3-methylquinoxaline-2-carboxylic acid artificial antigen and antibody obtained by the 3-methylquinoxaline-2-carboxylic acid artificial antigen

A technology of methylquinoxaline and carboxylic acid, applied in the field of 3-methylquinoxaline-2carboxylic acid artificial antigen and its prepared antibody, can solve the problems of carcinogenesis, endangering human and animal health, and mutagenicity

Active Publication Date: 2012-09-12
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have found that quinoxaline-based prototype drugs and their metabolites have obvious safety problems, and have obvious toxic and side effects such as teratogenicity, carcinogenicity, mutagenicity, photosensitivity, and adrenal cortex damage, which seriously endanger the health of humans and animals.

Method used

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  • 3-methylquinoxaline-2-carboxylic acid artificial antigen and antibody obtained by the 3-methylquinoxaline-2-carboxylic acid artificial antigen
  • 3-methylquinoxaline-2-carboxylic acid artificial antigen and antibody obtained by the 3-methylquinoxaline-2-carboxylic acid artificial antigen
  • 3-methylquinoxaline-2-carboxylic acid artificial antigen and antibody obtained by the 3-methylquinoxaline-2-carboxylic acid artificial antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, preparation 3-methylquinoxaline-2-carboxylic acid hapten

[0030] One, the preparation of 3-methylquinoxaline-2-carboxylic acid hapten

[0031] 1. Weigh 10 mg of 3-methylquinoxaline-2-carboxylic acid and place it in a 10mL reaction bottle; add an appropriate amount of acetone and a small amount of DMF to completely dissolve it (acetone is used as a reaction system to dissolve 3-methylquinoxaline-2 -Carboxylic acid, the role of DMF is to promote dissolution), add 10 μL of thionyl chloride (acting as activated carboxyl group), heat and reflux for 1 h; then add 0.5 ml of n-hexane and blow it with nitrogen until the volume is constant, then add 0.5 ml of n-hexane and blow it with nitrogen Blow until the volume is constant, then add 0.5 ml of n-hexane and blow with nitrogen until the volume is constant to obtain solution I.

[0032] 2. Weigh 20mg of γ-aminobutyric acid, dissolve it in 1ml of 2mol / L KOH aqueous solution (as a reaction system to dissolve γ-amino...

Embodiment 2

[0039] Preparation and characterization of embodiment 2, 3-methylquinoxaline-2-carboxylic acid artificial antigen

[0040] 1. Synthesis and characterization of 3-methylquinoxaline-2-carboxylic acid immunogen

[0041] 1. Preparation of 3-methylquinoxaline-2-carboxylic acid immunogen

[0042] (1) Dissolve 10 mg of the compound shown in formula (I) prepared in Example 1 in 2 mL of N, N'-dimethylamide, add 10 mg of N-hydroxysuccinimide and 10 mg of 1-ethyl-(3 -Dimethylaminopropyl) carbodiimide hydrochloride, magnetically stirred at room temperature for 2 h to obtain solution III.

[0043] (2) Add 30mg of bovine serum albumin into 2mL of PBS buffer solution and fully dissolve to obtain solution IV.

[0044] (3) Slowly add solution III to solution IV, stir slowly for 24 hours, put it into a dialysis bag, dialyze in normal saline at 4°C for 72h (change the water 6 times in the middle), then centrifuge at 8000rmp for 30min at 4°C, and take The supernatant, that is, the 3-methylquin...

Embodiment 3

[0058] Preparation of embodiment 3, 3-methylquinoxaline-2-carboxylic acid monoclonal antibody

[0059] Balb / c mice: purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.;

[0060] SP2 / 0 myeloma cells: purchased from Siggma-Aldrich Company, the product catalog number is 08060101.

[0061] 1. Animal immunity

[0062] The MQCA-BSA solution prepared in Example 2 was used to immunize Balb / c mice, and each mouse was immunized with 100 μg MQCA-BSA once, for a total of 4 times with an interval of two weeks between each time. Injection, the last three immunizations were intraperitoneal injection.

[0063] 2. Cell fusion and cloning

[0064] 1. Three days after the fourth immunization, splenocytes were collected and fused with SP2 / 0 myeloma cells at a ratio of 5:1 (quantity ratio). Cell supernatants were measured by indirect competitive ELISA, and positive wells were screened.

[0065] 2. Cloning the positive wells by using the limiting dilution method to obt...

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Abstract

The invention discloses a 3-methylquinoxaline-2-carboxylic acid artificial antigen and an antibody obtained by the 3-methylquinoxaline-2-carboxylic acid artificial antigen. The invention provides a compound shown in the formula (I). The compound shown in the formula (I) is prepared by structural modification of 3-methylquinoxaline-2-carboxylic acid, retains a feature structure of 3-methylquinoxaline-2-carboxylic acid as much as possible and has active groups which can couple with a carrier protein. The 3-methylquinoxaline-2-carboxylic acid artificial antigen provided by the invention is a conjugate prepared by coupling of the compound shown in the formula (I) and a carrier protein. The 3-methylquinoxaline-2-carboxylic acid artificial antigen is used for animal immunization so that high-specificity monoclonal and polyclonal antibodies are obtained. A preparation method of the high-specificity monoclonal and polyclonal antibodies is simple and practicable. The 3-methylquinoxaline-2-carboxylic acid artificial antigen can be used for detection of olaquindox metabolites. The antibodies obtained by the 3-methylquinoxaline-2-carboxylic acid artificial antigen can be used for detection of olaquindox metabolites.

Description

technical field [0001] The invention relates to a 3-methylquinoxaline-2 carboxylic acid artificial antigen and an antibody prepared therefrom. Background technique [0002] 3-Methylquinoxaline-2-carboxylic acid (MQCA) is a metabolite produced in animals by olaquindox, acetylmethaquine, and quinocetone. Because it can inhibit a variety of Gram-positive and negative bacteria, and has obvious growth-promoting effects on livestock, poultry, and fish, quinoxaline drugs (including olaquindox, acetylmethaquine, and quinocetone) are widely used in my country. The application is very extensive. Studies have found that quinoxaline-based prototype drugs and their metabolites have obvious safety problems, and have obvious toxic and side effects such as teratogenicity, carcinogenicity, mutagenicity, photosensitivity, and adrenal cortex damage, which seriously endanger the health of humans and animals. [0003] The European Union issued a document in 1998 to prohibit the addition of olaq...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D241/44C07K14/765C07K14/77C07K16/44C12N5/20
Inventor 沈建忠王战辉江海洋张素霞李建成
Owner CHINA AGRI UNIV
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