# US 8,465,988 B2 · Cell-based antioxidant protection assay

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- Number: US 8,465,988 B2
- Document ID: US8465988B2
- Status: Granted
- Kind: B2
- Application number: 12/740,776
- Filed: 2008-10-31
- Published: 2010-12-09
- Granted: 2013-06-18
- Earliest claimed: 2007-11-02
- Assignees: Natural Immune Systems, Inc.
- Applicants: Gitte S. Jensen
- Inventors: Gitte S. Jensen
- Web page: https://patentgrep.com/patent/US8465988B2

## CPC

- G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)}
- G01N 33/5014: {for testing toxicity}
- G01N 33/52: 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)}
- Y10T 436/105831: Protein or peptide standard or control [e.g., hemoglobin, etc.]
- Y10T 436/13: Tracers or tags
- Y10T 436/25125: Digestion or removing interfering materials
- Y10T 436/25375: Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
- 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)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

Methods are provided herein for determining antioxidant activity of a test sample in intact cells. The method includes determining the antioxidant capacity of a test sample in intact red blood cells, wherein the test sample is added to intact red blood cells and oxidative damage is measured by alteration of fluorescence intensity of an oxidation-sensitive fluorescent indicator dye.

## Claims

### Claim 1

A method for determining antioxidant activity of a sample comprising:
adding a sample to intact red blood cells under conditions which permit the sample to cross the cell membrane of the intact red blood cells;
washing the red blood cells to remove sample which has not crossed the cell membrane of the red blood cells;
lysing the washed red blood cells;
adding a fluorescent indicator dye to the lysed red blood cells, wherein the fluorescence indicator dye exhibits oxidation-sensitive changes in fluorescence intensity;
inducing oxidative stress in the lysed red blood cell mixture; and
detecting fluorescence intensity in the lysed red blood cell mixture using a microplate reader,
wherein a decrease in the fluorescence intensity of the fluorescence indicator dye in the lysed red blood cells in the presence of the sample as compared to a control comprising lysed red blood cells which have not been treated with the sample or lysed red blood cells treated with a standard with known antioxidant activity indicates the antioxidant activity of the sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein lysing the red blood cells comprises incubating in hypotonic buffer.

### Claim 3 (depends on 1)

The method of claim 1, wherein the fluorescent indicator dye comprises 5-(and -6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) or 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) diacetate.

### Claim 4 (depends on 1)

The method of claim 1, wherein inducing oxidative stress comprises adding a free radical generator in the presence of the fluorescent indicator dye.

### Claim 5 (depends on 4)

The method of claim 4, wherein the free radical generator comprises hydrogen peroxide, tert-butylhydroperoxide, cumene hydroperoxide, or 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH).

### Claim 6 (depends on 1)

The method of claim 1, wherein the sample comprises an extract.

### Claim 7 (depends on 6)

The method of claim 6, wherein the extract comprises an aqueous extract, an organic extract, or a combination thereof.

### Claim 8 (depends on 6)

The method of claim 6, further comprising dilution of the extract in a physiological buffer.

### Claim 9 (depends on 1)

The method of claim 1, wherein the sample comprises serum or plasma from a subject.

### Claim 10 (depends on 9)

The method of claim 9, wherein the subject has consumed a compound of interest.

### Claim 11 (depends on 1)

The method of claim 1, wherein the sample comprises a plurality of standards with known antioxidant activity.

### Claim 12 (depends on 11)

The method of claim 11, wherein the plurality of standards comprises one to six standards.

### Claim 13 (depends on 11)

The method of claim 11, wherein the plurality of standards comprises one or more of gallic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, ascorbic acid, or methyl sulfonyl methane.

### Claim 14 (depends on 1)

The method of claim 1, wherein the sample comprises a compound of interest.

### Claim 15 (depends on 1)

The method of claim 1, wherein the red blood cells comprise red blood cells from a subject.

### Claim 16 (depends on 1)

The method of claim 1, wherein adding the sample to intact red blood cells comprises:
administering the sample to a subject, thereby adding the sample to intact red blood cells; and
collecting the red blood cells from the subject prior to washing the red blood cells.

### Claim 17 (depends on 16)

The method of claim 16, further comprising adding a free radical generator in the presence of the fluorescent indicator dye.

### Claim 18 (depends on 1)

The method of claim 1, wherein lysing the washed red blood cells comprises exposing the cells to hypotonic conditions, detergent, or physical disruption.

### Claim 19 (depends on 1)

The method of claim 1, wherein the control comprises red blood cells which have not been treated with the sample or red blood cells treated with a standard with known antioxidant activity.

### Claim 20

A method for determining antioxidant activity of a sample comprising:
adding a sample to intact red blood cells under conditions which permit the sample to cross the cell membrane of the intact red blood cells;
washing the red blood cells to remove sample which has not crossed the cell membrane of the red blood cells;
lysing the washed red blood cells, thereby generating lysed red blood cells;
inducing oxidative stress in the lysed red blood cells; and
detecting fluorescence intensity of hemoglobin in the lysed red blood cells using a microplate reader, wherein hemoglobin exhibits oxidation-sensitive changes in fluorescence intensity,
wherein a decrease in the fluorescence intensity of the hemoglobin in the lysed red blood cells treated with the sample as compared to a control comprising lysed red blood cells which have not been treated with the sample or lysed red blood cells treated with a standard with known antioxidant activity indicates the antioxidant activity of the sample.

### Claim 21 (depends on 20)

The method of claim 20, wherein inducing oxidative stress comprises adding a free radical generator in the presence of the lysed red blood cells.

### Claim 22

A method for determining antioxidant activity of a sample comprising:
adding a sample to intact red blood cells under conditions which permit the sample to cross the cell membrane of the intact red blood cells;
washing the red blood cells to remove sample which has not crossed the cell membrane of the red blood cells;
lysing the washed red blood cells in water;
adding 5-(and-6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester to the lysed red blood cells, wherein the 5-(and-6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester exhibits oxidation-sensitive changes in fluorescence intensity;
adding hydrogen peroxide to the lysed red blood cell mixture; and
detecting fluorescence intensity of the 5-(and-6-)-chloromethyl-2′,7′dichlorodihydrofluorescein diacetate acetyl ester in the lysed red blood cell mixture using a microplate reader, wherein a decrease in the fluorescence intensity in the lysed red blood cells treated with the sample as compared to a control comprising lysed red blood cells which have not been treated with the sample or lysed red blood cells treated with a standard with known antioxidant activity indicates the antioxidant activity of the sample.