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Synthesis method of high-sensitivity carbendazol complete antigen

A technology of complete antigen and synthesis method, applied in the field of synthesis of complete antigen of carbendazim, to achieve the effect of high sensitivity and high specificity

Active Publication Date: 2014-09-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the present invention, a hapten H1 with a carboxyl group is obtained by reacting 2-chlorobenzimidazole, an analog of carbendazim, with 6-aminocaproic acid, and the hapten H1 is coupled with a carrier protein to prepare an immunogen; based on heterologous coating The sensitivity of the antibody can be further improved. Another hapten H2 is obtained from the reaction of the analogue of carbendazim, aminochlorobenzimidazole, and 6-aminocaproic acid. H2 is coupled with OVA to obtain the coating agent. These two haptens are currently There is no literature report yet

Method used

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  • Synthesis method of high-sensitivity carbendazol complete antigen
  • Synthesis method of high-sensitivity carbendazol complete antigen
  • Synthesis method of high-sensitivity carbendazol complete antigen

Examples

Experimental program
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Effect test

Embodiment 1

[0034] The preparation of embodiment 1 carbendazim hapten H1

[0035] (1) Synthesis of precursor 261: Aminocaproic acid methyl ester hydrochloride (obtained by refluxing aminocaproic acid and thionyl chloride in methanol) and equimolar amounts of 2-chlorobenzimidazole were mixed in diiso Microwave reaction in propylethylamine at 150°C overnight for 13h yielded about 10% of the product, and the intermediate was obtained through preparation and purification.

[0036] (2) Synthesis of hapten H1: Dissolve the above intermediate in tetrahydrofuran (THF) ， Stir the reaction overnight at room temperature, remove THF by rotary evaporation, adjust the pH to 6~7 with 1mol / L HCl solution, and purify the product by preparative chromatography.

Embodiment 2

[0037] Embodiment 2 Carbendazim hapten H1 prepares complete antigen

[0038] 1) Activation of hapten H1: Take 35 mg of carbendazim hapten H1, add 2 mL of DMF to dissolve, then add NHS and EDC respectively (the molar ratio of hapten, NHS, and EDC is 1: 1.5: 2), and stir at room temperature for 12 hours .

[0039] 2) Preparation of protein solution: Take 160mg of bovine serum albumin (the molar ratio of hapten H1 to bovine serum albumin is 60:1), add 10mL of 0.1M pH9.6 carbonate buffer.

[0040] 3) Reaction: slowly drop the activated hapten H1 solution prepared in step 1) into the protein solution prepared in step 2), and react at room temperature for 8 hours. Dialyze with PBS buffer solution for 3 days, during which the water was changed 6 times, and the complete antigen of carbendazim was obtained.

Embodiment 3

[0041] Implementation example 3, carbendazim hapten H2 preparation coating original

[0042] The structural formula of H2 is:

[0043]

[0044] Take 20 mg of carbendazim hapten H2, add 1 mL of DMF to dissolve, and pre-cool at 0°C for 30 min. At 0°C, add glutaraldehyde (the molar ratio of hapten to glutaraldehyde is 1:1.2), and react at 0°C for 20 minutes. Take 120mg of chicken ovalbumin (the molar ratio of hapten to chicken ovalbumin is 30:1), add 10mL of 0.1M pH9.6 carbonate buffer, and pre-cool at 0°C for 30 min. At 0°C, the activated hapten solution was slowly added dropwise to the protein solution, and reacted at 0°C for 4 hours. Dialyze with PBS buffer solution for 3 days, during which the water was changed 6 times to obtain the carbendazim-coated original.

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Abstract

The invention relates to a synthesis method of a high-sensitivity carbendazol complete antigen, belonging to the technical field of biochemical engineering. In the method, a carboxylic hapten H1 is synthesized by reacting carbendazol analog 2-chlorobenzimidazole and 6-aminocaproic acid, and is coupled with a carrier protein to prepare an immunogen; and on the basis that the heterologous coating can further enhance the sensitivity of the antibody, the hapten H2 obtained by reacting the carbendazol analog aminochlorobenzimidazole and 6-aminocaproic acid is coupled with OVA to obtain the coupled substance which can be used as a coating source. The complete antigen prepared by the method appears a specific carbendazol antigenic determinant, thereby providing possibilities for screening out high-specificity carbendazol monoclonal antibodies. The generated antibody has the advantages of high specificity and high sensitivity, and can be used for establishing an enzyme linked immunosorbent assay analysis method and a colloidal gold test paper quick detection method, thereby being used for quickly detecting carbendazol residues in food.

Description

technical field [0001] The invention relates to a high-sensitivity synthesis method of carbendazim complete antigen, which belongs to the technical field of biochemical industry. Background technique [0002] Carbendazim is a broad-spectrum, high-efficiency, low-toxic systemic benzimidazole fungicide. Its chemical structural formula is as follows: [0003] [0004] Its mechanism of action is that it interferes with cell division by interfering with the formation of the cell spindle, and has good protective and therapeutic effects on plants. It is widely used in various agricultural products, and has the effect of preventing and controlling the diseases of various crops caused by fungi (such as Deuteromycetes, polyascomycetes). Can be used for foliar spray, seed treatment and soil treatment etc. Carbendazim has potential damage to animal liver, kidney and reproductive system. [0005] At present, the methods for detecting carbendazim are mainly gas chromatography (GC),...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K14/795C07K14/77C07K1/107C07K16/44G01N33/53
CPCC07K14/765C07K14/77C07K14/795C07K16/44C07K19/00
Inventor 徐丽广胥传来严会娟匡华马伟刘丽强宋珊珊吴晓玲
Owner JIANGNAN UNIV
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