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Hybridoma cell line capable of secreting CHP (cyproheptadine) monoclonal antibody and preparation method

A technology of hybridoma cell lines and monoclonal antibodies, which is applied to tumor/cancer cells, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc. Detection and other issues

Inactive Publication Date: 2018-08-21
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the methods for detecting cyproheptadine are mainly liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS / MS), enzyme-linked immunosorbent assay, immunoaffinity chromatographic column and electrochemical sensor, etc., but These methods all have the disadvantages of cumbersome operation, time-consuming, expensive, etc., and cannot realize rapid detection of a large number of samples.

Method used

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  • Hybridoma cell line capable of secreting CHP (cyproheptadine) monoclonal antibody and preparation method
  • Hybridoma cell line capable of secreting CHP (cyproheptadine) monoclonal antibody and preparation method
  • Hybridoma cell line capable of secreting CHP (cyproheptadine) monoclonal antibody and preparation method

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Experimental program
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Effect test

Embodiment 1

[0073] Embodiment 1: the synthesis of cyproheptadine hapten

[0074] 50 mL of toluene solution was added to 5 g of compound 1 with a concentration of 17.4 mmol, and then 3 g of ethyl formate with a concentration of 34.8 mmol was added dropwise, and stirred at 120° C. overnight. The mixture was cooled to room temperature, added HCl and extracted 3 times with 50 mL ethyl acetate, the organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo to obtain 4 g of compound 2. Add 64mL of ethanol to 4g of compound 2 with a concentration of 11.6mmol, add 15 equivalents of aqueous potassium hydroxide dropwise to the mixture, and aqueous potassium hydroxide is dissolved in 16mLH by 10.3g of potassium hydroxide. Stir overnight at °C. The mixture was concentrated in vacuo, then extracted 3 times with 50 mL of ethyl acetate, the organic layer was dried over anhydrous Na2SO4, and concentrated in vacuo to obtain 2.5 g of compound 3. 2.5 g of compound 3 with a concentration of 9.2...

Embodiment 2

[0075] Embodiment 2: the synthesis of complete antigen of cyproheptadine

[0076] Weigh 5 mg of cyproheptadine hapten into a brown vial, add 1 mL of pH 4.7, 0.1 M MES buffer solution to dissolve, add 10 mg of EDC and 6 mg of NHS solid into the hapten reaction bottle, stir for 6-8 hours to obtain an activation solution, weigh The keyhole limpet hemocyanin KLH was dissolved in the CB solution, the activation solution was added to the protein solution, the pH was adjusted to 9.0 with 1M NaOH, and the reaction was carried out at room temperature overnight. The whole process was protected from light, and the complete antigen and unconjugated small molecules were separated by dialysis , and identify by the ultraviolet method, obtain the complete antigen of cyproheptadine (the ultraviolet characterization effect of the complete antigen of cyproheptadine of the present invention is as follows figure 2 ).

Embodiment 3

[0077] Embodiment 3: the preparation of the hybridoma cell line that secretes cyproheptadine monoclonal antibody

[0078] 1. Acquisition of animal immunity

[0079] Select healthy 6-8 week-old Balb / C mice for immunization, mix the obtained cyproheptadine complete antigen with an equal amount of oil, and then add emulsifier to obtain incomplete Freund's adjuvant after emulsification. Freund's adjuvant was added to Freund's adjuvant to obtain complete Freund's adjuvant; the obtained Freund's adjuvant was subcutaneously injected in the back to immunize BALB / c mice for several times. Complete Freund's adjuvant for 2 booster immunizations; on the 7th day after the 3rd immunization process, the mice were blood collected, and the immune titer and immunosuppressive ability of the mouse serum were detected by indirect ELISA, and the cyproheptadine in the serum was deleted. The immunized mice with high antibody content; the selected mice were boosted with incomplete Freund's adjuvant f...

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Abstract

The invention relates to a hybridoma cell line capable of secreting a CHP (cyproheptadine) monoclonal antibody and a preparation method, and belongs to the field of food safety immunodetection. The preservation number of the hybridoma cell line is CGMCC No.14697. The method comprises the following steps: a BALB / c mouse is immunized for the first time with a complete Freund's adjuvant, subjected tobooster immunization three times with an incomplete Freund's adjuvant and subjected to impact immunization once with an adjuvant-free CHP complete antigen, and the BALB / c mouse acquires immunity; splenocytes of the high-tilter and low-IC50 mouse which acquires immunity are fused with myeloma cells of the mouse with a PEG method, and the cell line is obtained through indirect competitive ELISA (enzyme-linked immunosorbent assay) screening and three times of subcloning. The monoclonal antibody secreted by the cell line has better specificity and detection sensitivity on CHP (IC50 is 0.370 ng / ml) and can be used for detecting CHP residues in food.

Description

technical field [0001] The invention relates to a hybridoma cell line secreting cyproheptadine monoclonal antibody and a preparation method thereof, belonging to the field of food safety immunoassay. Background technique [0002] Cyproheptadine (CHP) is an antihistamine drug and an H1 receptor antagonist. It also has the effect of anti-serotonin, which can inhibit the satiety center of the hypothalamus to stimulate appetite, increase body weight, promote Therefore, many unscrupulous manufacturers illegally add this drug to animal feed and animal drinking water to rapidly increase animal weight and obtain illegal profits. [0003] However, this illegal behavior will cause drug residues in animals, and cyproheptadine will accumulate in animals in large quantities. After people eat these animals, these residual drugs will accumulate in human bodies, because cyproheptadine will cause drowsiness and dizziness , fatigue, nausea and polyphagia, weight gain, and even anaphylactic s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44C07K14/795G01N33/577C07D211/70
CPCG01N33/577C07D211/70C07K14/795C07K16/44C07K19/00C07K2317/35G01N2430/00C12N5/0693G01N33/9406C12N5/163C07K16/065C07K16/1289G01N33/12
Inventor 匡华蒋薇胥传来徐丽广马伟刘丽强吴晓玲宋珊珊胡拥明
Owner JIANGNAN UNIV
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