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Hybridoma cell strain secreting ketoconazole monoclonal antibody and application of hybridoma cell strain

A hybridoma cell line and ketoconazole single technology, applied in the field of immunochemistry, can solve the problem that the sensitivity of ketoconazole antibody needs to be further improved, and achieve the effects of good detection sensitivity and good specificity

Active Publication Date: 2021-11-30
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] CN109305963 A discloses a ketoconazole hapten, an artificial antigen and its preparation method and application, and shows that the artificial antigen can be used for the preparation of ketoconazole antibody and the detection of ketoconazole residual drugs. The antigen is obtained by reacting its analog acetylketoconazole with succinic anhydride; however, the sensitivity of the ketoconazole antibody in this patent needs to be further improved

Method used

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  • Hybridoma cell strain secreting ketoconazole monoclonal antibody and application of hybridoma cell strain
  • Hybridoma cell strain secreting ketoconazole monoclonal antibody and application of hybridoma cell strain
  • Hybridoma cell strain secreting ketoconazole monoclonal antibody and application of hybridoma cell strain

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Effect test

Embodiment 1

[0042] Embodiment 1: the synthesis of ketoconazole hapten

[0043] Since the small molecule of ketoconazole is not immunogenic and cannot stimulate the immune response of mice to produce antibodies, it is necessary to couple ketoconazole to the protein through protein linkage technology to obtain immunogenicity; protein coupling Active groups commonly used in the technology include amino, carboxyl, hydroxyl, mercapto, etc. Since the molecular structure of ketoconazole does not contain amino, carboxyl, or hydroxyl groups, carboxyl groups need to be derived from its structure.

[0044] The ketoconazole hapten structure after derivation of the present invention is as follows:

[0045]

[0046] The derivatization process includes the following three reactions:

[0047]

[0048] 1. Weigh raw materials ketoconazole 1, Pd 2 (dba) 3 , cesium carbonate in a Schlenk tube, add PPh3 into the Schlenk tube, then add methyl acrylate 2, solvent 1,4-dioxane. Complete deoxygenation wa...

Embodiment 2

[0051] Embodiment 2: the synthesis of complete antigen of ketoconazole

[0052] Weigh 8 mg ketoconazole hapten (KCZ-HS), 5 mg N-hydroxysuccinimide (NHS), dissolve in 300 μL N,N-dimethylformamide (DMF), stir at room temperature for 10 min; weigh again Take 7mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), fully dissolve it with 100μL DMF, add it to the KCZ-HS solution, and stir at room temperature for 4-6h (called liquid A). Take 5mg of BSA, dilute it to 2mg / 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 KCZ-HS-BSA, which is identified by ultraviolet absorption scanning method.

Embodiment 3

[0053] Embodiment 3: the synthesis of ketoconazole coating former

[0054] Dissolve 4 mg of ketoconazole hapten (KCZ-HS) and 2.5 mg of N-hydroxysuccinimide (NHS) in 300 μL of anhydrous N,N-dimethylformamide (DMF), and stir at room temperature for 10 min to obtain ketone Conazole hapten (KCZ-HS) solution; 4.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in 100 μL of anhydrous DMF and added to KCZ -HS solution, stirred at room temperature and reacted for 4-6 hours to obtain liquid A; 5 mg of chicken ovalbumin (OVA) was diluted with 1 mL of carbonate buffer solution (CBS) with a concentration of 0.01 mmol / L to obtain liquid B; Slowly add solution A to solution B to react dropwise to obtain a reaction solution; dialyze the reaction solution with PBS solution to remove unreacted small molecule hapten to obtain the coating agent (KCZ-HS-OVA).

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Abstract

The invention provides a hybridoma cell strain secreting a ketoconazole monoclonal antibody and application of the hybridoma cell strain. The hybridoma cell strain of the invention has preserved in the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Academy 1, Beichen West Road, Chaoyang District, Beijing, on May 13, 2021, with a preservation number of CGMCC No.22327, and the hybridoma cell strain is classified and named as a monoclonal cell strain. The ketoconazole monoclonal antibody secreted by the hybridoma cell strain of the invention has high specificity and high detection sensitivity (an IC50 value is 0.7 ng / mL, and the cross-over rate for a ketoconazole analogue is less than 1%) on the ketoconazole, the detection limit of the ketoconazole in a sample is 0.1 ng / g, and a detection linear range is 0.1-2.5 ng / g. The hybridoma cell strain can be used for establishing an immunological detection method of ketoconazole to detect ketoconazole residues in foods.

Description

technical field [0001] The invention belongs to the technical field of immunochemistry, in particular to a hybridoma cell strain secreting ketoconazole monoclonal antibody and its application. Background technique [0002] Ketoconazole (Ketoeonazole, KCZ) is an imidazole antifungal drug, and the trade names on the market are Jinda Ke Ning, Wei Kang Ling and Ke Ning, which inhibit the activity of fungal cytochrome P-450 by highly selective interference. Biosynthesis of ergosterol on the cell membrane. It is effective for both superficial and deep fungal infections. It can not only inhibit the growth of fungi, but also inhibit the transformation of spores into mycelium and prevent further infection. Low concentration antibacterial, high concentration bactericidal. It has antibacterial effects on Candida, Chromomycetes, Coccidioides, Histoplasma, Sporothrix and Trichophyton. It is clinically applicable to the treatment of tinea manuum, tinea pedis, tinea dermatitis, tinea co...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44C07K14/765G01N33/53G01N33/577C07D405/06
CPCC07K16/44C07K14/765G01N33/5308G01N33/577C07D405/06Y02A50/30
Inventor 匡华程媛胥传来徐丽广孙茂忠刘丽强宋珊珊吴晓玲郝昌龙胡拥明
Owner JIANGNAN UNIV
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