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Hybridoma cell strain capable of secreting dicofol monoclonal antibody and application of hybridoma cell strain

A hybridoma cell line and a technology for dicofol, which is applied in the field of immunodetection, can solve the problems of high technical requirements for operators, unsuitable for on-site detection, and inability to produce immediate results, and achieves good detection sensitivity, good specificity, and easy availability of raw materials. Effect

Pending Publication Date: 2021-11-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In order to effectively supervise and monitor the use of dicofol in food, a determination method with good specificity and high sensitivity is needed, and the current detection methods such as gas chromatography, high performance liquid chromatography, high performance liquid chromatography tandem mass spectrometry, etc., cost Expensive, cumbersome pre-treatment of samples, high technical requirements for operators, and results cannot be obtained immediately, and there are many interfering substances in food, so the instrument method is not suitable for on-site detection

Method used

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  • Hybridoma cell strain capable of secreting dicofol monoclonal antibody and application of hybridoma cell strain
  • Hybridoma cell strain capable of secreting dicofol monoclonal antibody and application of hybridoma cell strain
  • Hybridoma cell strain capable of secreting dicofol monoclonal antibody and application of hybridoma cell strain

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Embodiment 1

[0050] The preparation of embodiment 1 hybridoma cell line JHJ

[0051] (1) Synthesis of hapten:

[0052]

[0053] The synthesis scheme of the dicofol hapten is shown in the above reaction formula. Weigh 4,4'-dichlorobenzophenone (15mg, 0.0597mmol) and carboxymethylhydroxylamine hemihydrochloride (30mg, 0.235mmol) into a 10mL screw-top brown glass bottle, add 1.8mL methanol, pyridine and a mixed solution of pure water (v:v:v=4:1:1), and then placed in a 70° C. water bath for continuous stirring for 6 hours. After the reaction was complete, take it out and let it stand overnight at room temperature. After the solution was blown dry with nitrogen, add 2 mL of dichloromethane to redissolve, and repeat the extraction with pure water 3-4 times (1 mL / time), discard the water layer, and dry the organic phase with nitrogen to obtain a viscous solid that is dicofol semi antigen.

[0054] (2) Preparation of immunogen and coating agent:

[0055] Preparation of immunogen: Weigh 5....

Embodiment 2 3

[0065] Example 2 IC of dicofol monoclonal antibody 50 Determination of

[0066] 1 Coating: Dilute the original coating prepared in step (2) of Example 1 with 0.05M pH9.6 carbonate buffer starting from 1 μg / mL, 100 μL / well, and react at 37°C for 2 hours;

[0067] 2 Washing: Pour off the solution in the plate, and wash 3 times with washing solution, 3 minutes each time;

[0068] 3 Blocking: After patting dry, add 200 μL / well blocking solution and react at 37°C for 2 hours. After washing, dry it for later use;

[0069] 4 Adding samples: Dilute the antiserum starting from 1:1000, and add it to the coated wells of each dilution, 100 μL / well, and react at 37°C for 30 minutes; after fully washing, add 1:3000 diluted HRP-sheep Anti-mouse IgG, 100 μL / well, react at 37°C for 30 minutes;

[0070] 5 Color development: Take out the microplate plate, after fully washing, add 100 μL of TMB color development solution to each well, and react in the dark at 37°C for 15 minutes;

[0071] 6 Te...

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Abstract

The invention discloses a hybridoma cell strain capable of secreting a dicofol monoclonal antibody and application of the hybridoma cell strain, and belongs to the technical field of immunodetection. The invention discloses a hybridoma cell strain JHJ capable of secreting a dicofol monoclonal antibody. The hybridoma cell strain JHJ is collected in the China General Microbiological Culture Collection Center (CGMCC) and is classified and named as the monoclonal cell strain JHJ with the collection date of May 13, 2021 and the collection number of CGMCC No.22329. The hybridoma cell strain is obtained by immunizing a BALB / c mouse with a dicofol complete antigen, taking splenocytes of the mouse with high titer and good inhibition, fusing with myeloma cells through a PEG method, and carrying out screening and three times of subcloning through an indirect competitive enzyme-linked immunosorbent assay. The monoclonal antibody secreted by the cell strain has good specificity and detection sensitivity (IC50 value is 2ng / mL) to dicofol, provides a raw material for immunodetection of dicofol residues in food, and has a practical application value.

Description

technical field [0001] The invention belongs to the technical field of immune detection, and in particular relates to a hybridoma cell strain secreting dicofol monoclonal antibody and application thereof. Background technique [0002] Dicofol, also known as 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethanol or Kaile San, is an organochlorine acaricide commonly used in agricultural production, and is often used in my country. It is used to control a variety of harmful mites in cotton, fruit trees and flowers. However, in the production process of dicofol, there may be a certain amount of DDT residue, which is harmful to the environment and human health. In recent years, more and more evidence has shown that its exposure in the environment has toxic and estrogenic effects on fish, reptiles, birds, mammals and humans, and is extremely toxic to aquatic organisms. Therefore, my country has banned the application of dicofol in agricultural production. On October 27, 2017, the list o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44C07K14/765C07K14/795C07K14/77C07C249/08C07C249/14C07C251/60G01N33/577G01N33/53C12R1/91
CPCC07K16/44C07K14/765C07K14/795C07K14/77C07C249/08C07C249/14G01N33/577G01N33/5308C07K2317/92C07C251/60
Inventor 宋珊珊林璐胥传来匡华徐丽广马伟刘丽强吴晓玲胡拥明
Owner JIANGNAN UNIV
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