ELISA (Enzyme Linked Immunosorbent Assay) test kit for testing difenoconazole residue and test method

A technology of difenoconazole and detection kit, which is applied in the directions of biological testing, chemical instruments and methods, measuring devices, etc., can solve the problem of no detection of difenoconazole residues, etc., and achieves low base and processing process. Simple, low processing time results

Inactive Publication Date: 2017-05-17
SHENYANG JINCHENG TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the detection of dife

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Difenoconazole hapten preparation

[0031] 350 g of anhydrous aluminum chloride and 249.5 mL of a 20% nitrobenzene solution were placed in a three-necked flask equipped with a stirrer, a reflux condenser and a separatory funnel, and the reaction temperature was controlled at 10 °C. Add 128.5 mL of ethyl chloride, then slowly drop in 150 g of difenoconazole suspending agent with a concentration of 30%, stir at a constant speed after the dropwise addition, and place it in the hydrogen fluoride solution at 10 °C, react for 2 hours to make it complete The hydrogen fluoride was evaporated completely. After the reaction, the organic layer was separated, and the solvent DMF was evaporated to purify the product. Weigh 20 g of potassium permanganate and dissolve it in the reaction solution, raise the temperature to 50 °C, take 15 g of the reactant from the previous step and add it to the reaction vessel, after adding all of it, raise the temperature to 65 °C, and stir until the...

Embodiment 2

[0033] Preparation of Difenoconazole Artificial Antigen

[0034] Weigh 2 g of difenoconazole hapten, dissolve in 2 mL of DMF, stir evenly, add 30 μL of tri-n-butylamine, ice-bath, add 30 μL of isobutyl chloroformate dissolved in DMF, and stir for 1 h. Weigh 3.2 g of BSA and dissolve it in 5 mL of citrate buffer with a pH of 9.6 and a concentration of 0.1 mol / L. Add the mixed solution of hapten and DMF to the BSA solution drop by drop, stir at 4 °C for 5 h, centrifuge the reaction solution at 5000 r / min for 30 min after stirring, discard the precipitate, keep the supernatant, and The dialysate was dialyzed for three days, and the dialysate was changed every 8 hours during the dialysis period, and finally the artificial antigen of difenoconazole was obtained.

Embodiment 3

[0036] Difenoconazole Antibody Preparation

[0037] Dissolve 3 mg of difenoconazole artificial antigen in 1 mL of normal saline, emulsify the diluted difenoconazole artificial antigen with an equal volume of Freund's complete adjuvant, and then inject the emulsified The artificial antigen of difenoconazole was injected into New Zealand white rabbits, and the dose of each rabbit was 0.5 mg / kg. After 15 days, the difenoconazole artificial antigen diluted with normal saline was emulsified with an equal volume of Freund's incomplete adjuvant, and the dose was 0.01 mg / kg. After 15 days, the difenoconazole artificial antigen diluted with benzene was used for the third time. After the artificial antigen of etheroconazole was emulsified with Freund's incomplete adjuvant, booster immunization was carried out. From the booster immunization, blood was collected 7 days after immunization, and the antiserum titer was determined until the titer reached a certain value, and the prepared Goo...

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Abstract

The invention discloses an ELISA (Enzyme Linked Immunosorbent Assay) test kit for testing difenoconazole residue and a test method. Chloroethane is utilized to substitute hydrogen atoms of three sites on a benzene ring in a difenoconazole molecular structure with ethyl groups, which are oxidized into carboxyl groups, afterwards, the mixed anhydride method is utilized to connect a hapten onto a carrier, so that an artificial antibody is formed, the carrier is bovine serum albumin, an artificial difenoconazole antigen is utilized to immunize New Zealand white rabbits to prepare an antibody, and along with related agents, such as standard difenoconazole, washing concentrate, substrate color-developing solution and stop solution, the antibodies compose the ELISA test kit. The main steps of a test include: sample preprocessing, kit testing, and test result analysis. Compared with a variety of current conventional test methods applied widely, the enzyme linked immunosorbent assay method disclosed by the invention has advantages, such as low cost and high testing sensitivity, and an effective method is provided for difenoconazole residue testing.

Description

technical field [0001] The invention belongs to the technology for detecting pesticide residues in vegetables and fruits, and more specifically relates to detecting the residues of difenoconazole in fruit boxes and vegetables by using an enzyme-linked immunoassay technology. [0002] technical background [0003] Difenoconazole (difenoconazole) is a triazole fungicide, which has the characteristics of stable chemical properties, low toxicity and broad-spectrum antibacterial properties. It can be transported into the crops and transported to the diseased part to exert its bactericidal effect. It has a good control effect on common diseases such as anthracnose, leaf blight, black rot, and powdery mildew in various fruits and vegetables, especially for vegetables. Leaf spot and pear scab have a good control effect. In addition, it can also stimulate the growth of crops and improve the yield and quality of fruits and vegetables. Although difenoconazole has a good control effect ...

Claims

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Application Information

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IPC IPC(8): C07K16/44C07K16/06G01N33/53
CPCC07K16/44C07K16/06G01N33/5308G01N2430/00
Inventor 马列纪明山魏松红闫恕姜震
Owner SHENYANG JINCHENG TECH CO LTD
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