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High coupling ratio holoantigen synthesis method of olaquindox residue marker

A synthesis method and whole antigen technology, applied in the field of immunochemical analysis, can solve the problems of low accuracy, inflammation, irritation, etc., and achieve the effect of less allergies, high safety, and less by-products

Inactive Publication Date: 2014-04-30
LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing 3-methylquinoxaline-2-carboxylic acid whole antigen synthesis methods all use N,N'-dicyclohexylcarbodiimide (DCC) as a catalyst, but the disadvantage of this catalyst is that during the synthesis process The easily produced by-product N,N'-dicyclohexyl urea is not easy to remove. This by-product is irritating to the skin and eyes and causes inflammation. The coupling ratio of the obtained whole antigen is generally only about 10:1
At present, there is no reasonable and reliable detection method for the coupling ratio of the whole antigen of the residual markers of olaquindox in China. The method for determining the coupling ratio is mainly ultraviolet spectrophotometry, which is not specific and accurate.

Method used

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  • High coupling ratio holoantigen synthesis method of olaquindox residue marker
  • High coupling ratio holoantigen synthesis method of olaquindox residue marker
  • High coupling ratio holoantigen synthesis method of olaquindox residue marker

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Experimental program
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Effect test

Embodiment 1

[0029] Synthesis of whole antigen (MQCA-BSA): Dissolve 25mmol of 3-methylquinoxaline-2-carboxylic acid (MQCA) in 0.5mL of dimethylformamide, then add 25mmol of N,N'-di Isopropylcarbodiimide (DIC), stirred for 30min, then added 40mmol of N-hydroxysuccinimide, stirred at 37°C in the dark for 16h, centrifuged to remove the precipitate, and the supernatant obtained by centrifugation was reaction solution A; 3.0×10 -4 Mmol bovine serum albumin (BSA, molecular weight 68000) was dissolved in 1.5mL of 0.02 mol / L phosphate buffer solution (pH=7.4) to obtain solution B; Stir overnight, place the reaction solution in an ultrafiltration tube (molecular cut-off 30 KD), centrifuge for 10 min, add 1 mL of 0.2 mol / L PBS buffer solution with pH=7.4, then centrifuge, repeat the operation 3 to 4 times, fully Wash off the unreacted small molecules, collect the filtrate, take 1mL of the filtrate to pass through a 0.22μm filter membrane, and use high-performance liquid chromatography to detect the...

Embodiment 2

[0031] Synthesis of whole antigen (MQCA-OVA): Dissolve 25mmol of MQCA in 0.5mL of dimethylformamide, then add 25mmol of DIC, stir for 30min, then add 40mmol of N-hydroxysuccinimide, and avoid Stir the reaction for 16 hours, centrifuge to remove the precipitate, and the supernatant obtained by centrifugation is the reaction solution A; 4.4×10 -4 Dissolve mmol ovalbumin (OVA, molecular weight 45,000) in 1.5mL 0.02 mol / L phosphate buffer solution (pH=7.4) to obtain solution B; add solution B dropwise to reaction solution A, and stir at 37°C Overnight, put the reaction solution in an ultrafiltration tube (molecular cut-off: 30 KD), centrifuge for 10 minutes, add 1 mL of 0.2mol / L PBS buffer solution with pH=7.4, centrifuge again, repeat the operation 3 to 4 times, and fully wash away Collect the filtrate for uncompletely reacted small molecular substances, take 1mL of the filtrate to pass through a 0.22μm filter membrane, and use high-performance liquid chromatography to detect the...

Embodiment 3

[0033] Synthesis of the whole antigen (MQCA-KLH): Dissolve 25mmol of MQCA in 0.5mL of dimethylformamide, then add 25mmol of DIC, stir for 30min, then add 40mmol of N-hydroxysuccinimide, and avoid Stir the reaction for 16 hours, centrifuge to remove the precipitate, and the supernatant obtained by centrifugation is the reaction solution A; 2.5×10 -6 One mmol of keyhole limpet hemocyanin (KLH, molecular weight 8,000,000) was dissolved in 1.5 mL of 0.02 mol / L phosphate buffer solution (pH=7.4) to obtain solution B; solution B was added dropwise to reaction solution A, Stir overnight at 37°C, place the reaction solution in an ultrafiltration tube (molecular cutoff: 30 KD), centrifuge for 10 minutes, add 1 mL of 0.2mol / L PBS buffer solution with pH=7.4, centrifuge again, and repeat the operation 3 to 4 times , fully wash away the unreacted small molecular substances, collect the filtrate, take 1mL of the filtrate to pass through a 0.22μm filter membrane, and use high-performance li...

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Abstract

The invention discloses a high coupling ratio holoantigen synthesis method of an olaquindox residue marker. The method comprises the following steps: performing an esterification reaction of 3-methylquinoxaline-2-carboxylic acid with excessive N-hydroxysuccinimide in the presence of N,N'-diisopropyl carbodiimide serving as a catalyst; and performing coupling reaction of ester with carrier protein to obtain the high coupling ratio holoantigen of the olaquindox residue marker. The catalyst used in the method has high safety and hardly causes allergy of operators, few side products are generated, the coupling ratio of the prepared holoantigen is between 32 and 62; and after a mouse is immunized with the antigen, an MQCA (methyl-3-quinoxaline-2-carboxylic acid) multi-clone antibody with valence of antibody being over 128,000 can be obtained, the olaquindox residue marker has less than 20 percent of cross reaction with olaquindox, quinoceton and maquinox, and no cross reaction with chloramphenicol, clenbuterol hydrochloride, antibiotics and the like.

Description

technical field [0001] The invention belongs to the technical field of immunochemical analysis, and in particular relates to a method for synthesizing a whole antigen with a high coupling ratio of residual markers of olaquindox. Background technique [0002] Lalaquindox, also known as quinamide alcohol, and its trade names are Beyonol and Kuaiyuling, have moderate to obvious cumulative toxicity, have obvious teratogenic effects on most animals, and have potential three properties to humans, that is, pathogenicity. Deformation, mutagenicity, carcinogenicity, and food safety issues caused by its abuse in feed and feed additives have attracted much attention. Research on rapid detection kits and colloidal gold test strips has become a domestic hotspot in recent years. 3-Methylquinoxaline-2-carboxylic acid is a small molecular substance, which cannot stimulate the animal body to produce an immune response to obtain antibodies, and needs to be linked with macromolecular substanc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/765C07K14/77C07K14/795G01N33/531G01N30/02
Inventor 张景艳李建喜张凯杨志强王磊王学智张宏孟嘉仁秦哲
Owner LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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