Infrared assay detecting secondary structure profiles of alpha-synuclein

Patent metadata

Field	Value
Publication number	US-11237176-B2
Application number	US-201716346841-A
Country	US
Kind code	B2
Filing date	Dec 1, 2017
Priority date	Dec 2, 2016
Publication date	Feb 1, 2022
Grant date	Feb 1, 2022

Abstract

The invention provides an infrared assay which allows the secondary structure analysis of alpha-synuclein from complex fluids like serum, blood plasma or cerebrospinal fluid without prior isolation, concentration or pretreatment. The secondary structure profile provides an indication of the proportion of alpha-synuclein in aggregated form and/or extent of aggregation of alpha-synuclein in aggregated form.

First claim

What is claimed is:

1. A method for determining a profile of secondary structure of alpha-synuclein in a sample, comprising:
a. introducing the sample into a cell comprising an infrared source, an infrared sensor element linked to an antibody, wherein the antibody is an antibody comprising CDRs H1, H2 and H3 of SEQ ID NOS:12-14 respectively and CDRs L1, L2 and L3 of SEQ ID NOS:15-17 respectively, and an infrared detector, whereby alpha-synuclein in the sample binds to the antibody on the surface of the infrared sensor element;
b. submitting an infrared beam from the infrared source through the infrared sensor to the infrared detector to obtain an infrared spectrum characterizing the alpha-synuclein of the sample; and
c. analyzing the obtained infrared spectrum to determine a secondary structure profile of the alpha-synuclein in the sample.

2. The method of claim 1 , wherein the secondary structure profile provides an indication of the proportion of alpha-synuclein in aggregated form and/or extent of aggregation of alpha-synuclein in aggregated form.

3. The method of claim 1 , wherein step (c) comprises analyzing the shift of an amide I band maximum of alpha-synuclein to determine the secondary structure of alpha-synuclein, an amide I band maximum frequency of 1646 cm −1 indicating monomeric alpha-synuclein and a lower amide I band maximum frequency of alpha-synuclein indicating aggregated alpha-synuclein.

4. The method of claim 3 , wherein the sample is from a patient with Parkinson's disease, and the amide I band maximum frequency of the sample occurs below 1646 cm −1 indicating aggregated alpha-synuclein.

5. The method of claim 4 , wherein the patient has prodromal Parkinson's disease.

6. The method of claim 5 , wherein the patient has mild Parkinson's disease.

7. The method of claim 5 , wherein the patient has moderate Parkinson's disease.

8. The method of claim 5 , wherein the patient has advanced Parkinson's disease.

9. The method of claim 1 , wherein step (c) comprises comparing the obtained infrared spectrum with a spectrum of alpha-synuclein with a known secondary structure and/or with a known concentration.

10. The method of claim 1 , wherein the infrared sensor element comprises a germanium internal reflection element of trapezoid or parallelogram shape transparent in the infrared with sufficient signal to noise ratio to detect an amide I band.

11. The method of claim 1 , wherein the antibodies are linked to the infrared sensor by silane or thiol linkers.

12. The method of claim 1 , further comprising: (i) detecting a signal, parallel to the infrared analysis, by another optical method, including UV/Vis-fluorescence, at different wavelengths; and/or (ii) combining immuno-ATR-IR vibrational spectroscopy with parallel fluorescence spectroscopy.

13. The method of claim 1 , wherein the antibody comprises a mature heavy chain variable region of SEQ ID NO:10 and a mature light chain variable region of SEQ ID NO:11.

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

15. The method of claim 14 , wherein the human has a Lewy body disease.

16. The method of claim 14 , wherein the human has Parkinson's disease.

17. The method of claim 16 , wherein the human is receiving immunotherapy for the disease.

18. The method of claim 1 , wherein the sample is from a transgenic mouse with a transgene expressing human alpha-synuclein.

19. The method of claim 18 performed multiple times on samples from the same human to detect changes in profile over time.

20. The method of claim 1 , wherein the sample is a body fluid.

21. The method of claim 20 , wherein the sample is cerebrospinal fluid (CSF) or blood of a human.

22. The method of claim 1 , wherein the sample is a brain homogenate of a human or transgenic animal.

23. The method of claim 22 , wherein the human is receiving a regime of immunotherapy and the method further comprises changing the regime in response to changes in the profile over time.

24. The method of claim 1 , wherein the sample is a medium used to culture cells wherein the cells express recombinant human alpha-synuclein.

25. The method of claim 1 , performed on a population of subjects, wherein a greater proportion of subjects with a level of the amide I band maximum frequency of alpha-synuclein below a threshold receive treatment for Parkinson's disease than subjects in which the level of the amide I band maximum frequency of alpha-synuclein is above the threshold.

26. The method of claim 25 , the threshold of the amide I band maximum frequency is 1643 cm −1 .

Assignees

• Prothena Biosciences Ltd

Inventors

• Barbour Robin

Classifications

• G01N2333/4709

  Amyloid plaque core protein · CPC title

• A61P25/16 Primary

  Anti-Parkinson drugs · CPC title

• G01N21/552

  Attenuated total reflection · CPC title

• C07K2317/34

  Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues · CPC title

• C07K16/18

  against material from animals or humans · CPC title