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A kind of paraquat monoclonal antibody hybridoma cell line and its application

A technology of monoclonal antibody and paraquat, which is applied in the application, fusion of cells, and cells modified by introducing foreign genetic material, etc., can solve the problems of inapplicable rapid detection of a large number of samples, complex pretreatment, and time-consuming

Active Publication Date: 2020-09-04
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional detection methods of paraquat include gas chromatography-mass chromatography, capillary electrophoresis, high performance liquid chromatography and capillary electrophoresis mass spectrometry, etc. However, the pretreatment of these methods is complicated and time-consuming, and is not suitable for rapid detection of a large number of samples

Method used

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  • A kind of paraquat monoclonal antibody hybridoma cell line and its application
  • A kind of paraquat monoclonal antibody hybridoma cell line and its application
  • A kind of paraquat monoclonal antibody hybridoma cell line and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0032] The preparation of embodiment 1 hybridoma cell line LH1

[0033] 1. Derivation of hapten: 4,4'-bipyridyl and methyl iodide as starting materials, synthesized N-methyl-N'-pentanoyl-dipyridine dibromide as the hapten of paraquat, record for Para-COOH.

[0034]2. Preparation of complete antigen Para-COOH-KLH: Weigh 2.7mg Para-COOH, 2.7N-hydroxysuccinimide (NHS), dissolve in 300μL anhydrous N,N-dimethylformamide (DMF) , stirred at room temperature for 10 min; then weighed 4.8 mg of N,N'-dicyclohexylcarbodiimide (DCC), dissolved it in 100 μL of anhydrous DMF, added it to the Para-COOH solution, and stirred at room temperature for 6-8 h ( called liquid A). Take 6.8mg KLH, add 1mL 0.01M phosphate buffer solution (PBS, pH=7.4) to dilute (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 Dialysis to remove the unreacted small molecule hapten to obtain the complete antigen Para-CO...

Embodiment 2

[0043] (1) Coating: The coated Para-COOH-OVA was diluted 3-fold with 0.05M pH9.6 carbonate buffer starting from 1 μg / mL, 100 μL / well, and reacted at 37°C for 2 hours.

[0044] (2) Washing: Pour off the solution in the plate, and wash 3 times with washing liquid, each time for 3 minutes.

[0045] (3) Blocking: After patting dry, add 200 μL / well blocking solution and react at 37°C for 2 hours. Wash and tumble dry for later use.

[0046] (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 - Goat anti-mouse IgG, 100 μL / well, react at 37°C for 30 minutes.

[0047] (5) Color development: Take out the ELISA plate, after washing thoroughly, add 100 μL of TMB color development solution to each well, and react in the dark at 37°C for 15 min.

[0048] (6) Termination and measurement: 50 μL of stop solution was added to each well to term...

Embodiment 3

[0050] Taking water resources as an example, detecting paraquat residues in them

[0051] (1) Sample pretreatment: Negative samples (named sample 1 and sample 2) were obtained from the Jiangsu Import and Export Inspection and Quarantine Bureau, added with Para and centrifuged, and 50 μL of the supernatant (or diluted to a certain extent) was used for detection.

[0052] (2) Sample detection: According to the steps of Example 2, label drawing and actual detection were carried out at the same time, and the results are shown in Table 1. The recovery rate is 83.4%-110.9%, the matrix interference is small, and the result is ideal.

[0053] Table 1. ic-ELISA detection of spiked water samples (n=6)

[0054]

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Abstract

The invention discloses a paraquat monoclonal antibody hybridoma cell line and an application thereof, belonging to the field of food safety immune detection. The invention obtains a monoclonal antibody hybridoma cell line through screening. The monoclonal antibody secreted by this cell line has good specificity and detection sensitivity to paraquat, IC 50 The value is 0.97ng / mL, which can realize the detection of paraquat residues in vegetables, water resources and soil, and provides raw materials for the immunological detection of paraquat residues in food, which has practical application value.

Description

technical field [0001] The invention relates to a paraquat monoclonal antibody hybridoma cell line and an application thereof, belonging to the field of food safety immune detection. Background technique [0002] Paraquat (paraquat, abbreviated as Para), the chemical name is 1-1-dimethyl-4-4-bipyridine cationic salt, is a quick-acting contact-killing herbicide, due to its strong effect on green plant tissue It has been popularized and applied in agriculture due to its destructive effect. At present, paraquat has been used in more than 100 countries around the world, and its consumption is second only to the herbicide glyphosate, and it is also one of the most imported pesticides in my country. Due to the strong adsorption of paraquat in the soil, it will remain in the soil and cause serious pollution to water resources. Moreover, paraquat is extremely toxic to humans, and there is no specific antidote. The mortality rate of oral poisoning can reach 90%. The above has been ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C12N15/06C07K16/44G01N33/577
CPCC07K16/44C12N5/163G01N33/577G01N2430/20
Inventor 胥传来李月匡华徐丽广马伟刘丽强吴晓玲宋珊珊胡拥明
Owner JIANGNAN UNIV
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