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Method for measuring concentration of asymmetric dimethylarginine and diagnostic reagent kit

A technology of dimethylarginine and dimethylarginine dimethylamino, applied in the field of determination of asymmetric dimethylarginine concentration and diagnostic kits

Active Publication Date: 2013-09-25
BEIJING STRONG BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because there is no mature method and kit for enzymatic detection of ADMA concentration in the prior art, there is a need for methods and kits that can easily, sensitively and accurately measure ADMA concentration

Method used

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  • Method for measuring concentration of asymmetric dimethylarginine and diagnostic reagent kit
  • Method for measuring concentration of asymmetric dimethylarginine and diagnostic reagent kit
  • Method for measuring concentration of asymmetric dimethylarginine and diagnostic reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1: Double reagent kit formula 1 and the comparative experiment with liquid chromatography-mass spectrometry

[0073] The first formula of kit of the present invention is double reagent kit, and its composition is respectively as follows:

[0074] Reagent 1 composition and concentration:

[0075] Good's buffer: 50mM,

[0076] ATP: 5mM,

[0077] ASS: 5kU / L,

[0078] L-Aspartic Acid: 10mM,

[0079] AMP deaminase: 20kU / L,

[0080] Deaminated oxidized coenzyme NAD: 2mM,

[0081] NAD synthetase: 20kU / L,

[0082] Glucose: 50mM,

[0083] Glucose dehydrogenase: 50kU / L,

[0084] Glutamate 5mM,

[0085] Glutamine Synthetase: 5kU / L,

[0086] PK: 5KU / L,

[0087] PEP: 5mM,

[0088] WST-3: 3mM,

[0089] BSA: 0.3%,

[0090] Reagent 2 composition and concentration:

[0091] Good's buffer: 50mM,

[0092] DDAH: 5KU / L,

[0093] Diaphorase: 10kU / L,

[0094] L-methionine yellow imide: 20mM,

[0095] BSA: 0.3%.

[0096] This detection method is a rate method. Af...

Embodiment 2

[0101] Embodiment 2: Double reagent kit formula 2 and its measurement repeatability and linear range

[0102] The second formula of the kit of the present invention is also a two-reagent kit, and its components are as follows:

[0103] Reagent 1 composition and concentration:

[0104] Phosphate buffer: 50mM,

[0105] ATP: 5mM,

[0106] ASS: 5kU / L,

[0107] L-Aspartic Acid: 10mM,

[0108] AMP deaminase: 20kU / L,

[0109] Deaminated oxidized coenzyme NAD: 2mM,

[0110] NAD synthetase: 20kU / L,

[0111] NADPH: 1mM,

[0112] 2-ketoglutarate: 1mM,

[0113] Glucose: 5mM,

[0114] Glucose dehydrogenase: 50kU / L,

[0115] Glutamate dehydrogenase: 5kU / L,

[0116] WST-3: 2mM,

[0117] BSA: 0.3%,

[0118] Reagent 2 composition and concentration:

[0119] Phosphate buffer: 50mM,

[0120] DDAH: 5KU / L,

[0121] Diaphorase: 10kU / L,

[0122] p-pyridinium chloromercuric benzoate: 20mM,

[0123]BSA: 0.3%.

[0124] The method of using this kit to measure the concentration of asym...

Embodiment 3

[0127] Embodiment 3: double reagent kit formula 3

[0128] The 3rd formula of test kit of the present invention is double reagent kit, and its composition is respectively as follows:

[0129] Reagent 1 composition and concentration:

[0130] Good's buffer: 50mM,

[0131] ATP: 6mM,

[0132] ASS: 0.3kU / L,

[0133] L-Aspartic Acid: 2mM,

[0134] AMP deaminase: 15kU / L,

[0135] Deaminated oxidized coenzyme NAD: 10mM,

[0136] NAD synthetase: 15kU / L,

[0137] Glucose: 5mM,

[0138] Glucose dehydrogenase: 20kU / L,

[0139] Glutamic acid: 2mM

[0140] Glutamine Synthetase: 2kU / L,

[0141] PK: 2kU / L,

[0142] PEP: 2mM,

[0143] WST-3: 1mM,

[0144] BSA: 0.05%,

[0145] Reagent 2 composition and concentration:

[0146] Good's buffer: 50mM,

[0147] DDAH: 2kU / L,

[0148] Diaphorase: 5kU / L,

[0149] L-methionine yellow imide: 5mM,

[0150] BSA: 0.05%.

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Abstract

The invention relates to a method for measuring the concentration of asymmetric dimethylarginine and a diagnostic reagent kit. The method comprises the following steps of: degrading asymmetric dimethylarginine (ADMA) by adopting dimethylarginine dimethylamino hydrolase to generate dimethylamine and citrulline; reacting the citrulline with adenosine triphosphate (ATP) and aspartate under the action of arginine succinate synthetase to generate adenosine monophosphate (AMP); generating inosine monophosphate (IMP) and ammonia by the AMP under the action of AMP deaminase; generating oxidization nicotinamide adenine dinucleotide (NAD) by the ammonia and deamination NAD under the action of NAD synzyme; and calculating the concentration of the ADMA by detecting the concentration of the NAD. The method and the reagent kit are accurate in results, and high in speed, repeatability and interference resistance, and are convenient to popularize and use.

Description

technical field [0001] The invention relates to a method for measuring the concentration of asymmetric dimethylarginine and a diagnostic kit. In particular, the present invention relates to the generation of oxidative enzymes using dimethylarginine dimethylaminohydrolase, ATP, aspartate, argininosuccinate synthase, AMP deaminase, NAD synthase, and the deamination oxidative coenzyme NAD. Type coenzyme, a method and a kit for measuring the concentration of asymmetric dimethylarginine by detecting the concentration of the coenzyme. Background technique [0002] Asymmetric dimethylarginine (ADMA), an endogenous molecule detectable in human blood and urine, is formed during the hydrolysis of methylated proteins in vivo. ADMA can play a biological role by inhibiting the synthesis of nitric oxide. More and more clinical studies have proved that there is a statistically significant and independent association between ADMA and the incidence of important adverse cardiovascular event...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/527C12Q1/25C12Q1/34C12Q1/32
Inventor 宋占科刘希刘瑶
Owner BEIJING STRONG BIOTECH INC
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