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ADMA (Asymmetric Dimethylarginine) immunoassay reagent and detection method

A reagent, glycine technology, applied in the field of medical immunology in vitro diagnosis, to achieve the effect of high specificity, high immunogenicity and strong specificity

Inactive Publication Date: 2017-01-04
北京德奥平生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is a lack of asymmetric dimethylarginine detection reagents with high sensitivity and specificity on the market, especially those suitable for automated testing

Method used

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  • ADMA (Asymmetric Dimethylarginine) immunoassay reagent and detection method
  • ADMA (Asymmetric Dimethylarginine) immunoassay reagent and detection method
  • ADMA (Asymmetric Dimethylarginine) immunoassay reagent and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Synthesis and structure confirmation of ADMA derivative 1

[0030] The biochemical structure of the ADMA derivative 1 used in the following examples is as figure 2 Shown in compound 3 in, the synthetic route is as follows figure 2 .

[0031] Concrete synthetic steps:

[0032] 1) Weigh 900mg of compound 1 (ADMA) and dissolve it in 90ml of pyridine, immediately add 360 mg of EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) and 540mg of NHS (N-hydroxysuccinimide), and put the solution at room temperature Under stirring for 30 hours, the synthetic solution of compound 2 was obtained;

[0033] 2) Add 126ul of β-mercaptoethanol to the above solution, stir at room temperature for 30min, inactivate excess EDC, and desalt into PBS with pH 7.2;

[0034] 3) Dissolve 15.00g of aminocaproic acid in 10mL of PBS solution with pH 7.2, take 50mL of the above synthetic solution and slowly add it dropwise to the solution of aminocaproic acid, stir at roo...

Embodiment 2

[0037] Example 2: Synthesis and structure confirmation of ADMA derivative 2

[0038] The biochemical structure of the ADMA derivative 2 used in the following examples is as image 3 Shown in compound 8, its synthetic route is as follows image 3 .

[0039] Concrete synthetic route is as follows:

[0040] 1) Weigh 100mg of ADMA and dissolve it in concentrated sulfuric acid, add 1200mg of methanol, and heat to reflux for 2h. After the synthetic solution was diluted with water, the solvent was evaporated by a reduced pressure method, and finally a white solid compound 4 was obtained;

[0041] 2) Weigh 500mg of compound 5 (aminocaproic acid) and dissolve it in 50ml of pyridine, immediately add 400mg of EDC (1-ethyl-3-3-dimethylaminopropyl]carbodiimide hydrochloride) and 600mg of NHS (N-hydroxysuccinimide), and put the solution at room temperature Under stirring for 30 hours, the synthetic solution of compound 6 was obtained;

[0042] 3) Add 90ul of β-mercaptoethanol to the ab...

Embodiment 3

[0048] Example 3: ADMA immunogen synthesis

[0049] ADMA immunogen is composed of rabbit albumin or mouse albumin with figure 1 The ADMA derivatives shown are connected by linking groups. In this embodiment, the synthesis method of the immunogen is described in detail by taking the ADMA derivative whose linking arm is aminocaproic acid as an example.

[0050] Rabbit albumin-ADMA carboxyl derivative A1 immunogen synthesis:

[0051] Specific steps are as follows:

[0052] 1) Dissolve 40mg of rabbit albumin in 4mL of 0.1M MES buffer at pH 5.0, and place the above solution in beaker A;

[0053] 2) Dissolve 30mg of ADMA derivative 1 in 6mL of 0.1M MES buffer at pH 5.0 and add to beaker A;

[0054] 3) Add 50 mg of EDC to beaker A to obtain a mixed solution, stir and react at room temperature for 2 hours; dialyze and purify the stirred mixed solution with neutral PBS buffer to obtain rabbit albumin-ADMA immunogen, which is stored at -70°C .

[0055] Synthesis of murine albumin-A...

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Abstract

The invention relates to a method and reagent for immunological detection of a homogeneous enzyme determination technology, and is used for determination of the ADMA (N<G>,N<G>,dimethyl-L-arginine, asymmetric dimethylarginine, ADMA) content in a detected sample. According to the invention, anti-ADMA specific antibodies and enzyme-ADMA complexes are used, the immunological reaction of the enzyme-ADMA complexes and the anti-ADMA specific antibodies is utilized, after antigen antibody complexes are formed, the activity of the enzyme-ADMA complexes is inhibited, the changes of enzymatic activity in a reaction system is determined to obtain the number of combined enzyme markers, so as to obtain the content of ADMA in the detected sample. The method has the advantage of capability of realizing immunoassay as the common biochemical enzyme method, so as to simultaneously determine multiple samples on a full-automatic biochemical analyzer.

Description

technical field [0001] The invention relates to the directional connection of small molecules and proteins, small molecule components and detection methods, and belongs to the field of medical immune in vitro diagnosis. It is a homogeneous immunological enzymatic assay. In particular, it relates to a detection method and the preparation of a kit, in particular to an ADMA detection reagent and its preparation and detection method. Background technique [0002] Asymmetric dimethylarginine (N G , N G , dimethyl-L-arginine, asymmetricdimethylarginine, ADMA) is also called asymmetric dimethylarginine. In 1970, Kakimoto first isolated asymmetric dimethylarginine from human urine. ADMA is methylated by proteins containing arginine residues under the action of protein arginine methyltransferases (protein arginine methyltransferases, PRMT), and ADMA can be released after the methylated protein is hydrolyzed. ADMA can competitively inhibit nitric oxide synthase (NOS) to reduce th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/531
CPCG01N33/6806G01N33/531
Inventor 周建平
Owner 北京德奥平生物技术有限公司
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