US-12624388-B2 - Methods and kits for detecting tau

Abstract

The invention relates to methods and kits for assessing brain injury, e.g., traumatic brain injury resulting from blast exposure. The invention provides methods of quantifying the amount of phosphorylated tau or total tau in a biological sample. The invention further provides a method of determining the number of blast exposures experienced by a subject. Also provided herein are kits for detecting phosphorylated tau or total tau in a biological sample.

Inventors

• Christopher Campbell
• Kimbra Kenney
• Jessica GILL

Assignees

• MESO SCALE TECHNOLOGIES, LLC.
• THE HENRY M. JACKSON FOUNDATION FOR THE ADVANCEMENT OF MILITARY MEDICINE, INC.
• THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES

Dates

Publication Date

20260512

Application Date

20211108

Claims (20)

1. 1 . A method of quantifying the amount of phosphorylated tau (p-tau) in a biological sample, wherein the method comprises: a) contacting the biological sample with: (i) a first capture reagent that binds to non-phosphorylated tau; (ii) a second capture reagent that binds to non-phosphorylated tau, wherein the first and second capture reagents are on a surface, and the surface further comprises an anchoring reagent; (iii) a first detection reagent that binds to non-phosphorylated tau, wherein the first detection reagent comprises a first nucleic acid probe; and (iv) a second detection reagent that binds to p-tau and does not bind to non-phosphorylated tau, wherein the second detection reagent comprises a second nucleic acid probe, thereby forming a complex on the surface comprising the first and second capture reagents, p-tau, and the first and second detection reagents; b) using an extension process that requires the first and second nucleic acid probes to be in proximity, extending the second nucleic acid probe to form an extended sequence comprising an anchoring region that binds to the anchoring reagent; c) binding the extended sequence to the anchoring reagent; and d) measuring the amount of extended sequence bound to the surface, thereby quantifying the amount of p-tau.
2. 2 . The method of claim 1 , wherein the p-tau is phosphorylated at amino acid position T175, T181, T212, S214, cis T231, trans T231, S293, S396, S610, or a combination thereof, wherein the amino acid position corresponds to SEQ ID NO:1.
3. 3 . The method of claim 1 , wherein each capture reagent and detection reagent comprises an antibody or antigen-binding fragment thereof, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or an aptamer.
4. 4 . The method of claim 1 , wherein the extending comprises polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), self-sustained synthetic reaction (3SR), isothermal amplification, or combination thereof.
5. 5 . The method of claim 1 , wherein the extending comprises binding the first and second nucleic acid probes to a template oligonucleotide and extending the second nucleic acid probe by PCR.
6. 6 . The method of claim 1 , wherein the anchoring reagent comprises an anchoring oligonucleotide, and wherein the extended sequence comprises an anchoring oligonucleotide complement that is complementary to the anchoring oligonucleotide, and a detection oligonucleotide complement that is complementary to a detection oligonucleotide; and wherein the measuring comprises contacting the extended sequence with a labeled probe comprising the detection oligonucleotide and a detectable label, and measuring the amount of detectable label bound to the surface.
7. 7 . The method of claim 1 , wherein the detection reagent comprises a detectable label, and wherein the detectable label is an electrochemiluminescence (ECL) label, and the measuring comprises measuring an ECL signal.
8. 8 . The method of claim 1 , wherein the surface comprises a particle.
9. 9 . The method of claim 1 , wherein the surface comprises an electrode, and the measuring further comprises applying a potential to the electrode and measuring electrochemiluminescence.
10. 10 . The method of claim 1 , wherein the surface comprises a plurality of distinct binding domains, and the first capture reagent, the second capture reagent, and the anchoring reagent are located on two or more distinct binding domains on the surface.
11. 11 . A method of determining the number of blast exposures experienced by a subject, comprising a) conducting the method of claim 1 on a biological sample of the subject to quantify the amount of p-tau in the biological sample; and b) determining the number of blast exposures experienced by the subject based on the amount of p-tau.
12. 12 . The method of claim 1 , wherein the biological sample is whole blood, blood serum plasma, cerebrospinal fluid, urine, saliva, or an extraction or purification therefrom, or dilution thereof; and/or wherein the biological sample comprises an exosome.
13. 13 . The method of claim 1 , wherein the biological sample is obtained from a subject exposed to blast, at risk of exposure to blast, or suspected of having been exposed to blast.
14. 14 . The method of claim 1 , wherein the extending comprises binding the first and second nucleic acid probes to a template oligonucleotide, forming a circular template oligonucleotide, and extending the second nucleic acid probe by rolling circle amplification (RCA).
15. 15 . The method of claim 1 , wherein the surface comprises a well of a multi-well plate.
16. 16 . The method of claim 1 , wherein the surface comprises a particle, and the method further comprises collecting the particle on an electrode, and the measuring further comprises applying a potential to the electrode and measuring electrochemiluminescence.
17. 17 . The method of claim 1 , wherein the surface comprises a plurality of distinct binding domains, and the first capture reagent, the second capture reagent, and the anchoring reagent are located on the same binding domains on the surface.
18. 18 . A method of quantifying the amount of phosphorylated tau (p-tau) in a biological sample, wherein the method comprises: a) contacting the biological sample with: (i) a first capture reagent that binds to non-phosphorylated tau; (ii) a second capture reagent that binds to non-phosphorylated tau, wherein the first and second capture reagents are on a surface, and the surface further comprises an anchoring reagent; (iii) a detection reagent that binds to p-tau and does not bind to non-phosphorylated tau, wherein the detection reagent comprises a nucleic acid probe, thereby forming a complex on the surface comprising the first and second capture reagents, p-tau, and the detection reagent; b) extending the nucleic acid probe to form an extended sequence comprising an anchoring region that binds to the anchoring reagent; c) binding the extended sequence to the anchoring reagent; and d) measuring the amount of extended sequence bound to the surface, thereby quantifying the amount of p-tau.
19. 19 . The method of claim 18 , wherein the extending comprises binding the nucleic acid probe to a template oligonucleotide, forming a circular template oligonucleotide, and extending the nucleic acid probe by rolling circle amplification (RCA).
20. 20 . A method of quantifying the amount of phosphorylated tau (p-tau) in a biological sample, wherein the method comprises: a) contacting the biological sample with: (i) a first capture reagent that binds to non-phosphorylated tau; (ii) a second capture reagent that binds to non-phosphorylated tau, wherein the first and second capture reagents are on a surface; (iii) a detection reagent that binds to p-tau and does not bind to non-phosphorylated tau, wherein the detection reagent comprises a detectable label, thereby forming a complex on the surface comprising the first and second capture reagents, p-tau, and the detection reagent; and b) measuring the amount of detectable label on the surface, thereby quantifying the amount of p-tau.

Description

GOVERNMENT LICENSE RIGHTS This invention was made with government support under award number W81XWH-17-1-0648 awarded by the United States Department of Defense. The government has certain rights in the invention. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 6, 2022, is named 0076-0030US1_SL.txt and is 4,305 bytes in size. FIELD OF THE INVENTION The invention relates to methods and kits for assessing brain injury, e.g., traumatic brain injury resulting from blast exposure. The invention provides methods of quantifying the amount of tau, e.g., phosphorylated tau and total tau in a biological sample. The invention further provides a method of determining the number of blast exposures experienced by a subject. Also provided herein are kits for detecting tau, e.g., phosphorylated tau and total tau in a biological sample. BACKGROUND Traumatic brain injury (TBI), specifically repeated blast exposures, can cause life-long symptoms in some individuals. Military personnel are particularly susceptible to TBI. In the most severe cases, individuals can develop chronic traumatic encephalopathy (CTE), which is progressive neurodegeneration similar to Alzheimer's disease. Currently, it is difficult to assess an individual's likelihood of developing CTE and to monitor the rate of progression in living donors. Since symptoms of CTE are non-specific, assessments of neurologic and cognitive function may be confounded by other conditions. A method of assessing risk for neurodegeneration and monitoring rate of neurodegeneration would be valuable for clinical trials of experimental treatments to halt neurodegeneration after TBI. Such methods would aid in enriching clinical trials with suitable participants and as a proximal indicator of efficacy. SUMMARY OF THE INVENTION In embodiments, the invention provides a method of quantifying the amount of phosphorylated tau (p-tau) in a biological sample, comprising: (a) contacting the biological sample with: (i) a first capture reagent that binds non-phosphorylated tau; (ii) a second capture reagent that binds non-phosphorylated tau, wherein the first and second capture reagents are on a surface, and the surface further comprises an anchoring reagent; (iii) a first detection reagent that binds non-phosphorylated tau, wherein the first detection reagent comprises a first nucleic acid probe; and (iv) a second detection reagent that binds p-tau, wherein the second detection reagent comprises a second nucleic acid probe, thereby forming a complex on the surface comprising the first and second capture reagents, p-tau, and the first and second detection reagents; (b) using an extension process that requires the first and second nucleic acid probes to be in proximity, extending the second nucleic acid probe to form an extended sequence comprising an anchoring region that binds to the anchoring reagent; (c) binding the extended sequence to the anchoring reagent; and (d) measuring the amount of extended sequence bound to the surface, thereby quantifying the amount of p-tau. In embodiments, the invention provides a method of quantifying the amount of phosphorylated tau (p-tau) in a biological sample, comprising: (a) contacting the biological sample with: (i) a first capture reagent that binds non-phosphorylated tau; (ii) a second capture reagent that binds non-phosphorylated tau, wherein the first and second capture reagents are on a surface, and the surface further comprises an anchoring reagent; (iii) a detection reagent that binds p-tau, wherein the detection reagent comprises a nucleic acid probe, thereby forming a complex on the surface comprising the first and second capture reagents, p-tau, and the detection reagent; (b) extending the nucleic acid probe to form an extended sequence comprising an anchoring region that binds to the anchoring reagent; (c) binding the extended sequence to the anchoring reagent; and (d) measuring the amount of extended sequence bound to the surface, thereby quantifying the amount of p-tau. In embodiments, the invention provides a method of quantifying the amount of phosphorylated tau (p-tau) in a biological sample, comprising: (a) contacting the biological sample with: (i) a first capture reagent that binds non-phosphorylated tau; (ii) a second capture reagent that binds non-phosphorylated tau, wherein the first and second capture reagents are on a surface; (iii) a detection reagent that binds p-tau, wherein the detection reagent comprises a detectable label, thereby forming a complex on the surface comprising the first and second capture reagents, p-tau, and the detection reagent; and (c) measuring the amount of detectable label on the surface, thereby quantifying the amount of p-tau. In embodiments, the invention provides a method of quantifying the amount of total tau (t-tau) in a biological