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Fat greenling vitellogenin sandwich ELISA kit and preparation method, detection method and application thereof

A protoprotein and taki six-line technology, which is applied in the field of preparation of a yolk protein sandwich ELISA kit for omega six-line fish, can solve problems such as toxic effects, carcinogenicity, biological harm, etc., and achieves improved concentration and purity, easy operation, The effect of increasing the concentration

Inactive Publication Date: 2015-02-11
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, so far, my country has not seen the research and development of POPs biomonitoring technology in the marine environment.

Method used

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  • Fat greenling vitellogenin sandwich ELISA kit and preparation method, detection method and application thereof
  • Fat greenling vitellogenin sandwich ELISA kit and preparation method, detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The preparation of embodiment 1 ELISA kit

[0022] The preparation of the sandwich ELISA kit for detecting the vitellogenin of Otaki six line fish is divided into the following steps:

[0023] (1) Separation and purification of vitellogenin

[0024] Intramuscular injection17 β - Estradiol (17 β -estradiol,E 2 ) to induce the production of vitellogenin in Otaki six line fish. One week after the injection, blood was collected, centrifuged at 5000 g for 10 minutes at 4 °C, and the supernatant was collected; an equal volume of saturated ammonium sulfate solution was added to the supernatant, shaken in an ice bath for 2 hours, centrifuged at 5000 g for 10 minutes, and added to the precipitate 25mM Tris-HCl (pH 7.5) to redissolve the pellet. For further purification, use a Sephadex G-200 chromatography column (1.5′ 20 cm), add 1 ml of the above solution, wash the chromatography column with 25mM Tris-HCl (pH 7.5) at a flow rate of 0.3 ml / min, and collect the first eluti...

Embodiment 2

[0041] Example 2 ELISA kit for the detection of pure Vitellogenin of Otaki six line fish

[0042] The ELISA kit prepared in Example 1 is divided into the following steps when used:

[0043] 1) Dilute the rabbit polyclonal antibody to 3 μg / ml with the coating solution, add 100 μl / well to a blank 96-well ELISA plate, and coat overnight at 4°C, or Incubate at 37°C for 2 hours. The solution in the well was discarded, and the solution was washed 3 times.

[0044] 2) Add blocking solution to 96-well ELISA plate, 300 μL / well, and incubate at room temperature for 1 hour. Discard the solution in the well and wash 3 times.

[0045] 3) Use the sample diluent to dilute the Otaki six line fish vitellogenin standard to 1.95, 3.9, 7.8, 15.62, 31.25, 62.5, 125 ng / ml. Add 100 μL / well of each standard and test sample to a 96-well ELISA plate, and incubate at 37°C for 1 hour. Discard the solution in the well and wash 5 times.

[0046] 4) Add horseradish peroxidase-labeled rabbit anti-Lix...

Embodiment 3

[0051] Example 3 The ELISA kit of the present invention detects the plasma of female / male Otaki hexalis

[0052] Intramuscular injection17 β - Estradiol (17 β -estradiol,E 2 ) to induce the production of vitellogenin in Otaki six line fish. One week after the injection, blood was taken, and the plasma was gradually diluted; it was detected by the method of Example 2, and the specific results can be found in figure 2 .

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Abstract

The invention discloses a fat greenling vitellogenin sandwich ELISA kit and a preparation method, a detection method and application thereof. The preparation method comprises the following steps: obtaining vitellogenin from 17 (i) beta ( / i) - estradiol induced fat greenling plasma through an ammonium sulfate precipitation and SephadexG-200 by purification to prepare a polyclonal antibody, and labeling a horseradish peroxidase enzyme to the purified antibody; putting a fat greenling vitellogenin purifying product, a rabbit anti- fat greenling vitellogenin polyclonal antibody, a horseradish peroxidase enzyme labeled rabbit anti-fat greenling vitellogenin polyclonal antibody, and a blank 96-well ELISA plate as well as a coating buffer, a scrubbing solution, a confining liquid, a sample diluent, a developing solution and a stop buffer in a kit body to obtain a sandwich ELISA kit for detecting the fat greenling vitellogenin. Through the kit provided by the invention, vitellogenins in fat greenling plasma, body surface mucus, a liver tissue and a liver cell culture medium can be sensitively, accurately and quantitatively detected in a detection range of 1.95 to 125ngmL<-1>, and an important tool is provided for the detection of an endocrine disruptor in an offshore area in China.

Description

technical field [0001] The invention belongs to the field of ecological detection, and in particular relates to the preparation and application of a Otaki hexaline fish vitellogenin sandwich ELISA kit. Background technique [0002] Persistent organic pollutants (Persistent Organic Pollutants, POPs) are difficult to decompose in the environment, can exist in the environment for a long time, can migrate and spread on a global scale through various transmission routes, and accumulate and amplify in organisms through the food chain , a class of organic pollutants that have toxic effects on the human body and the environment. These pollutants do not exist in nature themselves, but are the products of the human industrial revolution. The hazards of persistent organic pollutants to humans and the environment have already become a global problem. In order to solve this problem, the United Nations Environment Program (UNEP) and the Swedish government co-chaired a plenipotentiary meet...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/531G01N33/74
CPCG01N33/531G01N33/689G01N33/743
Inventor 王松王骏张铁军冷凯良苗钧魁姜勇罗忻吴振兴
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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