Preparation method and application of diethylstilbestrol cationization complete antigen

A diethylstilbestrol, complete antigen technology, applied in chemical instruments and methods, animal/human proteins, serum albumin, etc., can solve problems such as reputation, health risks, economic losses, etc., to improve the probability, increase the probability of coupling, The effect of improving the efficiency of antigen presentation

Inactive Publication Date: 2013-06-26
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, driven by huge economic interests, the illegal addition of diethylstilbestrol by farmers still exists, seriously affecting the international reputation of my country's aquatic products and livestock products, causing major economic losses and also bringing hidden dangers to people's health

Method used

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  • Preparation method and application of diethylstilbestrol cationization complete antigen
  • Preparation method and application of diethylstilbestrol cationization complete antigen
  • Preparation method and application of diethylstilbestrol cationization complete antigen

Examples

Experimental program
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Effect test

Embodiment 1c

[0021] Preparation and identification of embodiment 1cBSA

[0022] Under the condition of ice bath, add 670 μL ethylenediamine (EDA) dropwise to 670 μL ethanesulfonic acid coupling buffer (MES), adjust the pH value to about 4.75 with 4M HCl, this is solution A; (50mg dissolved in 500mL), EDC MES solution (30mg dissolved in 500μL) was added dropwise to solution A in an ice bath; the reaction was magnetically stirred at room temperature for 2h, and acetic acid (about 400μL) was added to stop; deionized water was dialyzed for 72h, freeze-dried spare. Synthetic route such as figure 1 As shown, it was identified by a UV spectrophotometer, and compared with BSA, the maximum absorption peak of cBSA was blue-shifted ( figure 2 ).

Embodiment 2

[0023] Example 2 Preparation and identification of diethylstilbestrol cationized complete antigen

[0024] Using the EDC method, accurately weigh 10.0 mg of diethylstilbestrol hemisuccinate (prepared in our laboratory), 7.8 mg of EDC, and 3.7 mg of NHS, dissolve in 2 mL of N, N-dimethylformamide (DMF), and shake at room temperature to avoid light 8h, this is solution A; accurately weigh 46.2mg of cBSA, dissolve it in 3mL PBS (pH=7.4), and this is solution B; slowly add solution A to solution B under vigorous shaking, shake and react overnight at 4°C; After completion, the reaction solution was dialyzed with PBS at 4°C for 72 hours, and the solution was changed every 4-6 hours. DES-HS-BSA was prepared in the same way as a control. Using SDS-PAGE electrophoresis to identify it from the perspective of molecular weight, it can be seen that the cBSA-DES band obviously lags behind BSA ( image 3 ), using mass spectrometry to identify and estimate the coupling ratio of small molecul...

Embodiment 3

[0025] Example 3 Animal immunization of diethylstilbestrol cationized complete antigen

[0026] Twelve 6-7-week-old female SPF Balb / C mice were randomly divided into two groups and immunized with DES-BSA and DES-cBSA respectively. For the first immunization, the immunogen was mixed and emulsified with the same amount of Freund's complete adjuvant, and injected intraperitoneally at a single point. Booster immunization was carried out, and the dose of immunization was increased step by step. Antibody titers in serum were determined by indirect ELISA ( Figure 5 ).

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Abstract

The invention discloses a preparation method and an application of a diethylstilbestrol cationization complete antigen, belonging to the technical field of biochemical engineering. According to the invention, a cationized carrier protein is synthesized and coupled with carboxylated diethylstilbestrol by an EDC method; through mass spectrum identification and SDS-PAGE analysis, a cationized diethylstilbestrol artificial complete antigen is successfully synthesized; the preparation method is simple and effective and low in cost; compared with a natural carrier protein, when the cationized complete antigen is used as an immunogen, the immunized mouse organism can generate immune response of a higher level; and the synthesized artificial complete antigen can be used for preparing an efficient antibody for specifically identifying diethylstilbestrol and for developing an immunization speed test kit for quickly detecting diethylstilbestrol.

Description

technical field [0001] The invention relates to a method for preparing a cationized complete antigen of diethylstilbestrol, which belongs to the technical field of biochemical industry and is used for preparing an efficient antibody specifically recognizing the chemical veterinary drug diethylstilbestrol and developing an immune rapid test kit capable of rapidly detecting diethylstilbestrol residue. Background technique [0002] Diethylstilbestrol (DES), also known as diethylstilbestrol and diethylstilbestrol, chemically named 1,2-bis(4-hydroxyphenyl)-1,2-diethylethylene, was synthesized by Dodds in London, England in 1938 , DES is a synthetic non-steroidal estrogen with a phenolic hydroxyl structure. It is a colorless crystal or white crystalline powder, almost odorless and tasteless, almost insoluble in water, soluble in ethanol, chloroform, ether, fatty oil, Dilute sodium hydroxide, etc., with a melting point of 169-172°C. [0003] In veterinary medicine, diethylstilbest...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K16/44G01N33/53
Inventor 宁保安柳明彭媛连璐高志贤孙思明白家磊张亦红
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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