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Hydrolase method for measuring NG, NG<'>dimethyl-L-arginine or asymmetric dimethylarginine and kit

A dimethylarginine and asymmetric technology, applied in the field of hydrolytic enzymatic determination of asymmetric dimethylarginine and kits, can solve the problem of ADMA enzymatic maturing kits coming out, not easy to obtain, citrulline Unknown sources of hydrolytic enzymes and other issues

Inactive Publication Date: 2016-03-23
BEIJING DIAGREAT BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The product citrulline is further decomposed under the action of citrulline hydrolase for determination. The key point is that the source of citrulline hydrolase is unknown and not easy to obtain
Therefore, there is still no mature kit for ADMA enzyme detection.

Method used

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  • Hydrolase method for measuring NG, NG&lt;'&gt;dimethyl-L-arginine or asymmetric dimethylarginine and kit
  • Hydrolase method for measuring NG, NG&lt;'&gt;dimethyl-L-arginine or asymmetric dimethylarginine and kit
  • Hydrolase method for measuring NG, NG&lt;'&gt;dimethyl-L-arginine or asymmetric dimethylarginine and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Implementation example one: the composition of the kit

[0031] According to the purpose and principle of the present invention, five types of kits including dry powder single reagent, dry powder double reagent, dry powder three reagents, liquid double reagent and liquid three reagents are assembled, wherein there are two types of three reagents and double reagent kits respectively, The difference lies in the two methods of measuring the ammonia concentration, which are composed of a colorimetric method or a rate method to measure the ADMA concentration kit.

[0032] ADMA standard:

[0033] Stabilizer: 20mMPH8.0 tris buffer; 0.5% bovine serum albumin; 1mM EDTA; 0.01% sodium azide; 10% glycerol;

[0034] When in use, add the pure ADMA standard product into the stabilizer to configure ADMA standard products of 0, 0.5, 1, 2, 3, 4, 5umol / L

[0035] 1. One of the dry powder single reagent formulas of the kit of the present invention has the following ingredients:

[0036]...

Embodiment 2

[0057] Implementation example two: diacetyl monoxime-thiosemicarbazide colorimetric method detects ADMA standard substance standard curve drawing

[0058] The calibrator was subjected to R1, R2, and R3 reactions in sequence, and the results obtained were as follows;

[0059] ADMA concentration (umol / L) 0 0.5 1 2 3 4 5 Absorbance (A) 0.0943 0.129 0.154 0.233 0.297 0.357 0.42

[0060] Standard curve see figure 2 .

Embodiment 3

[0061] Implementation Example 3: Kit Sensitivity Analysis

[0062] BSA protein was used as the negative control of the ADMA standard, diluted to 0, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, and 2 μM respectively, and then used as a sample for determination with a liquid three-reagent kit to detect the ADMA content.

[0063] Depend on image 3 It can be seen that the sensitivity of the detection kit is 0.1umol / L.

[0064] Implementation example three: Repeatability analysis of diacetyl monoxime-thiosemicarbazide colorimetric method kit

[0065] 10 clinical samples were collected and tested 20 times with the colorimetric liquid three-reagent kit, the mean value was obtained, and the coefficient of variation was calculated. When the coefficient of variation is less than 15%, it indicates that the kit has good repeatability.

[0066] serum Mean (μM) different coefficient sample 1 0.57 3.9％ sample 2 0.66 5.1％ sample 3 0.82 4.5％ Sample 4 0.96 6.0...

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Abstract

The invention relates to a biological enzyme method for measuring NG, NG<'>dimethyl-L-arginine or asymmetric dimethylarginine (ADMA) and kit development. Particularly, the biological enzyme method and a kit are characterized in that under the effect of dime-thylarginine dimethyl amino hydrolase (DDAH), the ADMA generates dimethylamine and L-citrulline which are equal in amount, and the L-citrulline is converted into L-ornithine, ammonia and carbon dioxide which are equal in amount through omithine transcarbamoylase, EC2.1.3.3 and carbamatekinase, EC2.7.2.2. The content of the ammonia is measured with a diacetyl monoxime-thiosemicarbazide colorimetric method or a glutamate dehydrogenase rate method, and then the concentration of the ADMA in serum is inferred. The biological enzyme method and the kit have the advantages that reaction steps and reaction conditions are simplified, good repeatability, good accuracy, good sensitivity and good stability are achieved, the rapid requirement, the mass requirement and the economical requirement of clinical detection are met, and application and popularization are facilitated.

Description

technical field [0001] The invention relates to the determination of asymmetric dimethylarginine (N G , N G’ dimethyl-L-arginine, asymmetricdimethylarginine, ADMA) and kit development. In particular, the present invention relates to the use of dimethylarginine dimethylaminohydrolase, ornithine carbamoyltransferase, and carbamoyl kinase to convert ADMA into equivalent amounts of ammonia, and then through diacetylmonoxime-amino Thiourea colorimetric method or glutamic acid dehydrogenase rate method to measure ammonia content, method and kit for inferring serum ADMA concentration. The invention provides an important basis for simple, accurate and sensitive monitoring of asymmetric dimethylarginine. Background technique [0002] Asymmetric dimethylarginine (aN G , N G’ dimethyl-L-arginine, asymmetricdimethylarginine,, ADMA), also known as asymmetric dimethylarginine, molecular formula see figure 1 , ADMA was first detected in plasma and urine in 1992 by Vallance (VallanceP...

Claims

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Application Information

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IPC IPC(8): G01N21/31G01N35/00
Inventor 许秀丽
Owner BEIJING DIAGREAT BIOTECH CO LTD
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