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Method for preparing 2 methyl 4 chlorine artificial half antigen, artificial antigen, specific antibody and application

A technology of artificial haptens and artificial antigens, applied in chemical instruments and methods, specific peptides, carrier-bound/immobilized peptides, etc., can solve problems such as unsuitable for large-scale sample detection and analysis, great influence, cumbersome process, etc., to achieve Easy on-site monitoring, strong specificity, and high sensitivity

Inactive Publication Date: 2006-06-07
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sensitivity of these methods is greatly affected by steps such as sample purification and concentration, and requires complex instruments that most laboratories do not have. The process is cumbersome and requires derivatization treatment, which is not suitable for the detection and analysis of large batches of samples.

Method used

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  • Method for preparing 2 methyl 4 chlorine artificial half antigen, artificial antigen, specific antibody and application
  • Method for preparing 2 methyl 4 chlorine artificial half antigen, artificial antigen, specific antibody and application
  • Method for preparing 2 methyl 4 chlorine artificial half antigen, artificial antigen, specific antibody and application

Examples

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Effect test

preparation example Construction

[0040] 3 Preparation of Antibody and Establishment of ELISA Method

[0041]Three immunogen complexes prepared by coupling hapten M, MC and MB to protein were immunized with three rabbits according to conventional methods. From the second time of booster immunization, blood was collected from the rabbit's ear vein on the 8th day after each immunization, and the titer of the serum was determined by indirect ELISA after appropriate dilution. After the fourth immunization, the rabbits obtained high-titer antibodies, and the titers of antiserum were 1:25600, 1:12800, 1:12800; 1:25600, 1:12800, 1:51200; 1:51200, 1:51200 and 1:102400. After the immune serum is qualified, blood is collected to separate the antiserum.

[0042] The ELISA method of 2A4Cl was established by using the 2A4C antigen antibody immunoreaction and enzymatic reaction. It has high specificity and sensitivity in the detection and analysis of 2-methyl-4-chlorine residues in food, plants, environmental soil and wa...

Embodiment 1

[0043] Example 1: Synthesis of Hapten MC

[0044] 1) According to the excess of thionyl chloride, SOCl 2 and 2-methyl-4-chlorine into a three-necked flask, heated to reflux for 1.5 h under magnetic stirring, and distilled off excess SOCl 2 , That is, the product 2-methyl-4-chlorophenoxyacetyl chloride.

[0045] 2) According to the feeding ratio of 6-aminocaproic acid: 2-methyl-4-chlorophenoxyacetyl chloride = 1.2: 1, weigh 6-aminocaproic acid and put it into a three-necked flask, add 3ml of 4mol / L NaOH under ice bath solution, stirred magnetically to dissolve, then added 2mL of 2-methyl-4-chlorophenoxyacetyl chloride solution dissolved in 10mL of dioxane, at an interval of 5min, added 2ml of the above solution and 1ml of 2mol / L NaOH solution per 1mmol of raw material, and added After completion, the reaction was stirred at 0-4°C for 2h. Rise to room temperature, adjust the pH value of the reaction solution to about 4.0 with concentrated hydrochloric acid, extract with ethyl...

Embodiment 2

[0048] Example 2: Synthesis of Hapten MB

[0049] 1) According to the excess of thionyl chloride, SOCl 2 and 2-methyl-4-chlorine into a three-necked flask, heated to reflux for 1.5 h under magnetic stirring, and distilled off excess SOCl 2 , That is, the product 2-methyl-4-chlorophenoxyacetyl chloride.

[0050] 2) According to the feeding ratio of γ-aminobutyric acid: 2-methyl-4-chlorophenoxyacetyl chloride = 1.5:1, weigh γ-aminobutyric acid into a three-necked flask, add 5ml of 4mol / L NaOH under ice bath solution, stirred magnetically to dissolve, then added 5 mL of 2-methyl-4-chlorophenoxyacetyl chloride solution dissolved in 25 mL of dioxane, at an interval of 10 minutes, added 5 mL of the above solution and 5 mL of 4 mol / L NaOH solution per 5 mmol of raw materials, and added After completion, the reaction was stirred at 0-4°C for 2h. Rise to room temperature, adjust the pH value of the reaction solution to about 4.0 with concentrated hydrochloric acid, extract with ethy...

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Abstract

The invention relates to a preparation method of 2-methyl-4-chloro artificial hapten, artificial antigen and specific antibody and its application. The hapten 4-(2-methyl-4 -chlorophenoxyacetyl)aminobutyric acid and 6-(2-methyl-4-chlorophenoxyacetyl)aminocaproic acid (referred to as MB and MC). 2-methyl-4-chloro (2-methyl-4-chlorophenoxycarboxylic acid) itself can also be used as a hapten (abbreviated as M). Jointly prepare immunizing antigen and coating antigen. Immunizing animals with the immunized antigen produces specific antibodies with high affinity for 2,4,4. This antibody does not readily cross-react with other compounds. The enzyme-linked immunosorbent assay method established with the antibody can be used for rapid, sensitive, convenient and cheap detection of trace amounts of 2-methyl-4-chlor residues in samples.

Description

technical field [0001] The invention relates to a preparation method of 2-methyl-4-chloro artificial hapten, artificial antigen and specific antibody and its application. Background technique [0002] Due to the continuous expansion of the scale of pesticide use, the environmental impact of pesticide residues and the chronic and long-term effects on human health are increasingly concerned and worried, and the restrictions on pesticide residues are therefore becoming more and more stringent. New requirements and higher standards have been put forward in various aspects such as indicators and indicators. For small molecular toxic chemicals with a molecular weight less than 1000 Daltons, such as pesticides and their metabolites, traditional residue analysis methods mainly rely on physical and chemical analysis methods such as gas chromatography (GC), liquid chromatography (HPLC) or mass spectrometry (MS). Traditional physical and chemical analysis methods a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07C235/34C07K16/00C07K17/02G01N33/02G01N33/00
Inventor 程敬丽吴慧明施海燕魏方林朱国念
Owner ZHEJIANG UNIV
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