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Acetamiprid hapten and preparation method thereof as well as application

A technology of acetamiprid and hapten, which is applied in the field of preparation of acetamiprid hapten and antigen, and achieves high sensitivity, high antibody specificity and rapid detection effect

Active Publication Date: 2019-01-01
ZHENGZHOU TOBACCO RES INST OF CNTC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation method of acetamiprid hapten and antigen has not been reported at present

Method used

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  • Acetamiprid hapten and preparation method thereof as well as application
  • Acetamiprid hapten and preparation method thereof as well as application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Preparation of acetamiprid hapten

[0023] 1. Synthesis of acetamiprid hapten

[0024] Take 0.50 g of N-desmethyl acetamiprid, add 50 mL of methanol to dissolve, add 0.30 g of aminobutyric acid, stir, add 0.37 mL of 37% formaldehyde solution, stir, mix well, react at 80°C for 4 h; stop the reaction, spin evaporate Remove methanol, add 50 mL of water, add 100 mL of ethyl acetate for extraction, extract three times, combine the organic phases, evaporate to dryness, put on a silica gel column, dichloromethane / methanol ( V / V , 10 / 1) was eluted and separated to obtain 0.69 g of the hapten product carboxyacetamiprid, with a yield of 92.81%.

[0025] 2. Identification of acetamiprid hapten

[0026] The above haptens were identified by H NMR spectroscopy, 1 H NMR (CDCl 3 , 300MHZ) δ: 11.0 (1H, -COOH), 8.53 (1H, s, ArH), 7.86 (1H, d, ArH), 7.21 (1H, d, ArH), 4.16 (1H, dd, =CH-) ,3.91 (1H, s, -CH 2 -), 3.21 (2H, d, -CH 2 -), 2.82 (2H, t, -CH 2 -), 2.49 (2H, t...

Embodiment 2

[0028] Example 2 Preparation of Acetamiprid Antigen

[0029] 1. Synthesis of Acetamiprid Immunogen

[0030] The acetamiprid hapten was conjugated with bovine serum albumin (BSA) to obtain the immunogen.

[0031] Take 11 mg of the hapten carboxyacetamiprid, add 1 mL of dimethyl sulfoxide to dissolve, add 0.18 mL of isobutyl chloroformate, and 0.1 mL of triethylamine, and react for 1 h at 0-4°C to obtain Hapten Activation Solution A ; Take 50 mg of BSA, add 3 mL of 0.8% saline to dissolve to obtain liquid B; add liquid A dropwise to liquid B, continue to stir for 5 h, 0.02 mol / L PB dialysis purification for 3 days, change the liquid 3 times a day, The immunogen was obtained, aliquoted, and stored at -20°C.

[0032] 2. Synthesis of acetamiprid coating agent

[0033] The acetamiprid hapten was coupled with ovalbumin (OVA) to obtain the coating source.

[0034] Take 5 mg of the hapten carboxyacetamiprid, add 1 mL of dimethyl sulfoxide to dissolve, add 9 mg of dicyclohexylcarbod...

Embodiment 3

[0037] Example 3 Preparation of Acetamiprid Monoclonal Antibody

[0038] 1. Obtaining hybridoma cells

[0039] 1) First immunization: Fully emulsify the acetamiprid hapten-BSA conjugate (immunogen) with an equal amount of Freund's complete adjuvant, and inject 6-week-old Balb / c mice subcutaneously, 0.2 mL each;

[0040] 2) Booster immunization twice: from the first immunization, booster immunization once every two weeks, with Freund's incomplete adjuvant instead of Freund's complete adjuvant, the method and dosage are the same as the first immunization;

[0041] 3) One week after the last booster immunization, the fundus vein blood was collected to measure the titer and inhibition. When there was inhibition and the titer reached more than 1:10000, the following last immunization was carried out: intraperitoneal injection of 0.1 mL of the immunogen solution without any adjuvant, and then executed three days later Mice, whose spleen was fused with myeloma cells;

[0042] 4) Us...

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Abstract

The invention discloses acetamiprid hapten and a preparation method thereof as well as application. The acetamiprid hapten is characterized in that the acetamiprid hapten is obtained by reacting N-noracetamiprid and aminobutyric acid; and the acetamiprid hapten is obtained by coupling acetamiprid hapten and carrier protein. The hapten prepared by the preparation method shows a specific acetamipridantigen determinant, so that a high-specificity acetamiprid monoclonal antibody is possibly screened out. The generated antibody is high in specificity and sensitivity, can be used for establishing an enzyme linked immunoadsorption assay method and a colloidal gold test paper quick measuring method, so that quick detection on acetamiprid in tobaccos and food is realized.

Description

technical field [0001] The invention relates to a preparation method and application of acetamiprid hapten and antigen. The invention belongs to the technical field of pesticide immunochemistry. Background technique [0002] Acetamiprid (acetamiprid) belongs to the neonicotinoid insecticide, which mainly acts on the acetylcholine receptors in the post-synaptic membrane of insects to effectively kill insects. It is effective against aphids, leafhoppers, whitefly, The control effect of leafminer and other pests is relatively ideal. It is a broad-spectrum, high-efficiency, and low-toxicity insecticide, which is widely used in actual production and life. [0003] As a smoking product, the safety of tobacco has attracted more and more attention, and pesticide residues are one of the main factors affecting the safety of tobacco leaves. Acetamiprid is a high-efficiency neonicotinoid insecticide. Although it is highly safe, its harm to the human body cannot be ignored. In addition...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D213/61C07K14/765C07K14/77C07K14/795G01N33/577G01N33/558
CPCC07D213/61C07K14/765C07K14/77C07K14/795C07K19/00G01N33/558G01N33/577
Inventor 陈黎范子彦刘惠民唐纲岭樊美娟崔华鹏郭吉兆鲁亚辉曹东山李旭
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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