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Monoclonal antibody capable of simultaneously detecting isofenphos-methyl and isocarbophos and application thereof

A technology of monoclonal antibody and isofenphos-methyl, which is applied in the direction of microorganism-based methods, measuring devices, chemical instruments and methods, etc., can solve the problem of low sensitivity and achieve the effect of high sensitivity and strong specificity

Active Publication Date: 2022-04-08
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] A Chinese patent discloses a monoclonal antibody against three organophosphorus pesticides and its application. The monoclonal antibody can be used for the detection of isocarbophos, isofenphos-methyl and isofenphos-ethyl in agricultural products and agricultural production environments , the detection sensitivity is not high
[0006] At present, there is no high-sensitivity enzyme-linked immunoassay for the simultaneous detection of isofenphos-methyl and isocarbophos

Method used

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  • Monoclonal antibody capable of simultaneously detecting isofenphos-methyl and isocarbophos and application thereof
  • Monoclonal antibody capable of simultaneously detecting isofenphos-methyl and isocarbophos and application thereof
  • Monoclonal antibody capable of simultaneously detecting isofenphos-methyl and isocarbophos and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Synthesis of isofenphos-methyl and isocarbophos hapten

[0045] 1. Experimental method

[0046] Synthesis of isofenphos-methyl and isocarbophos haptens, the specific synthesis process see figure 1 .

[0047] 1. Synthesis of hapten shown in formula (I)

[0048]

[0049] Take 3.6g of isopropyl salicylate, 3g of O-methyl phosphorothioate dichloride, 0.5g of TBAB dissolved in 10mL of dichloromethane (DCM) and stir, 0.5g of NaOH dissolved in 10mL of tertiary water, add the above DCM solution, stirred vigorously to make the organic-aqueous phase layer interface disappear, and stirred overnight. Transfer to a separatory funnel, and after the layers are separated, the organic phase of the lower layer is removed and rotary evaporated to obtain the intermediate product shown in formula (III). After 400-mesh silica gel chromatography, the mobile phase is petroleum ether, and the purified intermediate product is obtained after separation and purification. Take 0.5...

Embodiment 2

[0056] Example 2 Synthesis and Identification of Artificial Antigen

[0057] 1. Experimental method

[0058] 1. Artificial antigen synthesis shown in formula (IV)

[0059] Dissolve 9.4 mg of the hapten represented by formula (I) in 600 μL of N,N-dimethylformamide, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt under stirring acid salt and N-hydroxysuccinimide, and stirred at room temperature for 4 hours in the dark to obtain the activated hapten, which is called A solution. Wherein, the mol ratio of the hapten shown in formula (I), the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and the N-hydroxysuccinimide is 1:1.5:1.5. Dissolve 20mg of bovine lactoferrin (LF) in pH 9.6 carbonic acid buffer solution with a bovine lactoferrin concentration of 10mg / mL, which is called liquid B. The above liquid A was added dropwise to the liquid B under stirring in an ice bath, the molar ratio of the hapten in the liquid A having the structural formula shown in ...

Embodiment 3

[0072] Example 3 Preparation of Monoclonal Antibodies that Can Simultaneously Detect Isofenphos-methyl and Isocarbophos

[0073] 1. Experimental method

[0074] Get 6-8 weeks old Balb / c mice (Zhuhai Baishitong Biotechnology Co., Ltd.), and immunize the antigen with the artificial antigen shown in formula (IV-1) at a concentration of 1 mg / mL prepared in Example 2 and Freund's complete adjuvant Mix equal amounts of the same doses, and after complete emulsification, inject 100 μL into the abdomen and back of mice. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent booster immunizations, and immunization was performed every 2 weeks, and a total of 3 booster immunizations were performed. One week after the second booster immunization, blood was taken from the tail vein to test the titer and inhibition. After the third booster immunization, mice with higher titer and inhibition rate were selected for cell fusion...

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Abstract

The invention discloses a monoclonal antibody capable of simultaneously detecting isofenphos-methyl and isocarbophos and application of the monoclonal antibody, the monoclonal antibody is secreted by a hybridoma cell strain which is preserved in China Center for Type Culture Collection on May 28, 2019 and has a preservation number of CCTCC No: C 2019107, and on the basis of the monoclonal antibody, the isofenphos-methyl and isocarbophos-methyl can be simultaneously detected. The invention establishes a detection method for detecting isofenphos-methyl and isocarbophos based on indirect competitive enzyme-linked immunosorbent assay. The detection method for detecting pesticide residues of isofenphos-methyl and isocarbophos has the characteristics of rapidness, strong specificity and high sensitivity, the IC50 value of the detected isofenphos-methyl is 0.47 ng / mL, the detection limit (IC10 is 0.052 ng / mL, and the linear range is 0.12-1.90 ng / mL; the IC50 value of isocarbophos is detected to be 0.96 ng / mL, the detection limit (IC10 is 0.112 ng / mL, and the linear range is 0.25-3.75 ng / mL.

Description

technical field [0001] The invention relates to the technical field of pesticide detection, in particular to a monoclonal antibody capable of simultaneously detecting isofenphos-methyl and isocarbophos and its application. Background technique [0002] my country is a big country in the production and use of pesticides. The use of pesticides has created economic benefits for agriculture. At the same time, pesticide residues have caused many problems such as environmental pollution and food safety. Both isofenphos-methyl and isocarbophos are organophosphorus pesticides, which are currently one of the highly toxic pesticides restricted in use in my country. The harm of organophosphorus pesticides to the human body is mainly to inhibit the activity of cholinesterase in the body, leading to the accumulation of acetylcholine, thereby producing neurotoxicity. [0003] At present, for the detection of isofenphos-methyl and isocarbophos, instrumental analysis is the main detection ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/577G01N33/531C12R1/91
CPCY02A50/30
Inventor 徐振林袁健城陈子键罗林沈玉栋雷红涛肖治理王弘
Owner SOUTH CHINA AGRI UNIV
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