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Asymmetric dimethylarginine immunogen, antibody, detecting reagent and preparation method

A dimethylarginine and asymmetric technology, applied in the field of asymmetric dimethylarginine detection reagents and its preparation, can solve the problems of mature products without large-scale clinical application, inability to perform high-throughput analysis, and human factors Large impact and other issues, to achieve the effect of promoting clinical use, reducing detection costs, and high sensitivity

Inactive Publication Date: 2016-07-13
苏州博源医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The common features of high performance liquid chromatography and high performance liquid chromatography-mass spectrometry are good accuracy and high specificity, but the disadvantages are complex operation, cumbersome sample processing, long test time, special expensive instruments are required, and high-throughput analysis cannot be performed , it is difficult to apply clinically
Enzyme-linked immunoassay is slightly more convenient than chromatography. Its disadvantages are that it needs manual operation, takes a long time, is greatly affected by human factors, and is difficult to realize automation. At present, it is mainly used in experimental research.
In addition, the literature also reported possible detection methods such as fluorescence polarization immunoassay, radioimmunoassay, latex-enhanced immunoturbidimetric assay, cloned enzyme donor immunoassay, and enzymatic method for the determination of ADMA, but there are currently no Mature products without large-scale clinical application

Method used

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  • Asymmetric dimethylarginine immunogen, antibody, detecting reagent and preparation method
  • Asymmetric dimethylarginine immunogen, antibody, detecting reagent and preparation method
  • Asymmetric dimethylarginine immunogen, antibody, detecting reagent and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Synthesis of an asymmetric dimethylarginine immunogen

[0048] The asymmetric dimethylarginine immunogen is composed of bovine serum albumin (BovineSerumAlbumin, BSA) and asymmetric dimethylarginine represented by formula (II) The specific steps are as follows:

[0049] 1. Dissolve 200mg of bovine serum albumin in 50ml of 0.2M, pH8.5 phosphate buffer;

[0050] 2. Add the following chemicals into a small beaker and stir to dissolve: 200mg of asymmetric dimethylarginine, 3.5ml of dimethylformamide, 3.5ml of ethanol, 7.0ml of 10mM potassium phosphate buffer at pH 5.0, 200mg of 1- Ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 50mg N-hydroxyl sulfosuccinimide, these chemicals were stirred and dissolved at room temperature for 30 minutes;

[0051] 3. The dissolved solution was added dropwise to the BSA solution, and stirred overnight at 2-8°C to obtain the antigen; the synthesized antigen was purified by dialysis to obtain the asymmetric dimethylarginine immunog...

Embodiment 2

[0052] Example 2: Preparation of anti-asymmetric dimethylarginine specific antibody

[0053] The asymmetric dimethylarginine immunogen prepared in Example 1 was inoculated into experimental animal rabbits by conventional methods, and the antiserum was collected after booster immunization. The specific steps were as follows:

[0054] 1. Dilute the asymmetric dimethylarginine immunogen synthesized above to 1.0 mg / ml with PBS to obtain an antigen solution, then mix 1.0 ml of the antigen solution with an equal amount of Freund's complete adjuvant, and conduct the experimental animal rabbit injection.

[0055] 2. After 2-3 weeks, inject 1.0ml of the same antigen solution and the same amount of Freund's incomplete adjuvant to the above-mentioned experimental animal rabbit once, and then inject once every four weeks, a total of 4 injections.

[0056] 3. Taking blood from the experimental animal rabbit in step 2, separating and purifying to obtain an anti-asymmetric dimethylarginine ...

Embodiment 3

[0057] Embodiment 3: ELISA test of asymmetric dimethylarginine

[0058] 1. Establishment of standard curve for ELISA detection of asymmetric dimethylarginine

[0059] (1) Preparation of standard products

[0060] Asymmetric dimethylarginine powder (purchased from Sigma) was dissolved in methanol solution to prepare a 1000 μmol / L stock solution. The stock solution was diluted sequentially with ELISA buffer to 4.00 μmol / L, 2.00 μmol / L, 1.00 μmol / L, 0.5 μmol / L, 0.25 μmol / L and 0.00 μmol / L standard solutions. Among them, the ELISA buffer contains 50.0mM Tris, 145mM NaCl and 0.25% BSA.

[0061] (2) Utilize the ELISA test method of asymmetric dimethylarginine to prepare standard curve

[0062] Dilute the anti-asymmetric dimethylarginine specific antibody prepared in Example 2 with PBS to a final concentration solution of 1:6000, coat 100 μL / well on a 96-well enzyme-linked plate, and place at 4°C for 12- 24 hours; wash the above-mentioned 96-well ELISA plate coated with anti-asym...

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Abstract

The invention discloses asymmetric dimethylarginine immunogen, an antibody, a detecting reagent and a preparation method. The asymmetric dimethylarginine immunogen is high in immunogenicity, can be induced to obtain an anti-asymmetric dimethylarginine specific antibody with high valence, and does not have any cross reaction with common 62 medicines. The asymmetric dimethylarginine immunogen detecting reagent prepared from the antibody can be used for precisely and quickly determining the content of asymmetric dimethylarginine in biological samples such as blood serum, blood plasma and urea. Compared with a current detecting reagent on the market, the detecting reagent has the advantages of being simple and convenient to operate, high in sensitivity, strong in specificity and accurate in result, further can effectively reduce the detecting cost of asymmetric dimethylarginine, and is beneficial for clinical large-scale popularization and use.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an asymmetric dimethylarginine immunogen, an anti-asymmetric dimethylarginine specific antibody, an asymmetric dimethylarginine detection reagent and a preparation method thereof. Background technique [0002] Asymmetric dimethylarginine (ADMA), its structural formula is shown in formula (II): [0003] [0004] Formula (Ⅱ) [0005] Asymmetric dimethylarginine, an endogenous molecule detectable in human serum, plasma, and urine, is a methylated arginine that is composed of arginine that makes up intracellular proteins Residues are methylated under the catalysis of protein arginine methyltransferase, and then generated by hydrolysis reaction. It has been proved that there is an endogenous mechanism for ADMA to regulate the generation of nitric oxide (NO) in vivo, that is, ADMA can competitively inhibit the activity of nitric oxide synthase (NOS), thereby reducing the synthesis of ni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K14/795C07K14/47C07K1/34C07K16/44G01N35/00G01N33/53G01N33/535G01N33/533
CPCC07K14/765C07K14/47C07K14/795C07K16/44C07K2317/00G01N33/53G01N33/533G01N33/535G01N35/00G01N2800/044G01N2800/321G01N2800/323G01N2800/324G01N2800/325
Inventor 付卫虞留明
Owner 苏州博源医疗科技有限公司
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