# US 7,611,844 B2 · Methods for detecting asymmetric dimethylarginine in a biological sample

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- Number: US 7,611,844 B2
- Document ID: US7611844B2
- Status: Granted
- Kind: B2
- Application number: 10/713,674
- Filed: 2003-11-13
- Published: 2004-10-28
- Granted: 2009-11-03
- Earliest claimed: 2002-11-15
- Assignees: The Board of Trustees of the Leland Stanford Jr. University
- Inventors: Ken Y. Lin; John Cooke
- Web page: https://patentgrep.com/patent/US7611844B2

## CPC

- G01N 33/6812: {Assays for specific amino acids}
- G01N 33/538: by sorbent column, particles or resin strip {, i.e. sorbent materials}
- G01N 2800/52: Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
- 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.]
- Y10T 436/255: including use of a solid sorbent, semipermeable membrane, or liquid extraction
- 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

The present invention provides methods of detecting asymmetric dimethylarginine (ADMA) in a sample, particularly a sample that may contain symmetrical dimethylarginine (SDMA) and/or arginine. The methods generally involve modifying any SDMA and arginine in the sample such that SDMA and arginine are readily distinguishable from ADMA; and detecting ADMA. The invention further provides antibodies specific for ADMA; antibodies specific for modified SDMA; and antibodies specific for modified arginine. The invention further provides kits for practicing the subject methods.

## Claims

### Claim 1

A method of detecting asymmetric dimethylarginine (ADMA) in a sample comprising ADMA and at least one of symmetric dimethylarginine (SDMA) and arginine, the method comprising the steps of:
a) contacting the sample with an agent that protects the α-amino group of ADMA and the α-amino group of the at least one of SDMA and arginine, generating a sample comprising ADMA having a protected α-amino group and at least one of SDMA and arginine having a protected α-amino group;
b) contacting the sample generated in step (a) with an α-dicarbonyl compound, wherein said α-dicarbonyl compound modifies the guanidino nitrogens of SDMA and the guanidino nitrogens of arginine, producing modified SDMA and modified arginine, wherein said modified SDMA and said modified arginine are distinguishable from ADMA; and
c) detecting ADMA in the sample generated in step (b).

### Claim 2 (depends on 1)

The method of claim 1, wherein said α-dicarbonyl compound is phenylglyoxal.

### Claim 3 (depends on 1)

The method of claim 1, wherein the agent that protects the α-amino group is a dye that provides a detectable signal.

### Claim 4 (depends on 3)

The method of claim 3, wherein the dye is a fluorophore.

### Claim 5 (depends on 4)

The method of claim 4, wherein the fluorophore is fluoro-7-nitrobenzofurazan.

### Claim 6 (depends on 1)

The method of claim 1, wherein said detecting step comprises contacting the sample with an antibody that binds specifically to ADMA, to SDMA, or to both ADMA and SDMA, wherein said antibody does not bind to the modified SDMA.

### Claim 7 (depends on 6)

The method of claim 6, wherein the antibody is detectably labeled.

### Claim 8 (depends on 1)

The method of claim 1, wherein said detecting step comprises contacting the sample with an antibody that binds specifically to the α-amino group-modified ADMA.

### Claim 9 (depends on 8)

The method of claim 8, further comprising detecting one or more of modified SDMA and modified arginine, wherein said detection of one or more of modified SDMA and modified arginine comprises contacting the sample with an antibody that binds specifically to one or more of modified SDMA and modified arginine.

### Claim 10 (depends on 1)

The method of claim 1, wherein said ADMA is detected by high performance liquid chromatography.

### Claim 11 (depends on 1)

The method of claim 1, wherein said ADMA is detected by capillary electrophoresis.

### Claim 12 (depends on 1)

The method of claim 1, wherein the α-dicarbonyl compound is selected from biacetyl, pyruvic acid, glyoxal, methyglyoxal, deoxyosones, 3-deoxyosones, malondialdehyde, 2-oxopropanal, phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione.

### Claim 13 (depends on 1)

The method of claim 1, wherein the sample is a biological sample.

### Claim 14 (depends on 13)

The method of claim 13, wherein the biological sample is serum or plasma.

### Claim 15 (depends on 13)

The method of claim 13, wherein the biological sample is subjected to solid phase extraction before step (a) to clean up the sample.

### Claim 16 (depends on 1)

The method of claim 1, wherein the agent that protects the α-amino group is ortho-phthaldialdehyde.