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Hybridoma cell lines secreting diphenhydramine monoclonal antibodies and application thereof

A hybridoma cell line and monoclonal antibody technology, which is applied in the direction of analytical materials, microbial-based methods, preparation of organic compounds, etc., can solve the problems of high solvent, consumption, expensive equipment, etc., and achieve the effect of good detection sensitivity

Active Publication Date: 2021-07-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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 lines secreting diphenhydramine monoclonal antibodies and application thereof
  • Hybridoma cell lines secreting diphenhydramine monoclonal antibodies and application thereof
  • Hybridoma cell lines secreting diphenhydramine monoclonal antibodies and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Synthesis of diphenhydramine hapten

[0048] Since the diphenhydramine small molecule is not immunogenic, it cannot stimulate the mice to produce an immune response and then produce antibodies, so it is necessary to couple diphenhydramine to the protein through protein linkage technology to make it immunogenic; protein Active groups commonly used in coupling technology include amino, carboxyl, hydroxyl, mercapto, etc. Since diphenhydramine’s molecular structure does not contain these active groups, it needs to be derivatized. The specific steps are as follows:

[0049] Weigh 50 mg of 2-(benzhydryloxy)-N-methylethylamine, dissolve it in 5 mL of pyridine, then add 25 mg of succinic anhydride to the reaction solution, stir the reaction at 50°C for 4 h, then add 10 mL of ultrapure Water quenches the reaction to give the derivative product. Then use chloroform to extract three times, combine the chloroform phase and blow with nitrogen to obtain the diphenhydramin...

Embodiment 2

[0050] Example 2: Synthesis of Diphenhydramine Immunogen

[0051] Weigh 5.5 mg diphenhydramine hapten, 4.2 mg N-hydroxysuccinimide (NHS), dissolve in 300 μL N,N-dimethylformamide (DMF), stir at room temperature for 10 min to obtain diphenhydramine Ming hapten solution; then weighed 6.9mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), fully dissolved with 100μL DMF, added to diphenhydramine In the hapten solution, stir at room temperature for 6-8h (referred to as liquid A). Take 8 mg of BSA, dilute it to 4 mg / mL with 0.01M carbonate buffer (CBS) (referred to as solution B), then slowly add solution A to solution B drop by drop, react at room temperature overnight; then use 0.01M PBS solution Dialyzed to remove unreacted small molecular haptens, to obtain the diphenhydramine immunogen, which was identified by ultraviolet absorption scanning method.

Embodiment 3

[0052] Embodiment 3: the synthesis of diphenhydramine coating former

[0053] Dissolve 3.0 mg of diphenhydramine hapten and 2.3 mg of N-hydroxysuccinimide (NHS) in 300 μL of anhydrous N,N-dimethylformamide (DMF), and stir at room temperature for 10 minutes to obtain diphenhydramine Hapten solution; 3.8 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in 100 μL of anhydrous DMF, and added to diphenhydramine hapten solution , stirred at room temperature and reacted 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; The solution was slowly added into 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.

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Abstract

The invention discloses hybridoma cell lines capable of secreting diphenhydramine monoclonal antibodies and an application thereof, and belongs to the field of cosmetic safety immunodetection. The hybridoma cell lines SHS 5B11 capable of secreting diphenhydramine monoclonal antibodies are named as monoclonal cell lines by classification, and preserved on September 27, 2020 with the preservation number of CGMCCNo.20787. The monoclonal antibodies secreted by the cell lines have good specificity and detection sensitivity to diphenhydramine. According to the present invention, diphenhydramine complete antigens are synthesized, then subjected to mixed emulsification with equal amount of Freund's adjuvant, and afterwards subcutaneously injected to immune BALB / c mice through backs of the mice. The hybridoma cell lines SHS 5B11 are obtained through screening with indirect competitive enzyme-linked immunosorbent assay and subcloning three cloning. The monoclonal antibodies secreted by the cell lines have good detection sensitivity ( IC50 value of 2.93 ng / mL) to diphenhydramine, and can be used for detecting diphenhydramine residues in cosmetics.

Description

technical field [0001] The invention relates to a hybridoma cell strain secreting diphenhydramine monoclonal antibody and an application thereof, belonging to the field of cosmetics safety immune detection. Background technique [0002] Diphenhydramine is an antihistamine that is widely used in the field of medicine and has good effects on allergic reactions such as skin itching, allergic rhinitis, urticaria and contact dermatitis. Diphenhydramine can sterilize and remove acne, and has a certain anti-allergic effect, so it may be added to acne-removing cosmetics to achieve the effect of removing acne. Since the raw materials used in cosmetics or propylene glycol in the technological process can promote the absorption of diphenhydramine by the human body, when the content of diphenhydramine in the human body is too high, the central nervous system will be strongly inhibited, resulting in drowsiness, Dizziness, headache, nausea, vomiting and upper abdominal discomfort and oth...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44C07K14/77C07C233/18C07C231/02C07C231/24G01N33/577G01N33/53C12R1/91
CPCC07K16/44C07K14/77C07C233/18C07C231/02C07C231/24G01N33/577G01N33/5308
Inventor 胥传来刘杰匡华刘丽强宋珊珊胡拥明
Owner JIANGNAN UNIV
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