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Cyclosporin A immunogen, anti-cyclosporin A specific antibody and cyclosporin A detection reagent

A cyclosporine and immunogen technology, which is applied in the field of cyclosporine A detection reagents, and achieves the effects of high antibody specificity, high immunogenicity and easy operation

Active Publication Date: 2015-07-22
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

At present, there is a lack of cyclosporine A detection reagents with good stability, high sensitivity and strong specificity in the market, especially good quality automatic detection reagents

Method used

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  • Cyclosporin A immunogen, anti-cyclosporin A specific antibody and cyclosporin A detection reagent
  • Cyclosporin A immunogen, anti-cyclosporin A specific antibody and cyclosporin A detection reagent
  • Cyclosporin A immunogen, anti-cyclosporin A specific antibody and cyclosporin A detection reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Synthesis and Analysis of Cyclosporine A Derivatives

[0069] The chemical structure of cyclosporine A derivative is shown in formula (IV):

[0070]

[0071] The synthetic route and preparation steps of the above-mentioned cyclosporine A derivatives are as follows:

[0072]

[0073] 1. Weigh 200mg (0.16mmol) of cyclosporine A and 29.2mg (0.16mmol) of 2-(bromomethyl) methyl acrylate, and dissolve them in 3mL of DCM; weigh 56mg (0.24mmol) of benzyl triethyl Ammonium chloride was dissolved in 3 mL of 40% KOH aqueous solution; the above two solutions were mixed to form a reaction mixture solution.

[0074] 2. The above reaction mixture solution was stirred overnight at room temperature; the pH value of the reacted solution was adjusted to pH=6 with HCl, and then the organic layer was separated.

[0075] 3. The organic phase was concentrated and then purified by prep-TLC to obtain 9 mg of the final product as a white solid with a yield of 50%.

[0076] 4. ...

Embodiment 2

[0078] Example 2 Synthesis of Cyclosporine A Immunogen

[0079] The cyclosporine A immunogen is composed of bovine serum albumin (Bovine Serum Albumin, BSA) and the cyclosporine A derivative shown in formula (II) In this embodiment, taking n=1 as an example, the synthesis method of the immunogen is described in detail, and the specific steps are as follows:

[0080] 1. Dissolve 200mg of bovine serum albumin in 50ml of 0.2M, pH8.5 phosphate buffer;

[0081] 2. Add the following chemicals into a small beaker and stir to dissolve: 200mg of synthetic cyclosporine A derivatives, 3.5ml of dimethylformamide, 3.5ml of ethanol, 7.0ml of 10mM potassium phosphate buffer at pH 5.0, 200mg1 -Ethyl-3-(-3-dimethylaminopropyl)carbodiimide, 50mg N-hydroxysulfosuccinimide, these chemicals were stirred and dissolved at room temperature for 30min;

[0082] 3. The dissolved solution was added dropwise to the BSA solution, and stirred overnight at 2-8°C to obtain the antigen; the synthesized anti...

Embodiment 3

[0083] Example 3: Preparation of Anti-Cyclosporine A Specific Antibody

[0084] The cyclosporine A immunogen prepared in Example 2 was inoculated into experimental animal rabbits by conventional methods, and the antiserum was taken after booster immunization. The specific steps were as follows:

[0085] 1. Dilute the cyclosporine A 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 Freund's complete adjuvant, and inject the experimental animal rabbit.

[0086] 2. After 2-3 weeks, inject 1.0ml of the same antigen solution and incomplete Freund's adjuvant to the above-mentioned experimental animal rabbit once, and then inject once every four weeks, a total of 4 injections.

[0087] 3. Take blood from the experimental animal rabbit in step 2, separate and purify to obtain anti-cyclosporin A specific antibody with a titer of 1:30000-1:50000.

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Abstract

The invention discloses a cyclosporin A immunogen and a synthesis method thereof, as well as an anti-cyclosporin A antibody obtained from the immunogen, a detection reagent and a preparation method of the cyclosporin A immunogen. The cyclosporin A immunogen prepared by the preparation method disclosed by the invention has high immunogenicity and can obtain the anti-cyclosporin A specific antibody which has high valence and no cross reaction with common 62 medicines by induction; and the cyclosporin A detection reagent prepared from the antibody can precisely and rapidly determine the content of cyclosporin in a sample. Compared with the existing detection reagent in the market, the detection reagent disclosed by the invention has the advantages of simplicity and convenience in operation, high sensitivity, strong specificity, accurate result and the like, can effectively reduce the detection cost of cyclosporin A and is conductive to clinical large-scale popularization and application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to cyclosporine A immunogen, anti-cyclosporin A specific antibody and cyclosporine A detection reagent. Background technique [0002] Cyclosporin A (Cyclosporin A), its structural formula is shown in formula (Ⅲ): [0003] [0004] Cyclosporine A is a fungal-derived high fat-soluble cyclic peptide small-molecule drug. It is a powerful immunosuppressant that selectively inhibits helper T The proliferation and function of lymphocytes (Th) produce immune regulation, and it is one of the main drugs that play an immunosuppressive role after organ transplantation. Treatment with cyclosporine A can significantly improve the survival rate of transplanted organs such as skin, heart, kidney, liver, pancreas, bone marrow, lung, and small intestine. In addition, cyclosporine A can also be used in the treatment of various autoimmune diseases. The therapeutic effect and toxicity of cyclosporine A ...

Claims

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Application Information

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IPC IPC(8): C07K14/795C07K14/765C07K14/47C07K16/44G01N33/577
Inventor 虞留明邱玲娄金丽毛远丽
Owner 苏州博源医疗科技有限公司
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