Monoclonal antibody hybridoma cell line for paraquat and application of monoclonal antibody hybridoma cell line

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. It can solve the problems of complex pretreatment, inapplicability of rapid detection of a large number of samples, time-consuming, etc.

Active Publication Date: 2018-07-06
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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  • Monoclonal antibody hybridoma cell line for paraquat and application of monoclonal antibody hybridoma cell line
  • Monoclonal antibody hybridoma cell line for paraquat and application of monoclonal antibody hybridoma cell line
  • Monoclonal antibody hybridoma cell line for paraquat and application of monoclonal antibody hybridoma cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of Hybridoma Cell Line LH1

[0033] 1. Derivation of hapten: 4,4'-bipyridine and methyl iodide are the starting materials to synthesize N-methyl-N'-valeryl-dipyridine dibromide as the hapten of paraquat. It is Para-COOH.

[0034] 2. Preparation of complete antigen Para-COOH-KLH: Weigh 2.7mg Para-COOH, 2.7N-hydroxysuccinimide (NHS), and dissolve in 300μL anhydrous N,N-dimethylformamide (DMF) Stir the reaction at room temperature for 10min; then weigh 4.8mg N,N'-dicyclohexylcarbodiimide (DCC), dissolve it with 100μL of anhydrous DMF, add it to the Para-COOH solution, stir at room temperature for 6-8h ( Called A liquid). Take 6.8mg KLH, add 1mL 0.01M phosphate buffer solution (PBS, pH=7.4) to dilute (called B solution), then slowly add A solution to B solution drop by drop, react overnight at room temperature; then use 0.01M PBS solution Dialysis, remove the unreacted small molecule hapten, obtain the complete antigen Para-COOH-KLH, and identify it by u...

Embodiment 2

[0043] (1) Coating: Dilute the coated original Para-COOH-OVA with 0.05M pH9.6 carbonate buffer from 1μg / mL at a 3-fold ratio, 100μL / well, and react at 37°C for 2h.

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

[0045] (3) Blocking: After patted dry, add 200μL / well blocking solution and react at 37°C for 2h. Dry it for later use after washing.

[0046] (4) Adding samples: Dilute the antiserum from 1:1000 in multiples and add it to the coated wells of each dilution, 100μL / well, react at 37°C for 30min; after thorough washing, add 1:3000 diluted HRP -Goat anti-mouse IgG, 100μL / well, react at 37°C for 30min.

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

[0048] (6) Termination and determination: Add 50μL of termination solution to each well to terminate the reaction, and then me...

Embodiment 3

[0050] Take water resources as an example to test the residual amount of paraquat

[0051] (1) Sample pretreatment: Obtain negative samples (named sample 1 and sample 2) from Jiangsu Import and Export Inspection and Quarantine Bureau, add Para and centrifuge, and take 50μL of supernatant (or make a certain dilution) for testing.

[0052] (2) Sample detection: According to the steps of Example 2, the label pull and the actual detection are carried out at the same time. 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 test spiked water samples (n=6)

[0054]

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Abstract

The invention discloses a monoclonal antibody hybridoma cell line for paraquat and application of the monoclonal antibody hybridoma cell line, and belongs to the field of immunodetection of food safety. The monoclonal antibody hybridoma cell line is obtained by screening. A monoclonal antibody secreted by the cell line is relatively high in specificity and detection sensitivity to the paraquat; the IC50 value is 0.97 ng/mL, and detection of paraquat residues in vegetables, water resources and soil can be realized; raw materials are provided for immunodetection of the paraquat residues in food;the monoclonal antibody hybridoma cell line has an actual application value.

Description

Technical field [0001] The invention relates to a paraquat monoclonal antibody hybridoma cell strain and its application, and belongs 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 cation salt, is a quick-acting contact kill type biocidal herbicide, because it has a strong effect on green plant tissues The destructive effect is promoted and applied in agriculture. At present, paraquat is 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 pesticide varieties in my country. Because paraquat has a strong adsorption effect in the soil, it produces residues in the soil and seriously pollutes water resources. 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 banned or stri...

Claims

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Application Information

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Patent Type & Authority Applications(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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