scaffold protein 4(DLG 29, DLGH4, PSD 3995, PSD 4642, SAP-90, SAP A).

In some of the methods, the detecting comprises histochemical analysis. In some of the methods, a primary antibody capable of binding phosphorylated a-synuclein detects a phosphorylated a-synuclein at residue S129.

In some of the methods, the sample is fixed. In some embodiments, the sample is a formalin fixed, paraffin embedded (FFPE) sample. In some of the methods, the sample is a frozen sample. In some of the methods, the sample comprises a section of the neural characteristic. In some of the methods, the sample is selected from the group consisting of: skin tissue, colon tissue and submandibular glands.

In some of the methods, the primary antibody capable of binding phosphorylated alpha-synuclein and the primary antibody capable of binding a neurological feature are from the same host species, wherein the host species is a mouse or a rabbit.

In some of the methods, step (a) further comprises contacting the sample with a first secondary antibody having a first label conjugated thereto, wherein the first secondary antibody is immunoreactive with a primary antibody capable of binding phosphorylated alpha-synuclein. In some embodiments, the method comprises contacting the sample with a set of reagents reactive with a first label of a first secondary antibody to generate a first detectable signal in the sample in proximity to phosphorylated a-synuclein.

In some of the methods, prior to contacting the sample with a primary antibody capable of binding a neural characteristic, the method comprises denaturing the immune complexes in the sample by incubating the sample at 100 ℃ for at least 15 minutes. In some embodiments, the method further comprises contacting the sample with a second secondary antibody having a second label conjugated thereto, wherein the second secondary antibody is immunoreactive with a primary antibody capable of binding the neurological feature. In some embodiments, the method includes contacting the sample with a set of reagents reactive with a second label of a second secondary antibody to generate a second detectable signal in the sample proximate to the neural feature. In some embodiments, the first detectable signal and the second detectable signal are different. In some embodiments, the first detectable signal is silver stain and the second detectable signal is QM-Dabsyl or fast red.

In some of the methods, the subject is suspected of having PD.

Also provided herein is a kit comprising: (a) a primary antibody capable of binding phosphorylated alpha-synuclein; and (b) a primary antibody capable of binding to a neural characteristic. In some embodiments, the kit further comprises: (c) a first secondary antibody having a first label conjugated thereto, wherein the first secondary antibody is immunoreactive with a primary antibody capable of binding phosphorylated a-synuclein; (d) a set of reagents that generates a first detectable signal when reacted with a first label of a first secondary antibody; (e) a second secondary antibody having a second label conjugated thereto, wherein the second secondary antibody is immunoreactive with a primary antibody capable of binding a neurological feature; and (f) a set of reagents that, when reacted with a second label of a second secondary antibody, produces a second detectable signal; wherein the first detectable signal and the second detectable signal are different. In some embodiments, the first detectable signal is silver stain and the second detectable signal is QM-Dabsyl or fast red.

In some of the kits, the primary antibody capable of binding a neurological characteristic is selected from an antibody capable of binding a protein selected from the group consisting of: ubiquitin C-terminal hydrolase L1(PGP9.5, UCHL1, NDGOA; PARK 5; PGP 95; SPG 79; Uch-L1; HEL-117; PGP 9.5; HEL-S-53), RNA-bound FOX-1 homolog 3(RBFOX3, FOX3, NEUN, FOX-3, HRNBP3), microtubule-associated protein 2(MAP2, MAP2A, MAP2B, MAP2C), 160kDa neurofilament medium chain (NEFM, NFM, NEF3, NF-M), 200kDa neurofilament heavy chain (NEFH, NFH, CMT2CC), synapsin (SYP, MRX96, MRYP) and Discs lager MAGGUK scaffold protein 4(DLG 29, DLGH4, PSD 3995, PSD 4642, SAP-90, SAP A).

Also provided herein are methods for determining whether a subject has Parkinson's Disease (PD), the method comprising: (a) preparing a fixed or frozen section of a biological sample from a subject suspected of having PD; (b) detecting whether the phosphorylated alpha-synuclein is within a neural signature of the slice; and (c) diagnosing the subject as having PD when the phosphorylated alpha-synuclein is located within a neural signature. In some embodiments, step (c) further comprises: (d) the sections are contacted with a primary antibody capable of binding phosphorylated alpha-synuclein and a primary antibody capable of binding a neurological feature.

In some of the methods, the primary antibody capable of binding a neurological feature is selected from an antibody capable of binding to a protein selected from the group consisting of: ubiquitin C-terminal hydrolase L1(PGP9.5, UCHL1, NDGOA; PARK 5; PGP 95; SPG 79; Uch-L1; HEL-117; PGP 9.5; HEL-S-53), RNA-bound FOX-1 homolog 3(RBFOX3, FOX3, NEUN, FOX-3, HRNBP3), microtubule-associated protein 2(MAP2, MAP2A, MAP2B, MAP2C), 160kDa neurofilament medium chain (NEFM, NFM, NEF3, NF-M), 200kDa neurofilament heavy chain (NEFH, NFH, CMT2CC), synapsin (SYP, MRX96, MRYP) and Discs lager MAGGUK scaffold protein 4(DLG 29, DLGH4, PSD 3995, PSD 4642, SAP-90, SAP A).

In some of the methods, the method further comprises: contacting the section with a first secondary antibody having a first label conjugated thereto, wherein the first secondary antibody is immunoreactive with a primary antibody capable of binding phosphorylated alpha-synuclein, and contacting the section with a second secondary antibody having a second label conjugated thereto, wherein the second secondary antibody is immunoreactive with a primary antibody capable of binding a neural feature. In some embodiments, the method further comprises: contacting the section with a set of reagents reactive with a first label of a first secondary antibody to generate a first detectable signal in proximity to phosphorylated alpha-synuclein in the sample; and contacting the section with a set of reagents reactive with a second label of a second secondary antibody to generate a second detectable signal in the sample proximal to the neural feature. In some embodiments, the method further comprises denaturing the immune complex in the sample after contacting the section with a primary antibody capable of binding phosphorylated alpha-synuclein and before contacting the section with a primary antibody capable of binding a neurological feature.

In some of the methods, the sections are contacted with at least one protease prior to contacting with the primary antibody. In some embodiments, the method further comprises contacting the section with at least one phosphatase.

In some of the methods, the primary antibody capable of binding phosphorylated alpha-synuclein and the primary antibody capable of binding a neurological feature are from the same host species.

In some of the methods, the first detectable signal and the second detectable signal are different. In some embodiments, the first detectable signal is silver stain and the second detectable signal is QM-Dabsyl or fast red.

Also provided herein are methods of diagnosing PD in a subject, the method comprising: (a) obtaining a biological sample comprising at least one neurological feature from a subject suspected of having PD; (b) detecting whether the phosphorylated alpha-synuclein is within the neural signature of the sample by contacting the sample with an anti-pgp.5 antibody and determining co-localization between the phosphorylated alpha-synuclein and PGP 9.5; and (c) diagnosing the subject with PD when the presence of co-localization between phosphorylated alpha-synuclein and PGP9.5 in the neurological signature is positively determined.

Also provided herein are methods of diagnosing and treating PD in a subject, the method comprising: (a) obtaining a biological sample comprising at least one neurological feature from a subject suspected of having PD; (b) detecting whether the phosphorylated alpha-synuclein is within the neural signature of the sample by contacting the sample with an anti-pgp.5 antibody and determining co-localization between the phosphorylated alpha-synuclein and PGP 9.5; (c) diagnosing the subject with PD when the presence of co-localization between phosphorylated alpha-synuclein and PGP9.5 in the neurological signature is positively determined; and (d) administering a therapy to treat PD in a subject diagnosed with PD.

Also provided herein are methods of treating a subject diagnosed with PD, comprising administering to the subject an effective regime of an antibody to alpha-synuclein, wherein the antibody capable of binding phosphorylated alpha-synuclein and the antibody capable of binding a neurological feature have been shown to co-localize in the neurological feature in a skin sample from the subject.

Also provided herein are methods of treating a subject determined to have PD, comprising administering to the subject an effective regime of an alpha-synuclein antibody, wherein the subject is determined to have PD by any of the methods disclosed herein.

In some such methods, the effective regime of an α -synuclein antibody comprises an α -synuclein antibody selected from the group consisting of: monoclonal antibodies that bind to residues 1-20 of alpha-synuclein, residues 1-10 of alpha-synuclein, residues 4-15 of alpha-synuclein, residues 91-99 of alpha-synuclein, residue 117-123 of alpha-synuclein, residue 118-126 of alpha-synuclein, prasuzuzumab (PRX002), humanized antibodies having the CDRs of antibody clone 1H7(ATCC accession No. PTA-8220), humanized antibodies having the CDRs of antibody clone 9E4(ATCC accession No. PTA-8221), the CDRs of antibody clone NI-202.21D11 and the CDRs of antibody clone NI-202.12F4, for example, alpha-synuclein antibodies disclosed in U.S. patent nos. 8,092,801, 8,609,820, 8,790,644, 8,940,276, 9,580,493, which are incorporated herein by reference in their entirety. Some such antibodies comprise VH CDR1 comprising residues 31-35 of SEQ ID NO. 49, VH CDR2 comprising residues 50-68 of SEQ ID NO. 49, VH CDR3 comprising residues 101-102 of SEQ ID NO. 49, VL CDR1 comprising residues 23-33 of SEQ ID NO. 50, VL CDR2 comprising residues 49-55 of SEQ ID NO. 50, and VL CDR3 comprising residues 88-98 of SEQ ID NO. 50. Some such antibodies comprise VH CDR1 comprising SEQ ID NO. 51, VH CDR2 comprising SEQ ID NO. 52, VH CDR3 comprising SEQ ID NO. 53, VL CDR1 comprising SEQ ID NO. 54, VL CDR2 comprising SEQ ID NO. 55, and VL CDR3 comprising SEQ ID NO. 56. Some such antibodies comprise a heavy chain comprising SEQ ID NO 57, and a light chain comprising SEQ ID NO 58.

In some such methods, the antibody is prasuzumab (PRX 002). In some such methods, the antibody comprises three light CDRs, designated SEQ ID NOS: 18-20, respectively, and three heavy chain CDRs, designated SEQ ID NOS: 22-24, respectively. In some such methods, the antibody comprises a light chain designated SEQ ID NO 17 and a heavy chain designated SEQ ID NO 21

In some such methods, the antibody is 9E4, as disclosed in U.S. patent No. 8,609,820, which is incorporated herein by reference in its entirety. In some such methods, the antibody comprises three light CDRs, designated SEQ ID NOS: 26-28, respectively, and three heavy chain CDRs, designated SEQ ID NOS: 30-32, respectively. In some such methods, the antibody comprises one light chain, designated SEQ ID NO:25, and one heavy chain, designated SEQ ID NO: 29.

In some such methods, the antibody is NI-202.21D 11. In some such methods, the antibody comprises three light CDRs, designated SEQ ID NOS: 34-36, respectively, and three heavy chain CDRs, designated SEQ ID NOS: 38-40, respectively. In some such methods, the antibody comprises a light chain variable region designated SEQ ID NO 33 and a heavy chain variable region designated SEQ ID NO 37.

In some such methods, the antibody is NI-202.12F 4. In some such methods, the antibody comprises light and heavy CDRs, designated SEQ ID NOS: 42-44, respectively, and three heavy chain CDRs, designated SEQ ID NOS: 46-48, respectively. In some such methods, the antibody comprises a light chain variable region designated SEQ ID NO:41 and a heavy chain variable region designated SEQ ID NO: 45.

Also provided herein are methods for detecting phosphorylated alpha-synuclein, the methods comprising: contacting the biological sample with a primary antibody capable of binding phosphorylated alpha-synuclein; and detecting a primary antibody capable of binding phosphorylated alpha-synuclein. In some of the methods, the sample is contacted with at least one protease prior to contacting with the primary antibody capable of binding phosphorylated alpha-synuclein. In some methods, the method further comprises contacting the sample with at least one phosphatase. In some methods, the detecting comprises histochemical analysis. In some methods, a primary antibody capable of binding phosphorylated a-synuclein detects a-synuclein phosphorylated at residue S129. In certain methods, the primary antibody capable of binding phosphorylated alpha-synuclein is a 7E2 antibody clone or a 3G2 antibody clone. In some methods, the sample is fixed. In certain methods, the sample is a formalin-fixed, paraffin-embedded (FFPE) sample. In some methods, the sample is a frozen sample

In some methods, the method further comprises contacting the sample with a first secondary antibody having a first label conjugated thereto, wherein the first secondary antibody is immunoreactive with a primary antibody capable of binding phosphorylated alpha-synuclein. In some methods, the method includes contacting the sample with a set of reagents reactive with a first label of a first secondary antibody to generate a first detectable signal in the sample in proximity to the phosphorylated alpha-synuclein.

Drawings

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FIG. 1A shows exemplary staining of alpha-synuclein phosphorylated at S129 in brain tissue from subjects with PD using anti-alpha-synuclein antibodies and the indicated concentrations (MJF-R13(8-8) top panel; P-syn/81A top panel; 5H5 bottom panel).

FIG. 1B shows exemplary staining in brain tissue from subjects with PD using anti-phosphorylated S129 α -synuclein antibody and indicated concentrations (panel on 2G 11; panel in 7E 2; panel under 3G 2).

FIG. 1C shows exemplary staining in brain tissue from subjects with PD using anti-phosphorylated S129 α -synuclein antibody and indicated concentrations (11A5 upper panel; pSyn #64(WAKO) lower panel).

FIG. 2A shows that no staining was observed for 3G2 and 7E2 phosphorylated S129 α -synuclein antibody without antigen repair in brain tissue of non-PD subjects (panel in 7E2 (0.08 μ G/mL); panel under 3G2 (0.08 μ G/mL)). The upper panel shows total alpha-synuclein stained by antibody 5C12 (0.5. mu.g/ml). There was no protease or phosphatase treatment. Images were taken at 10x magnification. Antibodies were diluted in a discover coat Ig Block.

FIG. 2B shows that no staining was observed for 3G2 and 7E2 phosphorylated S129 α -synuclein antibody without antigen repair in brain tissue of non-PD subjects (panel in 7E2 (0.08 μ G/mL); panel under 3G2 (0.08 μ G/mL)). The upper panel shows total alpha-synuclein stained by antibody 5C12 (0.5. mu.g/ml). There was no protease or phosphatase treatment. Images were taken at 10x magnification. Antibodies were diluted in a discover coat Ig Block.

Figure 3A shows very weak cytoplasmic staining in non-PD brain tissue with anti-phosphorylated S129 α -synuclein 7E2 antibody after absence of antigen repair (cellular regulation) after phosphatase treatment (lower panel) (upper panel). Alkaline phosphatase treatment was 50. mu.g/mL for 2 hours.

Fig. 3B shows very weak cytoplasmic staining in non-PD brain tissue with anti-phosphorylated S129 α -synuclein 3G2 antibody (upper panel) after absence of antigen repair (cellular regulation) after phosphatase treatment (lower panel). Alkaline phosphatase treatment was 50. mu.g/mL for 2 hours.

FIG. 4A shows that the staining of lewy bodies by MJF-R13(8-8) antibody in cortical brain tissue from subjects with PD was severely reduced by alkaline phosphatase treatment (lower panel; 50. mu.g/mL, 2 hours). Images were taken at 10x magnification. MJF-R13(8-8) antibody (0.4. mu.g/mL) was diluted in DISCOVERY coat Ig Block.

FIG. 4B shows that staining of Lewy bodies by 81A antibody in cortical brain tissue from subjects with PD was severely reduced by alkaline phosphatase treatment (lower panel; 50 μ g/mL, 2 hours). Images were taken at 10x magnification. The 81A antibody (2.0. mu.g/mL) was diluted in DISCOVERY coat Ig Block.

FIG. 4C shows a moderate reduction in the staining of lewy bodies by the 11A5 antibody in cortical brain tissue from subjects with PD by alkaline phosphatase treatment (lower panel; 50. mu.g/mL, 2 hours). Images were taken at 10x magnification. The 11A5 antibody (0.4. mu.g/mL) was diluted in DISCOVERY coat Ig Block.

FIG. 4D shows a moderate reduction in Lewis body staining by 7E2 antibody in cortical brain tissue from subjects with PD by alkaline phosphatase treatment (lower panel; 50. mu.g/mL, 2 hours). Images were taken at 10x magnification. 7E2 antibody (0.08. mu.g/mL) was diluted in DISCOVERY coat Ig Block.

FIG. 4E shows a moderate reduction in the staining of lewy bodies by 3G2 antibody in cortical brain tissue from subjects with PD by alkaline phosphatase treatment (lower panel; 50. mu.g/mL, 2 hours). Images were taken at 10x magnification. The 3G2 antibody (0.08. mu.g/mL) was diluted in DISCOVERY coat Ig Block.

FIG. 5A shows protease treatment ((II))Protease 1 at 36 ℃ for 4 minutes) reduced non-lewy body staining by the 11a5 antibody in cortical brain tissue from subjects with PD without reducing lewy body staining. Images were taken at 10x magnification. The 11A5 antibody (0.4. mu.g/mL) was diluted in DISCOVERY coat Ig Block.

FIG. 5B shows protease treatment: (Protease 1 at 36 ℃ for 4 minutes) reduced non-lewy body staining by the 7E2 antibody in cortical brain tissue from subjects with PD without reducing lewy body staining. Images were taken at 10x magnification. 7E2 antibody (0.08. mu.g/mL) was diluted in DISCOVERY coat Ig Block.

FIG. 5C shows protease treatment: (Protease 1 at 36 ℃ for 4 minutes) reduced non-lewy body staining by the 3G2 antibody in cortical brain tissue from subjects with PD without reducing lewy body staining. Images were taken at 10x magnification. The 3G2 antibody (0.08. mu.g/mL) was diluted in DISCOVERY coat Ig Block.

Fig. 6 shows that, in the absence of protease treatment, phosphorylation-specific alpha-synuclein antibodies 7E2 (middle panel) and 3G2 (lower panel) produce the same staining pattern in brain tissue from subjects with PD as total alpha-synuclein antibody 5C12 (upper panel) after protease treatment. Images were taken at 10x magnification. All antibodies were diluted in DISCOVERY coat Ig Block (0.5. mu.g/mL for 5C12 antibody; 0.08. mu.g/mL for 7E2 antibody and 0.08. mu.g/mL for 3G2 antibody).

FIG. 7A shows that anti-phosphorylated S129 α -synuclein 7E2 antibody (lower panel) detects aggregated α -synuclein in skin sections from subjects with PD in the absence of protease-based antigen repair, similar to using anti-total α -synuclein 5C12 antibody (upper panel) withProtease 1 treatment (4 min at 36 ℃) coupled with the results detected.

FIG. 7B shows that anti-phosphorylated S129 a-synuclein 11A5 antibody (top panel) and 3G2 antibody detect aggregated a-synuclein in skin sections from subjects with PD in the absence of protease-based antigen repair, similar to using anti-total a-synuclein 5C12 antibody (top panel in FIG. 7A) andprotease 1 treatment (4 min at 36 ℃) coupled with the results detected.

FIG. 7C shows that anti-phosphorylated S129 a-synuclein MJF-R13 antibody (top panel) and P-Syn/81A antibody detect aggregated a-synuclein in skin sections from subjects with PD in the absence of protease-based antigen repair, similar to the use of anti-total a-synuclein 5C12 antibody (top panel in FIG. 7A) andprotease 1 treatment (4 min at 36 ℃) coupled with the results detected.

FIG. 8A shows that the DISCOVERY coat Ig Block produced minimal background while retaining the staining intensity of the anti-phosphorylated S129 α -synuclein 7E2 antibody (1.0 μ g/mL) in PD samples. The images were taken at 10x magnification in the upper image and at 40x magnification in the lower image. The images in the upper and lower panels are from different fields of view of the same tissue section.

Figure 8B shows that the 90103 diluent produced minimal background while retaining the staining intensity of the anti-phosphorylated S129 α -synuclein 7E2 antibody (1.0 μ g/mL) in the PD sample. The images were taken at 10x magnification in the upper image and at 40x magnification in the lower image. The images in the upper and lower panels are from different fields of view of the same tissue section.

Figure 9 shows the results of testing anti-phosphorylated S129 α -synuclein 7E2 antibody above and below the indicated concentration of 1.0 μ g/mL (guard band experiment) where quantitative measurements were performed as a percentage of the neurological features stained per slide. For this experiment, tissue sections were not treated with protease or phosphatase prior to antibody application and chromophore detection. Staining of skin tissue from subjects with PD with anti-phosphorylated S129 α -synuclein 7E2 antibody diluted in DISCOVERY coat Ig Block at 0.5 μ g/mL, 0.75 μ g/mL, 1.0 μ g/mL, 1.25 μ g/mL and 1.5 μ g/mL indicates that the staining was robust with antibody concentrations varying +/-50% around the 1.0 μ g/mL marker.

Figure 10A shows the nerve signature in skin samples from normal subjects (non-PD subjects) stained with anti-PGP 9.5 antibody EPR4118(0.2 μ g/mL).

Figure 10B shows neurological features in skin samples from normal subjects (non-PD subjects) stained with anti-PGP 9.5 antibody 13C4/I3C4(0.2 μ g/mL).

Fig. 10C shows polyclonal anti-PGP 9.5 antibody by Cell Marque (1.0 μ g/mL;catalog number 760-4434) staining neurological features in skin samples from normal subjects (non-PD subjects).

Figure 11A shows intense background staining (about 24-48 hours) of collagen fibers in freshly cut tissue slides from non-PD subjects. Staining was performed using 0.1 μ g/mL anti-phosphorylated S129 α -synuclein 7E2 antibody and the VENTANA OptiView DAB IHC detection kit. Tissue sections were not treated for CC1 antigen retrieval. Similar results were obtained when the anti-phosphorylated S129 α -synuclein 7E2 antibody and OptiView HQ linker were not applied (data not shown), demonstrating that collagen background staining was produced by OptiView multimers.

Figure 11B shows minimal background staining of collagen fibers in tissue slides from non-PD subjects stained about 5 months after cutting the slides.

Fig. 12A shows that background staining of collagen fibers in tissue slides by antigen repair can be exacerbated with proteases in tissue slides from non-PD subjects. Tissues were tested with anti-phosphorylated S129 α -synuclein 7E2 antibody (0.1 μ g/mL) diluted in DISCOVERY coat Ig Block.

Fig. 12B shows that background staining of collagen fibers in tissue slides can be reduced by CC1 antigen repair in tissue slides from non-PD subjects. Tissues were incubated with anti-phosphorylated S129 α -synuclein 7E2 antibody (1.0 μ g/mL) diluted in DISCOVERY coat Ig Block on a VENTANA BenchMark ULTRA autostainer without prior antigen repair with CC1 (top panel) or after 48 minutes of antigen repair with CC1 at 100 ℃ (bottom panel).

Figure 13 shows that the ultraView Universal DAB detection kit (right panel) results in lower staining intensity of PGP9.5 compared to the OptiView DAB IHC detection kit (left panel) in PD skin (top panel) and control skin (bottom panel) samples. And (4) carrying out OptiView detection: HQ linker + HRP multimer. And (3) ultraView detection: anti Rb/Ms HRP multimers. Tissue samples were pretreated with CC1 for 32 min at 100 ℃ and PGP9.5 EPR4118 antibody (0.5. mu.g/mL) was diluted in DISCOVERY coat Ig Block. Images were taken at 40x magnification.

Figure 14 shows that the amplification process with ultraView Universal DAB detection kit results in an increase in non-specific background staining in PD skin (upper panel) and control skin (lower panel) samples. Amplification: ms anti-Rb followed by Rb anti-Ms IgG. And (3) ultraView detection: anti Rb/Ms HRP multimers. Anti-phosphorylated S129 α -synuclein 7E2 antibody (1.0 μ g/mL) was diluted in DISCOVERY coat Ig Block. No pretreatment with CC1 or protease was performed. Images were taken at 40x magnification.

Figure 15 shows that both the ultraView SISH DNP detection kit (top panel) and the discover pure kit (middle and bottom panel) can detect aggregated anti-phosphorylated S129 α -synuclein in skin samples from subjects with PD with little background when used with goat anti-rabbit HRP conjugate staining deposits. Phosphorylated S129 α -synuclein is stained black (silver stain) or purple (TAMRA). Anti-phosphorylated S129 α -synuclein 7E2 antibody was present at 1.0 μ g/mL in a DISCOVERY coat Ig Block. And (3) detection: goat anti-rabbit HRP with silver (top panel) or TAMRA (middle panel), or OptiView with TAMRA (HQ-conjugated goat anti-rabbit antibody/anti HQ-HRP, bottom panel). PGP9.5 antibody EPR4118 at 0.5. mu.g/mL in a DISCOVERY coat Ig Block stained yellow. And (3) detection: goat anti-rabbit-AP with Dabsyl (yellow). No pretreatment with CC1 or protease was performed.

FIG. 16 shows that black silver staining deposited in cutaneous nerve features by anti-phosphorylated α -synuclein 7E2 antibody and ultraView SISH DNP detection kit provides for the use of EPR4118 antibody and theVisualization of high contrast of aggregated phosphorylated S129 α -synuclein on a yellow background with PGP9.5 of DISCOVERY UltraMap anti-Rb alkaline phosphatase and QM-DABSYL. The tissue sample is a skin sample from a subject with PD (upper panel), and the skin sample is from a normal subject (non-PD) (lower panel).

Fig. 17A shows immunohistochemical detection of phosphorylated S129 α -synuclein (black/silver staining) and PGP9.5 (yellow) in the neurological features of large blood vessels in skin samples from subjects with PD.

Fig. 17B shows immunohistochemical detection of phosphorylated S129 α -synuclein (black/silver staining) and PGP9.5 (yellow) in nerve features of small nerve bundles in skin samples from subjects with PD.

Fig. 17C shows immunohistochemical detection of phosphorylated S129 α -synuclein (black/silver staining) and PGP9.5 (yellow) in neurological features of eccrine glands in skin samples from subjects with PD.

Fig. 17D shows immunohistochemical detection of phosphorylated S129 α -synuclein (black/silver staining) and PGP9.5 (yellow) in neurological features of large nerve bundles in skin samples from subjects with PD.

Fig. 17E shows immunohistochemical detection of phosphorylated S129 α -synuclein (black/silver staining) and PGP9.5 (yellow) in the neural features of the pilus muscle in skin samples from subjects with PD.

Figure 18 shows an example of diffuse granular and discrete types of silver-phosphorylated S129 α -synuclein staining that can be observed in cutaneous nerve tracts from non-PD control subjects (top panel) and PD subjects (bottom panel). Sections of scalp biopsies were stained using the phosphorylated S129 α -synuclein and PGP9.5 silver/yellow dual IHC assay without protease or phosphatase treatment (scheme 1). The yellow-stained structure is a nerve expressing PGP 9.5.

Figure 19 shows a similar pattern of peripheral neuronal staining between granular phosphorylated S129 α -synuclein (upper panel) and myelin basic protein (lower panel), indicating the potential localization of diffuse pS129 α -synuclein in Schwann cells.

Fig. 20A shows staining of phosphorylated S129 α -synuclein by 11a5 antibody (0.4 μ g/mL) without CC1 treatment (upper panel), decreasing after 32 minutes of treatment with ULTRA CC1 at 100 ℃ (lower panel).

Fig. 20B shows staining of phosphorylated S129 α -synuclein by 7E2 antibody (0.08 μ g/mL) without CC1 treatment (upper panel), decreasing after 32 minutes of treatment with ULTRA CC1 at 100 ℃ (lower panel).

Fig. 20C shows staining of phosphorylated S129 α -synuclein by 3G2 antibody (0.08 μ G/mL) without CC1 treatment (upper panel), decreasing after 32 min treatment with ULTRA CC1 at 100 ℃ (lower panel).

FIG. 20D shows staining of phosphorylated S129 α -synuclein by MJF-R13 antibody (0.4 μ g/mL) without CC1 treatment (top panel), decreased after 32 min treatment with ULTRA CC1 at 100 ℃ (bottom panel).

FIG. 20E shows staining of phosphorylated S129 α -synuclein by P-Syn/81A antibody (2.0 μ g/mL) in the absence of CC1 treatment (top panel), decreasing after 32 min treatment with ULTRA CC1 at 100 ℃ (bottom panel).

FIG. 21A shows aProtease 2 treatment for 4 minutes (right panel) resulted in a moderate increase in staining of discrete phosphorylated S129 α -synuclein (detected with the 7E2 antibody clone) in skin sections from subjects with PD (compared to untreated; left panel). By usingProtease 2 treatment for 4 minutes also significantly reduced granular phosphorylated S129 α -synuclein staining and PGP9.5 staining in skin sections from subjects with PD.

FIG. 21B shows aProtease 2 treatment for 4 min (right panel) significantly reduced (compared to untreated; left panel) staining of phosphorylated S129 α -synuclein (detected with the 7E2 antibody clone) in skin sections from non-PD subjects that might be mistaken for a discrete type.

Figure 22 shows the effect of protease 3 treatment for 4 minutes (middle panel) or 12 minutes (lower panel) on staining with phosphorylated S129 α -synuclein antibody in skin from subjects with PD compared to no protease treatment (upper panel). Phosphorylated S129 α -synuclein stained black. The 7E2 antibody clone was cloned at 1.0. mu.g/mL in a DISCOVERY coat Ig Block (detection: Goat anti-rabbit HRP with silver). PGP9.5 antibody EPR4118 was stained yellow at 0.5. mu.g/mL in DISCOVERY coat Ig Block (detection: Goat anti-rabbit AP with Dabsyl). No pretreatment with CC1 or protease was performed.

Figure 23A shows that alkaline phosphatase (purified calf intestinal alkaline phosphatase from NEB) pretreatment for 8 minutes at pH9 (lower panel) removed diffuse granular phosphorylated alpha-synuclein staining without affecting the dense discrete staining typically observed in tissues not treated with alkaline phosphatase (upper panel).

Figure 23B shows that alkaline phosphatase (purified calf intestinal alkaline phosphatase from NEB) pretreatment at pH9 for 24 minutes (upper panel) or 40 minutes (lower panel) removed diffuse granular phosphorylated alpha-synuclein staining without affecting the dense discrete staining typically observed in tissues not treated with alkaline phosphatase.

FIG. 24 shows that alkaline phosphatase (recombinant alkaline phosphatase from Roche; 30. mu.g/mL) treatment at pH8 for 8 min resulted in a reduction in staining of discretely phosphorylated S129. alpha. -synuclein (lower panel) compared to no AP treatment (upper panel) or AP treatment at pH10 (middle panel).

Figure 25 shows that pretreatment with alkaline phosphatase followed by protease 3 abolished diffuse granular phosphorylated alpha-synuclein staining without significant loss of dense discrete staining.

Figure 26 shows that PGP9.5 detection using EPR4118 antibody (1.0 μ g/mL) is enhanced with increasing antigen repair time using the ULTRA CC1 bulk solution (0 min-top panel; 16 min-middle panel; or 64 min-bottom panel).

Figure 27 shows that antigen repair with ULTRA CC2 for 16 minutes at 100 ℃ produced an enhancement in PGP9.5 staining using EPR4118 antibody.

Figure 28 shows an overview of two protocols for the simultaneous detection of alpha-synuclein (aSyn) and PGP9.5 for parkinson's disease characterization in skin samples.

Figure 29 shows individual value plots showing the percentage of neurological features containing phosphorylated S129 α -synuclein staining in 4 μm sections from scalp biopsies of subjects with PD and normal non-PD subjects. 15 PD and 9 non-PD control scalp skin samples were stained for the same cohort with two different phosphorylated S129 α -synuclein and PGP9.5 silver/yellow dual IHC assay protocols: no re (scheme 1) and (scheme 2) protease/phosphatase.

Figure 30A shows individual value plots showing the percentage of neurological features with discretely phosphorylated S129 α -synuclein staining in 4 μm sections from scalp biopsies of subjects with PD and normal, non-PD subjects. 15 PD and 7 non-PD control scalp samples were stained contemporaneously with the phosphorylated S129 α -synuclein and PGP9.5 silver/yellow dual IHC assay protocol (without protease/phosphatase (protocol 1) and with protease/phosphatase (protocol 2)) and also with the protease resistant α -synuclein DAB IHC assay developed by pRED/Prothena.

Figure 30B shows individual value plots showing the percentage of neurological features with diffuse granular phosphorylated S129 α -synuclein staining in 4 μm sections from scalp biopsies of subjects with PD and normal non-PD subjects. 15 PD and 7 non-PD control scalp samples were stained contemporaneously with the phosphorylated S129 α -synuclein and PGP9.5 silver/yellow dual IHC assay protocol (without protease/phosphatase (protocol 1) and with protease/phosphatase (protocol 2)) and also with the protease resistant α -synuclein DAB IHC assay developed by pRED/Prothena.

Figure 31 shows individual value plots showing the percentage of neurological features containing phosphorylated S129 α -synuclein staining in 4 μm sections from abdominal skin biopsies of subjects with PD and normal non-PD subjects. Staining of 20 PD and 20 non-PD control abdominal skin biopsy samples cohort with two different phosphorylated S129 α -synuclein and PGP9.5 silver/yellow dual IHC assay protocols: without (scheme 1) and with (scheme 2) protease/phosphatase.

Fig. 32A shows the difference in staining of normal colon tissue by protocol 1 (top panel) compared to protocol 2 (bottom panel). The scale bar is 50 μm.

Fig. 32B shows the difference in staining of normal colon tissue by protocol 1 (top panel) compared to protocol 2 (bottom panel). The scale bar is 50 μm.

Fig. 33A shows an exemplary staining of submandibular gland tissue from a subject with PD using regimen 2. Images were taken at 10x magnification.

Fig. 33B shows an exemplary staining of sigmoid colon tissue from a subject with PD using regimen 2. Images were taken at 20x magnification.

Fig. 33C shows an exemplary staining of scalp tissue from a subject with PD using regimen 2. Images were taken at 20x magnification.

FIG. 34 shows that in pediatric skin tissue, protocol 1 (P1; top panel) generated a re-phosphorylated α -synuclein silver stain with a discrete morphology that was not present in the case of protocol 2 (P2; bottom panel).

Detailed Description

The present disclosure relates to methods and compositions for accurately and unambiguously identifying the presence of Parkinson's Disease (PD) in subjects who would otherwise be undiagnosed to have PD, as well as subjects at risk of or suspected of having parkinson's disease. The methods and compositions may also be used to exclude parkinson's disease in patients with dyskinesias (sometimes associated with parkinson's-like symptoms) including, but not limited to: progressive supranuclear palsy, multiple system atrophy, viral parkinson's disease, essential tremor, drug or toxin induced parkinson's disease, post-traumatic parkinson's disease, arteriosclerotic parkinson's disease, parkinson-dementia syndrome of guam, corticobasal ganglionic degeneration or normal pressure hydrocephalus. The disclosed methods comprise contacting a biological sample comprising at least one neurological characteristic from a subject with a primary antibody capable of binding phosphorylated alpha-synuclein. By detecting whether a primary antibody capable of binding phosphorylated alpha-synuclein is located within the neural signature of the sample, it can be determined whether the living subject has PD. Subjects may include patients before onset of symptoms of PD and patients in early stages of the disease who are currently unable to routinely diagnose PD accurately. In addition, post-mortem analysis can be performed with biological samples collected post mortem.

As used herein, the term "biological sample" includes samples that can be tested by the methods and kits of the present disclosure, and includes human and animal bodies (fixed or frozen), tissue specimens, and fixed cell specimens. The term "tissue" refers to a collection of interconnected cells that perform a similar function within an organism. The biological sample may comprise a sample from a healthy or apparently healthy human subject, or a sample from a human subject affected or suspected to be affected by a disorder or disease to be diagnosed or treated, such as parkinson's disease. The biological sample may be a sample obtained from any organ or tissue, including biopsies, such as tumor biopsies. Also, the sample may be from a deceased patient, such as a sample taken during an autopsy. The biological sample may also include a cytological sample (in some examples, the cytological sample may be derived from a tissue, such as a tissue section from skin tissue, colon tissue, submandibular gland tissue, or brain). In other examples, the sample may be a cell or cell pellet prepared from a biological sample obtained from a subject.

In some examples, the biological sample can include normal or cancerous tissue, e.g., skin tissue (from, e.g., the scalp, abdomen, or torso of a subject), colon tissue, submandibular gland tissue, brain tissue, olfactory bulb, lung tissue, ovarian tissue, pancreatic tissue, mesothelial tissue, gastrointestinal tissue, head and neck tissue, breast tissue, liver tissue, kidney tissue, prostate tissue, uterine tissue, cerebrospinal fluid (or cells and/or neural features isolated from cerebrospinal fluid), bone, lung cells, ovarian cells, pancreatic or mesothelial cells, colon cells, head and neck cells, breast cells, liver cells, kidney cells, skin cells, prostate cells, uterine cells, bone cells, brain cells, and lung cells. For example, samples from skin, colon, submandibular gland, olfactory bulb, lung, ovary, liver, or pancreas or other tumors containing cellular material can be obtained by surgical resection of all or part of the tumor, by collection of a fine needle brain biopsy, a fine needle aspirate or needle biopsy from the tumor, and other methods known in the art.

As used herein, the term "neural feature" refers to neural cells and tissue structures known to be innervated. For example, the neural characteristic may be a portion of a neural cell, a portion of a precursor neural cell, or both a neural cell and a precursor neural cell and/or portions thereof. In some of the methods, the neural feature is adjacent to a neural cell, e.g., the precursor neural cell is adjacent to a healthy neural cell. Other types of neural features include tissue structures known to be innervated, including but not limited to: non-medullary or thin intramedullary intra-epidermal nerve fibers widely distributed in the dermis; adrenergic, noradrenergic, cholinergic sympathetic fibers or vasodilatory peptidergic fibers that innervate autonomic structures, including but not limited to sweat glands, hair follicles, pili muscles, and blood vessels. In some methods, the neural feature may comprise a component of a precursor nerve cell or a necrotic or apoptotic nerve cell.

In the context of the present disclosure, the term "primary antibody" refers to an antibody binding agent, e.g., an intact antibody molecule, a fragment or derivative of said molecule (e.g., a conjugate comprising an antibody or a polymeric antibody), which specifically binds to a "target" (e.g., a phosphorylated a-synuclein or a component of a neurological signature), more specifically to a single unit of the target of a sample (e.g., an epitope of the target molecule). In some of the methods disclosed herein, the primary antibody may be an antibody capable of binding to alpha-synuclein (pS129 aSyn) phosphorylated at residue S129. In some of the methods disclosed herein, the primary antibody can be an antibody capable of binding a neural characteristic component. In the following disclosure, primary antibodies are described in more detail under the heading "antibodies

To detect whether a primary antibody is located within a neural feature, the sample is contacted with an antibody and the sample is examined for the presence of the antibody. For example, when a primary antibody is labeled with a detectable label, determination of the location of the primary antibody can be accomplished. Detection of the marker in the sample corresponds to localization of the antibody within the neural feature.

Some of the methods disclosed herein further include contacting the biological sample from the subject with a second antibody capable of binding to a protein other than dephosphorylated a-synuclein within the neurological signature. The use of the second primary antibody allows the location of the first primary antibody and the second primary antibody within the neural feature to be determined. The determination of PD can be accurately and unambiguously determined from the co-localization of the two antibodies within the detected neural characteristics.

The second antibody may, for example, be selected from antibodies capable of binding to a protein that is a constituent of a neurological trait, and may, for example, be selected from one of: ubiquitin C-terminal hydrolase L1(PGP9.5, UCHL1, NDGOA; PARK 5; PGP 95; SPG 79; Uch-L1; HEL-117; PGP 9.5; HEL-S-53), RNA-bound FOX-1 homolog 3(RBFOX3, FOX3, NEUN, FOX-3, HRNBP3), microtubule-associated protein 2(MAP2, MAP2A, MAP2B, MAP2C), 160kDa neurofilament medium chain (NEFM, NFM, NEF3, NF-M), 200kDa neurofilament heavy chain (NEFH, NFH, CMT2CC), synapsin (SYP, MRX96, MRYP) and discos large MAGGUK scaffold protein 4(DLG 29, DLGH4, PSD 3995, PSD 4642, SAP-90, SAP A).

In some of the methods disclosed herein, the primary antibody capable of binding phosphorylated alpha-synuclein and the primary antibody capable of binding a neurological feature are from the same host species, and the host species may be a mouse or a rabbit. In some methods, the primary antibody capable of binding phosphorylated alpha-synuclein and the primary antibody capable of binding a neural characteristic are from different host species, such as mice or rabbits.

The terms "localization" and "co-localization" refer to the location of one or more target proteins within the same neural feature. In some methods, when a signal independently indicative of a protein is identified in a neural feature, the co-localization of the protein within the neural feature is determined when compared to the respective background of the signal. Signals indicative of peptides located outside of a neural feature are not indicative of proteins located within the neural feature. As an example, the signal indicative of phosphorylated a-synuclein localized in a neurological feature is one, two, three, or four standard deviations higher than background signal, or a signal at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold higher than background signal.

Methods for determining localization and co-localization of antibodies include, for example, histochemical analysis. As used herein, the term "immunohistochemistry" or "IHC" refers to a method of determining the presence or distribution of an antigen in a fixed or frozen sample by detecting the interaction of the antigen with a specific binding agent, such as an antibody, within the sample. Accordingly, the present disclosure provides methods for determining whether a subject has Parkinson's Disease (PD) by using fixed or frozen sections of a biological sample from a subject suspected of having PD. When phosphorylated a-synuclein localizes within a neural feature, phosphorylated a-synuclein localized within a neural feature in the slice can be detected, and the subject can be diagnosed with PD. The methods of the present disclosure include contacting the slice with a first primary antibody capable of binding phosphorylated alpha-synuclein. The method may further include contacting the slice with a second primary antibody capable of binding a neural feature.

Some of the methods disclosed herein may also include an automated histochemical staining platform, such as an automated IHC/ISH slide stainer. Automated IHC/ISH slide stainers typically include at least: the system includes a reservoir of various reagents used in the staining protocol, a reagent dispensing unit in fluid communication with the reservoir for dispensing the reagents onto the slides, a waste disposal system for removing used reagents or other waste from the slides, and a control system for coordinating the actions of the reagent dispensing unit and the waste disposal system. In addition to performing staining steps, many automated slide stainers can also perform staining assist steps (or be compatible with a separate system that performs such assist steps), including: slide baking (to adhere the sample to the slide), dewaxing (also known as deparaffinization), antigen retrieval, counterstaining, dehydration and removal, and coverslipping. Prichard, Overview of Automated immunology chemistry, Arch Pathol Lab Med., Vol.138, pp.1582 1578 (2014), the entire contents of which are incorporated herein by reference, describe several specific examples of Automated IHC/ISH slide stainers and various features thereof, including the intelliPATH(Biocare Medical)、(Celerus Diagnostics)、DAKOAnd DAKO AUTOSTATAINER48(Agilent Technologies)、(Ventana Medical Systems,Inc.)、LeicaAnd Lab Vision^TMAutostainer (thermo scientific) automatic slide stainer. Additionally, Ventana Medical Systems, inc. is the assignee of a number of U.S. patents disclosing Systems and methods for performing automated analysis, including U.S. patent nos.: 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901 and 6,943,029, and U.S. published patent application nos.: 20030211630 and 20040052685, each of which is incorporated by reference herein in its entirety. Commercially available dyeing apparatuses generally operate according to one of the following principles: (1) open single slide staining with slides placed horizontally and reagent dispensed as a puddle on the slide surface containing the tissue sample (such as on a DAKO AUTOSTAINER)48(Agilent Technologies) and(Biocare Medical) stainers); (2) liquid coating techniques, in which reagents are coated or distributively deposited on a sample by a layer of inert fluid (such as, for example, inBenchMark andperformed on a stainer); (3) capillary gap staining, in which a slide surface is placed in proximity to another surface (possibly another slide or coverslip) to create a narrow gap through which capillary forces draw and hold liquid reagents in contact with the sample (such as by DAKO)LeicaAnd DAKODyeing principle used by stainers). Some iterations of capillary gap staining will not mix the fluid in the gap (such as in DAKO)And LeicaAbove). In a variation of capillary gap staining, known as dynamic gap staining, a sample is applied to a slide using capillary force, and then parallel surfaces are translated relative to each other to agitate the reagents during incubation to achieve reagent mixing (such as in DAKO)Staining principle performed on a slide stainer). In translational gap staining, a translatable head is positioned on a slide. The lower surface of the head is spaced from the slide by a first gap that is small enough to allow a meniscus of liquid to be formed by the liquid on the slide during translation of the slide. A mixing extension having a lateral dimension less than a width of the slide extends from a lower surface of the translatable head to define a second gap less than the first gap between the mixing extension and the slide. During head translation, the lateral dimension of the mixing extension is sufficient to generate lateral motion in the liquid on the slide in a direction generally extending from the second gap to the first gap. See WO 2011-. Recently it has been suggested to use ink jet technology to deposit reagents on glass slides. See US 2016-. The staining technique list is not intended to be comprehensive and any fully or semi-automated system for performing biomarker staining may be integrated into the histochemical staining platform.

Detection of antibody binding

In some of the methods disclosed herein, step (a) further comprises contacting the sample with a first secondary antibody having a first label conjugated thereto, wherein the first secondary antibody is immunoreactive with a primary antibody capable of binding phosphorylated alpha-synuclein. In some of the methods disclosed herein, the method comprises contacting the sample with a set of reagents reactive with a first label of a first secondary antibody to generate a first detectable signal in the sample proximal to phosphorylated alpha-synuclein.

In some of the methods disclosed herein, the method further comprises contacting the sample with a second secondary antibody having a detectable second label conjugated thereto, wherein the second secondary antibody is immunoreactive with a primary antibody capable of binding the neurological feature. In some of the methods disclosed herein, the method comprises contacting the sample with a set of reagents reactive with a second label of a second secondary antibody to generate a second detectable signal in the sample proximal to the neural feature. In some of the methods disclosed herein, the first detectable signal and the second detectable signal are different. In certain methods disclosed herein, the first detectable signal is silver staining and the second detectable signal is QM-Dabsyl or fast red.

In the context of the present disclosure, the term "secondary antibody" refers to an antibody binding agent, e.g., an intact antibody molecule, a fragment or derivative of such a molecule (e.g., a conjugate comprising an antibody or polymeric antibody) that has an antigen binding domain that specifically binds a primary antibody that binds to a target antigen. Since multiple secondary antibodies bind to the primary antibody, the secondary antibodies can help to improve sensitivity and signal amplification. In some embodiments, the secondary antibody may be conjugated to an enzyme, such as horseradish peroxidase (HRP) or Alkaline Phosphatase (AP); or fluorescent dyes such as Fluorescein Isothiocyanate (FITC), rhodamine derivatives, Alexa Fluor dyes; or other molecular conjugation for various applications.

In some of the methods disclosed herein, the sample is contacted with at least one protease prior to contacting with the primary antibody capable of binding phosphorylated alpha-synuclein. In certain methods disclosed herein, the method further comprises contacting the sample with at least one phosphatase. In some of the methods, the sample is contacted with a protease and a phosphatase. The use of a protease or a protease and a phosphatase may be according to the method wherein the determination of PD is done independently using the first primary antibody, or wherein the determination is done by using the first primary antibody and the second primary antibody. In some of the methods disclosed herein, the use of a phosphatase enhances the detection of phosphorylated alpha-synuclein that is unique to a subject with parkinson's disease by removing phosphorylated alpha-synuclein that is common in subjects without parkinson's disease. The use of proteases enhances the detection of phosphorylated alpha-synuclein by improving the accessibility of phosphorylated alpha-synuclein to primary antibodies.

In some of the methods disclosed herein, prior to contacting the sample with a primary antibody capable of binding a neural characteristic, the method comprises denaturing the immune complexes in the sample, for example, by incubating the sample at 100 ℃ for at least 15 minutes. In certain methods, denaturation can be carried out at about 80 ℃ to at least about 110 ℃ for about 5 minutes to at least about 60 minutes. For example, denaturation can be performed at about 80 ℃, about 85 ℃, about 90 ℃, about 95 ℃, about 100 ℃, about 105 ℃, or about 110 ℃ for at least about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, or about 48 minutes, or about 50 minutes, or about 55 minutes, or at least about 60 minutes.

As used herein, the term "protease" or "peptidase" or "proteolytic enzyme" refers to an enzyme that undergoes proteolysis or protein catabolism by hydrolysis of peptide bonds. Proteases can be classified into seven major classes based on catalytic residues. For example, serine proteases (using serine alcohols), cysteine proteases (using cysteine thiols), threonine proteases (using threonine secondary alcohols), aspartic proteases (using aspartic acids), glutamic proteases (using glutamic acids), metallo-proteases (using metals, usually zinc) and asparagines peptide-cleaving enzymes (using asparagine to perform the elimination reaction). Examples of proteases include, but are not limited to: trypsin, chymotrypsin, enterokinase, intracellular protease GluC, proteolytic enzyme K, thrombin, factor Xa, and spinachUsnin, alkaline protease (e.g., alkaline VIII protease), papain, collagenase, dispase, pepsin, cathepsin D, and carboxypeptidase A. Examples of commercially available proteases include, but are not limited to:protease 1 (P/N760) -2018,Protease 2 (P/N760) -2019 orProtease 3 (P/N760-. The incubation time of the protease can be determined by one of skill in the art, but the incubation time can range from about 2 minutes or less to at least about 20 minutes, and the temperature can range from about 15 ℃ or less to at least about 45 ℃. For example, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, or at least about 20 minutes, at about 15 ℃, at about 20 ℃, at about 25 ℃, at about 30 ℃, at about 35 ℃, at about 36 ℃, at about 37 ℃, at about 38 ℃, at about 39 ℃, or at least at about 40 ℃. In certain examples, the incubation can be at about 36 ℃ for about 4 minutes, or at about 40 ℃ for about 4 minutes 30 seconds, or at about 40 ℃ for about 5 minutes, or at about 36 ℃ for about 12 minutes.

As used herein, the term "phosphatase" refers to an enzyme that uses water to cleave phosphate monoesters into phosphate ions and alcohol (the phosphatase removes the phosphate group from the molecule). Examples of phosphatases may include, but are not limited to: alkaline phosphatase, acid phosphatase, phosphoprotein phosphatase 1(PP1), phosphoprotein phosphatase 2A (PP2A), phosphoprotein phosphatase 2B (PP2B), phosphoprotein phosphatase 2C (PP2C), and lambda protein phosphatase. Examples of commercially available phosphatases include, but are not limited to: bovine alkaline phosphatase (Roche P/N3359123001) from Pichia pastoris (Pichia pastoris). The incubation time of the phosphatase may be determined by one skilled in the art, but the incubation time may range from about 5 minutes to at least about 12 hours, and the temperature may range from about 20 ℃ to at least about 50 ℃. For example, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or at least about 12 hours at about 20 ℃, about 21 ℃, about 22 ℃, about 23 ℃, about 24 ℃, about 25 ℃, about 30 ℃, about 36 ℃, about 37 ℃, about 38 ℃, about 39 ℃, or at least about 40 ℃. In certain examples, the incubation can be at about 36 ℃ for about 2 hours, or at about 37 ℃ for about 10 minutes, or at about 37 ℃ for about 60 minutes, or at about 37 ℃ for about 45 minutes, or at about 37 ℃ for about 60 minutes.

In some of the methods disclosed herein, the incubation time will vary and depend on the incubation temperature. Incubation periods for substantially intact antibody binding, development and other steps of the method are well known. Many of the steps, e.g., antigen exposure, antibody binding and visualization, are performed at elevated temperatures of at least about 35 ℃, preferably from about 40 ℃ to about 45 ℃ or up to 100 ℃, for a period of about 4 minutes to about 90 minutes. For example, in some methods, the primary antibodies may be incubated with the sample for 32 minutes at 36 ℃, or they may be incubated for 20 minutes at about 22 ℃. In another example, the protease may be incubated with the sample at 36 ℃ for 12 minutes, or the protease may be incubated with the sample at 36 ℃ for 4 minutes. In yet another example, the antigen exposure may be performed at least at about 100 ℃ for about 30-90 minutes. However, if the incubation time is appropriately prolonged, most steps can be performed at a temperature as low as 4 ℃ except for the steps depending on the enzyme activity (antigen exposure and color development).

Where the antibody staining reagent is specific for the antigen to be exposed, the tissue section may be treated with proteolytic enzymes or proteases prior to addition of the primary antibody. Proteolytic enzymes or proteases may be added to the evaporation inhibitor liquid covering the tissue section and sink by the evaporation inhibitor liquid into the underlying tissue section. After a sufficient incubation time for antigen exposure, e.g., about 4 minutes to about 30 minutes, the slide can be washed and the evaporation inhibitor liquid can be reapplied.

In some of the methods, a sufficient amount of protease solution (typically about 100-. Treatment with proteases may help expose the antigen to the labeling reagent, thereby obtaining more accurate results. In some of the methods, the incubation period is sufficient to disrupt the cross-linking, but not so long as to disrupt the antigen. The time period depends on temperature, enzyme concentration, tissue thickness, tissue type and length of time in formalin. The appropriate time can be readily determined by one skilled in the art. For example, it may be effective at about 36 ℃ for about 4 minutes, or at about 40 ℃ for about 4 minutes and 30 seconds, or at about 40 ℃ for about 5 minutes, or at about 36 ℃ for about 12 minutes. After incubation, the protease solution can be washed away and the next reagent used in the staining/labeling process. When these ranges are not observed, excessive and/or insufficient digestion of the tissue by proteases is likely to occur, which may result in disrupted/masked antigens, respectively. After incubation, the slides can be washed.

Antibodies

As used herein, the term "antibody" refers to a polypeptide ligand comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds an epitope of an antigen. Antibodies include at least light or heavy chain immunoglobulin variable regions or immunoglobulin-like molecules (including, by way of example and not limitation, IgA, IgD, IgE, IgG, and IgM, combinations thereof and the like produced during the immune response of any vertebrate, e.g., mammal such as human, goat, rabbit, and mouse), and non-mammalian species such as shark immunoglobulin. Antibodies include monoclonal antibodies, polyclonal antibodies or fragments of antibodies, as well as other antibodies known in the art. In some examples, the antibody is labeled with a detectable label (such as an enzyme or fluorophore). Antibodies also include antibody fragments that specifically bind to a target molecule (or a group of highly similar target molecules) thereby substantially excluding binding to other molecules.

The term "antibody" also includes antigen-binding fragments of naturally occurring antibodies or recombinant antibodies. The term antibody in vivo covers the binding fragment of non-limiting examples include Fab, (Fab')₂Fv and single chain Fv (scFv). Fab is a fragment containing a monovalent antigen-binding fragment of an antibody molecule produced by papain digestion of an intact antibody to produce an intact light chain and a portion of one heavy chain or, equivalently, by genetic engineering. Fab' is a fragment of an antibody molecule obtained by treatment of an intact antibody with pepsin followed by reduction to give an intact light chain and a partial heavy chain; two Fab' fragments were obtained per antibody molecule. (Fab')₂Is an antibody fragment obtained by treating an intact antibody with pepsin without subsequent reduction or equivalently by genetic engineering. F (Ab')₂Is a dimer of two FAb' fragments linked together by disulfide bonds. Fv is a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains. Single chain antibodies ("SCAs") are genetically engineered molecules that contain a variable region of a light chain, a variable region of a heavy chain, linked as genetically fused single chain molecules by suitable polypeptide linkers. Methods for preparing these fragments are conventional in the art.

Antibodies and antibody binding fragments have binding constants to target molecules (i.e., phosphorylated alpha-synuclein and neural characteristic endogenous proteins) that are at least 10 greater than the binding constants of other molecules in a biological sample³M^-1At least 10⁴M^-1Or at least 10⁵M^-1The binding constant of (c). In some examples, the antibody has a high binding affinity for phosphorylated alpha-synuclein or PGP9.5, such as at least about 1x10^-8M, at least about 1.5x10^-8M, at least about 2.0x10^-8M, at least about 2.5x10^-8M, at least about 3.0x10^-8M, at least about 3.5x10^-8M, at least about 4.0x10^-8M, at least about 4.5x10^-8M or at least about 5.0x10^-8Binding affinity of M. In certain embodiments, an antibody that binds phosphorylated alpha-synuclein or PGP9.5 has a dissociation constant (Kd) of ≦ 10⁴nM、≤10³nM, ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM, or ≦ 0.0001nM, or ≦ 0.00001nM (e.g., 10nM^-4M or to 10^-6M, e.g. from 10^-7M to 10^-9M, e.g. from 10^-10M to 10^-12M, or, for example, from 10^-13M to 10^-15M). In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) with the Fab form of the antibody of interest and its antigen. In another example, Kd may be measured using a label-free optical scanner for microarray detection based on polarization-modulated oblique incidence reflectance difference (OI-RD). In yet another example, a CM5 chip with immobilized antigen at 25 ℃ was used at approximately 10 Response Units (RU)Or(BIAcore, inc., Piscataway, n.j.) Kd is measured by surface plasmon resonance assay.

An antibody consists of a heavy and a light chain, each having a variable region, referred to as the variable heavy chain (V)_H) Domains and variable light chains (V)_L) And (4) a zone. V_HRegion and V_LThe regions are collectively responsible for binding to the antigen recognized by the antibody. The heavy and light chains each contain a constant region and a variable region (these regions are also referred to as "domains"). In combination, the heavy and light chain variable regions specifically bind antigen. The light and heavy chain variable regions contain "framework" regions, also referred to as "complementarity determining regions" or "CDRs," interrupted by three hypervariable regions. The framework regions and the extent of CDRs have been defined (see Kabat et al, Sequences of Proteins of Immunological Interest, U.S. department of Health and Human Services, 1991). The Kabat database can now be maintained online. The sequences of the framework regions of different light or heavy chains are within one speciesAre relatively conservative. The framework regions of an antibody, i.e., the combined framework regions that make up the light and heavy chains, are used to locate and align the CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of the antigen. The CDRs of each chain are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and are also commonly identified by the chain in which the particular CDR is located. Thus, V_HCDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, and V_LCDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Antibodies that bind phosphorylated alpha-synuclein will have a specific V_HRegion and V_LRegion sequences, and thus specific CDR sequences. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although CDRs vary from antibody to antibody, only a limited number of amino acid positions in a CDR are directly involved in antigen binding. These positions in the CDRs are called Specificity Determining Residues (SDRs).

Several antibodies against alpha-synuclein (also known as SNCA, PD1, NACP, PARK1, PARK4, aSyn) and phosphorylated alpha-synuclein are known. Alpha 0-synuclein is a member of the synuclein family, which also includes beta-and gamma-synuclein. Synuclein is expressed in the brain, and alpha-and beta-synuclein selectively inhibit phospholipase D2. Alpha-synuclein may be used to integrate presynaptic signaling and membrane trafficking. Mutations in alpha-synuclein have been implicated in the pathogenesis of parkinson's disease. Alpha-synuclein peptide is a major component of amyloid plaques in the brain of patients with Alzheimer's disease. In certain methods, phosphorylated alpha-synuclein can be detected. The alpha-synuclein sequence is publicly available, e.g., from GenBank^TMObtained in sequence databases (e.g., amino acid accession number NP _000336.1 encoded by accession number NM _ 000345.3). One of ordinary skill in the art can identify additional alpha-synuclein nucleic acid and protein sequences, including alpha-synuclein variants and isoforms. Antibodies for detecting alpha-synuclein are known in the art and may include, but are not limited to: rabbit monoclonal antibody clone 7E2 directed to phospho-S129 alpha-synucleinAnd 3G2, which is CPS R by Roche Diagnostics GmbH&D Early Development&Reagent Design (DXREAA) was generated with phosphopeptides corresponding to human alpha-synuclein residues 122-135 as immunogens. Additional monoclonal antibodies directed against phosphorylated S129 α -synuclein (aSyn) may be purchased, for example, from Abcam (clone MJF-R13(8-8), P/N ab168381 and P-syn/81A, P/N ab184674) or WAKO (clone pSyn #64, P/N015-25191). The mouse monoclonal antibody clone LB509 to alpha-synuclein was purchased from Abcam (P/N ab27766) regardless of the phosphorylation state of S129. An anti- α -synuclein mouse monoclonal antibody from Prothena Corporation, clone 5C12 (independent S129 phosphorylation;no. PTA-9197) and anti-phosphorylated S129-alpha-synuclein mouse monoclonal antibody clone 11A5(No. PTA-8222). See also table 10 below.

An example of an anti-phosphorylated S129 α -synuclein antibody is the rabbit monoclonal antibody clone 7E2, which was generated by Roche Diagnostics GmbH CPS R & D Early Development & Reagent Design (DXREAA) using the phosphopeptide corresponding to human α -synuclein residue 122-135 as an immunogen. The 7E2 antibody or antigen-binding fragment thereof comprises a Light Chain (LC) variable region comprising the amino acid sequence of SEQ ID NO: 01. The 7E2 antibody or antigen-binding fragment thereof comprises a Heavy Chain (HC) variable region comprising the amino acid sequence of SEQ ID NO: 05.

01(7E2 light chain):

AQVLTQTPSPVSAAVGGTVTISCQSSQSVYNNNNLVWYQQKPGQPPKQVIYKASKVASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCLGGYSGDIYTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

SEQ ID NO:02 7E2 LC CDR1:QSVYNNNN

SEQ ID NO:03 7E2 LC CDR2:KASKVAS

SEQ ID NO:04 7E2 LC CDR3:LGGYSGDIYT

05(7E2 heavy chain):

CQSVEESGGRLVTPGTPLTLTCTASGFTISSYHMSWVRQAPGKGLEWIGYISTSGNIYYASWAKGRFTISKTSSTTVDLRMTSLTTEDTATYFCARLGIATGYSFWGHGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

SEQ ID NO:06 7E2 HC CDR1:GFTISSYHMS

SEQ ID NO:07 7E2 HC CDR2:ISTSGNI

SEQ ID NO:08 7E2 HC CDR3:ARLGIATGYSF

in some embodiments, antibodies are provided having a light chain variable region with an amino acid sequence having at least 90% sequence identity to the amino acid sequence represented by SEQ ID NO: 01. In a further embodiment, an antibody having a heavy chain variable region with an amino acid sequence having at least 90% sequence identity to the amino acid sequence represented by SEQ ID No. 05 is provided.

Another example of an anti-phosphorylated S129 α -synuclein antibody is the rabbit monoclonal antibody clone 3G2, which was generated by Roche Diagnostics GmbH CPS R & D Early Development & Reagent Design (DXREAA) using the phosphopeptide corresponding to human α -synuclein residue 122-135 as an immunogen. The 3G2 antibody or antigen-binding fragment thereof comprises a Light Chain (LC) variable region comprising the amino acid sequence of SEQ ID NO: 09. The 3G2 antibody or antigen-binding fragment thereof comprises a Heavy Chain (HC) variable region comprising the amino acid sequence of SEQ ID NO: 13.

09(3G2 light chain):

AQVLTQTPSPVSAAVGGTVTISCQSSQSVYNNNNLVWFQKKPGQPPKQLIYKASKVASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCLGGYSGDIYTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

SEQ ID NO:10 3G2 LC CDR1:QSVYNNNN

SEQ ID NO:11 3G2 LC CDR2:KASKVAS

SEQ ID NO:12 3G2 LC CDR3:LGGYSGDIYT

13(3G2 heavy chain):

QEQLKESGGGLVTPGGTLTLTCTASGFTISSYHMSWVRQAPGKGLEWIGYISTSGNIYYATWAKGRFTISKTSSTTVDLRMTSLTTEDTATYFCARLGIATGYSFWGHGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

SEQ ID NO:14 3G2 HC CDR1:GFTISSYHMS

SEQ ID NO:15 3G2 HC CDR2:ISTSGNI

SEQ ID NO:16 3G2 HC CDR3:ARLGIATGYSF

in some embodiments, antibodies are provided having a light chain variable region with an amino acid sequence having at least 90% sequence identity to the amino acid sequence represented by SEQ ID NO: 09. In a further embodiment, an antibody having a heavy chain variable region with an amino acid sequence having at least 90% sequence identity to the amino acid sequence represented by SEQ ID NO. 13 is provided.

The present disclosure also provides methods of detecting one or more proteins that are part of a neural characteristic, as defined herein. Such proteins may be detected by an antibody capable of binding to a protein selected from the group consisting of: ubiquitin C-terminal hydrolase L1(PGP9.5, UCHL1, NDGOA; PARK 5; PGP 95; SPG 79; Uch-L1; HEL-117; PGP 9.5; HEL-S-53; and accession No. NM-004181.4 → NP-004172.2), RNA-bound FOX-1 homolog 3(RBFOX3, FOX3, NEUN, FOX-3, HRNBP 3; and accession No. NM-001082575.2 → NP-001076044.1), microtubule-associated protein 2(MAP2, MAP2A, MAP2B, MAP 2C; and accession No. NM-001039538.1 → NP-001034627.1), 160kDa neurofilament medium chain (NEFM, NFM, NEF3, NF-M; and accession No. NM-005382.2 → NP _005373.2), 200kDa neurofilament heavy chain (NEFH, NFH, CMT2 CC; and accession numbers NM _021076.3 → NP _066554.2), synaptophysin (SYP, MRX96, MRXSYP; and accession No. NM _003179.2 → NP _003170.1) or discs large MAGUK scaffold protein 4(DLG4, DLGH4, PSD-95, PSD95, SAP-90, SAP90, SAP 90A; and accession number NM _001365.4 → NP _ 001356.1). One of ordinary skill in the art can identify additional PGP9.5, RBFOX3, MAP2, NEFM, NEFH, SYP and DLG4 nucleic acid and protein sequences, including variants and isomers. Antibodies for detecting PGP9.5, RBFOX3, MAP2, NEFM, NEFH, SYP and DLG4 are known in the art. For example, antibodies capable of binding PGP9.5 include, but are not limited to: anti-human PGP9.5 monoclonal antibodies from rabbits (clone EPR4118, P/N ab108986) or mice (clone 13C/I3C4, P/N ab8189) were purchased from Abcam. Rabbit polyclonal antibody with RTD P/N760-4434 can be obtained from Cell Marque^TMAnd (4) obtaining.

As used herein, the term "contacting" refers to allowing association, particularly direct physical association, between two or more moieties, for example placement in solid form and/or in liquid form (e.g., placement of a biological sample, such as a biological sample immobilized on a slide, in contact with an antigen releasing solution).

As used herein, the term "specific binding" refers to the binding of an agent that preferentially binds to a defined target (such as an antibody to a specific antigen or a nucleic acid probe to a specific nucleic acid sequence). The target may be any molecule whose presence, location and/or concentration is or can be determined. Examples of target molecules include proteins and nucleic acids. Target molecules are typically detected using one or more conjugates of a specific binding molecule and a detectable label. With respect to antigens, "specific binding" refers to preferential association of an antibody or other ligand, in whole or in part, with a specific polypeptide. With respect to nucleic acid sequences, "specifically binds" refers to the preferential association of a nucleic acid probe, in whole or in part, with a specific nucleic acid sequence. A specific binding agent binds essentially only to a defined target. It is recognized that small degrees of non-specific interactions between molecules may occur, such as specific binders, as well as non-target polypeptide or non-target nucleic acid sequences. Although the selectively reactive antibody binds to an antigen, it can bind with low affinity. Specific binding of an antibody to an antigen typically results in a greater than 2-fold increase, such as greater than 5-fold, 10-fold, or greater than 100-fold, in the amount (per unit time) of antibody or other ligand bound to the target polypeptide, as compared to the non-target polypeptide. A variety of immunoassay formats are suitable for selecting antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein.

A sample comprising an antigen (such as a target antigen) is incubated with the antibody under conditions that allow antibody-antigen binding. Antibody-antigen binding can be detected by means of a detectable label conjugated to the antibody (direct detection) or a detectable label conjugated to a secondary antibody raised against the primary antibody (e.g., indirect detection). Detectable labels may include, but are not limited to, radioisotopes, fluorescent dyes (such as fluorescein, fluorescein isothiocyanate, and rhodamine), and chromogenic molecules.

As used herein, the term "detecting" refers to determining whether an agent (such as a signal or a particular antigen or protein) is present or absent in a sample. In some instances, this may further include quantization. As used herein, the term "detecting" refers to any method of determining whether a substance is present, or not, such as determining whether a target molecule is present in a biological sample. For example, the detection may include using visual or mechanical means to determine whether the sample exhibits a particular characteristic. In certain examples, detection refers to visual observation that the probe binds to the target, or observation that the probe does not bind to the target. For example, for the methods described herein, light microscopy and other microscopic means are commonly used to detect chromogenic precipitates.

Marker substance

As used herein, a "detectable label" refers to a molecule or material that can produce a detectable signal (such as visually, electronically, or otherwise) indicative of the presence and/or concentration of a target in a sample. When conjugated to a specific binding molecule, a detectable label can be used to locate and/or quantify the target to which the specific binding molecule is directed. Thus, the presence and/or concentration of the target in the sample can be detected by detecting the signal generated by the detectable label. The detectable label may be detected directly or indirectly, and several different detectable labels conjugated to different specific binding molecules may be used in combination to detect one or more targets. Multiple detectable labels, which can be separately detected, can be conjugated to different specific binding molecules that specifically bind different targets to provide a multiplex assay that can provide detection of multiple targets in a sample. Detectable signals may be generated by any known or yet to be discovered mechanism, including absorption, emission, and/or scattering of photons, including radio frequency, microwave frequency, infrared frequency, visible frequency, and ultraviolet frequency photons.

Detectable labels may include colored, fluorescent, phosphorescent, and luminescent molecules and materials that convert one substance into another to provide a detectably different catalyst (such as an enzyme) (such as by converting a colorless substance into a colored substance and vice versa, or by producing a precipitate or increasing the turbidity of the sample). Specific examples of detectable labels include: enzymes such as horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, or beta-glucuronidase; fluorophores such as fluorescein, luminophores, coumarins, BODIPY dyes, resorufin, and rhodamine (many other examples of Fluorescent molecules can be found in The Handbook-a Guide to Fluorescent Probes and laboratory technologies, Molecular Probes, Eugene, OR); nanoparticles, such as quantum dots; metal chelates, e.g. radioactive or paramagnetic metal ions (e.g. Gd)³⁺) DOTA and DPTA chelates of (a); and liposomes, e.g., liposomes containing entrapped fluorescent molecules. Where the detectable label comprises an enzyme, a detectable substrate (such as a chromophore, a fluorescent compound, or a luminescent compound) is used in combination with the enzyme to generate a detectable signal (a wide variety of such compounds are commercially available, for example, from Life Technologies, Carlsbad, Calif.).

As used herein, the term "chromophore" refers to a substance, such as a pigment or dye, that is capable of being converted to a colored product. Some chromophores are electron donors and, when oxidized, become colored products. The generation of colored products, or the property of becoming insoluble upon chemical transformation (such as by oxidation), makes chromophores useful for IHC. Non-limiting examples of chromogenic compounds include Diaminobenzidine (DAB), 4-chloro-2-methyl-diazobenzene cation (fast Red), Nitro Blue Tetrazolium (NBT), AP orange, Tetramethylbenzidine (TMB), 2' -diazo-bis- [ 3-ethylbenzothiazolinesulfonate ] (ABTS), New fuchsin, Iodonitrotetrazolium (INT), tetrazolium blue, and tetrazolium violet. Further chromophores are known to the person skilled in the art, for example 4-nitrophenyl phosphate (pNPP), bromochloroindole phosphate (BCIP), nitroblue tetrazolium (NBT), AP blue, o-dianisidine, 4-chloronaphthol (4-CN), nitrophenyl-beta-D-galactoside (ONPG), o-phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-beta-galactopyranoside (X-Gal), methylumbelliferyl-beta-D-galactoside (MU-Gal), p-nitrophenyl-alpha-D-galactoside (PNP), 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazole (C), Fuchsin, Iodonitrotetrazole (INT), tetrazole blue, and tetrazole violet. DAB is a chromophore that produces a brown end product that is highly insoluble in ethanol and other organic solvents. Oxidation of DAB results in polymerization and thus the ability to react with osmium tetroxide, thus increasing its staining intensity and electron density.

Alternatively, the enzyme may be used in a metallographic detection scheme. Metallographical detection methods include the use of oxidoreductases (such as horseradish peroxidase) and water soluble metal ions (e.g., silver or gold), oxidizing agents and reducing agents, again to form detectable precipitates. Under appropriate conditions (i.e., addition of a labeling enzyme) soluble metal ions such as silver (+1) or gold (+3) will be reduced to silver or gold atoms, such that under bright field optical microscopy it can serve as a specific point visible to the eye.

Reagent kit

The present disclosure also provides a kit comprising: a primary antibody capable of binding phosphorylated alpha-synuclein; and a primary antibody capable of binding a neural characteristic. In some of the kits disclosed herein, the kit further comprises: (a) a first secondary antibody having a first label conjugated thereto, wherein the first secondary antibody is immunoreactive with a primary antibody capable of binding phosphorylated a-synuclein; (b) a set of reagents that generates a first detectable signal when reacted with a first label of a first secondary antibody; (c) a second secondary antibody having a second label conjugated thereto, wherein the second secondary antibody is immunoreactive with a primary antibody capable of binding a neurological feature; and (d) a set of reagents that, when reacted with a second label of a second secondary antibody, produces a second detectable signal; wherein the first detectable signal and the second detectable signal are different. In some of the kits, the first detectable signal is silver stain and the second detectable signal is QM-Dabsyl or fast red.

As used herein, the term "kit" refers to a packaged combination of one or more vessels, containers, devices, etc. containing the necessary reagents for detecting a target analyte. The kit is accompanied by written or computer instructions for performing the method. The kit may contain labeled antibodies, nucleic acids, ligands, and the like. A kit for detecting a target analyte in a biological sample may comprise one or more containers each adapted to contain a specific binding member for the target analyte, a redox inactive reducing species, an enzyme label for rendering the reducing species active, a metal ion and a metal enhancing reagent. In some embodiments, the specific binding member is immobilized on a solid support.

In some of the methods disclosed herein, the primary antibody capable of binding to alpha-synuclein may be one of the antibodies disclosed in table 10. For example, antibodies for detecting alpha-synuclein may include, but are not limited to: rabbit monoclonal antibodies against phosphorylated S129 α -synuclein clone 7E2 and 3G 2. Additional monoclonal antibodies directed against phosphorylated S129 α -synuclein may include, for example, the Abcam clone MJF-R13(8-8), P/N ab168381 and P-syn/81A, P/N ab184674 or the WAKO clone pSyn #64, P/N015-25191. Regardless of the phosphorylation state of S129, a mouse monoclonal antibody directed against α -synuclein can be used as clone LB509 (e.g., Abcam (P/N ab 27766)). Also useful are those from Prothena CorporThe anti- α -synuclein mouse monoclonal antibody at the end of the elongation clone 5C12 (independent S129 phosphorylation;no. PTA-9197) and anti-phosphorylated S129-alpha-synuclein mouse monoclonal antibody clone 11A5(No.PTA-8222)。

In some of the methods disclosed herein, the primary antibody capable of binding a neurological feature is selected from an antibody capable of binding to a protein selected from the group consisting of: ubiquitin C-terminal hydrolase L1(PGP9.5, UCHL1, NDGOA; PARK 5; PGP 95; SPG 79; Uch-L1; HEL-117; PGP 9.5; HEL-S-53), RNA-bound FOX-1 homolog 3(RBFOX3, FOX3, NEUN, FOX-3, HRNBP3), microtubule-associated protein 2(MAP2, MAP2A, MAP2B, MAP2C), 160kDa neurofilament medium chain (NEFM, NFM, NEF3, NF-M), 200kDa neurofilament heavy chain (NEFH, NFH, CMT2CC), synapsin (SYP, MRX96, MRYP) and discos large MAGGUK scaffold protein 4(DLG 29, DLGH4, PSD 3995, PSD 4642, SAP-90, SAP A).

In some of the methods disclosed herein, the method further comprises: contacting the section with a first secondary antibody having a first label conjugated thereto, wherein the first secondary antibody is immunoreactive with a primary antibody capable of binding phosphorylated alpha-synuclein, and contacting the section with a second secondary antibody having a second label conjugated thereto, wherein the second secondary antibody is immunoreactive with a primary antibody capable of binding a neural feature.

In some of the methods disclosed herein, the method further comprises: contacting the section with a set of reagents reactive with a first label of a first secondary antibody to generate a first detectable signal in proximity to phosphorylated alpha-synuclein in the sample; and contacting the section with a set of reagents reactive with a second label of a second secondary antibody to generate a second detectable signal in the sample proximal to the neural feature.

In some of the methods disclosed herein, the method further comprises denaturing the immune complex in the sample after contacting the section with a primary antibody capable of binding phosphorylated alpha-synuclein and before contacting the section with a primary antibody capable of binding a neurological feature.

In some of the methods disclosed herein, the section is contacted with at least one protease prior to contacting with the primary antibody. In certain methods, the method further comprises contacting the section with at least one phosphatase.

In some of the methods disclosed herein, the primary antibody capable of binding phosphorylated alpha-synuclein and the primary antibody capable of binding a neurological feature are from the same host species.

In some of the methods disclosed herein, the first detectable signal and the second detectable signal are different. In some of the methods disclosed herein, the first detectable signal is silver stain and the second detectable signal is QM-Dabsyl or fast red.

Also provided herein are methods for diagnosing PD in a subject, the methods comprising: (a) obtaining a biological sample comprising at least one neurological feature from a subject suspected of having PD; (b) detecting whether the phosphorylated alpha-synuclein is within the neural signature of the sample by contacting the sample with an anti-pgp.5 antibody and determining co-localization between the phosphorylated alpha-synuclein and PGP 9.5; and (c) diagnosing the subject with PD when the presence of co-localization between phosphorylated alpha-synuclein and PGP9.5 in the neurological signature is positively determined. In some cases, where neural features can be identified without a signal associated with an anti-PGP 9.5 antibody, the method can be accomplished without the use of an anti-PGP 9.5 antibody. For example, an experienced pathologist may be able to identify neural features in a tissue sample without such a signal. A pathologist may diagnose a subject with PD by the presence of a signal associated with localized phosphorylated alpha-synuclein within a neurological feature.

Some methods may include, for example, scoring the phosphorylated alpha-synuclein and neurological features detected in the sample. In some methods, the number of neural features in the entire slide is counted and expressed as a percentage of neural features containing phosphorylated alpha-synuclein. In certain methods, wherein the neural characteristic is identified by the presence of a neural characteristic protein of interest, the presence of phosphorylated alpha-synuclein and the target neural characteristic protein may comprise determining the absolute number of neural characteristic stains with the phosphorylated alpha-synuclein antibody in the sample using a 5x5 ocular grid having an area of 0.25 square millimeters, determining the absolute number of neural characteristic stains with the target neural characteristic antibody in the sample using a 5x5 ocular grid having an area of 0.25 square millimeters, extrapolating the absolute number of cell stains with the phosphorylated alpha-synuclein antibody to the number of neural characteristics in a1 square millimeter region, and extrapolating the absolute number of neural feature staining with the neural feature of the target neural feature antibody to the number of neural features in the 1 mm square region, thereby generating a score for phosphorylated alpha-synuclein and the target neural feature protein. Thus, the method can include counting the number of neural feature stains positive for phosphorylated a-synuclein and the number of neural feature stains positive for the target neural feature protein in the grid (such as phosphorylated a-synuclein and/or the target neural feature positive neural feature). In one particular example, these values are inserted into the following equation: cell count/(# of 0.0156x grid). The formula depends on the diameter of the objective lens, which is variable between models even at equal magnifications. Other formulas may be used if different magnifications or different objectives are used. In some examples, randomly selected regions of the sample are selected for scoring. Thus, as shown in fig. 32, each co-localized black and yellow dot in the image represents a neural feature stain with phosphorylated a-synuclein and a target neural feature protein.

Provided herein are methods for determining whether a subject has Parkinson's Disease (PD), methods for diagnosing PD in a subject, and methods of diagnosing and treating PD in a subject by partially diagnosing a subject with PD when it is positively determined that there is co-localization between phosphorylated alpha-synuclein and neural features in a biological sample from the subject. For example, a subject may be determined or diagnosed as having parkinson's disease if a sample obtained from the subject is analyzed or scored using the methods provided herein because of the co-localization of phosphorylated alpha-synuclein within the neural feature that is increased relative to a normal sample (e.g., a non-PD sample of the same tissue type). For example, phosphorylated a-synuclein within a neural feature in a tissue sample is increased by at least 20%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, or at least 500% (such as by at least 2-fold, at least 5-fold, or at least 10-fold) compared to phosphorylated a-synuclein within a neural feature in a normal sample (such as a reference value or range of values for such a sample). In contrast, if a biological sample obtained from a subject is analyzed or scored using the methods provided herein, the subject may be determined or diagnosed as not likely to have parkinson's disease because of having phosphorylated alpha-synuclein within a similar or reduced neurological signature relative to a normal sample (e.g., a non-PD sample). In some examples, the disclosed methods may further comprise administering one or more therapies or treatments to the subject if the subject is determined or diagnosed as having parkinson's disease.

Also provided herein are methods of treating a subject diagnosed with PD, comprising administering to the subject an effective regime of an antibody to alpha-synuclein, wherein the antibody capable of binding phosphorylated alpha-synuclein and the antibody capable of binding a neurological feature have been shown to co-localize in the neurological feature in a skin sample from the subject. In certain methods, a subject determined to have PD is treated by administering to the subject an effective regime of an alpha-synuclein antibody, wherein the subject is determined to have PD by any of the methods disclosed herein.

As used herein, the terms "treat," "treating," and "treating" refer to the alleviation of one or more of the major or minor symptoms of parkinson's disease and related movement disorders. The major symptoms of parkinson's disease include, but are not limited to, tremor of the extremities at rest, slow movement of the body (bradykinesia), increased muscle rigidity or stiffness, gait or balance problems (abnormal posture). Minor symptoms of parkinson's disease include, but are not limited to: difficulty initiating or resuming movement, loss of small motor skills, lack of arm swing on the affected side of the body while walking, dragging on the affected side of the body, reduced facial expression, voice and/or language changes, cognitive disorders, sleep disorders, gastrointestinal dysfunction, and feelings of depression or anxiety.

Related movement disorders that may also be treated by the methods of the present disclosure include, but are not limited to: akathisia, akinesia (lack of movement), athetosis (twisting or torsion), ataxia, toseisis (involuntary, rapid and irregular movement of violence), cerebral palsy, chorea (e.g., sydenham's chorea, rheumatic chorea, huntington's chorea), dystonia (e.g., muscular dystonia, blepharospasm, writer's cramp, spasmodic torticollis), geniospasm (intermittent involuntary upward and downward movement of the chin and lower lip), myoclonus, Restless Leg Syndrome (RLS), spasticity, stereotypy, tardive dyskinesia, tic disorders (tourette's syndrome, postural tremor, motor tremor, essential tremor, cerebellar tremor, physiological tremor), and wilson's disease.

All subjects should be evaluated and managed by a multidisciplinary team with expertise and experience in neurodegenerative disorders and parkinson's disease prior to initiation of treatment and/or therapy. Subjects with PD typically have a medical professional and multidisciplinary health care team consisting of doctors from different specialties, including but not limited to: neurologists, occupational therapists, physical therapists, consultants, socioecians, registered dieticians and language therapists. After diagnosis of PD in a subject, a medical professional or team of medical professionals typically advises one or more treatment options, including one or more prescription drugs (e.g., benzalkonium mesylate (Cogentin), entacapone (comban), dopa, lardopa, levodopa and carbidopa (Sinemet), pramipexole (Mirapex), rasagiline (Azilect), ropinirole hydrochloride (Requip), rotigotine (neupriro), safinamide (Xadago), mei or trihexylphenyl (Artane)), surgery (e.g., deep brain stimulation, globulotomy, thalamotomy or gamma knife), substitution therapy (coenzyme Q10, massage, acupuncture, taiji, yoga, alexander technique or meditation), and/or healthy diet and exercise. The treatment regimen is determined by a medical professional or team of medical professionals and may be for each subject. Those skilled in the art are familiar with various other treatments for PD.

In some such methods, the effective regime of an α -synuclein antibody comprises an α -synuclein antibody selected from the group consisting of: monoclonal antibodies that bind to residues 1-20 of alpha-synuclein, residues 1-10 of alpha-synuclein, residues 4-15 of alpha-synuclein, residues 91-99 of alpha-synuclein, residue 117-123 of alpha-synuclein, residue 118-126 of alpha-synuclein, prasuzuzumab (PRX002), humanized antibodies having the CDRs of antibody clone 1H7(ATCC accession No. PTA-8220), humanized antibodies having the CDRs of antibody clone 9E4(ATCC accession No. PTA-8221), the CDRs of antibody clone NI-202.21D11 and the CDRs of antibody clone NI-202.12F4, for example, alpha-synuclein antibodies disclosed in U.S. patent nos. 8,092,801, 8,609,820, 8,790,644, 8,940,276, 9,580,493, which are incorporated herein by reference in their entirety.

Some such antibodies comprise VH CDR1 comprising residues 31-35 of SEQ ID NO. 49, VH CDR2 comprising residues 50-68 of SEQ ID NO. 49, VH CDR3 comprising residues 101-102 of SEQ ID NO. 49, VL CDR1 comprising residues 23-33 of SEQ ID NO. 50, VL CDR2 comprising residues 49-55 of SEQ ID NO. 50, and VL CDR3 comprising residues 88-98 of SEQ ID NO. 50. Some such antibodies comprise VH CDR1 comprising SEQ ID NO. 51, VH CDR2 comprising SEQ ID NO. 52, VH CDR3 comprising SEQ ID NO. 53, VL CDR1 comprising SEQ ID NO. 54, VL CDR2 comprising SEQ ID NO. 55, and VL CDR3 comprising SEQ ID NO. 56. Some such antibodies comprise a heavy chain comprising SEQ ID NO 57, and a light chain comprising SEQ ID NO 58.

SEQ ID NO:49 (SEQ ID NO:9 of US 8,940,276)

EVQLVQSGGGLVEPGGSLRLSCAVSGFDFEKAWMSWVRQAPGQGLQWVARIKSTADGGTTSYAAPVEGRFIISRDDSRNMLYLQMNSLKTED

TAVYYCTSAHWGQGTLVTVSS

SEQ ID NO:50 (SEQ ID NO:12 of US 8,940,276)

QSVLTQPPSVSVSPGQTARITCSGEALPMQFAHWYQQRPGKAPVIVVYKDSERPSGVPERFSGSSSGTTATLTITGVQAEDEADYYCQSPDSTNTYEVFGGGTKLTVL

SEQ ID NO:51 (SEQ ID NO:16 of US 9,580,493; NI-202.21D11-VHCDR1)

NYAMH

SEQ ID NO:52 (SEQ ID NO:17 of US 9,580,493; NI-202.21D11-VHCDR2)

WINAGNGKRKYSQKFQD

53 (SEQ ID NO:18 of US 9,580,493; NI-202.21D11-VHCDR3)

EEDHAGSGSYLSMDV

SEQ ID NO:54(US 9,580,493 SEQ ID NO: 23; NI-202.21D 11-VKPCR 1)

KSSQNVLYSSNNKNYLA

SEQ ID NO:55(US 9,580,493 SEQ ID NO: 24; NI-202.21D 11-VKPCR 2)

WASTRES

SEQ ID NO:56(US 9,580,493 SEQ ID NO: 25; NI-202.21D 11-VKPCR 3)

QQYYSSPLT

SEQ ID NO:57 (SEQ ID NO:10 of US 8,609,820)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASISSGGGSTYYPDNVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARGGAGIDYWGQGTLVTVSS

SEQ ID NO:58 (SEQ ID NO:5 of US 8,609,820)

DIQMTQSPSSLSASVGDRVTITCKSIQTLLYSSNQKNYLAWFQQKPGKAPKLLIYWASIRKSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYYSYPLTFGGGTKLEIK

In some such methods, the antibody is prasuzumab (PRX 002). In some such methods, the antibody comprises three light CDRs, designated SEQ ID NOS: 18-20, respectively, and three heavy chain CDRs, designated SEQ ID NOS: 22-24, respectively. In some such methods, the antibody comprises one light chain, designated SEQ ID NO 17, and one heavy chain, designated SEQ ID NO 21.

17>10680L Prazolizumab humanized LC calvital cells

DIQMTQSPSSLSASVGDRVTITCKSIQTLLYSSNQKNYLAWFQQKPGKAPKLLIYWASIRKSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYYSYPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

18 Prasilizumab LC CDR1 KSIQTLLYSSNQKNYLA SEQ ID NO

19 Prasilizumab LC CDR2 WASIRKS SEQ ID NO

20 Prasilizumab LC CDR3 QQYYSYPLT SEQ ID NO

21>10680H Prazolizumab humanized HC | R C

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASISSGGGSTYYPDNVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARGGAGIDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

22 Pralazinzumab HC CDR1 NYGMS of SEQ ID NO

23 Prasilizumab HC CDR1 SISSGGGSTYYPDNVKG SEQ ID NO

24 Prasizumab ozogamicin HC CDR1 GGAGIDY SEQ ID NO

In some such methods, the antibody is 9E4, as disclosed in U.S. patent No. 8,609,820, which is incorporated herein by reference in its entirety. The method provides an antibody comprising a humanized heavy chain comprising three Kabat CDRs of SEQ ID NO:29 and a humanized light chain comprising three CDRs of SEQ ID NO:25, provided that position L36(Kabat numbering) is occupied by F or Y and/or position L83(Kabat numbering) is occupied by L or F and/or position H73(Kabat numbering) is occupied by D or N, and/or position H93(Kabat numbering) is occupied by S or a. In some such antibodies, position L36(Kabat numbering) is occupied by F, and position H73(Kabat numbering) is occupied by D, and position H93(Kabat numbering) is occupied by S. In some such antibodies, position L36 is occupied by F. In some such antibodies, position L83 is occupied by L. In some such antibodies, position H73 is occupied by D. In some such antibodies, position H93 is occupied by a. In some such antibodies, position L36 is occupied by F and position L83 is occupied by L. In some such antibodies, position L36 is occupied by F and position H73 is occupied by D. In some such antibodies, position L36 is occupied by F and position H93 is occupied by a. In some such antibodies, position L36 is occupied by F, position L83 is occupied by L, and position H73 is occupied by D. In some such antibodies, position L36 is occupied by F, position L83 is occupied by L, and position H93 is occupied by a. In some such antibodies, position L36 is occupied by F, position L83 is occupied by L, position 1173 is occupied by D, and position H93 is occupied by a. In some such antibodies, the residues at positions L36, L83, H73, and H93(Kabat numbering) are occupied by F, and position H73(Kabat numbering) is occupied by D, and position H93(Kabat numbering) is occupied by a. In some such antibodies, position L36(Kabat encoding) is occupied by F and position H93(Kabat encoding) is occupied by S. In some such antibodies, position H73(Kabat numbering) is occupied by D and position H93(Kabat numbering) is occupied by S. In some such antibodies, position 1173(Kabat encoding) is occupied by D and position 1193(Kabat encoding) is occupied by a. In some such antibodies, position H93(Kabat encoding) is occupied by S. In some such antibodies, position 1173(Kabat encoding) is occupied by N. In some such antibodies, position L36(Kabat numbering) is occupied by F, position L83(Kabat numbering) is occupied by L, and position H73(Kabat numbering) is occupied by D, and position H93(Kabat numbering) is occupied by S. In some such antibodies, position L36(Kabat numbering) is occupied by F, position L83(Kabat numbering) is occupied by L, and position H93(Kabat numbering) is occupied by S. In some such methods, the antibody comprises three light CDRs, designated SEQ ID NOS: 26-28, respectively, and three heavy chain CDRs, designated SEQ ID NOS: 30-32, respectively. In some such methods, the antibody comprises a light chain variable region designated SEQ ID NO:25 and a heavy chain variable region designated SEQ ID NO: 29.

259E 4 LC variable region

DIQMTQSPSSLSASVGDRVTITCKSIQTLLYSSNQKNYLAWYQQKPGKAPKLLIYWASIRKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSYPLTFGGGTKLEIK

SEQ ID NO:26 9E4 LC CDR1 KSIQTLLYSSNQKNYLA

SEQ ID NO:27 9E4 LC CDR2 WASIRKS

SEQ ID NO:28 9E4 LC CDR3 QQYYSYPLT

299E 4 HC variable region

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASISSGGGSTYYPDNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGAGIDYWGQGTLVTVSS

SEQ ID NO:30 9E4 HC CDR1 NYGMS

SEQ ID NO:31 9E4 HC CDR2 SISSGGGSTYYPDNVKG

SEQ ID NO:32 9E4 HC CDR3 GGAGIDY

In some such methods, the antibody is NI-202.21D 11. In some such methods, the antibody comprises three light CDRs, designated SEQ ID NOS: 34-36, respectively, and three heavy chain CDRs, designated SEQ ID NOS: 38-40, respectively. In some such methods, the antibody comprises a light chain variable region designated SEQ ID NO 33 and a heavy chain variable region designated SEQ ID NO 37.

33NI-202.21D11 LC variable region of SEQ ID NO

DVVMTQSPDSLAVSLGERATINCKSSQNVLYSSNNKNYLAWYQQKPGHPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTITSLQTEDVAVYYCQQYYSSPLTFGGGTKVEIK

SEQ ID NO:34NI-202.21D11 LC CDR1 KSSQNVLYSSNNKNYLA

SEQ ID NO:35NI-202.21D11 LC CDR2WASTRES

SEQ ID NO:36NI-202.21D11 LC CDR3 QQYYSSPLT

SEQ ID NO 37NI-202.21D11 HC variable region

EVQLVESGAEVKKPGASVKVSCKASGYTFTNYAMHWVRQAPGQRLEWMGWINAGNGKRKYSQKFQDRVTINRDTSASTIYMELSSLGSEDTAVYYCAREEDHAGSGSYLSMDVWGQGTLVTVSS

SEQ ID NO:38NI-202.21D11 HC CDR1 NYAMH

SEQ ID NO:39NI-202.21D11 HC CDR2 WINAGNGKRKYSQKFQD

SEQ ID NO:40NI-202.21D11 HC CDR3 EEDHAGSGSYLSMDV

In some such methods, the antibody is NI-202.12F 4. In some such methods, the antibody comprises light and heavy CDRs, designated SEQ ID NOS: 42-44, respectively, and three heavy chain CDRs, designated SEQ ID NOS: 46-48, respectively. In some such methods, the antibody comprises a light chain variable region designated SEQ ID NO:41 and a heavy chain variable region designated SEQ ID NO: 45.

41NI-202.12F4 LC variable region of SEQ ID NO

QSVLTQPPSVSVSPGQTARITCSGEALPMQFAHWYQQRPGKAPVIVVYKDSERPSGVPERFSGSSSGTTATLTITGVQAEDEADYYCQSPDSTNTYEVFGGGTKLTVL

SEQ ID NO:42NI-202.12F4LC CDR1 SGEALPMQFAH

SEQ ID NO:43NI-202.12F4LC CDR2 KDSERPS

SEQ ID NO:44NI-202.12F4LC CDR3 QSPDSTNTYEV

SEQ ID NO 45NI-202.12F4 HC variable region

EVQLVQSGGGLVEPGGSLRLSCAVSGFDFEKAWMSWVRQAPGQGLQWVARIKSTADGGTTSYAAPVEGRFIISRDDSRNMLYLQMNSLKTEDTAVYYCTSAHWGQGTLVTVSS

SEQ ID NO:46NI-202.12F4HC CDR1 KAWMS

SEQ ID NO:47NI-202.12F4HC CDR2 RIKSTADGGTTSYAAPVEG

SEQ ID NO:48NI-202.12F4HC CDR3 TSAH

Also provided herein are methods for detecting phosphorylated alpha-synuclein, the methods comprising: contacting the biological sample with a primary antibody capable of binding phosphorylated alpha-synuclein; and detecting a primary antibody capable of binding phosphorylated alpha-synuclein. In some of the methods, the sample is contacted with at least one protease prior to contacting with the primary antibody capable of binding phosphorylated alpha-synuclein. In some methods, the method further comprises contacting the sample with at least one phosphatase. In some methods, the detecting comprises histochemical analysis. In some methods, a primary antibody capable of binding phosphorylated a-synuclein detects a-synuclein phosphorylated at residue S129. In certain methods, the primary antibody capable of binding phosphorylated alpha-synuclein is a 7E2 antibody clone or a 3G2 antibody clone. In some methods, the sample is fixed. In certain methods, the sample is a formalin-fixed, paraffin-embedded (FFPE) sample. In some methods, the sample is a frozen sample

In some methods, the method further comprises contacting the sample with a first secondary antibody having a first label conjugated thereto, wherein the first secondary antibody is immunoreactive with a primary antibody capable of binding phosphorylated alpha-synuclein. In some methods, the method includes contacting the sample with a set of reagents reactive with a first label of a first secondary antibody to generate a first detectable signal in the sample in proximity to the phosphorylated alpha-synuclein.

Examples of the invention

Parkinson's disease is a progressive neurodegenerative disease characterized by the presence of lewy bodies and lewy neurites in the brain of a subject suffering from the disease. Lewy bodies are rich in aggregated form of alpha-synuclein (aSyn), a poorly characterized functional protein. Accumulation of aSyn is also found in peripheral nerves of subjects with parkinson's disease. Aggregated aSyn can be detected using Immunohistochemistry (IHC) and can be distinguished from unaggregated aSyn due to its resistance to protease treatment. However, prolonged protease treatment may reduce tissue morphology in IHC assays, resulting in the need for an alternative method to distinguish aggregated aSyn from unaggregated aSyn. An abnormally high proportion of aggregated aSyn was found to be phosphorylated at the Ser129 residue (pS129) compared to unaggregated aSyn. As shown herein, a highly sensitive and specific assay for aggregated aSyn using pS129-aSyn as a surrogate marker for aSyn aggregation was developed.

Material

A tissue source. Scalp, abdominal region skin, colon and submandibular gland samples were obtained from the Banner Sun health institute (BSHRI) with the help of the brain and body donation program, Thomas g. In addition to standardized clinical assessments, the diagnosis of parkinson's disease is confirmed in tissue donors by the presence of lewy bodies. Likewise, the status of normal non-PD control subjects was determined by the absence of a lewy body. Samples (16 men, 8 women; age 65-95; 15 PD, 9 non-PD) were obtained post-mortem and fixed with formalin. Formalin-fixed, paraffin-embedded (FFPE) cortical brain blocks from normal individuals and subjects with PD were obtained from the Roche Tissue Diagnostics (RTD) internal Tissue bank and the Folio Biosciences, respectively. A small cohort of FFPE skin biopsy blocks was obtained from 8 individuals 1 to 16 years old (3 men, 5 women) by Folio Biosciences to assess the extent of pS129-aSyn staining, which is different from aggregated aSyn. A larger cohort of 72FFPE skin biopsy patches from 36 subjects was obtained from bon Postuma doctor of the general hospital neurology of montreal. Based on clinical examination of the symptoms exhibited by the subjects, 15 of 36 subjects were diagnosed with PD, and another 5 were diagnosed with atypical parkinson's disease. The remainder (16 subjects) were control subjects without PD. Two needle skin biopsies were taken from the subjects during the clinical visit and the FFPE blocks were transferred to the Ventana medical system via prophena. All other tissue specimens were obtained from an internal tissue bank of RTDs. All FFPE tissue sections were 4 μm thick unless otherwise noted.

Antibodies against alpha-synuclein (aSyn) and PGP 9.5. Rabbit monoclonal antibodies against phospho-S129 aSync clones 7E2 and 3G2 were generated from Roche Diagnostics GmbH CPS R & D Early Development & Reagent Design (DXREAA) with phosphopeptide corresponding to aSync residue 122-135 of human as immunogen. Additional monoclonal antibodies against the phospho-S129 aSyn were purchased from Abcam (clone MJF-R13(8-8), P/N ab168381 and P-syn/81A, P/N ab184674) or WAKO (clone pSyn #64, P/N015-25191). The mouse monoclonal antibody clone LB509 to aSyn was purchased from Abcam (P/N ab27766) regardless of the phosphorylation state of S129. The anti-aSyn mouse monoclonal antibody clone 5C12 (independent S129 phosphorylation) and the anti-phosphorylated S129 α -synuclein mouse monoclonal antibody clone 11a5 are gifts from prophena Corp. See also table 10.

Three anti-human PGP9.5 antibodies were used in this study. Two monoclonal antibodies, a rabbit (clone EPR4118, P/N ab108986) and another mouse (clone 13C/I3C4, P/N ab8189), were purchased from Abcam. Rabbit polyclonal antibody with RTD P/N760-4434Cell Marque^TMAnd (4) obtaining.

An enzyme. In an automated protease-resistant alpha-synuclein DAB IHC assay developed by pRED/Prothena, use was madeThe unaggregated aSyn was removed by protease 1 (P/N760-2018). In one particular embodiment of an automated pS129-aSyn and PGP9.5 silver/yellow Dual IHC assay (bumblebee assay protocol 2 described below), recombinant bovine alkaline phosphatase (Roche at 30. mu.g/mL in a stable diluent (Roche P/N06002919001)) and recombinant bovine alkaline phosphatase (Roche, at 30. mu.g/mL) were used, respectivelyProtease 3 (P/N760-.

Commercially available and user-supplied reagents for automated phospho-S129-aSyn and PGP9.5 silver/yellow dual IHC assay and protease resistant alpha-synuclein DAB IHC assay. Table 1 below contains a list of reagents used in bumblebee assay protocols 1 and 2 and DAB-based pled/prophena assay protocols for the detection of protease-resistant aSyn, including bulk solutions, enzymes, antibodies, detection kits and auxiliary reagents (see methods below).

TABLE 1

List of potential reagents used

An apparatus. A BenchMark ULTRA instrument with the following serial number was used: 310520, 310841, 310934, 310940, 311000, 311112, 311276, 311279, and 311311.

Method

DAB staining of FFPE brain and skin sections from donors with or without Parkinson's disease Using antibodies against aSynn or PGP9.5

Slicing FFPE brainAutomation of ultraView Universal DAB assay kit or OptiView DAB IHC assay kit with standard supplemental bulk solutionStaining was performed in a BenchMark ULTRA instrument. After deparaffinization, the slides were subjected to one of three antigen retrieval procedures, either alone or in combination, or none at all, as indicated. The three antigen retrieval procedures were: 1) diluted about 3.7 times on glass slides with reaction bufferProtease 1 (P/N760) -2018) and incubation at 36 ℃ for 4 min, 2) unless otherwise statedULTRA CC1 solution (P/N950-224) at 100 ℃ for 32 min (for antibodies against. alpha. -synuclein) or 64 min (for antibodies against PGP9.5), and 3) alkaline phosphatase (AP, recombinant from Pichia pastoris, high activity EIA grade, Roche) at 36 ℃ for 2 hours. Alkaline phosphatase was mixed in a stable solution (Roche P/N06002919001) at 50. mu.g/mL in a dispenser and 1 drop of a 1:1 mixture of 0.5M Tris pH10 and reaction buffer and 1 drop of 20mM MgCl2 were applied sequentially after 1 drop was applied to the dipTissue slides in reaction buffer. The primary antibody may be applied manually or automatically from a dispenser at a specified concentration. Unless otherwise stated, all primary antibody incubations were performed at 36 ℃ for 32 minutes. When detecting useultraView UniverWhen the sal DAB detection kit (P/N760-. The counterstain is used on the instrumentHematoxylin II (P/N790-2208 at 36 ℃ for 4 min) and a blue staining reagent (P/N760-2037 at 36 ℃ for 4 min). After alcohol dehydration in a Tissue-Tek automated slide stainer and coverslipper (Sakura), slides were coverslipped in xylene.

Automated phospho-S129-alpha-synuclein and PGP9.5 silver/yellow dual IHC assay (bumblebee assay protocol). Placing FFPE skin sections into a container with standard make-up bulk solutionIn the BenchMark ULTRA instrument, the bulk solution included ultraView Silver Wash II (Ventana P/N780-003). Two protocols (protocol 1 and protocol 2) based on the validated U Triplex IHC Silver _ QM _ DAB program were used to stain consecutive skin sections. After deparaffinization, the bumblebee assay protocol 1 (see table 2 below) was free of antigen retrieval steps prior to incubation with 7E2 anti-phosphorylated S129 α -synuclein antibody. In contrast, alkaline phosphatase (Roche, diluted to 30. mu.g/mL in a stable solution (Roche P/N06002919001)) andprotease 3 (P/N760-. The conditions for alkaline phosphatase and protease treatment were 8 minutes at 37 ℃ and 4 minutes at 36 ℃ respectively. Bumblebee assay protocol 1 and protocol 2 have the same antibody incubation and detection steps: 1) incubation with 7E2 antibody for 32 min at 36 deg.C, 2) useUltraView SISH DNP kit detection of phospho-aYn Using HRP-conjugated goat anti-rabbit antibodies (48 min incubation at 36 ℃) and precipitation of metallic silver, 3) useULTRA CC2 bulk solution (P/N950-223) was tested for primary and secondary antibodies at 100 ℃ for 16 min, which also served as antigen retrieval for PGP9.5, 4) incubated with EPR4118 anti-PGP 9.5 antibody at 36 ℃ for 32 min, 5) goat anti-rabbit antibody conjugated with AP ((P/N950-223))DISCOVERY UltraMap anti Rb Alk Phos, P/N760-. After dilution with the previously used 1 Xreaction buffer (Ventana P/N950-300), the 7E2 and ERP4118 antibodies were approximately at concentrations of 0.27. mu.g/mL and 0.14. mu.g/mL on the slides, respectively. The counterstain is used on the instrumentHematoxylin II (P/N790-2208 at 36 ℃ for 8 min) and a blue staining reagent (P/N760-2037 at 36 ℃ for 8 min). After alcohol dehydration in a Tissue-Tek automated slide stainer and coverslipper (Sakura), slides were coverslipped in xylene. See table 2 for a summary of scheme 1; see table 3 for a summary of scheme 2.

TABLE 2

Summary of scheme 1

Step # of	Description
		1	Paraffin removal [ selection ]]
2	The slide was heated from a moderate temperature to [72 deg.C ]](Paraffin removal)
		3	Preproperoxidase inhibition. [ selection]
4	Inhibitor [ selection]
		5	First antibody [ selection ]]
6	Use of Individual reagents [ selection]
		7	Applying one drop of [ antibody 2 ]](antibodies), application of coverslip, and incubation [32 min]
8	Silver detection [ selection ]]
		9	Detection of ultraVIEW SISH [ selection ]]
10	One drop of SIL, ISH, DNP, HRP was applied, a cover slip was applied, and incubated [48 min]
		11	One drop of SIL, ISH, DNP, CHRC was applied and incubated [12 min ]]
12	2 nd detection [ selection]
		13	Antibody denaturation [ selection ]]
14	The slides were heated from all temperatures to [100 deg.C ]](antibodies)
		15	Incubate [16 min](antibodies)
16	First antibody (2) [ selection]
		17	Application of Individual reagents (2) [ selection]
18	Applying one drop of [ antibody 5 ]](antibody 11), application of coverslip, and incubation [32 min]
		19	Second antibody (2) [ selection]
20	Individual application (2) [ selection]
		21	Blocking agent (2) [ selection]
22	Applying one drop [ option 1 ]](option 2), cover slip was applied and incubated for 4 minutes
		23	Applying one drop of [ antibody 10 ]](antibody 12), and incubation [32 min]
24	Chromophore (2) [ selection]
		25	Using two drops [ antibody 11 ]](antibody 15), application of coverslip, and incubation for 4 min
26	Applying one drop of [ antibody 12 ]](antibody 16), and incubation [32 min]
		27	Counterdyeing [ selection ]]
28	Applying a drop of [ hematoxylin II ]](counterstaining), application of coverslips, and incubation [8 min]
		29	After counter dyeing [ selection]
30	Applying a drop of blue staining reagent](after counterstaining), counterstaining was applied and incubated [8 min]

TABLE 3

Summary of scheme 2

Automated protease resistant alpha-synuclein DAB IHC assay (pRED/Prothena assay protocol). Since aggregated α -synuclein is known to be a poor substrate for proteases, histochemical staining of aggregated aSyn after removal of unaggregated α -synuclein by proteases can be used to detect lewy bodies. Assays based on this principle were developed by Roche pRED in cooperation with Prothena Biosciences to detect aggregated α -synuclein in skin biopsies (pRED/Prothena assay protocol). The assay relies on the 5C12 antibody, whose epitope has been mapped to residues 109 to 120 of alpha-synuclein (region outside the Ser129 phosphorylation site). The pRED/Prothena protocol is shown in Table 4, and the protocol used in this study is shown in Table 5. For the pRED/Prothena assay protocol, use was made ofProtease 1 (P/N760-2018) was treated with protease at 36 ℃ for 12 minutes and with the previously applied 1XAfter dilution with reaction buffer (P/N950-300), 5C12 antibody was incubated at ambient temperature (with the instrument slide heater disabled) for 20 minutes at an approximate slide concentration of 0.27. mu.g/mL. As shown in Table 4, the 5C12 antibody was diluted at 1. mu.g/mL in the dispenser formulationIn antibody diluent (P/N251-018, antibody diluent without Brij-35, internal P/N95028). After incubation with primary antibody, useThe OptiView DAB IHC detection kit (P/N760-700) detects the aggregated alpha-synuclein. Counterstaining on the instrumentUse ofHematoxylin II (P/N790-2208 at 36 ℃ for 8 min) and a blue staining reagent (P/N760-2037 at 36 ℃ for 8 min). After alcohol dehydration in a Tissue-Tek automated slide stainer and coverslipper (Sakura), slides were coverslipped in xylene.

TABLE 4

Summary of protocols for protease resistant alpha-synuclein

TABLE 5

Summary of protocols for protease resistant alpha-synuclein

Slide evaluation and aSyn signal scoring. Slides were scored by professionally verified pathologists or assay-developed scientists with extensive experience in assessing the pattern of aSyn staining in skin. For pS129-aSyn and PGP9.5 silver/yellow dual IHC assays, the number of total neural features with yellow PGP9.5 staining, as well as the number of neural features exhibiting discrete and/or diffuse granular silver pS129-aSyn staining were recorded. In a typical skin section, yellow PGP9.5 staining was observed in the nerve bundles and pili muscles and also in the surrounding secretory/sebaceous glands and blood vessels. At least 25 neural features are counted per slide, but more are typically counted to ensure that assay sensitivity is not adversely affected by the low number of neural features evaluated. In slides with limited skin area and a total number of neural features available for evaluation of less than 25, the entire skin area is scanned and the total number of neural features is counted. The results are expressed as a percentage of the neural features with a particular type of pS129-aSyn staining. Slides stained using the protease resistant aSyn DAB IHC assay were scored similarly except that neurological features were identified by morphology. In the case of tissue morphology significantly affected by treatment with protease 1, the number of total neurological features was derived from adjacent slides stained using the pS129-aSyn and PGP9.5 silver/yellow dual IHC assay.

And (5) carrying out statistical analysis. The correlation between the presence of phosphorylated a-synuclein signal and the clinical condition of the subject was assessed by the chi-square test using Minitab 17 statistical software.

EXAMPLE 1 characterization of 7E2 Rabbit monoclonal antibodies to pS 129-aSynn

Anti-phosphorylated S129 α -synuclein rabbit monoclonal antibody 7E2 was generated by Roche Diagnostics GmbH CPS R & D Early Development & Reagent Design. The immunogen used to generate 7E2 was a peptide conjugated to KLH via the N-terminal Cys residue. The sequence of this peptide, NEAYEMPPSEEEGYQD (SEQ ID NO:59), corresponds to residue 122-135 of human α -synuclein. Surface plasmon resonance (Biacore) experiments showed that the 7E2 antibody has high specificity for the α -synuclein (122-135, pS129) immunogenic peptide phosphorylated at Ser129 compared to the same peptide not phosphorylated at Ser 129. The 7E2 antibody showed a fast association rate and a slow linear dissociation rate for the alpha-synuclein (122-135, pS129) peptide.

The specificity of the 7E2 anti-phosphorylated S129 α -synuclein antibody in an immunohistochemical background was examined using a human tissue microarray containing 30 cores of normal tissue and 29 cores of cancer tissue from various anatomical sites (superbichips Laboratories, Seoul, Korea, P/N BC 8). Analysis of the body's journey/tumour journey (ToB/ToT) showed that the only non-neuronal staining observed with the 7E2 antibody was macrophages (alpha-synuclein has previously been reported to be expressed in macrophages). For a summary of the ToB/ToT analysis, see tables 6-9. ToB/ToT results demonstrate that the protocol is both accurate and specific.

TABLE 6

Summary of the physical analysis trip of protocol 1

TABLE 7

Summary of physical analysis travel of protocol 2

TABLE 8

Summary of tumor analysis journey of scheme 1

TABLE 9

Summary of tumor analysis journey of scheme 2

Example 2.7 immunohistochemical staining performance of E2 on FFPE tissue compared to other pS129-aSyn antibodies.

The detection of aggregated alpha-synuclein based on the different sensitivities of aggregated and unaggregated alpha-synuclein to proteolytic degradation provides a basis for the identification of lewy bodies in the brain and the definitive diagnosis of parkinson's disease. To evaluate the ability to detect lewy bodies against phosphorylated S129 α -synuclein antibodies (including 7E2), use was made ofThe OptiView DAB IHC assay kit stained cortical brain sections from two donors with parkinson's disease. All 8 anti-phosphorylated S129 α -synuclein antibodies (81A, #64, MJF-R13(8-8), 5H5, 2G11, 7E2, 3G2, and 11A5) produced DAB staining for lewy body characterization in PD brain sections (see table 10 for a list of non-limiting, exemplary α -synuclein antibodies tested). Of the 8 anti-phosphorylated α -synuclein antibody clones tested, 7E2, 3G2, 11A5, and MJF-R13(8-8) showed higher sensitivity to pS129-aSyn (see FIGS. 1A-1C).

Watch 10

Non-limiting, exemplary list of alpha-synuclein antibodies

In brain sections from non-PD individuals, no staining was observed with the 3G2 and 7E2 antibodies without antigen retrieval (see fig. 2A and 2B). In the case of ULTRA CC1 antigen repair, 3G2 and 7E2 antibodies produced weak cytoplasmic staining, but were absent when the samples were treated with phosphatase (see fig. 3A and 3B). Tests 81A, 7E2, 3G2, 11A5 and MJF-R13 (8) by prolonged phosphatase or moderate protease treatment-8) specificity for aggregated pS129-aSyn in Lewy bodies. The degree of lewy body staining in cortical brain sections from PD donors was either severe (with MJF-R13(8-8) and 81A) or moderate (with 11A5, 7E2 and 3G2) decreased after 2 hours at 36 ℃ in 8.8 μ G/mL of highly active recombinant alkaline phosphatase (see fig. 4A-4E). Thus, phosphatase treatment appeared to reduce non-lewy body staining without significantly affecting lewy body staining. In contrast, at 36 ℃ with VENTANA^TMProtease 1 treatment for 4 minutes had negligible effect on lewy body staining by 11a5, 7E2, or 3G2 antibodies in PD brain sections (see fig. 5A-5C). As a control, the same protease treatment removed most of the α -synuclein staining in PD brain by two different antibodies against S129 phosphorylated or non-S129 phosphorylated aSyn (LB509 and 5C12 monoclonal antibodies), except lewy bodies (data not shown).

Overall, in the absence of protease treatment, phosphorylation-specific α -synuclein antibodies 7E2 and 3G2 produced the same staining pattern in brain sections from subjects with PD as total α -synuclein antibody 5C12 after protease treatment (see fig. 6). However, 3G2 generally showed a higher background than 7E2 (data not shown). In addition to poor staining performance, #64 by 81A and WAKO of Abcam showed lower specificity compared to other antibodies against phosphorylated S129 α -synuclein. Unlike 3G2 and 7E2, 81A resulted in neurite staining in brain sections from non-PD individuals (data not shown). This likely reflects the reported cross-reactivity of 81A to the neurofilament light chain (NFL) phosphorylated at Ser 473. At low titers (1:1000), after phosphatase treatment, the #64 antibody clone from WAKO resulted in non-specific nuclear staining in brain sections from PD and non-PD subjects, and was not observed in the case of the other 7 antibody clones (data not shown).

In addition to the brain, the ability of the anti-pS 129-aSyn antibody clone to detect aggregated aSyn in skin samples from subjects with PD was also evaluated. All anti-pS 129-aSyn antibodies were able to detect aggregated aSyn in skin sections from subjects with PD in the absence of protease-based antigen repairAnalogous to the use of a combination with VENTANA^TMConditions for detection of protease 1 treated anti-total aSyn 5C12 antibody (see fig. 7A-7C). Antibody clones 81A, 7E2 and MJF-R13(8-8) were further tested on skin sections from subjects with PD and non-PD control subjects. It was not tested due to similar staining intensity and pattern of 3G2 and higher background compared to 7E2 antibody on brain samples. Staining with MJF-R13(8-8) was associated with non-specific background in smooth muscle and collagen, whereas 81A resulted in non-specific nuclear staining (data not shown).

Dilution and titration experiments were performed to optimize phosphorylated S129 α -synuclein staining using 7E2 antibody. The results of the diluent test showed that diluent 90040 and diluent 90103 had minimal background while maintaining staining intensity (see fig. 8A and 8B). Titration experiments showed that 7E2 dispensers at a concentration of 1. mu.g/mL had near maximum staining intensity and minimal background (data not shown). The titration experiment results were enhanced by a guard band experiment in which quantitative measurements were performed as a percentage of neuronal features stained per slide (see figure 9). Accelerated stability tests performed using the dual silver/yellow and single DAB detection protocols showed that 7E2 predicted a shelf life of 24 months at 4 ℃ (data not shown).

EXAMPLE 3 selection of immunohistochemically stained PGP9.5 antibody

Protein gene product 9.5(PGP9.5), more descriptively known as ubiquitin carboxy-terminal hydrolase L1(UCHL1), is a thioesterase essential for deubiquitination of ubiquitin-conjugated proteins and maintenance of free monoubiquitin pools 20-24. It is highly expressed in the brain and is estimated to account for 1% to 5% of total neuronal protein. PGP9.5 is commonly used as a marker for neuronal and neuroendocrine cells, but the expression of PGP9.5 is not limited to these cell types. Three different antibodies against PGP9.5 were evaluated for immunohistochemical detection of neurological features, neurons or neuroendocrine cells in skin, one from Cell Marque^TMThe RTD catalog of (P/N760-4434), and two monoclonal antibodies from Abcam (rabbit (EPR4118) and mouse (13C/I3C 4))). All three antibodies were able to specifically stain structures recognizable as neurons (see fig. 10A-10C). After a series of titrations of the three antibody concentrations, it was shown that 0.2 μ g/mL of EPR4118 and 13C/I3C4 had comparable staining intensity to the polyclonal antibody from Cell Marque at a dispenser concentration of 1.13 μ g/mL (see FIGS. 10A-10C). However, at these concentrations, the compounds reacted with EPR4118 or Cell Marque^TMThe 13C/I3C4 antibody showed a large amount of non-specific background staining compared to the polyclonal antibody (compare fig. 10B with fig. 10A and 10C).

The specificity of the EPR4118 antibody in the immunohistochemical background of phospho-S129-alpha-synuclein and PGP9.5 silver/yellow dual IHC assay was evaluated using a BC8 human tissue microarray containing 30 normal tissue cores and 29 cancer tissue cores from various anatomical sites (SuperBioChips Laboratories, Seoul, Korea). ToB/ToT analysis demonstrated staining of PGP9.5 in perivascular and glandular and neural and ganglion cells from various tissue sites. The specialized structures with innervation (islets) also stained positive for PGP9.5 (see tables 6-9). PGP9.5 staining was observed in renal tubules, rare stromal cells in the lung, germ cells in the testis, trophoblast cells in the placenta, and tumor cells from various types of cancer. These findings are consistent with the previously reported expression of PGP9.5 in various types of tumor and non-neuronal cell types.

Results of diluent testing, titration, guard bands and accelerated stability (data not shown) resulted in a aliquotte formulation of EPR4118 at 0.5. mu.g/mL in a DISCOVERY coat Ig Block (VENTANA P/N760-one 6008). In this formulation, EPR4118 predicted a shelf life of 24 months at 4 ℃ based on the accelerated stability test. The results of the inter-batch testing (data not shown) demonstrate that the performance of the EPR4118 antibody is consistent across four different production batches generated by two different manufacturers (Abcam and Spring).

Example 4 selection of aSyn and PGP9.5 assay and detection configurations

Protease resistance is a hallmark of many collectins including alpha-synuclein, and treatment with proteases enhances alpha in pathological forms-detection of synuclein. Roche pRED in cooperation with Prothena developed an assay with high specificity for aggregated a-synuclein in a cohort of PD and non-PD samples from BSHRI (subsequent transfer to Targos) (see table 4 and table 5). The pRED/Prothena aSyn assay is based on a 5C12 antibody that recognizes phosphorylated and unphosphorylated S129-aSyn byProtease 1 removes unaggregated aSyn and uses highly sensitiveAnd detecting by using an OptiView DAB IHC detection kit. However, while minimal background was observed with the pled/prophena aSyn assay protocol using skin sections that have been cut for at least several weeks, strong staining of collagen fibers was observed with freshly cut slides (see fig. 11A and 11B). Additional antigen repair with protease 1 (see figure 12A) exacerbates collagen background staining, but use of Ultra CC1 can reduce collagen background staining (see figure 12B). The reason for background staining of collagen was not the 5C12 antibody, since the 7E2 antibody produced the same collagen staining using the same detection protocol (data not shown). The background was also not due to non-specific adsorption of DAB by collagen fibers, as the ultraView DAB IHC detection kit did not generate any collagen staining (data not shown), but deposition of tyramide-TAMRA using the OptiView HQ Universal linker and HRP multimers (VENTANA pure kit, DISCOVERY, P/N760-. Finally, it was determined that OptiView HRP multimers were responsible for collagen fiber staining (data not shown). Physiological crosslinking of collagen fibers results in the formation of many types of adducts. anti-HQ IgG may cross-react with one or more of these adducts. This hypothesis is consistent with the reduction of reversed collagen background staining associated with CC1 treatment.

Investigation of a collection of scalp samples from BSHRI cohorts showed that the staining of collagen background DAB varied from light to heavy enough to interfere with the interpretation of specific signals from aggregated aSyn (data)Not shown). This prompted the search for an alternative detection method with sensitivity comparable to the OptiView DAB IHC detection kit for staining of aggregated aSyn. As shown in fig. 13, the ultraView Universal DAB detection kit consistently reduced the staining intensity for PGP9.5 compared to the OptiView DAB IHC detection kit. As a result, the ultraView Universal DAB detection kit was coupled with amplification using Ms anti-Rb IgG followed by Rb anti-Ms IgG ((S))Amplification kit, P/N760-080) was used to stain pS 129-aSyn. Even though the ultraView Universal DAB detection kit with amplification may have a similar staining intensity as the OptiView DAB IHC detection kit, the amplification process still produces a significant non-specific background (see fig. 14). High background staining was also observed when pS129-aSyn or PGP9.5 was detected using the VENTANA iView DAB detection kit (P/N760 and 091) (data not shown).

Using the 7E2 antibody, ultraView SISH DNP detection kit (P/N760-098) and DISCOVERY^TMThe Purple kit (P/N760. sup. + 229) was used to evaluate the staining of pS 129-aSynn in skin sections by two additional detection systems. As shown in fig. 15, both systems were able to detect aggregated pS129-aSyn with little background as long as the staining deposition was performed using goat anti-rabbit-HRP conjugate instead of OptiView HQ Universal linker and HRP multimer. In particular, using methods as described in the methods sectionDISCOVERY^TMUltraMap anti-Rb Alk Phos (P/N760-. The morphology of silver/black pS129-aSyn and yellow PGP9.5 staining in different types of neurological features in skin from subjects with PD is shown in fig. 17A-17E. Using the hornet assay configuration, two different types of pS129-aSyn staining were observed in the large nerve bundles of skin sections of subjects with PD: particulate dispersivityDyeing and discrete densitometric dyeing (see fig. 18). As described more fully in the subsequent sections, granular pS129-aSyn staining was observed in FFPE skin sections of both subjects with PD and non-PD subjects of the BSHRI cohort. In contrast, discrete pS129-aSyn staining was almost exclusively restricted to skin from subjects with PD. The subsequent section also describes the sensitivity of the 7E2 antibody and optimized bumblebee assay protocol against aggregated aSyn compared to the pled/prophena assay based on the 5C12 antibody, protease 1 and OptiView DAB IHC detection kit. Granular pS129-aSyn staining was shown generally as surrounding axons in sagittal nerve sections (see fig. 18) and peripheral neurons in transverse nerve sections (upper panel of fig. 19). Such staining pattern was similar to Schwann cell marker myelin basic protein (lower panel of fig. 19), indicating that pS129-aSyn protein responsible for the appearance of granular staining can be localized in Schwann cells. Accumulation of pS129-aSyn in Schwann cells has been previously described in patients with multiple system atrophy.

One assay configuration that has not been tested is antigen retrieval with protease 1 followed by incubation with 5C12 antibody and detection using the ultraView SISH DNP detection kit. This assay configuration potentially has similar sensitivity to aggregated aSyn with 7E2 antibody detected using the ultraView SISH DNP detection kit. However, it is not compatible with the dual assay incorporating PGP9.5 as a neuronal marker, since PGP9.5 requires antigen repair using Cell Conditioning (CC)1 or 2 bulk solutions, and the combination of protease 1 with CC1 or CC2 treatment results in a substantial loss of tissue morphology (data not shown).

Example 5 optimization of antigen repair conditions for phospho-aSyn staining with 7E2 antibody clone and PGP9.5 staining with EPR4118 antibody clone

Immunohistochemical detection of many epitopes is enhanced after antigen retrieval procedures. The effect of two different antigen retrieval procedures on the detection of pS129-aSyn in FFPE skin sections using the 7E2 antibody was evaluated:ULTRA cell conditioning (ULTRA CC1) bulk solutionAnd a protease. The effect of treatment with ULTRA CC1 or ULTRA CC2 solutions on PGP9.5 staining with EPR4118 antibody was also evaluated.

In contrast to most targets, staining of pS129-aSyn was abnormally reduced after only 32 minutes of treatment with ULTRA CC1 at 100 ℃ (see fig. 20A-20E). The negative effect of ULTRA CC1 on pS129-aSyn staining was observed with five different antibodies against pS 129-aSyn: 11A5, 7E2, 3G2, MJF-R13(8-8) and 81A (see FIGS. 20A-20E) indicate that phosphorylation of the S129 residue itself may have been affected by this treatment.

Protease treatment can improve detection of aggregated aSyn, but the underlying cause of this phenomenon is not clear. This may be due to removal of unaggregated aSyn after protease digestion or improved antibody access of antibodies into buried epitopes in protein aggregates or a combination of both. At 36 ℃ withProtease 2 treatment for 4 min (see FIGS. 21A and 21B) or withProtease 3 treatment for 4 or 12 minutes resulted in a moderate increase in discrete pS129-aSyn staining in skin sections from subjects with PD (see figure 22). A more significant effect of protease treatment was complete (protease 2 for 4 minutes or protease 3 for 12 minutes) or partial (protease 3 for 4 minutes) removal of granular pS129-aSyn staining in skin samples from subjects with PD or non-PD subjects (see fig. 21 and 22). This finding provides an opportunity for the pS129-aSyn assay, which has potentially higher specificity for aggregated aSyn. However, when the tissue was subsequently subjected to PGP9.5 detection with CC2 at 100 ℃, short term protease 3 treatment (4 minutes) failed to completely eliminate granular pS 129-aSynn staining, whereas protease 2 or extended protease 3 treatment (S) ((II))>4 minutes) resulted in a deterioration of tissue morphology (data not shown).

The possibility that the granular pS129-aSyn staining present in PD and non-PD samples could be completely eliminated while retaining a more specific detection scheme for the discrete pS129-aSyn staining visible only in PD samples led to the search for alternative methods to remove granular pS129-aSyn staining. Once the S129 residue is phosphorylated in the aggregated aYn, the attached phosphate group is more resistant to removal by protein phosphatases than phospho-S129 in soluble aYn (Waxman & Giasson, J Neuropathol Exp neuron 67(5):402- > 416(May 2008)). Mammalian alkaline phosphatase, a phosphatase with broad substrate specificity and exhibiting activity on serine phosphorylated proteins, was tested for its ability to selectively remove granular pS129-aSyn staining in FFPE skin sections. Treatment of deparaffinized skin sections with purified endogenous (New England Biolabs, NEB) or recombinant (Roche) calf intestinal alkaline phosphatase at pH9 or higher prior to incubation with 7E2 antibody prevented the appearance of granular pS129-aSyn staining without significantly affecting the level of discrete pS129-aSyn staining (see fig. 23A and 23B). Interestingly, alkaline phosphatase treatment at pH8 did result in reduced staining of discrete pS129-aSyn (see fig. 24). Sequential treatment with alkaline phosphatase to remove S129 phosphorylation in soluble aSyn followed by protease-mediated enhancement of antigen repair formed the basis of a surrogate pS129-aSyn and PGP9.5 silver/yellow dual IHC assay protocol (bumblebee assay protocol 2) with potentially higher specificity for aggregated aSyn (see figure 25).

Unlike pS129-aSyn, PGP9.5 detection using EPR4118 antibody increased with the duration of antigen repair using the ULTRA CC1 bulk solution (see figure 26). Antigen repair with ULTRA CC2 produced a similar enhancement in PGP9.5 staining (see fig. 27). Automated brightfield dual IHC assays require sequential antibody incubation and chromophore deposition cycles, as shown in figure 28. When two primary antibodies of the same species are used in a dual IHC assay protocol, a heat inactivation (HD) step in the form of ULTRA CC2 incubation at 100 ℃ is required to remove the primary and secondary antibodies deposited in the first cycle to prevent potential cross-reaction of the primary antibody in the first cycle with the secondary antibody in the second cycle. Although no heat inactivation step was required when the chromophore deposited in the first cycle was silver/black (the yellow stain deposited on top of the black stain remained black), treatment with CC2 for 16 minutes at 100 ℃ served as antigen retrieval using the EPR4118 antibody for PGP9.5 staining.

Example 6 analysis of reproducibility and accuracy

The experimental results described in the previous section led to two optimized protocols for automated phospho-S129-alpha-synuclein and PGP9.5 silver/yellow dual IHC assays (bumblebee assay protocol 1 and protocol 2). As detailed in the above section, scheme 1 does not contain an antigen retrieval step for the pS129-aSyn assay. Protocol 2 is identical to protocol 1 in all respects except that the samples were treated with alkaline phosphatase followed by protease after deparaffinization and prior to incubation with the 7E2 antibody. The in-and inter-run reproducibility of the bumblebee assay protocol 1 and protocol 2 was evaluated in scalp samples from three different subjects with PD. For on-the-fly reproducibility, slides from 5 serial sections of each PD subject were stained using protocol 1 or protocol 2, as described in the methods, and the number of neurological features with or without discrete pS129-aSyn was enumerated by the pathologist. PD subjects each had a discrete pS129-aSyn stained cutaneous nerve characteristic quantity as a percentage of total nerve characteristics averaged from 5 stained sections and are shown in table 11.

TABLE 11

Reproducibility of protocol 1 and protocol 2 in skin samples (on-the-fly)

For run-to-run reproducibility, triplicate slides from each of three different subjects with PD were stained on three different instruments using either protocol 1 or protocol 2 over discrete days. In operation, three slides from each PD subject were serially sectioned; however, between different runs, the slices are not continuous. The average percentage of neural features with discrete pS129-aSyn staining in each from the three runs is shown in table 12.

TABLE 12

Reproducibility of protocol 1 and protocol 2 in skin samples (run-to-run)

In the case of CV in excess of 15%, the most likely source of variation is the difference in the number of neurological features between consecutively sliced slides. Studies of the continuity of neural features between slides of successive slices led to the conclusion that in 5 to 10 4 μm slices, a difference of 10% to 20% in the number of neural features was attributable to the variation between slides in the feature distribution (see table 13). The intensity of PGP9.5 stained with the yellow DABSYL chromophore was 2.5 in all slides stained for the assay reproducibility study.

Watch 13

Continuity of neural features in skin tissue

Experiments were performed to analyze the accuracy of pS129-aSyn and PGP9.5 silver/yellow dual IHC assays and to score the percentage of characteristic staining of each in sections (approximately 25 characteristics). To analyze the inter-run accuracy, three runs of three slides were tested on the same instrument at least two days apart (protocol 1 CV: 2.77% dispersion, 5.13% dispersion; protocol 2 CV: 3.99% dispersion, 0.00% dispersion). To analyze the inter-instrument precision, two additional runs of three slides were performed on the new instrument and compared to the first run of the first instrument (protocol 1 CV: 3.22% dispersion, 11.86% dispersion; protocol 2 CV: 3.45% dispersion, 0.00% dispersion). In summary, the CV of the interplant and interplant accuracy of both the bumblebee assay protocol 1 and protocol 2 were below the 15% threshold required for acceptable transfer.

Example 7 assay verification-scalp samples

During the optimization of the phosphorylation- α -synuclein and PGP9.5 silver/yellow dual IHC assay (the nickname bumblebee assay), it was observed that a smooth discrete phosphorylation- α -synuclein staining pattern was only associated with a small cohort of PD scalp samples for feasibility testing. To determine whether this trend extends to a larger cohort of scalp samples from PD and non-PD subjects, scalp sections from 15 PD and 9 non-PD subjects were stained using protocol 1 and protocol 2 of the pS129-aSyn and PGP9.5 silver/yellow dual IHC assay (bumblebee assay). In addition, sections of a subset of this cohort containing 10 PD and 4 non-PD scalp samples were stained using an automated protease resistant aSyn DAB IHC assay (prad/prophena assay protocol). As shown in figure 29, 13 of the 15 samples from PD subjects (87%) exhibited a discrete pS129-aSyn after staining using either protocol 1 or protocol 2 of the bumblebee assay. Of the 9 non-PD scalp samples, 5 (56%) showed discrete pS129-aSyn staining using hornet protocol 1. In contrast, none of the 9 non-PD scalp samples showed discrete pS129-aSyn staining using bumblebee protocol 2 and mild protease and phosphatase treatment. Only diffuse granular type pS129-aSyn staining was observed with scheme 1, but not with scheme 2.

In a subset of subjects from which the results of the bumblebee assay protocol 1, the bumblebee assay protocol 2, and the prad/prophena assay protocol were available, discrete aSyn signals were observed in 14, and 13 PD scalp samples, respectively, of 15 stains (see fig. 30A). In 4 non-PD scalp samples stained using all three protocols, no discrete aSyn staining was observed with either the bumblebee protocol 2 or the prad/prophena protocol (see fig. 30A). Diffuse granular aSyn staining was observed in both PD and non-PD scalp samples using hornet protocol 1 (see fig. 30B). These results indicate that the detection of aggregated aSyn using a highly sensitive detection chemistry (metallic silver precipitation) in combination with antibodies against pS129-aSyn and methods based on antibodies against modified or unmodified aSyn combined with protease removal of unaggregated aSyn may have similar sensitivity.

Example 8 assay verification-Abdominal skin samples

To further evaluate the sensitivity and specificity of the phospho-aSyn and PGP9.5 silver/yellow dual IHC assay, abdominal skin samples from a cohort of 20 PD and 20 non-PD control subjects were stained using hornet assay protocol 1 and protocol 2. The results of the analysis are shown in fig. 31. Among the 20 PD abdominal skin samples stained using the hornet assay protocol 1 and protocol 2, discrete pS129-aSyn staining was observed in 19 and 17 samples, respectively. In contrast, none of the 20 non-PD abdominal skin samples showed any discrete pS129-aSyn signal. As also shown in fig. 31, the diffuse granular pS129-aSyn staining was present in most PD and non-PD abdominal skin samples stained using hornet assay protocol 1, and in PD samples stained at low levels using hornet assay protocol 2. These results indicate that there is a high correlation between the presence of discrete pS129-aSyn staining and PD clinical status in an extended cohort of abdominal skin from 20 PD and 20 non-PD subjects (36.2 and 29.6 pearson chi-square values for both protocols <0.0001 for bumblebee assay protocol 1 and protocol 2, respectively).

A cohort of 4 PD and 4 non-PD abdominal skin samples were provided as FFPE blocks and used for testing using the hornet assay protocol and the pled/prophena assay protocol based on 5C12 antibody and protease 1. The results of this analysis are shown in table 14 below.

TABLE 14

Percentage of neural features with discrete pS129-aSyn

Three of the four PD abdominal skin samples tested using the pled/prophena protocol showed a reduction in the percentage of neurological features with discrete pS129-aSyn compared to samples tested using either of the two bumblebee assay protocols. This was not observed in scalp samples stained using all three protocols (fig. 30). A possible explanation for the differences is that the tissue morphology of the abdominal skin sample is more susceptible to degradation by protease 1 treatment than the tissue morphology of the scalp sample. The reason for the significantly different sensitivity to protease 1 treatment is not clear at present. The abdominal slide was cut closer to its staining time than the scalp slide except for the anatomical site. In general, slides that had been cut at room temperature and left for a longer period (>30 days) prior to staining were observed to have a lower sensitivity to protease-induced morphological degradation (data not shown).

Example 9 assay validation scalp, colon and submandibular gland samples before S4

In addition to the skin, the colon and submandibular glands (SMG) are highly innervated and may be biopsy sites for detection of aggregated alpha-synuclein. Duplicate scalp, colon and SMG samples from 3 PD and 3 non-PD subjects collected at necropsy were stained using bumblebee assay protocol 2. Since previous studies on normal colon and SMG samples showed that protocol 1 generated heavily discrete phosphorylated alpha-synuclein stains, which can be removed by protease and phosphatase treatment using protocol 2 (see fig. 32A and 32B), protocol 1 was not employed. Typical images of discrete phosphorylated a-synuclein staining in three different tissue sites are shown (see fig. 33A-33C). The stained slides were assessed blindly by three independent readers (data not shown). Even if one of the readers used a different scoring method than the other readers, PD could be completely separated from non-PD samples based on the presence of discrete phosphorylated alpha-synuclein staining using the hornet protocol 2. Specificity and sensitivity were both 100%.

EXAMPLE 10 assay validation-skin biopsy samples

BSHRI scalp and abdominal skin samples for assay development and validation were obtained during necropsy. To evaluate the potential use of the phosphorylated S129 α synuclein and PGP9.5 silver/yellow dual IHC assay in diagnostic applications, FFPE sections from skin punch biopsies obtained during clinical visits with and without diagnosed PD symptoms were stained using two bumblebee assay protocols 1 and 2. Tissue sections from a total of 72FFPE blocks from 36 subjects were stained, as each subject had duplicate blocks. The stained slides were reviewed by a pathologist who was not aware of the clinical condition of the subjects. Since the samples are small needle biopsies of the skin, the number of neural features available per slide is limited (about 5 to 10). As a result, no quantitative measurements were made as a percentage of the silver stained nerve characteristics. Slides were considered positive for phosphorylated aSyn if there were discrete silver stains that were visibly non-grainy and of sufficient size to distinguish them from background psoriasis. The subject is considered overall positive if sections from one of the duplicate blocks are positive for phospho-aSyn staining. As in the pediatric skin biopsy samples, bumblebee protocol 1 generated a hyperphosphorylated-aSyn silver stain with a discrete morphology that was not present in the case of protocol 2 (see fig. 34). This resulted in 93% of 15 PD and 50% of 16 control subjects (69% in 36 subjects including 5 atypical parkinson's disease) developing positive discrete phosphorylated aSyn staining using regimen 1. In the case of regimen 2, 60% of 15 PD and 6% of 16 control subjects (31% of all 36 subjects) showed discrete phosphorylated aSyn signals (see tables 15 and 16). The respective non-blind results and corresponding PD status of individual tissue blocks of all 36 subjects are shown in table 16. Chi-square analysis of the results from all 36 subjects demonstrated a highly significant correlation between the presence of discrete phosphorylated aSyn signals and the assignment of PD status based on clinical symptoms (pearson chi-square value 10.506, DF 1, P0.001). When 5 patients with atypical parkinson's disease were excluded from the analysis, the association was still significant (pearson chi-square value 10.236, DF 1, P value 0.001).

Watch 15

phospho-Ser 129 alpha-synuclein (pS129-aSyn) staining in skin tissue of non-PD subjects using protocol 1 and protocol 2

TABLE 16

Non-blind phospho-Ser 129 a-synuclein (pS 129-aSynn) staining in skin tissue of non-PD subjects using protocol 1 and protocol 2

The results demonstrate that automated bisphosphorylation-aSyn and PGP9.5 immunohistochemistry assays have high sensitivity and specificity based on the presence of discrete phosphorylation-aSyn staining in skin biopsies for distinguishing subjects with PD from non-PD control subjects. The morphological appearance of the discrete phosphorylated aSyn stain and its resistance to protease and phosphatase treatment was consistent with aggregated aSyn. The assay may be of potential value in identifying subjects with PD and/or candidates for therapeutic approaches that target aggregation of aSyn.

As used herein, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "comprising a cell" includes a single or a plurality of cells, and is considered equivalent to the phrase "comprising at least one cell". The term "or" refers to a single element or a combination of two or more elements of the recited alternative element, unless the context clearly dictates otherwise. As used herein, "comprising" means "including". Thus "comprising a or B" means "including A, B, or a and B", without excluding other elements.

The foregoing is provided for exemplary purposes only and is not intended to limit the scope of the present disclosure, which is broadly described above. All references cited in this disclosure are incorporated herein by reference.

Although various specific embodiments of the present disclosure have been described herein, it is to be understood that the present disclosure is not limited to those precise embodiments, and that various changes or modifications may be effected therein by one skilled in the art without departing from the scope or spirit of the present disclosure.

The examples given above are merely illustrative and are not meant to be an exhaustive list of all possible embodiments, applications or modifications of the disclosure. Thus, various modifications and variations of the described methods and systems of the present disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the present disclosure has been described in connection with specific embodiments, it should be understood that the present disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure which are obvious to those skilled in molecular biology, immunology, chemistry, biochemistry or related fields are intended to be within the scope of the following claims.

It is to be understood that this disclosure is not limited to the particular methodology, protocols, reagents, etc., described herein as these may vary, as will be recognized by the skilled artisan. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure.

Embodiments of the present disclosure and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments and/or illustrated in the accompanying drawings and detailed in the following description. It should be noted that the features illustrated in the drawings are not necessarily drawn to scale and features of one embodiment may be employed with other embodiments as the skilled artisan will recognize, even if not explicitly stated herein.

Any numerical value recited herein includes all values from the lower value to the upper value, in increments of one unit, provided that there is a separation of at least two units between any lower value and any upper value. For example, if it is stated that the concentration of a component or a value of a process variable such as, for example, magnitude, angular magnitude, pressure, time, etc., is from 1 to 90, specifically from 20 to 80, more specifically from 30 to 70, it is intended that equivalents such as 15 to 85, 22 to 68, 43 to 51, 30 to 32, etc., be expressly enumerated in this specification. For values less than 1, one unit is considered to be 0.0001, 0.001, 0.01, or 0.1, as appropriate. These are only examples of what is specifically intended, and all possible combinations of numerical values between the minimum and maximum values recited should be considered to be expressly stated in this application in a similar manner.

Specific methods, devices, and materials are described, although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. The disclosures of all references and publications cited herein are expressly incorporated by reference in their entirety as if each were incorporated by reference.

Sequence listing

<110> F. HOFFMANN-LA ROCHE AG

HOFFMANN LA-ROCHE, INC.

VENTANNA MEDICAL SYSTEMS, INC.

ROCHE DIAGNOSTICS OPERATIONS, INC.

PROTHENA BIOSCIENCES LIMITED

ROCHE DIAGNOSTIS GMBH

<120> determination of Parkinson's disease

<130> 17-1472-WO

<150> US 62/716,504

<151> 2018-08-09

<160> 59

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Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 18

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 18

Lys Ser Ile Gln Thr Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 19

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 19

Trp Ala Ser Ile Arg Lys Ser

1 5

<210> 20

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 20

Gln Gln Tyr Tyr Ser Tyr Pro Leu Thr

1 5

<210> 21

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 21

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Asn Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ala Gly Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 22

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 22

Asn Tyr Gly Met Ser

1 5

<210> 23

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 23

Ser Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Asn Val Lys

1 5 10 15

Gly

<210> 24

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 24

Gly Gly Ala Gly Ile Asp Tyr

1 5

<210> 25

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ile Gln Thr Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg Lys Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 26

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 26

Lys Ser Ile Gln Thr Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 27

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 27

Trp Ala Ser Ile Arg Lys Ser

1 5

<210> 28

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 28

Gln Gln Tyr Tyr Ser Tyr Pro Leu Thr

1 5

<210> 29

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 29

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Asn Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ala Gly Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 30

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 30

Asn Tyr Gly Met Ser

1 5

<210> 31

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 31

Ser Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Asn Val Lys

1 5 10 15

Gly

<210> 32

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 32

Gly Gly Ala Gly Ile Asp Tyr

1 5

<210> 33

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 33

Asp Val Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Asn Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly His

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Thr Ser Leu Gln Thr Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 34

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 34

Lys Ser Ser Gln Asn Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 35

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 35

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 36

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 36

Gln Gln Tyr Tyr Ser Ser Pro Leu Thr

1 5

<210> 37

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 37

Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Ala Gly Asn Gly Lys Arg Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Asp Arg Val Thr Ile Asn Arg Asp Thr Ser Ala Ser Thr Ile Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Gly Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Glu Asp His Ala Gly Ser Gly Ser Tyr Leu Ser Met Asp

100 105 110

Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 38

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 38

Asn Tyr Ala Met His

1 5

<210> 39

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 39

Trp Ile Asn Ala Gly Asn Gly Lys Arg Lys Tyr Ser Gln Lys Phe Gln

1 5 10 15

Asp

<210> 40

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 40

Glu Glu Asp His Ala Gly Ser Gly Ser Tyr Leu Ser Met Asp Val

1 5 10 15

<210> 41

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 41

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Glu Ala Leu Pro Met Gln Phe Ala

20 25 30

His Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Val Ile Val Val Tyr

35 40 45

Lys Asp Ser Glu Arg Pro Ser Gly Val Pro Glu Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Thr Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Pro Asp Ser Thr Asn Thr Tyr

85 90 95

Glu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 42

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 42

Ser Gly Glu Ala Leu Pro Met Gln Phe Ala His

1 5 10

<210> 43

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 43

Lys Asp Ser Glu Arg Pro Ser

1 5

<210> 44

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 44

Gln Ser Pro Asp Ser Thr Asn Thr Tyr Glu Val

1 5 10

<210> 45

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 45

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Glu Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Asp Phe Glu Lys Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Gln Trp Val

35 40 45

Ala Arg Ile Lys Ser Thr Ala Asp Gly Gly Thr Thr Ser Tyr Ala Ala

50 55 60

Pro Val Glu Gly Arg Phe Ile Ile Ser Arg Asp Asp Ser Arg Asn Met

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Ser Ala His Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser

<210> 46

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 46

Lys Ala Trp Met Ser

1 5

<210> 47

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 47

Arg Ile Lys Ser Thr Ala Asp Gly Gly Thr Thr Ser Tyr Ala Ala Pro

1 5 10 15

Val Glu Gly

<210> 48

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 48

Thr Ser Ala His

1

<210> 49

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 49

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Glu Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Asp Phe Glu Lys Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Gln Trp Val

35 40 45

Ala Arg Ile Lys Ser Thr Ala Asp Gly Gly Thr Thr Ser Tyr Ala Ala

50 55 60

Pro Val Glu Gly Arg Phe Ile Ile Ser Arg Asp Asp Ser Arg Asn Met

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Ser Ala His Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser

<210> 50

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 50

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Glu Ala Leu Pro Met Gln Phe Ala

20 25 30

His Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Val Ile Val Val Tyr

35 40 45

Lys Asp Ser Glu Arg Pro Ser Gly Val Pro Glu Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Thr Thr Ala Thr Leu Thr Ile Thr Gly Val Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Pro Asp Ser Thr Asn Thr Tyr

85 90 95

Glu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 51

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 51

Asn Tyr Ala Met His

1 5

<210> 52

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 52

Trp Ile Asn Ala Gly Asn Gly Lys Arg Lys Tyr Ser Gln Lys Phe Gln

1 5 10 15

Asp

<210> 53

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 53

Glu Glu Asp His Ala Gly Ser Gly Ser Tyr Leu Ser Met Asp Val

1 5 10 15

<210> 54

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 54

Lys Ser Ser Gln Asn Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 55

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 55

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 56

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 56

Gln Gln Tyr Tyr Ser Ser Pro Leu Thr

1 5

<210> 57

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 57

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Asn Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ala Gly Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 58

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 58

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ile Gln Thr Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg Lys Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Leu Ala Thr Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 59

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> residue 122-135 corresponding to human alpha-synuclein

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> serine residue is phosphorylated

<400> 59

Asn Glu Ala Tyr Glu Met Pro Ser Glu Glu Gly Tyr Gln Asp

1 5 10