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Preparation method of zeranol monoclonal antibody and monoclonal antibody prepared by using same and application of monoclonal antibody

A technology of zearalanol and monoclonal antibody, which is applied in the field of biological immunology, can solve the problems of limiting the preparation of ZER monoclonal antibody, few methods of synthesis of whole antigen, and high price, and achieve good binding ability, simple and effective preparation method, and method simple effect

Inactive Publication Date: 2018-03-06
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ZER standard products are expensive, and there are few synthesis methods for the whole antigen, or the steps are cumbersome or the highly toxic chemical n-tributylamine is used, which limits the preparation of ZER monoclonal antibody to a certain extent.

Method used

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  • Preparation method of zeranol monoclonal antibody and monoclonal antibody prepared by using same and application of monoclonal antibody
  • Preparation method of zeranol monoclonal antibody and monoclonal antibody prepared by using same and application of monoclonal antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0034] The preparation of embodiment 1 whole antigen

[0035] Synthesis of the immunogen ZEN-BSA:

[0036]Weigh 1 mg of zearalenone and dissolve it in 1.2 mL of pyridine, add 2 mg of O-carboxymethyl hydroxylamine, and stir at room temperature for 16 h. After vacuum drying, dissolve in 1 mL of deionized water, adjust the pH to 8.0, add 1 mL of benzene three times, remove unreacted zearalenone by benzene, and keep the water phase. The pH of the aqueous phase was adjusted to 3.0, and precipitation appeared. Extracted with 10 mL of ethyl acetate for 4 times, retained the lipid phase, filtered through anhydrous sodium sulfate, and dried with a nitrogen blower to obtain the hapten ZEN-oxime. Synthesis of ZEN-BSA conjugates by carbodiimide method: dissolve the hapten ZEN-oxime, 0.7mg N-hydroxysuccinimide and 0.6mg soluble carbodiimide (EDC) in 1mL dimethyl Formamide, stirred at room temperature for 1h. Bovine serum albumin (BSA) was added dropwise to the above solution three times...

Embodiment 2

[0039] Embodiment 2 animal immunization and titer determination

[0040] Take a few healthy BALB / c mice aged 6-8 weeks (purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.) and number them separately. Take the ZEN-BSA conjugate and make 0.1μg / μL~10μg / μL with normal saline. Antigen solution, mixed with an equal volume of complete Freund's adjuvant, fully emulsified, 100 μg / immunized mice for initial immunization, and then boosted immunization once every 2 to 4 weeks. The compound was mixed with physiological saline to make 0.1μg / μL~10μg / μL antigen solution, mixed with an equal volume of incomplete Freund's adjuvant, fully emulsified, and injected subcutaneously at 100μg / mouse at multiple points on the back. 7 days after the 3rd-4th booster immunization, blood was collected by tail docking to obtain serum after immunization; ZEN-OVA was used as the coating agent, and the serum was serially diluted 2-3 times with PBS solution of pH 7.4 containing 5% skim milk , De...

Embodiment 3

[0041] Example 3 Preparation and fusion of immunized mouse splenocytes and myeloma cells

[0042] The mouse with the highest affinity between the serum of Example 2 and ZER was taken, and 3 days after the booster immunization, the eyeballs were picked for blood collection, the serum was collected for use, the mice were sacrificed, the spleen was aseptically removed, and the spleen was ground to obtain a spleen cell suspension. Wash 2-3 times with DMEM medium, add a certain amount of DMEM medium to resuspend the cells, stain and count.

[0043] The resuscitated SP2 / 0 myeloma cells (donated by the Sinopharm R&D Center of Jiangsu Academy of Agricultural Sciences) were cultured in complete culture medium containing 10% (v / v) fetal bovine serum. One day before confluence, cells were exchanged and cells were kept in logarithmic growth phase. On the day of fusion, collect the myeloma cells and centrifuge to discard the supernatant, add incomplete medium to resuspend the cells, stain...

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Abstract

The invention discloses a preparation method of zeranol monoclonal antibody and a monoclonal antibody prepared by using the same and application of the monoclonal antibody. The method comprises the following steps: preparation of antigen, determination of animal immunity and potency, preparation and fusion of splenocyte and myeloma cells of immune mice, screening of hybridoma cells, subcloning ofthe hybridoma cells, and preparation of a monoclonal antibody. The preparation method disclosed by the invention is simple and effective, overcomes the problems of high price of ZER standard products,less holoantigen synthesis methods, complicated steps or use of highly toxic chemical, tributylamine, and the like; by adopting ZEN standard products as raw material to prepare a ZER monoclonal antibody, the cost is low, the holoantigen synthesis method is simple, and the synthesis efficiency is high. According to the invention, the ZER monoclonal antibody is prepared, which is high in potency, has good combining capacity with ZER, has no cross reaction with other toxin such as aflatoxin, and can be used for establishing immunological detection methods such as ELISA.

Description

technical field [0001] The invention relates to the field of biological immunology, in particular to a preparation method of a zearalanol monoclonal antibody, the prepared monoclonal antibody and applications thereof. Background technique [0002] Zearalenol (Zeranol, ZER) is a semi-synthetic non-steroidal estrogen, which is a secondary metabolite produced by a variety of Fusarium fungi during the growth process - the product of the reduction of zearalenone . ZER can directly or indirectly act on the pituitary gland and pancreas, increase the level of growth hormone and insulin in the body, promote the synthesis of protein in the animal body, improve the utilization rate of feed, and thus produce the effect of promoting weight gain. Therefore, ZER has been used as a weight gain agent for cattle and sheep to bring high economic benefits to farmers. However, in the process of livestock and poultry breeding, ZER will remain in various edible tissues of animals, such as beef a...

Claims

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Application Information

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IPC IPC(8): C07K16/26C12N5/20G01N33/577
CPCC07K16/26G01N33/577
Inventor 宋素泉闫丽萍李丽
Owner NANJING AGRICULTURAL UNIVERSITY
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