Triclocarban hapten and antigen and preparation method and application

A technology of triclocarban and hapten, which is applied in the preparation methods of peptides, chemical instruments and methods, animal/human proteins, etc., can solve the problems of complicated operation, expensive equipment, and complicated operation, and achieve high purity and yield. High, easy to synthesize effect

Inactive Publication Date: 2019-07-02
北京维德维康生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have disadvantages such as poor selectivity, large amount of organic solvent used, complicated operation and poor reproducibility among different matrices, and the operation is cumbersome and the instruments are expensive.

Method used

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  • Triclocarban hapten and antigen and preparation method and application
  • Triclocarban hapten and antigen and preparation method and application
  • Triclocarban hapten and antigen and preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1、 3

[0031] Embodiment 1, the preparation of triclocarban hapten

[0032] One, the preparation of triclocarban hapten

[0033] Weigh equal amounts of 3,4-dichlorophenyl isocyanate and methyl 4-aminophenylbutyrate into a 50 mL round bottom flask. Add 10 mL THF and react under nitrogen protection. TLC was used to monitor the reaction. After confirming that the reaction was complete, the reaction solution was placed in a 40°C water bath and evaporated to dryness. After the product was weighed, it was dissolved in 7 mL of methanol, and 1 M NaOH solution was added according to twice the amount of the substance, heated at 40°C, and TLC Monitor hydrolysis. After complete hydrolysis, the pH was adjusted to 2 with 6 M HCl. The reaction solution was extracted twice with 50 mL ethyl acetate. The extracted aqueous phase and organic phase were spotted by TLC, and after confirming that there was no target product in the aqueous phase, anhydrous sodium sulfate was added to the collected organ...

Embodiment 2、 3

[0040] Embodiment 2, preparation and structural identification of triclocarban artificial antigen

[0041] 1. Preparation of triclocarban artificial antigen

[0042] 1. Synthesis of immunogen

[0043] (1) Dissolve 20mg of triclocarban hapten in 1.5mL DMF, stir at 200rpm for 10min, add 21.5mg of EDC to dissolve, then add 13mg of NHS, stir at room temperature (500rpm) to activate for 2-3h.

[0044] (2) Weigh 50mg of BSA and dissolve it in 3.5mL of 0.1M sodium bicarbonate solution, stir at 200rpm for 10min to fully dissolve, cool down in an ice bath to 0-4°C, stir at 1000rpm, add the reaction solution in step 1 dropwise (1mL / min), stirred at 500rpm for 24h.

[0045] (3) Put the reaction product into distilled water and rinse the dialysis bag (10cm), dialysis with 1L of 0.01M PBS (1×, pH7.2) at 4°C (100rpm) for 3 days, and change the solution 3 times a day (morning, middle and evening) , changing the medium 9 times in total, centrifuge the dialysis product at 5000rpm for 6min,...

Embodiment 3、 3

[0052] Example 3, Triclocarban Artificial Antigen Immunized Animals Prepared Monoclonal Antibody

[0053] 1. Animal immunity

[0054] Use the immunogen (triclocarban-BSA) prepared in Example 2 at 100 μg per mouse, dissolve the immunogen in normal saline and mix it with Freund’s complete adjuvant in equal volumes, and inject subcutaneously on the back of the neck to immunize 6-8 week-old Balb / c female mice, on the 7th, 14th, and 28th day after the initial immunization, the immunogen and Freund's incomplete adjuvant were mixed in equal volumes, and each additional immunization was given once, and the immune complex was 100 μg / rat 3 days before fusion, without Freund's adjuvant. Adjuvant was used for booster immunization once again.

[0055] 2. Cell fusion and cloning

[0056] According to the conventional method, the splenocytes of the immunized mice were mixed with the mouse myeloma cells (SP2 / 0) in the logarithmic growth phase, and then the preheated fusion agent (PEG4000) ...

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Abstract

The invention discloses an endocrine disruptor triclocarban hapten and an antigen and a preparation method thereof and application. The triclocarban artificial antigen provided in the invention is anantigen obtained by coupling a triclocarban hapten represented by a formula I with a carrier protein. The triclocarban artificial antigen synthesis method is simple, the purity and the yield are high,and is of great importance for triclocarban antibody preparation and residue detection of triclocarban in food.

Description

technical field [0001] The invention belongs to the field of purification and detection of endocrine interferon in food, and relates to a triclocarban hapten and antigen, a preparation method and application thereof. Background technique [0002] Endocrine disrupting chemicals (Environmental endocrine disrupting chemicals, EDCs) are exogenous chemical substances that exist in the environment and interfere with the human or animal endocrine system. Most of them enter the organism through the food chain, causing the organism's own hormones to be disturbed. Circulation and accumulation of fat in the body lead to abnormal physiological systems such as the normal reproduction, development, immunity, and behavioral activities of the organism. Triclocarban (TCC) is a kind of endocrine disruptor. As a high-efficiency broad-spectrum antibacterial agent, it is widely added to personal care products and daily chemical products. It can enter the human body through skin contact and oral ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535C07K14/765C07K14/77C07K14/795C07K14/47C07K1/107C07K16/44
CPCC07K14/47C07K14/765C07K14/77C07K14/795C07K16/44G01N33/535
Inventor 马立才聂靖东姚凯丁亚芳邵兵李淑芳王照鹏杨柳秦誉覃丹凤贾良曦
Owner 北京维德维康生物技术有限公司
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