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Furosemide monoclonal antibody, hybridoma cell strain and application

A technology of hybridoma cell line and furosemide, which is applied in the direction of using micro-injection, instruments, peptides, etc., can solve the problems of expensive instruments, application limitations, time-consuming, etc., and achieve simplified synthesis steps, good detection sensitivity and affinity Effect

Pending Publication Date: 2022-07-15
无锡迪腾敏生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have some disadvantages in varying degrees: time-consuming, expensive instruments and extensive sample pretreatment procedures, etc.
Analytical systems are therefore required, which means that the application of these methods in field analysis is limited

Method used

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  • Furosemide monoclonal antibody, hybridoma cell strain and application
  • Furosemide monoclonal antibody, hybridoma cell strain and application
  • Furosemide monoclonal antibody, hybridoma cell strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The preparation of embodiment 1 furosemide artificial antigen:

[0039]Synthesis of furosemide complete antigen: take 4.5 mg of furosemide, add 5.0 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 3.7 mg of NHS (N-hydroxysuccinimide), dissolved in DMF (N,N-dimethylformamide), stirred at room temperature, and activated for 6 h to obtain furosemide hapten; another 15 mg of BSA (bovine serum albumin) was dissolved in 3 mL , 0.05M, pH9.6 CB (carbonate buffer solution) solution; add the above activation solution dropwise to the BSA solution, after stirring at room temperature for 8h, then dialyze with 0.01MPBS for 3 days to remove unreacted small molecules Haptens, complete furosemide antigens were obtained, and stored in aliquots at -20°C.

Embodiment 2

[0040] Example 2: Preparation of hybridoma cell line secreting furosemide monoclonal antibody

[0041] 2.1 Acquisition of animal immunity

[0042] Healthy 6-8 week old Balb / C mice were selected for immunization. After the ergot ethylenediamine immunogen was mixed and emulsified with an equal amount of Freund's adjuvant, BALB / c mice were immunized with multiple subcutaneous injections on the back of the neck (except for sprint immunization). For the first immunization, complete Freund's adjuvant is used at a dose of 100ug / a; for multiple booster immunizations, incomplete Freund's adjuvant is used, and the dose is halved, that is, 50ug / a; for sprint immunization, no adjuvant is used, and it is directly diluted with normal saline and injected intraperitoneally , the dose is then halved to 25ug / pc. The interval between the first immunization and the second booster immunization was one month, the interval between multiple booster immunizations was 21 days, and the interval betwee...

Embodiment 3

[0050] Example 3: Preparation of furosemide monoclonal antibody

[0051] 8-10 weeks old BALB / c mice were taken, and each mouse was intraperitoneally injected with 1 mL of paraffin oil; 7 days later, each mouse was intraperitoneally injected with 1 × 10 6 For hybridoma cells, ascites was collected from the 7th day, the ascites was purified by the octanoic acid-saturated ammonium sulfate method, and the obtained monoclonal antibody was stored at -20°C.

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PUM

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Abstract

The invention provides a furosemide monoclonal antibody, a hybridoma cell strain and application, and belongs to the field of food safety immunodetection. The preservation number of the hybridoma cell strain is CGMCC (China General Microbiological Culture Collection Center) The furosemide complete antigen is synthesized, mixed and emulsified by using a Freund's adjuvant, and injected to immunize a BALB / c mouse. Mouse splenocytes with high titer and low IC50 are screened out, the mouse splenocytes are fused with mouse myeloma cells through a PEG method, a selective culture medium is adopted, and hybridoma cells obtained after fusion of the two cells are screened out; and screening cells by an indirect competitive enzyme-linked immunosorbent assay and subcloning for multiple times to obtain the monoclonal antibody hybridoma cell strain. The monoclonal antibody secreted by the cell strain has good detection sensitivity to the furosemide, the 50% inhibitory concentration IC50 to the furosemide is 0.49 ng / mL, and the monoclonal antibody is used for an immunodetection kit and a colloidal gold test strip and provides a powerful detection means for detection of the furosemide added in health food.

Description

technical field [0001] The invention belongs to the field of food safety immune detection, and specifically relates to a furosemide monoclonal antibody, a hybridoma cell line and an application. Background technique [0002] Furosemide is also known as furanilide, diuretic, diuretic sulfonamide, and its chemical name is 2-[(2-furanmethyl)amino]-5-(sulfamoyl)-4-chlorobenzoic acid. It has the pharmacological effects of diuresis and vasodilation. It is clinically used to treat peripheral edema caused by cardiac edema, renal edema, ascites due to cirrhosis, dysfunction or vascular disorder, and can promote the discharge of upper urethral calculi. It can be used to treat diseases such as acute pulmonary edema, cerebral edema, acute renal failure and hypertension, especially in cases in which other diuretics are ineffective; with rehydration, this product can promote the excretion of toxins. With the change of people's aesthetic concept and the love of beauty, the demand and use...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44C12N15/89G01N33/577G01N33/543G01N33/58
CPCC07K16/44C12N15/89G01N33/577G01N33/54313G01N33/543G01N33/581G01N2430/00
Inventor 刘丽强严婕妤胥传来匡华徐丽广孙茂忠郝昌龙宋珊珊吴爱红郭玲玲胥欣欣倪萍毕雪威郭鹏飞
Owner 无锡迪腾敏生物科技有限公司