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Bensulfuron-methyl universal hapten, artificial antigen, preparation method and application thereof

A bensulfuron-methyl, artificial antigen technology, applied in the preparation of sulfonic acid amides, biochemical equipment and methods, chemical instruments and methods, etc. Chemically stable effect

Inactive Publication Date: 2013-06-19
CHINA JILIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing synthesized bensulfuron-methyl artificial antigen retains the entire molecular structure of bensulfuron-methyl (benzene ring part, heterocycle part and urea bridge part), as recorded in the invention patent of 200910234686.X, which is prepared by immunization antibody recognizes only one bensulfuron-methyl molecule
[0005] On the method for preparing coupling bensulfuron-methyl artificial antigen, the activated ester method that usually adopts in the prior art couples sulfuron-methyl compound and carrier protein, and Zhang Lijie (Zhang Lijie, Zhou Yingxia, Wang Lei, etc. organophosphorus pesticide Synthesis and identification of universal antigen [J]. Pesticides, 2010, 49(3): 164-166, 169) and others found that if the reaction time is too long, the activated ester will be partially decomposed, and the yield will decrease instead. The polarity of the solvent used can also affect the yield of activated esters
The invention patent with the patent number 200910234686.X proposes a bensulfuron-methyl artificial immunogen BE-BSA and its preparation and use. This patent document chooses the activated ester method for the synthesis of artificial antigens. This method is time-consuming and expensive. Low efficiency, only the coupling reaction of antigen and carrier protein takes more than 5 days

Method used

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  • Bensulfuron-methyl universal hapten, artificial antigen, preparation method and application thereof
  • Bensulfuron-methyl universal hapten, artificial antigen, preparation method and application thereof
  • Bensulfuron-methyl universal hapten, artificial antigen, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Preparation and Identification of Universal Hapten

[0045] Dissolve 200mg of 2-methyl benzylsulfonamide 2-formate and 87mg of succinic anhydride in 5mL of acetonitrile, place in a water bath at 22°C, add 265.5mg of DBU dropwise within 10min, stir for 2h, then pour the reaction solution into 50mL of distilled water, 50mL Wash with ethyl acetate, remove the organic phase, adjust the pH of the aqueous phase to 2 with HCl, extract with ethyl acetate (2×50 mL), combine the organic phase with anhydrous MgSO 4 Dry, evaporate the solvent to dryness, and recrystallize from ethyl acetate to obtain Hapten, which is firstly detected by thin-layer chromatography (TLC), and then identified by NMR.

[0046] H NMR (CD 3 OD): δ7.91(d, 1H, J=7.5Hz Ar), 7.61-7.42(m, 3H, Ar), 4.78(s, 2H, CH 2 SO 2 ), 3.85(s, 3H, OCH 3 ), 2.48 (m, 2H, COCH 2 CH 2 COOH), 2.43(m, 2H, COCH 2 CH 2 COOH). See Figure 4 . Among them, some miscellaneous peaks appeared at 5.0-5.5 and 11-13 res...

Embodiment 2

[0047] Example 2 Synthesis and Identification of Artificial Antigen

[0048] Dissolve 16.45mg of Hapten in 1mL of tetrahydrofuran, add 13μL of tri-n-butylamine while stirring, after cooling to 0°C, add 7μL of isobutyl chloroformate, and stir for 60min under ice bath to obtain liquid A. Weigh 33.5 mg BSA (or 21.5 mg OVA) and dissolve it in borate buffer solution (0.2 mol / L, Ph=9.0) to form B solution. Slowly add solution A to solution B dropwise at 4°C, and stir overnight with magnetic force. The next day, the reaction solution was dialyzed in PBS buffer (pH=7.4) at 4°C for 3 days, the solution was changed 3 times / d, the dialysate was centrifuged at 10,000 r / min for 5 minutes, the supernatant was freeze-dried, and stored at -20°C.

[0049] The ultraviolet scanner scans the dialyzed sample at a wavelength of 190-400nm to identify the coupling situation. According to the characteristic absorption peak wavelength and absorbance value of each substance, the coupling ratio is calc...

Embodiment 3

[0052] Example 3 Preparation of Multiple Antiserum

[0053] The tail blood of Balb / c mice was observed for one week and kept as a negative control. The normal saline solution of the immunogen was mixed with Freund's complete adjuvant in equal amounts, and after the emulsification was complete, the mice were subcutaneously injected into the abdomen at multiple points, and the immune dose was 100 μg / 0.2ml. Two weeks later, incomplete Freund's adjuvant was used as emulsifier, and the mice were boosted with the same dose and method. The immunization process was once every two weeks, and immunized 4 times. 10 days after the third immunization, the mouse tail blood was collected, and the titer was measured by indirect non-competitive ELISA.

[0054] The inhibition rate of polyclonal antibodies against common haptens was determined by indirect competition ELISA (CI-ELISA). According to the optimal working concentration of antigen and antibody and optimized conditions, the general ...

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Abstract

Belonging to the technical fields of immunodetection and biochemical engineering, the invention relates to a bensulfuron-methyl universal hapten, an artificial antigen, a preparation method and application thereof. The artificial antigen prepared from the bensulfuron-methyl universal hapten provided in the invention can be immunized to obtain an antibody, which can achieve spectral identification of bensulfuron-methyl pesticide molecular with the artificial hapten structure, so that the indirect Elisa method adopting the artificial antigen and the antibody prepared therefrom can realize a wide detectable spectrum range. In the invention, the preparation of the artificial antigen employs an improved mixed anhydride method, which has high coupling efficiency and is simple.

Description

technical field [0001] The invention belongs to the technical field of immunoassay and biochemical industry, and relates to a bensulfuron-methyl general-purpose hapten, an artificial antigen, a preparation method and application thereof. Background technique [0002] Sulfur-methyl herbicides are currently the largest class of herbicides in the world. Since DuPont first developed the wheat field herbicide chlorsulfuron-methyl in 1982, through derivatization methods such as modification at both ends of sulfonylureas and urea bridges, currently Nearly 40 varieties have been commercialized. Sulfur-methyl herbicides inhibit acetolactate synthase (ALS) / acetate hydroxyacid synthase (AHAS), thereby destroying protein synthesis, interfering with DNA synthesis and cell division and growth, causing the normal growth of weeds to be destroyed and die [1] . Because of its high efficiency, low dosage, low toxicity and short half-life time, it is widely used in rice, wheat, corn and other...

Claims

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Application Information

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IPC IPC(8): C07C311/51C07C303/40C07K14/765C07K14/77C07K16/06C07K16/44C12N5/20G01N33/53
Inventor 王磊睢珂王志伟潘家荣赵杰郑月明
Owner CHINA JILIANG UNIV
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