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Digitonin monoclonal antibody hybridoma cell strain and application thereof

The technology of hybridoma cell line and digoxigenin is applied in the directions of biochemical equipment and methods, material testing products, instruments, etc., which can solve the problems of cumbersome steps, rapid detection, time-consuming and the like, and achieve the effect of good detection sensitivity

Active Publication Date: 2021-12-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The detection methods of digoxigenin are usually high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS) and other instrumental methods. These detection methods have the disadvantages of time-consuming, cumbersome steps, inability to perform rapid on-site detection, and high cost. , so it is of great significance to establish a rapid and simple method for the detection of digoxigenin

Method used

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  • Digitonin monoclonal antibody hybridoma cell strain and application thereof
  • Digitonin monoclonal antibody hybridoma cell strain and application thereof
  • Digitonin monoclonal antibody hybridoma cell strain and application thereof

Examples

Experimental program
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Embodiment 1

[0038] The preparation of embodiment 1 hybridoma cell line FJN

[0039] (1) Preparation of immunogen Digoxigenin-BSA:

[0040] Weigh 21.4mg of digoxigenin, dissolve in 1mL dimethylsulfoxide (DMSO), add 0.1M sodium periodate (NaIO 4 ) solution 1mL, stirred at room temperature for 1h; added 1M ethylene glycol 60μL, continued to stir for 5min; 2 CO 3 ) to adjust the pH to 9, continue to stir for 1h until the pH is stable; weigh 15mg of sodium borohydride (NaBH 4 ), dissolved in water and added to the above reaction solution to react for 16h; add 1M formic acid, adjust the pH to 6.5, and place it at room temperature for 1h; add ammonia water (NH4 OH), adjust pH to 8.5; dialyze the above reaction solution for 24h; after dialysis, adjust pH to 5 with 0.1M hydrochloric acid (HCl), precipitate at room temperature for 1h, 4°C for 3h; centrifuge at 12000rpm for 8min, and precipitate with hydrogen carbonate Sodium (NaHCO 3 ) was dissolved, and the complete antigen and unconjugated sm...

Embodiment 2

[0050] Example 2 IC of digoxigenin monoclonal antibody 50 determination of

[0051] (1) Coating: The coated protodigoxin-OVA was diluted with 0.05M pH 9.6 carbonate buffer starting from 1 μg / mL, 100 μL / well, and reacted at 37° C. for 2 h.

[0052] (2) Washing: Pour off the solution in the plate, and wash with washing solution 3 times, 3min each time.

[0053] (3) Blocking: After patting dry, add 200 μL / well of blocking solution, and react at 37° C. for 2 h. Dry after washing.

[0054] (4) Sampling: The antiserum (antiserum after the mouse is tail-broken for blood collection, diluted with the antibody diluent to the corresponding multiples) is diluted from 1:1000, and added to the coated wells of each dilution. 100 μL / well, react at 37 °C for 30 min; after thorough washing, add 1:3000 diluted HRP-goat anti-mouse IgG, 100 μL / well, and react at 37 °C for 30 min.

[0055] (5) Color development: Take out the ELISA plate, wash it thoroughly, add 100 μL of TMB color development s...

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Abstract

The invention belongs to the field of food safety immunodetection, and particularly relates to a digitonin monoclonal antibody hybridoma cell strain FJN and application thereof. The digitonin monoclonal antibody hybridoma cell strain FJN is preserved in the China General Microbiological Culture Collection Center (CGMCC), the address is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, West Beichen Road, Chaoyang District, Beijing, the preservation date is May 13, 2021, and the preservation number is CGMCC NO. 22325. A monoclonal antibody secreted by the digitonin monoclonal antibody hybridoma cell strain has relatively good specificity and detection sensitivity (IC50 value is 0.51ng / mL) to digitonin, and an immunological method is provided for detecting the digitonin content in a clinical blood sample. The digitonin monoclonal antibody hybridoma cell strain and the monoclonal antibody secreted by the digitonin monoclonal antibody hybridoma cell strain can be prepared into a kit for detecting digitonin, and the digitonin monoclonal antibody hybridoma cell strain has practical application value.

Description

technical field [0001] The invention belongs to the field of food safety immunoassay, and in particular relates to a digoxigenin monoclonal antibody hybridoma cell line FJN and its application. Background technique [0002] Digitoxin, also known as Digitoxin, Digitoxin or Digitoxin, is an oral cardiac glycoside preparation, which is a white crystalline powder, odorless, slightly soluble in chloroform, slightly Soluble in ethanol, ether, insoluble in water. Cardiac glycosides are mainly used clinically to treat heart failure and arrhythmia, belonging to Na + / K + ATPase inhibitors. Digoxigenin and Na + / K + ATPase binds reversibly on the cell membrane, inhibits the active transport of sodium ions and potassium ions, increases the concentration of sodium ions in the cell, which in turn promotes the exchange of sodium ions and calcium ions, and increases the concentration of calcium ions in the cell. Calcium ions play an important role in cardiac excitation and contractio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/577G01N33/53C12R1/91
CPCC07K16/44G01N33/577G01N33/9453C07K2317/33C07K2317/92Y02A50/30
Inventor 胥传来姜晓倩匡华徐丽广孙茂忠刘丽强吴晓玲宋珊珊胡拥明郝昌龙马伟吴爱红高巍
Owner JIANGNAN UNIV
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