Kit for detecting 3-methyl-quinoxaline-2-carboxylic acid

A methylquinoxaline and kit technology, which is applied in the field of kits for detecting 3-methylquinoxaline-2-carboxylic acid, can solve photosensitivity and adrenal cortex damage, endanger human and animal health, cause mutagenesis, etc. problems, to achieve the effect of convenient and easy inspection methods, long shelf life and high accuracy

Active Publication Date: 2012-08-01
北京维德维康生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Through research, it is found that quinoxaline prototype drugs and 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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  • Kit for detecting 3-methyl-quinoxaline-2-carboxylic acid
  • Kit for detecting 3-methyl-quinoxaline-2-carboxylic acid
  • Kit for detecting 3-methyl-quinoxaline-2-carboxylic acid

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1, preparation 3-methylquinoxaline-2-carboxylic acid hapten

[0041] One, the preparation of 3-methylquinoxaline-2-carboxylic acid hapten

[0042] 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.

[0043] 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

[0050]Preparation and characterization of embodiment 2, 3-methylquinoxaline-2-carboxylic acid artificial antigen

[0051] 1. Synthesis and characterization of 3-methylquinoxaline-2-carboxylic acid immunogen

[0052] 1. Preparation of 3-methylquinoxaline-2-carboxylic acid immunogen

[0053] (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.

[0054] (2) Add 30mg of bovine serum albumin into 2mL of PBS buffer solution and fully dissolve to obtain solution IV.

[0055] (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-methylquino...

Embodiment 3

[0069] Preparation of embodiment 3, 3-methylquinoxaline-2-carboxylic acid monoclonal antibody

[0070] Balb / c mice: purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.;

[0071] SP2 / 0 myeloma cells: purchased from Sigma-Aldrich Company, the product catalog number is 08060101.

[0072] 1. Animal immunity

[0073] 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.

[0074] 2. Cell fusion and cloning

[0075] 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.

[0076] 2. Cloning the positive wells by using the limiting dilution method to obta...

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Abstract

The invention discloses a kit for detecting 3-methyl-quinoxaline-2-carboxylic acid. The enzyme linked immunosorbent assay (ELISA) kit for detecting 3-methyl-quinoxaline-2-carboxylic acid comprises a monoclonal antibody which has an anti-preservation number of CGMCC No.5778 and is excreted by an olaquindox metabolite monoclonal antibody hybrid tumor 5A2. The kit comprises the monoclonal antibody, and a conjugate of a compound and a barrier protein, which is shown as a formula (I). The kit has the advantages of high sensitivity, high accuracy, high precision, low cost, simplicity for operation, short detection time, suitability for use by various units, simplicity for storage and long shelf life, can be used for quickly detecting a large number of samples at the same time, and can realize on-site high-flux quick detection. The ELISA kit is suitable for determining the residual quantity of olaquindox metabolites in feeds, animal tissues or excrements. The antibody, the kit and a detection method can play a major role in detection of the olaquindox metabolites.

Description

technical field [0001] The invention relates to a kit for detecting 3-methylquinoxaline-2-carboxylic acid. Background technique [0002] 3-Methylquinoxaline-2-carboxylic acid (MQCA) is a metabolite produced in animals by olaquindox, acetylmethaquine, and quinocetone. Through the research, it is found that the quinoxaline 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. International regulations on the maximum residue limits of quinoxaline drugs are very strict. The European Union stipulates that carbadox, olaquindox and their metabolites shall not be detected in animal food and that the sale of these two veterinary drugs is prohibited. my country's Ministry of Agriculture Announcement No. 235 stipulates that the maximum residue limit of olaquindox is olaquindo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577C12N5/20C12R1/91
Inventor 江海洋王世恩王照鹏何丹婷李淑芳潘静茹
Owner 北京维德维康生物技术有限公司
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