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Method for preparing artificial hapten, artificial antigen and specific antibody of chlorpyrifos and its usage

An artificial hapten, artificial antigen technology, applied in the fields of botanical equipment and methods, applications, animal repellents, etc. The effect of on-site monitoring, high sensitivity and low detection cost

Inactive Publication Date: 2003-07-23
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sensitivity of these methods is greatly affected by steps such as sample purification and concentration; moreover, these methods require complex instruments that most laboratories do not have, and the process is cumbersome, which is not suitable for the detection and analysis of large batches of samples

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The synthesis of hapten is the key point of the method. In order to highlight the specific antigenic determinant of chlorpyrifos molecule, linking arms are respectively introduced from two different sites of chlorpyrifos molecule to synthesize the hapten of chlorpyrifos. The hapten O, O-diethyl-O-[3,5-dichloro-6-(2-carboxyethyl ) Thio-2-pyridyl] phosphorothioate (abbreviated as AR); with phosphorus trichloride as raw materials, through four-step reaction, the hapten O-ethyl-O-[3,5,6- Trichloro-(2-pyridyl)]-O-(3-carboxypropyl) phosphorothioate (referred to as PO), prepared by coupling with BSA with carbodiimide method to prepare an immunogenic artificial antigen, immunized animals High-affinity antibodies to carbofuran can be produced with corresponding specificity. The ELISA method based on this antibody can be used to detect chlorpyrifos residues in water, soil and vegetables, etc. Compared with conventional GC and HPLC, it is fast, simple, sensitive and cheap. Embod...

Embodiment 2

[0044] Embodiment 2: the synthesis of artificial antigen

[0045] The basic method is to first activate the hapten and then couple it to the carrier protein in aqueous medium and mild conditions. The conjugation method depends on the active group of the hapten containing -COOH and -NH 2 The hapten generally uses the method of synthesizing peptides, and the hapten containing -SH and -OH needs to use a bifunctional reagent to generate -COOH and -NH 2 Compounds are then coupled. When the fat-soluble hapten is coupled, an appropriate amount of water-soluble organic solvent, such as N,N-dimethylformamide, should be added to the reaction medium to improve the solubility and reaction efficiency of the hapten in the medium. The groups involved in the reaction of various proteins are the same, such as amino group, carboxyl group, phenol group, imidazolyl group and so on. Commonly used proteins include bovine serum albumin, human serum albumin, and ovalbumin.

[0046] 2.1 Synthesis ...

Embodiment 3

[0054] Embodiment 3: the preparation of antibody

[0055] 3.1 Preparation of antiserum from immunized animals

[0056] The experiment selects healthy male rabbits about half a year old and weighing 2-3 kg. Each immunogen was used to immunize three rabbits (Zhejiang College of Traditional Chinese Medicine is responsible for raising the rabbits), numbered rabbits 1-6 respectively.

[0057] The experimental immunization dose was 0.25 mg / kg for basic immunization, and 0.5 mg / kg for booster immunization. Dilute appropriate amounts of AR-BSA and PO-BSA complexes with normal saline, respectively, and add an equal volume of Freund's complete adjuvant (Freund's adjuvant was used for booster immunization). Shi's incomplete adjuvant), fully emulsified until the emulsion droplets are not dispersed when dropped into water. A combination of subcutaneous multi-point injection on the back and intramuscular injection on the thigh was used. 6 points of subcutaneous immunization on the back, ...

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Abstract

A process for preparing artificial antigen, artificial hapten and specific antibody of chlorpyrifos is disclosed. A hapten O,O-diethyl-O[3,5-dichloro-6(2-carboxyethyl) thio-2- pyridyl] thionophosphate (AR) is prepared through reaction between O,O-diethyl-O(3,5,6-trichloro-2-pyridyl) thionophosphate and 3-hydroxypropionic acid. A hapten O-ethyl-O-[3,5,6-trichloron-(2-pyridyl)]-O- (3-carboxypropyl)thionophosphate (PO) is prepared from trichlorothion through 4-step reaction. An artificial antigen is prepared from said hapenjs and protein by coupling. A specific antibody is generated by immunizing animal with said antigen. It can be used to detect the residue of chlorpyrifos.

Description

technical field [0001] The invention relates to a preparation method of chlorpyrifos artificial hapten, artificial antigen and specific antibody and its application. Background technique [0002] The invention belongs to the technical field of immunochemistry and residue analysis of small-molecule pesticide compounds (molecular weight less than 1000 Daltons), relates to organic synthesis, immunochemistry and biochemistry, and relies on basic principles of immunology and immunochemistry and biotechnological means to design and synthesize Small molecule target analyte hapten, and coupled with carrier protein to prepare effective artificial antigen, immunize animals to prepare specific antibody for small molecule analyte, use the specific immunological reaction of antigen and antibody and the identification of markers that are easily detected and identified Amplification, quantitative detection of ultra-trace small molecule target analytes in samples, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N57/16C07F9/09
Inventor 朱国念吴刚程敬丽吴慧明魏方林
Owner ZHEJIANG UNIV
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