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Hybridoma cell strain CBC secreting monoclonal antibody of dicofol and application of hybridoma cell strain CBC

A hybridoma cell line, dicofol technology, applied in analytical materials, biochemical equipment and methods, instruments and other directions, can solve problems such as excessive residues, high solvent consumption, aquatic toxicity, etc., and achieve the effect of good detection sensitivity

Inactive Publication Date: 2019-12-24
江苏权正检验检测有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Extensive use of dicofol will lead to excessive residues. In recent years, more and more evidences have shown that its exposure in the environment has toxic and estrogenic effects on fish, reptiles, birds, mammals and humans. Very toxic to aquatic organisms
Despite the high sensitivity and specificity of these chromatography-based methods, there are some disadvantages such as the need for thorough sample cleanup, high solvent consumption, expensive equipment, and skilled technicians

Method used

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  • Hybridoma cell strain CBC secreting monoclonal antibody of dicofol and application of hybridoma cell strain CBC

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Synthesis of dicofol hapten

[0046] Since small molecules of dicofol are not immunogenic and cannot stimulate mice to generate immune responses and then produce antibodies, it is necessary to couple dicofol to proteins through protein linkage technology to obtain immunogenicity; commonly used in protein coupling techniques Active groups include amino, carboxyl, hydroxyl, mercapto, etc. Since the molecular structure of dicofol does not contain these active groups, derivation is required.

[0047] Put dicofol (250 mg, 0.67 mmol), ethyl cyanoacetate (1 mL), and acetic acid (1 mL) together in a round bottom flask, and slowly add 96% sulfuric acid (4 mL). After the addition was complete, the mixture was heated in a 50 °C water bath for 15 min. The mixture turned dark red, and the reaction was stirred at room temperature for 20 h. The mixture was extracted with ether. The ether phase was washed with water and sodium bicarbonate solution, dried and concentrated....

Embodiment 2

[0049] Example 2: Synthesis of dicofol complete antigen

[0050] Weigh 5.5mg dicofol hapten (DCF-COOH), 4.2mg N-hydroxysuccinimide (NHS), dissolve in 300μL N,N-dimethylformamide (DMF), stir at room temperature for 10min; weigh again Take 6.9 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), fully dissolve it in 100 μL DMF, add it to the DCF-COOH solution, and stir at room temperature to react 6- 8 h (referred to as liquid A). Take 8mg KLH, dilute it to 4mg / mL with 0.01M carbonate buffer solution (CBS) (called solution B), then slowly add solution A to solution B drop by drop, react at room temperature overnight; then use 0.01M PBS solution Dialyze to remove the unreacted small molecule hapten to obtain the complete antigen DCF-COOH-KLH, which is identified by the ultraviolet absorption scanning method.

Embodiment 3

[0051] Embodiment 3: the synthesis of dicofol coating original

[0052] Dissolve 3.0 mg of dicofol hapten (DCF-COOH) and 2.3 mg of N-hydroxysuccinimide (NHS) in 300 μL of anhydrous N,N-dimethylformamide (DMF), and stir at room temperature for 10 min to obtain dicofol semi- Antigen (DCF-COOH) solution; 3.8 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in 100 μL of anhydrous DMF and added to DCF-COOH solution, stirred at room temperature for 6-8 hours to obtain liquid A; dilute 10 mg of chicken ovalbumin (OVA) with 1 mL of carbonate buffer solution (CBS) with a concentration of 0.01 mmol / L to obtain liquid B; Solution A was slowly added to solution B for reaction to obtain a reaction solution; the reaction solution was dialyzed with PBS solution to remove unreacted small molecule haptens to obtain the coating original (DCF-COOH-OVA).

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Abstract

The invention discloses a hybridoma cell strain CBC secreting a monoclonal antibody of dicofol and application of the hybridoma cell strain CBC, and belongs to the field of food safety immunoassay. The cell strain CBC is preserved in the China General Microbiological Culture Collection Center, the address is Institute of Microbiology, Chinese Academy of Sciences, No.3, Beichen West Road, ChaoyangDistrict, Beijing, the preservation date is March 7, 2019, and the preservation number is CGMCC No.17398. Complete antigens of the dicofol are mixed and emulsified with equivalent freund adjuvants, and BALB / c mice are immunized by subcutaneous multipoint injection of neck and back through first immunization, multiple strengthening immunization and sprint immunization. Spleen cells of the mice withhigh titer and low IC50 are fused with mouse myeloma cells by a PEC method, selective culture is conducted, then cells are screened and sub-cloned for three times through indirect competitive enzyme-linked immunosorbent assay, finally the cell strain CBC is obtained, the monoclonal antibody secreted by the cell strain CBC has good detection sensitivity to the dicofol (value of the IC50 is 19.09 ng / mL), and can be used for the detection of dicofol residues in food.

Description

technical field [0001] The invention relates to a hybridoma cell line CBC secreting dicofol monoclonal antibody and application thereof, which belongs to the field of food safety immunological detection. Background technique [0002] Dicofol [2,2,2-trichloro-1,1-bis(4-chlorophenyl)ethanol], the English name dicofol (hereinafter abbreviated as DCF), is a broad-spectrum, low- Toxic organochlorine acaricide, which has contact and stomach poisoning effects on mites, and is effective on adults, larvae and eggs. The production and synthesis of dicofol uses DDT as the raw material. During the production process, the conversion rate of the chlorination section is generally about 70%. If the subsequent purification is not carried out or the purification is not complete, the finished product will contain DDT to varying degrees. [0003] Extensive use of dicofol will lead to excessive residues. In recent years, more and more evidences have shown that its exposure in the environment ha...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44C07K14/795G01N33/577
CPCC07K14/795C07K16/44G01N33/577
Inventor 胥传来张荣荣章晓萍匡华徐丽广刘丽强宋珊珊吴晓玲胡拥明曹玉朋
Owner 江苏权正检验检测有限公司
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