# US 9,040,248 B2 · Kits for detecting and monitoring endocrine disrupting chemicals (EDCs)

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- Number: US 9,040,248 B2
- Document ID: US9040248B2
- Status: Granted
- Kind: B2
- Application number: 13/912,071
- Filed: 2013-06-06
- Published: 2013-12-12
- Granted: 2015-05-26
- Earliest claimed: 2012-06-06
- 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: Gordon L. Hager; Diana A. Stavreva
- Web page: https://patentgrep.com/patent/US9040248B2

## CPC

- G01N 33/5035: {on sub-cellular localization}
- C07K 14/435: from animals; from humans
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)}
- G01N 33/5014: {for testing toxicity}
- G01N 33/68: involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)}
- G01N 33/743: {Steroid hormones}
- C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
- G01N 2520/00: Use of whole organisms as detectors of pollution
- 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)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Described herein are compositions, a system, and kits for detection of endocrine disruptor chemicals (EDCs) in environmental samples, such as samples of water including but not limited to waste water treatment plant effluent, using a live-cell fluorescence-based nuclear translocation reporter system. Upon binding of a ligand to a fluorescent-labeled reporter protein, the protein (and therefore the fluorescence) is translocated in a ligand level-dependent manner from the cytoplasm to the nucleus of live mammalian cells; this translocation is detectable as diffuse (cytoplasmic) fluorescence converting to localized, brightly fluorescent nuclei. The described kits can be used to reliably detect very low levels of EDC contamination, including in high throughput analysis systems as described.

## Claims

### Claim 1

A method for detecting or quantifying endocrine disruptor chemical (EDC) ligands in an environmental sample, the method comprising:
contacting with the environmental sample:
a first mammalian cell expressing a first traceable fusion protein;
a second mammalian cell expressing a second traceable fusion protein;
a third mammalian cell expressing a third traceable fusion protein;
a fourth mammalian cell expressing a fourth traceable fusion protein; and
a fifth mammalian cell expressing a fifth traceable fusion protein,
wherein the first, second, third, fourth, and fifth traceable fusion proteins independently comprise either:
(1) a superfamily receptor protein, and a marker protein domain; or
(2) the cytoplasmic/nuclear translocation domain of glucocorticoid receptor, the ligand binding domain of a superfamily receptor protein, and a marker protein domain; and
detecting cytoplasmic to nuclear translocation of the first, second, third, fourth, and/or fifth fusion proteins in response to one or more ligands of the ligand binding domain in the environmental sample,
wherein the first, second, third, fourth, and fifth traceable fusion proteins each bind a different ligand or class of ligands.

### Claim 2 (depends on 1)

The method of claim 1, wherein the environmental sample comprises a water sample, soil sample, or air sample.

### Claim 3 (depends on 2)

The method of claim 2, wherein the sample comprises a water sample comprising surface water, sub-surface (ground) water, rain, run-off, well water, spring water, drinking water (processed or not), river water, estuary water, ocean water, effluent, treated sewage or untreated sewage.

### Claim 4 (depends on 1)

The method of claim 1, wherein detecting the cytoplasmic to nuclear translocation of the first, second, third, fourth, and/or fifth fusion proteins in response to one or more ligands of the ligand binding domain in the environmental sample comprises:
scanning one or more of the mammalian cell(s) to obtain signal data from the marker of the traceable fusion protein;
converting the signal data to obtain the cellular location of the traceable fusion protein in the mammalian cell(s); and
analyzing the signal data using an analysis system having an algorithm to calculate changes in distribution of the labeled traceable protein between the cytoplasm and the nucleus of the mammalian cell(s), the analysis system having the capability of providing an accurate reading of the presence in the environmental sample of the ligand(s) which binds to that traceable fusion protein.

### Claim 5 (depends on 1)

The method of claim 1, wherein the marker protein domain of each traceable fusion protein is different.

### Claim 6 (depends on 1)

The method of claim 1, wherein the marker protein domain is a fluorescent protein domain.

### Claim 7 (depends on 1)

The method of claim 1, wherein the first, second, third, fourth and/or fifth mammalian cell is a human cell.

### Claim 8 (depends on 1)

The method of claim 1, wherein at least one traceable fusion protein binds a glucocorticoid, at least one traceable fusion protein binds an androgen, at least one traceable fusion protein binds a progestin, at least one traceable fusion protein binds an aryl hydrocarbon, and at least one traceable fusion protein binds a thyroid hormone.