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Anti-chlorothalonil monoclonal antibody hybridoma cell strain and application thereof

A hybridoma cell line and antibody cloning technology, applied in biochemical equipment and methods, instruments, microorganisms, etc., can solve the problems of long drug effect period, inability to realize rapid sample detection, cumbersome operation, etc., and achieve good detection sensitivity and specificity sexual effect

Inactive Publication Date: 2014-09-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chlorothalonil has no systemic conduction effect, but once it is sprayed on the plant body, it can have good adhesion on the surface of the plant and is not easily washed away by rain, so the drug has a long period of efficacy. In addition, it is widely used , therefore, chlorothalonil and its metabolites have relatively large residues in plants and soil, and are even detected in arctic regions
[0003] At present, the methods for detecting chlorothalonil are mainly gas chromatography (GC), liquid chromatography (HPLC), spectrophotometry, derivative synchronous fluorescence method, thin layer chromatography-spectrophotometry, gas chromatography and other ultraviolet instrument methods, However, these methods have the disadvantages of cumbersome operation, time-consuming, and expensive costs, and cannot achieve rapid detection of a large number of samples. Therefore, it is of great significance to establish a fast and simple detection method for chlorothalonil.

Method used

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  • Anti-chlorothalonil monoclonal antibody hybridoma cell strain and application thereof
  • Anti-chlorothalonil monoclonal antibody hybridoma cell strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Preparation of Anti-Chlorothalin Clonal Antibody Hybridoma Cell Line D

[0026] (1) Synthesis of complete antigen:

[0027] Weigh 500mg (1.9mmol) of chlorothalonil, 106.6mg (1.9mmol) of KOH, 262mg (2mmol) of 6-aminocaproic acid, dissolve these three drugs in 20mL of ethanol, react at 60°C for 12h, then react at 90°C for 12h . Rotate the reactants, then dissolve them in 20mL of water, adjust the pH to 4-5 with hydrochloric acid, produce a precipitate, and dry the precipitate to obtain the hapten CTNH, which is identified and analyzed by LC-MS.

[0028] Weigh 35.8 mg of CTNH, 56.9 mg of EDC, and 35.6 mg of NHS, dissolve in 1 mL of anhydrous DMF (referred to as solution A), and stir at room temperature for 8 h. Weigh 112 mg of BSA and dissolve it in 20 mL of 0.01 mol / L PBS with pH 7.2 (referred to as solution B), add solution A to solution B drop by drop at room temperature, and react overnight at room temperature to obtain the conjugate CTNH- BSA mixture, com...

Embodiment 2

[0039] Example 2 Preparation and identification of monoclonal antibody No. D of the anti-Chlorella monoclonal antibody hybridoma cell line

[0040] (1) Preparation of monoclonal antibody: 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 fluid was collected from the seventh day, and the ascites fluid was purified by octanoic acid-ammonium sulfate method, and the obtained monoclonal antibody was stored at -20°C.

[0041] (2) Detection: use indirect competitive ELISA to determine the IC of the monoclonal antibody chlorothalonil 50 They are: 1.5μg / L, indicating that it has good sensitivity to chlorothalonil and can be used for chlorothalonil immunoassay detection.

Embodiment 3

[0042] Example 3 Application of Monoclonal Antibody

[0043] The monoclonal antibody prepared by the anti-Chlorothalonil clonal antibody hybridoma cell line D of Example 1 through ascites in vivo was applied to the ELISA addition and recovery test of chlorothalonil, and the specific steps were as follows:

[0044] (1) Coating: Dilute the coated original CTNH-OVA with 0.05M pH9.6 carbonate buffer starting from 2μg / mL, 100μL / well, and react at 37°C for 2h;

[0045] (2) Washing: Pour off the solution in the plate, spin dry, and wash 3 times with washing liquid, 3 minutes each time;

[0046] (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;

[0047] (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 1 hour; after fully washing, add 1:3000 diluted HRP - Goat anti-mouse IgG, 100 μL / well, react at 37°C ...

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Abstract

The invention relates to an anti-chlorothalonil monoclonal antibody hybridoma cell strain and application thereof, belonging to the technical field of immunologic detection in food safety. The anti-chlorothalonil monoclonal antibody hybridoma cell strain D is collected in China General Microbiological Culture Collection Center, and the collection number is CGMCC No.9304.After the chlorothalonil complete antigen and equivalent Freund's adjuvant are mixed and emulsified, the emulsion is subjected to back subcutaneous injection to immunize a BALB / c mouse. The complete Freund's adjuvant is utilized for the first immunization, and the incomplete Freund's adjuvant is utilized afterwards. The high-titer IC50 mouse splenocyte and mouse myeloma cell are fused by a PEG process, and are subjected to indirect competitive ELISA screening and three-time subcloning to obtain the hybridoma cell strain D. The monoclonal antibody secreted by the cell strain D has favorable specificity and detection sensitivity for chlorothalonil (the IC50 value is 1.5 mu g / L), and can be used for chlorothalonil residue detection in food safety.

Description

technical field [0001] The invention relates to a hybridoma cell strain resistant to clonal antibody clone and application thereof, and belongs to the technical field of food safety immunology detection. Background technique [0002] Chlorothalonil is a broad-spectrum organochlorine fungicide, which can prevent fungal diseases of various crops and green lawns. Chlorothalonil has no systemic conduction effect, but once it is sprayed on the plant body, it can have good adhesion on the surface of the plant and is not easily washed away by rain, so the drug has a long period of efficacy, and it is widely used , therefore, chlorothalonil and its metabolites have relatively large residues in plants and soil, and have even been detected in arctic regions. [0003] At present, the methods for detecting chlorothalonil are mainly gas chromatography (GC), liquid chromatography (HPLC), spectrophotometry, derivative synchronous fluorescence method, thin layer chromatography-spectrophoto...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/577C12R1/91
Inventor 匡华胥传来严会娟徐丽广马伟刘丽强宋珊珊吴晓玲
Owner JIANGNAN UNIV
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