# US 11,906,530 B2 · Methods for the detection of tau protein aggregates

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- Number: US 11,906,530 B2
- Document ID: US11906530B2
- Status: Granted
- Kind: B2
- Application number: 16/474,040
- Filed: 2017-12-29
- Published: 2020-08-06
- Granted: 2024-02-20
- Earliest claimed: 2016-12-30
- Assignees: The United States of America, as represented by the Secretary, Department of Health and Human Services
- Applicants: THE UNITED STATES OF AMERICA, as represented by the Secretary, Department of Health and Human Services
- Inventors: Byron Winslow Caughey; Eri Saijo; Allison Lindsey Kraus; Michael Anthony Metrick, II
- Web page: https://patentgrep.com/patent/US11906530B2

## CPC

- G01N 33/6896: {Neurological disorders, e.g. Alzheimer's disease}
- G01N 2800/2821: Alzheimer
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Methods are disclosed herein for determining whether a subject has a Tauopathy, such as Pick disease, Alzheimer disease, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) or argyrophilic grain disease (AGD). These methods utilize an amyloid seeding assay.

## Claims

### Claim 1

A method of determining whether a subject has a 3R/4R Tauopathy, comprising:
(a) performing a seeded Tau polymerization assay on a biological sample from the subject, comprising:
(i) contacting the biological sample with a purified recombinant truncated Tau protein, wherein the recombinant truncated Tau protein consists of SEQ ID NO: 43 or SEQ ID NO: 44, to form a reaction mixture;
(ii) incubating the reaction mixture to permit coaggregation of disease-associated Tau protein (TD) present in the biological sample with the recombinant truncated Tau protein;
(iii) maintaining incubation conditions that promote coaggregation of the recombinant truncated Tau protein with the TD to result in a conversion of the recombinant truncated Tau protein to amyloid Tau protein aggregates while inhibiting development of spontaneously forming Tau protein aggregates (rT(spon)); and
(iv) agitating amyloid Tau protein aggregates formed during step (iii), wherein the agitating comprises shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking; and
(b) detecting amyloid Tau protein present in the reaction mixture, and wherein detection of amyloid Tau protein in the reaction mixture indicates that the subject has the 3R/4R Tauopathy.

### Claim 2 (depends on 1)

The method of claim 1, wherein the biological sample is a brain tissue sample or a cerebral spinal fluid sample.

### Claim 3 (depends on 1)

The method of claim 1, wherein the biological sample is a nasal brushing, saliva, blood, serum, plasma, cerebral spinal fluid, feces, urine or tissue sample.

### Claim 4 (depends on 1)

The method of claim 1, wherein detecting the presence of Tau amyloid in the biological sample comprises the use of an amyloid-sensing dye.

### Claim 5 (depends on 1)

The method of claim 1, wherein agitating aggregates in step (iv) comprises agitating aggregates in the absence of sonication.

### Claim 6 (depends on 1)

The method of claim 1, wherein the shaking cycle in step (iv) comprises a period of rest that precedes the period of shaking, and wherein the period of rest and the period of shaking are equal.

### Claim 7 (depends on 1)

The method of claim 1, wherein the shaking cycle in step (iv) includes the period of rest and the period of shaking at a ratio of about 1:2 to about 2:1.

### Claim 8 (depends on 1)

The method of claim 1, wherein the shaking cycle in step (iv) is 20 to 180 seconds in length.

### Claim 9 (depends on 8)

The method of claim 8, shaking cycle in step (iv) is 120 seconds in length.

### Claim 10 (depends on 1)

The method of claim 1, wherein the reaction mix includes thioflavin T (ThT), and wherein detecting amyloid Tau protein comprises detecting fluorescence.

### Claim 11 (depends on 1)

The method of claim 1, wherein the recombinant truncated Tau protein has an N-terminus or a C-terminus, and the recombinant Tau protein comprises at least six consecutive histidine residues at or near the N-terminus or the C-terminus.

### Claim 12 (depends on 11)

The method of claim 11, wherein the recombinant truncated Tau protein is purified using immobilized metal ion affinity chromatography.

### Claim 13 (depends on 1)

The method of claim 1, wherein the reaction mix comprises a carrier lacking human Tau.

### Claim 14 (depends on 13)

The method of any claim 13, wherein the carrier is a brain homogenate lacking human Tau.

### Claim 15 (depends on 14)

The method of claim 14, wherein the human Tau-free brain homogenate is a murine brain homogenate from a mouse deficient for the production of murine Tau protein.

### Claim 16 (depends on 1)

The method of claim 1, wherein the reaction mixture further comprises an effective amount of N2.

### Claim 17 (depends on 1)

The method of claim 1, wherein steps (a)-(b) are performed in the absence of added anionic detergent.

### Claim 18 (depends on 1)

The method of claim 1, wherein agitating amyloid Tau protein aggregates formed during step (iii) is performed for at least about 10 hours.

### Claim 19 (depends on 1)

The method of claim 1, wherein agitating amyloid Tau protein aggregates formed during step (iii) is performed for at least about 15 hours.

### Claim 20 (depends on 4)

The method of claim 4, wherein the amyloid sensing dye is thioflavin T or thioflavin S.

### Claim 21 (depends on 12)

The method of claim 12, wherein the recombinant truncated Tau protein is purified by elution from a column comprising an immobilized metal ion using between about 46 mM and about 200 mM imidazole.

### Claim 22 (depends on 1)

The method of claim 1, wherein the reaction mixture comprises 1 to 10 beads per 50 μl or 100 μl of reaction mixture.

### Claim 23 (depends on 22)

The method of claim 22, wherein the reaction mixture comprises 1 bead per 50 μl of reaction mixture.

### Claim 24 (depends on 22)

The method of claim 22, wherein the beads are about 0.5 mm to about 3 mm in diameter.

### Claim 25 (depends on 24)

The method of claim 24, wherein the beads are about 800 μm in diameter.

### Claim 26 (depends on 1)

The method of claim 1, wherein the 3R/4R Tauopathy is Alzheimer disease.

### Claim 27 (depends on 1)

The method of claim 1, wherein step (i) comprises contacting the biological sample with a first purified recombinant truncated Tau protein consisting of SEQ ID NO: 43 and the reaction mixture further comprises a second purified recombinant truncated Tau protein.

### Claim 28 (depends on 27)

The method of claim 27, wherein the second recombinant truncated Tau protein has three microtubule binding domains.

### Claim 29 (depends on 28)

The method of claim 28, wherein the second recombinant truncated Tau protein comprises SEQ ID NO: 14.

### Claim 30 (depends on 1)

The method of claim 1, wherein the reaction mixture comprises heparin.

### Claim 31

A method of detecting 3R/4R Tau protein aggregates, comprising:
(a) performing a seeded Tau polymerization assay on a biological sample from a subject, comprising:
(i) contacting the biological sample with a purified recombinant truncated Tau protein, wherein the recombinant truncated Tau protein consists of SEQ ID NO: 43 or SEQ ID NO: 44, to form a reaction mixture;
(ii) incubating the reaction mixture to permit coaggregation of 3R/4R disease-associated Tau protein (TD) present in the biological sample with the recombinant truncated Tau protein;
(iii) maintaining incubation conditions that promote coaggregation of the recombinant truncated Tau protein with the TD to result in a conversion of the recombinant truncated Tau protein to amyloid Tau protein aggregates while inhibiting development of spontaneously forming Tau protein aggregates (rT(spon)); and
(iv) agitating amyloid Tau protein aggregates formed during step (iii), wherein the agitating comprises shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking; and
(b) detecting amyloid Tau protein present in the reaction mixture.

### Claim 32 (depends on 31)

The method of claim 31, wherein step (i) comprises contacting the biological sample with a first purified recombinant truncated Tau protein consisting of SEQ ID NO: 43 and the reaction mixture further comprises a second purified recombinant truncated Tau protein.

### Claim 33 (depends on 32)

The method of claim 32, wherein the second recombinant truncated Tau protein has three microtubule binding domains.

### Claim 34 (depends on 33)

The method of claim 33, wherein the second recombinant truncated Tau protein comprises SEQ ID NO: 14.

### Claim 35 (depends on 31)

The method of claim 31, wherein the biological sample comprises a brain tissue sample or a cerebral spinal fluid sample.