# US 11,079,392 B2 · Methods and compositions for detecting misfolded proteins

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- Number: US 11,079,392 B2
- Document ID: US11079392B2
- Status: Granted
- Kind: B2
- Application number: 16/406,942
- Filed: 2019-05-08
- Published: 2019-11-21
- Granted: 2021-08-03
- Earliest claimed: 2014-04-10
- Assignees: Yale University
- Applicants: Yale University
- Inventors: Irina Buhimschi; Catalin S. Buhimschi; Michael Choma; Hemant Tagare; Stephan Michael Jonas
- Web page: https://patentgrep.com/patent/US11079392B2

## CPC

- G01N 33/689: {related to pregnancy or the gonads}
- G01N 33/52: Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper {and including single- and multilayer analytical elements (immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72)}
- G01N 33/68: involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)}
- G01N 33/6839: {involving dyes, e.g. Coomassie blue, bromcresol green}
- C09B 69/106: {containing an azo dye}
- G01N 21/78: producing a change of colour
- G01N 2800/368: Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
- 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)
- C09B: ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES {, e.g. PIGMENTS}; MORDANTS; LAKES (fermentation or enzyme using processes to synthesise a desired chemical compound C12P)

## Abstract

Various aspects and embodiments of the present disclosure are directed to methods and compositions (e.g., kits) for the identification of subjects with misfolded proteins in their urine. For example, methods and compositions for determining that a urine sample from a pregnant woman contains or does not contain misfolded are provided. In some embodiments, the presence of misfolded proteins in a urine sample from a pregnant woman is an indication of preeclampsia.

## Claims

### Claim 1

A method of assessing a urine sample from a pregnant woman, comprising:
(a) applying a solution to a first region of a surface that comprises cellulose, wherein the solution comprises (i) a urine sample from a pregnant woman and (ii) a dye that binds protein aggregates and intercalates with cellulose fibers; and
(b) assessing over a period of time whether the dye diffuses from the first region to a second region of the surface, wherein diffusion of the dye from the first region to the second region of the surface is indicative of protein aggregates in the urine sample, and lack of diffusion of the dye from the first region to the second region of the surface is indicative of no protein aggregates in the urine sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein the dye is selected from the group consisting of Congo Red, Thioflavin S, Thioflavin T, Evans Blue, Trypan blue, amino-8-napthalene sulfonate (ANS), and bis-azo ANS.

### Claim 3 (depends on 2)

The method of claim 2, wherein the dye is Congo Red.

### Claim 4 (depends on 1)

The method of claim 1, wherein the concentration of the dye in the solution is 0.05% to 0.2%.

### Claim 5 (depends on 1)

The method of claim 1, wherein the surface that comprises cellulose is a paper surface.

### Claim 6 (depends on 1)

The method of claim 1, further comprising:
(d) diluting an additional urine sample from the pregnant woman with a dye that binds protein aggregates and intercalates with cellulose fibers to produce a solution comprising dye-labeled protein aggregates to produce a dilute urine sample; and repeating steps (a) and (b) with the dilute urine sample.

### Claim 7 (depends on 6)

The method of claim 6, wherein the additional urine sample is diluted 5-fold to 15-fold.

### Claim 8 (depends on 1)

The method of claim 1 further comprising diagnosing the pregnant woman as having preeclampsia, or at risk of preeclampsia, if the urine sample contains protein aggregates.

### Claim 9

A method of assessing a urine sample from a pregnant woman, comprising:
(a) applying a solution to a first region of a surface that comprises cellulose, wherein the solution comprises (i) a urine sample from a pregnant woman, (ii) a first dye that binds protein aggregates and intercalates with cellulose fibers, and (iii) a second dye that is a different color from the first dye and does not bind protein aggregates or cellulose; and
(b) assessing over a period of time whether the first dye and the second dye diffuse from the first region to a second region of the surface, wherein diffusion of both the first dye and the second dye from the first region to the second region of the surface is indicative of protein aggregates in the urine sample, and diffusion of the second dye, but not the first dye, from the first region to the second region of the surface is indicative of no protein aggregates in the urine sample.

### Claim 10 (depends on 9)

The method of claim 9 further comprising acquiring an image of the surface.

### Claim 11 (depends on 10)

The method of claim 10, wherein the image is acquired using a mobile telephone.

### Claim 12 (depends on 9)

The method of claim 9 further comprising quantifying the difference between the extent to which the first dye diffuses from the first region to the second region of the surface and the extent to which the second dye diffuses from the first region to the second region of the surface.

### Claim 13 (depends on 9)

The method of claim 9, wherein the first dye is selected from the group consisting of Congo Red, Thioflavin S, Thioflavin T, Evans Blue, Trypan blue, amino-8-napthalene sulfonate (ANS), and bis-azo ANS.

### Claim 14 (depends on 13)

The method of claim 13, wherein the first dye is Congo Red.

### Claim 15 (depends on 14)

The method of claim 14, wherein the second dye is erioglaucine.

### Claim 16 (depends on 9)

The method of claim 9, wherein the concentration of the first dye in the solution is 0.05% to 0.2%.

### Claim 17 (depends on 9)

The method of claim 9, wherein the concentration of the second dye in the solution is 0.05% to 0.2%.

### Claim 18 (depends on 9)

The method of claim 9, wherein the surface that comprises cellulose is a paper surface.

### Claim 19 (depends on 9)

The method of claim 9, further comprising:
(d) diluting an additional urine sample from the pregnant woman with a first dye that binds protein aggregates and intercalates with cellulose fibers and a second dye that is a different color from the first dye and does not bind protein aggregates or cellulose to produce a dilute urine sample; and repeating steps (a)-(c) with the dilute urine sample.

### Claim 20 (depends on 19)

The method of claim 19, wherein the additional urine sample is diluted 5-fold to 15-fold.

### Claim 21 (depends on 9)

The method of claim 9 further comprising diagnosing the pregnant woman as having preeclampsia, or at risk of preeclampsia, if the urine sample contains protein aggregates.

### Claim 22

A method of assessing a urine sample from a pregnant woman, comprising:
(a) combining a urine sample from a pregnant woman with a dye that binds protein aggregates and intercalates with cellulose fibers to produce a solution comprising dye-labeled protein aggregates to form a solution comprising dye-labeled protein aggregates;
(b) applying the solution to a surface that comprises cellulose to produce on the surface a spot comprising the dye; and
(c) assessing whether the solution that passes through the surface has color or not, wherein a solution having color is indicative of protein aggregates in the urine sample, and a solution not having color is indicative of no protein aggregates in the urine sample.

### Claim 23 (depends on 22)

The method of claim 22, wherein the first dye is selected from the group consisting of Congo Red, Thioflavin S, Thioflavin T, Evans Blue, Trypan blue, amino-8-napthalene sulfonate (ANS), and bis-azo ANS.

### Claim 24 (depends on 23)

The method of claim 23, wherein the first dye is Congo Red.

### Claim 25

A method of determining whether a pregnant woman has, or is at risk of, preeclampsia, the method comprising:
(a) applying a solution to a first region of a surface that comprises cellulose, wherein the solution comprises (i) a urine sample from a pregnant woman and (ii) Congo Red; and
(b) determining that the pregnant woman has, or is at risk of, preeclampsia if the Congo Red diffuses from the first region to the second region of the surface.