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Diethylstilbestrol detection kit and detection method

A detection kit and a technology of diethylstilbestrol, applied in the field of bioengineering, can solve problems such as difficult to meet the requirements of fast and simple on-site detection, the sensitivity of banned drugs cannot meet the requirements, and the price of instruments is expensive, so as to reduce subjectivity and low cost , Easy and fast operation

Inactive Publication Date: 2010-08-11
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Traditional drug residue analysis methods include gas chromatography, liquid chromatography, mass spectrometry, etc. The instruments required by these methods are expensive, and the experimental process is cumbersome, and it is difficult to meet the requirements of fast and simple on-site detection
At present, microbial methods and enzyme-linked immunoassays are widely used in the rapid detection of drug residues. However, these two methods often fail to meet the sensitivity requirements for the detection of banned drugs.
The rapid screening method represented by Chemiluminescence Immunoassay (CLIA) is the mainstream technology in immunology, but there is no report on the detection of diethylstilbestrol by Chemiluminescence Immunoassay (CLIA) at home and abroad.

Method used

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  • Diethylstilbestrol detection kit and detection method
  • Diethylstilbestrol detection kit and detection method

Examples

Experimental program
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Embodiment 1

[0027] The preparation of embodiment 1 monoclonal antibody

[0028] (1) Preparation of immunogen

[0029] Coupling diethylstilbestrol (DES) with bovine serum albumin to obtain a DES-BSA conjugate is the immunogen.

[0030] (2) Monoclonal antibody preparation

[0031] Immunize the mice with the above-mentioned synthetic immunogen, mix the immunogen with an equal amount of Freund's complete adjuvant to make an emulsifier, and inject it subcutaneously at multiple points in the abdomen of the mouse, and then add the immunogen to the mice every two weeks. The emulsifier made by mixing the same amount of Freund's incomplete adjuvant was used to boost the immunization once, and the immunization site was subcutaneous on the back of the neck. From the third immunization, blood was collected from the orbit of the mice one week after each immunization to detect the serum titer. A total of 5 immunizations were performed. One week after the last immunization, the splenocytes of the immun...

Embodiment 2

[0033] Preparation of detection plate and reagent in embodiment 2 diethylstilbestrol detection kit

[0034] In this Example 2, the diethylstilbestrol detection kit is equipped with a detection plate, a diethylstilbestrol standard solution, a diethylstilbestrol monoclonal antibody, an enzyme-labeled secondary antibody, a substrate reaction solution, a sample diluent, and a concentrated washing solution. The specific preparations are as follows:

[0035] (1) Preparation of diethylstilbestrol microwell detection plate

[0036] Use 0.2M carbonate buffer with pH 9.6 as the coating solution, dilute diethylstilbestrol and ovalbumin conjugate (DES-OVA) to 200ng / ml, add 100μl / well into the chemiluminescent well plate, and keep at 4°C Pack overnight. Spin dry, add 200 μl / well containing 1% gelatin, pH 7.4 phosphate buffer for blocking for 2 hours at 37°C, wash 3 times with 1% sodium azide, pH 7.4 phosphate washing solution, spin dry, pack Store in bags containing desiccant.

[0037] ...

Embodiment 3

[0044] Embodiment 3 utilizes kit of the present invention to detect diethylstilbestrol

[0045] (1) Sample pretreatment

[0046] Pretreatment of animal tissue (chicken, pig), fish, shrimp meat samples:

[0047] ①Put 3g of sample in a centrifuge tube, add 9ml of ethyl acetate, and crush it fully under the homogenizer;

[0048] ② Then add 0.3gCaCl 2 , shake at the maximum speed for 3 minutes; at room temperature, centrifuge at 6000rpm for 5 minutes;

[0049] ③Pipete 6ml supernatant to another new test tube, blow dry with nitrogen in a water bath at 60-70°C;

[0050] ④ Dissolve the residue with 2.0ml of n-hexane, then add 1.0ml of 1X PBS-methanol (3:2, v / v), and vortex for 2min;

[0051] ⑤ Centrifuge at 6000rpm for 10min at room temperature, remove the upper layer of n-hexane, and take 50 μl of the lower layer solution for detection.

[0052] (2) The detection steps of the kit are as follows:

[0053] ① Dilute the 10× concentrated washing solution 10 times to be the washing...

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Abstract

The invention discloses a diethylstilbestrol chemiluminescent detection kit, comprising a detection plate and a reagent. The reagent comprises an antibody which is a monoclonal antibody prepared by using a conjugate of diethylstilbestrol and bovine serum albumin as an immunogen. The diethylstilbestrol detection kit can realize quick and large-scale detection and has very high specificity and sensitivity, wherein the detection sensitivity reaches 0.016 ppb.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a diethylstilbestrol detection kit and a detection method thereof. Background technique [0002] Diethylstibestrol (DES) is an artificially synthesized estrogen. Because it can promote animal weight gain, increase protein deposition and reduce fat deposition, it has been widely used as a fish feed additive to improve the conversion rate of fish feed and promote fish growth. . Since the 1970s, people have found through experiments that DES has teratogenic and carcinogenic toxicity. At present, many countries in the world have banned DES in livestock and poultry and aquaculture. In 2002, the Ministry of Agriculture of my country stipulated to cancel all uses of DES and its salt and ester preparations in all food animals. However, driven by economic interests, the illegal addition of diethylstilbestrol by farmers still exists, seriously affecting the international reputatio...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/543G01N21/76
Inventor 王权蒋蔚陈永军赵新宇顾惠明刘迎春
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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