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Preparation and application of mass-selective monoclonal antibody capable of resisting 8 triazin type agricultural chemicals

A technology of monoclonal antibodies and triazines, applied in the field of rapid detection of pesticide residues, can solve the problems of less research on triazine pesticides, and achieve the effects of high sensitivity, low detection cost, and simple operation

Active Publication Date: 2019-06-11
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although triazine pesticide antibodies and related immunoassay methods have been reported, most studies have focused on atrazine and other chlorinated triazine pesticides, and less research has been done on thio- and oxo-triazine pesticides. The mass-selective monoclonal antibodies and enzyme-linked immunoassay methods of amethazine, prometone, azizine, zhongdingtong, atraton, diquat, sizazone, and amethazine have not been reported yet.

Method used

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  • Preparation and application of mass-selective monoclonal antibody capable of resisting 8 triazin type agricultural chemicals
  • Preparation and application of mass-selective monoclonal antibody capable of resisting 8 triazin type agricultural chemicals
  • Preparation and application of mass-selective monoclonal antibody capable of resisting 8 triazin type agricultural chemicals

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: the preparation of hapten

[0047] (1) Synthesis of the immune hapten 4-(4-isopropylamino-6-methylthio-1,3,5-triazine-2-)aminobutyric acid

[0048]①In a 50mL three-neck flask, dissolve 2,4,6-trichloro-1,3,5-triazine (2g) and isopropylamine (644mg) in dry dichloromethane (20mL), and cool to -30°C under nitrogen protection. ℃, slowly drop 10mL of diisopropylethylamine (DIPEA) (2.1g) diluted with dichloromethane into the flask, react at -20℃~-10℃ for 2h, concentrate the reaction solution, spin dry, and disperse in 50mL Water, filtered, collected filter cake, and recrystallized with ethanol to obtain 2.1 g of intermediate a (4,6-dichloro-N-isopropyl-1,3,5-triazin-2-amine) as a white solid.

[0049] ②In a 225mL three-neck flask, dissolve the above-mentioned intermediate a (1g), 4-aminobutyric acid (400mg) and DIPEA (939mg) in 10mL of absolute ethanol, and react at 60°C for 12 hours under the protection of nitrogen. Concentrate the reaction solution and spin dr...

Embodiment 2

[0059] Embodiment 2: the preparation of artificial antigen

[0060] (1) Preparation of immune antigen

[0061] Weigh the immune hapten H10.015mmol, NHS 0.03mmol, DCC 0.03mmol, dissolve in 200μL dimethylformamide (DMF), seal the reaction at room temperature under magnetic stirring overnight, centrifuge at 10000rpm for 5 minutes, take the supernatant and slowly add it to 1mL In 10mg / mL OVA-PBS solution, stirred at 4°C for 12h. After the reaction was completed, the reaction solution was placed in a dialysis bag and dialyzed in 0.01 mol / L pH 7.4 PBS buffer solution at 4°C for 72 hours, and the dialysis solution was changed every 6 hours. After the dialysis, the white emulsion (H1-OVA) in the dialysis bag was taken out and stored in a refrigerator at -20°C.

[0062] (2) Preparation of coated antigen

[0063] The coating antigen is prepared by coupling the coating hapten H2 with BSA, and the method is the same as the coupling of the immune antigen.

[0064] (3) Artificial antige...

Embodiment 3

[0067] Example 3: Animal Immunization

[0068] H1-OVA was used as an immunogen to immunize 6-8 week-old Balb / c female mice. Each mouse was given 100 μg of immunogen subcutaneously in the abdominal cavity and back of each mouse, once every two weeks, and immunized four times in total. For the first immunization, an equal volume of immunogen was emulsified with Freund's complete adjuvant, and for subsequent immunizations, an equal volume of immunogen was emulsified with Freund's incomplete adjuvant. One week after the third immunization, blood was collected from the orbit of the mice, and the titer and specificity of the serum were detected by indirect ELISA.

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Abstract

The invention discloses a preparation method and application of a mass-selective monoclonal antibody capable of resisting 8 triazin type agricultural chemicals, and belongs to the technical field of biology. According to the preparation method disclosed by the invention, an appropriate activity arm is introduced into a 1,3,5- triazine ring, immune half-antigen and coating half-antigen are prepared, by an activity ester method, ovalbumin (OVA) and bovine serum albumin (BSA) are coupled to prepare immunogen and coating antigen, a Balb / c mouse is immunized by the immunogen, and a monoclonal antibody is prepared through a hybridoma technique; based on the antibody, the coating antigen is used as detection antigen, heterogenous indirect competitive enzyme-linked immunosorbent assay (ELISA) is established, and the mass-selective monoclonal antibody can be used for detecting 8 triazin type agricultural chemicals including prometryn, prometon, mesoran, secbumeton, atratone, desmetryn, simetrynand ametryn. The method is low in detection cost, high in sensitivity and simple and convenient in operation, and is suitable for on-site quick massive screening of the 8 triazin type agricultural chemicals.

Description

technical field [0001] The present invention relates to the preparation method of group selective monoclonal antibody against eight kinds of triazine pesticides and its application in enzyme-linked immunoassay method. The on-site rapid screening of eight triazine pesticides including atraton, diquat, sigrazine and ametraz belongs to the field of rapid detection of pesticide residues. Background technique [0002] my country's agricultural production continues to grow steadily. At the same time, the long-term, large-scale and irrational use of agricultural chemicals such as chemical fertilizers, chemical pesticides, and herbicides has a negative impact on my country's agricultural ecological environment, safe production of agricultural and livestock products, and human health and life. Pesticide residues have caused serious threats to human and animal poisoning and death incidents frequently occur. In the export of vegetables and tea in my country, incidents such as rejection...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/577C07K14/77C07D251/52C12R1/91
Inventor 杨美华刘聪敏豆小文
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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