Diethylstilbestrol artificial antigen and antibody prepared therefrom

A diethylstilbestrol and antibody technology, applied in the field of diethylstilbestrol artificial antigen and its prepared antibody, can solve the problems of limited popularization and application, complex processing process, and long detection time

Inactive Publication Date: 2016-08-03
哈尔滨市兽药饲料监察所 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, a variety of physical and chemical detection methods for the detection of DES residues have been established at home and abroad, mainly including high-performance liquid chromatography (HPLC) and liquid chromatography-mass chromatography (LC-MS), but the sample pretreatment process of the instrument method is complicated and the detection time is long , the instruments and equipment are expensive, and professional personnel are required to operate, which seriously limits the promotion and application of this method.

Method used

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  • Diethylstilbestrol artificial antigen and antibody prepared therefrom
  • Diethylstilbestrol artificial antigen and antibody prepared therefrom
  • Diethylstilbestrol artificial antigen and antibody prepared therefrom

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, preparation diethylstilbestrol hapten

[0037] 1. Preparation process

[0038] according to figure 1 The synthetic circuit diagram shown is prepared, and the specific steps are as follows:

[0039] (1) Dissolve 536 mg of diethylstilbestrol as a bulk drug in 5 mL of DMF, stir for 15 min, add 68.57 mg of 70% sodium hydride at room temperature, and react with magnetic stirring for 1 hour. Then 390 mg of ethyl 4-bromobutyrate was added dropwise to react with magnetic force for 1 hour, the reaction liquid was added dropwise to 45 ml of ice water to precipitate off-white solid, filtered, the filter cake was washed with 10 ml of water, and the collected filter cake was air-dried at 45° C. for 4 hours to obtain Diethylstilbestrol butyrate ethyl ester intermediate.

[0040] (2) Add 500mg of diethylstilbestrol butyrate intermediate and 10ml THF to a round bottom flask, stir magnetically at 25°C for 20 minutes, add dropwise 3.5ml of 1M lithium hydroxide aqueous so...

Embodiment 2

[0043] Embodiment 2, preparation diethylstilbestrol artificial antigen

[0044] 1. Diethylstilbestrol immunogen

[0045] 1. Preparation process of diethylstilbestrol immunogen

[0046] Prepare the immunogen (DES-BBAE-BSA) as follows:

[0047] (1) Dissolve 15.86 mg of the diethylstilbestrol hapten prepared in Example 1 with 1.5 mL of DMF, stir at 200 rpm for 10 min, add 21.5 mg of EDC, add 13 mg of NHS after dissolving, and stir at room temperature (500 rpm) to activate for 2 h.

[0048] (2) Weigh 50mg of bovine serum albumin and dissolve it in 3.5mL of 0.1M sodium bicarbonate solution, stir at 200rpm for 10min to fully dissolve, put the reaction solution of step 1 in the reaction box in an ice bath (<4°C) and stir (1000rpm ) was added dropwise (1mL / min), stirred (500rpm) for 24h.

[0049](3) Put the reaction product into a clean dialysis bag, and dialyze in 1L of 0.01MPBS (pH7.2) dialysate at 4°C with stirring (100rpm) for 3 days, change the solution 3 times a day, and cent...

Embodiment 3

[0059] Embodiment 3, the preparation of polyclonal antibody

[0060] The DES-BBAE-BSA prepared in Example 2 was used as an immunogen to immunize 3-4 month old New Zealand white rabbits. For the first immunization, the prepared immunogen was emulsified with an equal volume of Freund's complete adjuvant, and injected subcutaneously at multiple points on the armpit and back of the neck. For booster immunization, Freund's complete adjuvant was replaced with Freund's incomplete adjuvant, and the immunization dose and immunization method were the same as above. The interval between each immunization was 3 weeks. From the fourth immunization, blood was collected from the ear vein on the seventh day of each immunization, and the antibody titer and IC were detected by indirect competitive ELISA method. 50 . After the antibody performance parameters meet the requirements, blood is collected from the heart, and the polyantisera (antibodies) are separated by centrifugation.

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PUM

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Abstract

The invention discloses a diethylstilbestrol artificial antigen and an antibody prepared from the antigen. The structural formula of a diethylstilbestrol half antigen is shown in a formula I. The original stereochemical structure of a DES molecule is remained, and production of a specific antibody is also facilitated. The diethylstilbestrol artificial antigen is a conjugate of a compound shown in the formula I and carrier protein. The invention also provides an antibody by taking the diethylstilbestrol artificial antigen as an immunogen and a kit for detecting diethylstilbestrol. The kit comprises the diethylstilbestrol half antigen and / or the diethylstilbestrol artificial antigen and / or antibody. The formula (I) is described in the specification.

Description

technical field [0001] The invention relates to an artificial antigen of diethylstilbestrol and an antibody prepared therefrom. Background technique [0002] Diethylstibestrol (DES) is a synthetic estrogen that can promote animal growth, increase protein deposition, improve feed conversion rate and reduce fat deposition, so it has been widely used as a growth promoter in livestock and poultry farming. However, DES can lead to metabolic disorders and abnormal development of the body. Research by the International Agency for Research on Cancer (IARC) also found that DES is a carcinogen that can remain in animal-derived foods such as meat, eggs, and milk, and endanger human health through the food chain. Therefore, the European Union and other countries prohibit the use of DES in food animal breeding. The Ministry of Agriculture of my country issued Announcements No. 176 and No. 193 in 2002, clearly prohibiting the use of diethylstilbestrol in all food animal breeding to promot...

Claims

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Application Information

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IPC IPC(8): C07C27/02C07C59/68C07C67/31C07C69/712C07K14/765C07K14/77C07K16/44G01N33/68G01N33/53
CPCC07C51/09C07C59/68C07C67/31C07K14/765C07K14/77C07K16/44G01N33/53G01N33/6854C07C69/712
Inventor 霍峰林晓莉董艳峰秦誉覃丹凤贾良曦李宁彭玮于静泉张巍张治成梅岩松张磊刘中成梁代华
Owner 哈尔滨市兽药饲料监察所
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