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Dursban monoclonal antibody preparing method

A monoclonal antibody, chlorpyrifos technology, applied in biochemical equipment and methods, anti-animal/human immunoglobulin, instruments, etc., can solve the problem of no successful preparation of chlorpyrifos monoclonal antibody, and achieve effective monitoring and sensitive on-site monitoring Effect

Inactive Publication Date: 2007-05-23
SOUTHEAST UNIV
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Problems solved by technology

[0006] At present, although there are foreign reports on the immunoassay of chlorpyrifos pesticide residues, the only domestic report is the chlorpyrifos polyclonal antibody made from rabbit antiserum, and there is no report on the successful preparation of chlorpyrifos monoclonal antibody

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Embodiment Construction

[0018] I) Synthesis and identification of chlorpyrifos complete antigen:

[0019] Under alkaline conditions, the original drug of chlorpyrifos is heated and refluxed with 3-mercaptopropionic acid, so that the 6th chlorine atom on the pyridine ring of chlorpyrifos molecule is replaced by 3-mercaptopropionic acid with 3 straight-chain carbon atoms through a nucleophilic substitution reaction The hapten O, O-diethyl-O-[3,5-dichloro-6-(2-carboxyethyl)thio-2-pyridyl] phosphorothioate was synthesized, followed by the Chlorpyrifos artificial immune antigen and coating antigen were synthesized by amine method using bovine serum albumin (BSA) and ovalbumin (OVA) as carrier proteins, respectively.

[0020] The above-mentioned artificially synthesized antigens were identified by ultraviolet spectroscopy and molybdenum blue colorimetry, and the results showed that the maximum ultraviolet absorption peaks of the artificially synthesized complete antigens were changed compared with their re...

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Abstract

This invention relates to a preparation method for chlorpyrifos monoclonal antibody, which is against to a specific pesticide. The said method includes (1) introducing a coupling bridge on the sixth chlorine atom in pyridine ring of chlorpyrifos through nucleophilic substitution reaction, and combining with macromolecule carrier protein to obtain completely artificial antigen; (2) preparing mice spleen cells solution by immunizing chlorpyrifos fully, fusing mice spleen cells and mice myeloma cells SP2 / 0 with PEG, and obtaining positive hybridoma with stable secretion of chlorpyrifos antibody (which are IgG1 subclass) through hybridoma antibody detection and multiple cloning; and (3) establishing chlorpyrifos indirect competitive ELISA test by using monoclonal antibodies secreted by high antibody titer cell lines. The detection limit is 0 .03 mug / mL. The antibody has specificity and no cross-reaction with fenitrothion, malathion, parathion, etc. pesticides, and can be used for immunoassay of chlorpyrifos.

Description

technical field [0001] The invention relates to a method for preparing a monoclonal antibody directed at a specific pesticide, and belongs to the technical field of on-site immune monitoring reagent manufacturing for pesticide pollution. Background technique [0002] Since the middle of the last century, with the wide application of various pesticides in agricultural production, food storage and daily life, there have been more and more reports on the pollution of water sources, food, crops and the environment caused by pesticide residues. These pesticides and their metabolism Many of the products belong to persistent organic compounds (POPs), "three-caused" substances and environmental endocrine disruptors (EDCs), which are serious and long-lasting harm to the environment and human beings. Therefore, the problem of pesticide pollution has become one of the focuses of urgent monitoring and governance. my country is the world's second largest pesticide producer and also the l...

Claims

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Application Information

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IPC IPC(8): C07K16/18G01N33/577C12N5/18
Inventor 尹立红浦跃朴梁戈玉李云晖刘冉吴巍
Owner SOUTHEAST UNIV
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