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Detection reagent kit and detection method for diethyl stilbestrol

A technology of diethylstilbestrol and kits, applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of low sensitivity, high false positive rate, instability, etc., and achieve the effect of convenient use, high sensitivity, and simple structure

Inactive Publication Date: 2009-07-15
JIANSGU INST OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ELISA is usually used as a rapid screening test method for on-site sampling, and HPLC or GC-MS is used as a confirmatory test method. At present, most of the diethylstilbestrol ELISA test kits used by my country's testing institutions are imported from abroad, which are expensive and difficult to promote and apply in China.
Domestic diethylstilbestrol ELISA kits generally have problems such as low sensitivity, high false positive rate, and instability

Method used

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  • Detection reagent kit and detection method for diethyl stilbestrol
  • Detection reagent kit and detection method for diethyl stilbestrol
  • Detection reagent kit and detection method for diethyl stilbestrol

Examples

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Embodiment 1

[0013] Example 1: Preparation of kit and detection of animal tissue samples

[0014] Preparation of immune antigen:

[0015] The artificial immunization antigen of the present invention is to transform diethylstilbestrol (DES) into diethylstilbestrol hemisuccinate (DES-HS), and then use the mixed acid anhydride method to combine with the macromolecular carrier protein, and use the synthetic DES-HS-BSA as the immune antigen Animals are immune.

[0016] Weigh 20mg to 100mg of diethylstilbestrol (DES), add 5mL of pyridine, add 10mg to 50mg of succinic acid, stir overnight at room temperature, the reaction is complete, wash with acidic water, suction filter the precipitate, and dry to obtain diethylstilbestrol hemisuccinate (DES- HS).

[0017] The carrier protein is bovine serum albumin (BSA). Weigh 30mg~300mg bovine serum albumin, dissolve in 6mL~10mL of 50% dioxane aqueous solution, adjust the pH value to 9~10, and make A liquid; weigh 10mg~50mg DES-HS, dissolve in 2mL dioxan...

Embodiment 2

[0060] Implementation example 2 preparation kit

[0061] DES-BSA antigen preparation:

[0062] Weigh 20mg to 100mg of diethylstilbestrol (DES), add 5mL of pyridine, add 10mg to 50mg of succinic acid, stir overnight at room temperature, the reaction is complete, wash with acidic water, suction filter the precipitate, and dry to obtain diethylstilbestrol hemisuccinate (DES- HS).

[0063] Weigh 12mg of DES-HS, 40mg of N-hydroxysuccinamide (NHS), 35mg of carbodiimide (EDC) and dissolve in N,N'-dimethylformamide (DMF), stir at room temperature for 24h to prepare A solution; Weigh 50mg of BSA and dissolve it in 3mL of 0.01mol / mL pH7.4 PBS to make solution B. Add solution A dropwise to solution B, stir while adding, react at room temperature for 3 hours, dialyze the reaction solution in distilled water for 72 hours, and change distilled water 5 times during the period. Or purified by Sephadex G50 column and eluted with 0.05mol / L pH8.0 carbonate buffer solution. Collect the dialys...

Embodiment 4

[0074] The reagents provided by the kit are the same as those in Example 1, and are used to detect feed samples.

[0075] Reagents that should be prepared by the laboratory:

[0076] (1) 0.2mol / L ammonium acetate buffer solution: dissolve 15.4g ammonium acetate in 900mL water, adjust the pH with glacial acetic acid to pH 5.2p±H0.2, and dilute to 1L;

[0077] (2) 1.0mol / L sodium hydroxide solution;

[0078] (3) 0.1mol / L sodium hydroxide methanol solution + water (7+3);

[0079] (4) MTBE+methanol (9+1, containing 2% formic acid) solution;

[0080] (5) Solid-phase extraction column: OasisMAX column (60 mg, 3 mL) or equivalent.

[0081] Others are with embodiment 1.

[0082] The specific detection steps are as follows:

[0083] Accurately weigh 5.0g±0.1g of animal feed into a 50mL capped centrifuge tube, add 10mL of 0.2mol / L ammonium acetate buffer, homogenize for 1min, add 10mL of ether, vortex for 3min, and centrifuge at 3500r / min for 5min , Pipet the ether layer into anot...

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Abstract

The invention provides a kit for detecting diethylstilbertrol and detection method thereof, belonging the the time resolution fluoroimmunoassay (TRFIA) technical field, capable of detecting the content of diethylstilbestrol (DES) in animal-source food, blood, urine and feedstuff. The kit uses the TRFIA to detect the DES and the mark immune response is the base of the detection. A micropore plate is coated by DES-carrier protein and added with DES standard or sample and then added with DES antibody. The free DES and the DES-carrier protein on the micropore plate complete the DES antibody and the DES antibody which is not bonded with the free DES or the DES-carrier protein is cleared away and added with the EU3+-goat anti-rabbit antibody, after labeling the immune reaction, the non-bonded EU3+-goat anti-rabbit antibody is cleared away. After adding the reinforcement liquid, the fluorescence intensity cps is detected using a time resolution fluorescence instrument and the fluorescence intensity is inversely proportional to the DES concentration in sample and the content of DES in sample can be determined according to the standard curve. The DES detection kit has features of simple structure, convenient use, cheapness, high sensitivity up to 1ng / mL.

Description

technical field [0001] The invention relates to a detection kit for detecting diethylstilbestrol (DES) and a detection method thereof, belonging to the technical field of time resolved fluoroisnmunoassay (TRFIA), and used for detecting the residual amount of diethylstilbestrol in food and feed. Background technique [0002] Diethylstilbestrol is a synthetic stilbene estrogen with phenolic hydroxyl structure. It has been widely used all over the world since 1938. It is mainly used as a growth promoter in animal husbandry to improve the utilization rate of feed. Part of the diethylstilbestrol obtained by animals through food is excreted into the environment, and part remains in the animal body and is enriched in the human body through the food chain. Residues of hormone drugs will disturb the physiological functions of normal people, and more seriously affect the normal growth and development of children. In the late 1970s, it was discovered that diethylstilbestrol can cause ...

Claims

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Application Information

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IPC IPC(8): G01N33/543
Inventor 宓晓黎李红陆茂林金坚李利东黄飚袁建兴司宏飞杜姝莲
Owner JIANSGU INST OF MICROBIOLOGY
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