Hybridoma cell strain secreting thiamethoxam monoclonal antibody and application thereof

A hybridoma cell line, monoclonal antibody technology, applied in hybrid peptides, analytical materials, microorganism-based methods, etc., can solve the problems of time-consuming, complicated pretreatment, not suitable for rapid detection of a large number of samples, etc., and achieve good detection sensitivity. , good specificity effect

Inactive Publication Date: 2018-12-14
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Traditional detection methods for thiamethoxam include gas chromatography-mass chromatography, capillary electrophoresis, high performance liquid chromatography, and capillary electrophoresis-mass spectrometry. To protect the interests of consumers, it is necessary to establish an efficient and rapid detection method for thiamethoxam

Method used

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  • Hybridoma cell strain secreting thiamethoxam monoclonal antibody and application thereof
  • Hybridoma cell strain secreting thiamethoxam monoclonal antibody and application thereof
  • Hybridoma cell strain secreting thiamethoxam monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Synthesis of Thiamethoxam Hapten

[0066] Synthetic routes such as figure 1 .

[0067] Since small molecules of thiamethoxam are not immunogenic and cannot stimulate mice to produce immune responses and then produce antibodies, it is necessary to couple thiamethoxam to proteins through protein linkage technology to obtain immunogenicity; protein coupling Active groups commonly used in technology include amino, carboxyl, hydroxyl, mercapto, etc. Since the molecular structure of TMX does not contain these active groups, it needs to be derivatized.

[0068] Dissolve 20mL dimethyl sulfoxide (DMSO) in 5.8g thiamethoxam (TMX), add 2.25g potassium hydroxide, add β-mercaptopropionic acid solution dropwise while stirring (β-mercaptopropionic acid solution is obtained by adding 2.1g β-mercaptopropionic acid was dissolved in 10mL of DMSO), and the oil bath was heated to 100°C for reaction to obtain a reaction solution; after the reaction solution was naturally cooled ...

Embodiment 2

[0069] Embodiment 2: the synthesis of complete antigen of thiamethoxam

[0070] Dissolve 2.8 mg of thiamethoxam hapten (TMX-COOH) and 2.8 mg of N-hydroxysuccinimide (NHS) in 300 μL of anhydrous N,N-dimethylformamide (DMF), and stir at room temperature for 10 minutes to obtain Thiamethoxam hapten (TMX-COOH) solution; 4.8 mg N,N'-dicyclohexylcarbodiimide (DCC) was dissolved in 100 μL anhydrous DMF, added to the TMX-COOH solution, stirred at room temperature for reaction After 6-8 hours, liquid A was obtained; 6.8 mg of keyhole limpet hemocyanin (KLH) was diluted with 1 mL of phosphate buffered saline (PBS) with a concentration of 0.01 mmol / L and a pH of 7.4 to obtain liquid B; solution was slowly added to solution B for reaction to obtain a reaction solution; the reaction solution was dialyzed with PBS solution to remove unreacted small molecule hapten to obtain a complete antigen (TMX-COOH-KLH).

Embodiment 3

[0071] Embodiment 3: the synthesis of thiamethoxam coating former

[0072] Dissolve 5 mg of thiamethoxam hapten (TMX-COOH) and 4.8 mg of N-hydroxysuccinimide (NHS) in 300 μL of anhydrous N,N-dimethylformamide (DMF), and stir at room temperature for 10 min to obtain thiamethoxam Methoxazine hapten (TMX-COOH) solution; 7.6 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in 100 μL of anhydrous DMF, and added to In the TMX-COOH solution, stir at room temperature and react for 6-8 hours to obtain liquid A; dilute 10 mg of chicken ovalbumin (OVA) with 1 mL of phosphate buffered saline (PBS) with a concentration of 0.01 mmol / L and a pH of 7.4 to obtain liquid B solution; slowly add solution A to solution B drop by drop for reaction to obtain a reaction solution; dialyze the reaction solution with PBS solution to remove unreacted small molecule haptens to obtain the original coating (TMX-COOH-OVA).

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Abstract

The invention relates to a hybridoma cell strain secreting thiamethoxacin monoclonal antibody and application thereof, belonging to the field of food safety immunodetection. The accession number of the hybridoma cell strain is CGMCC No. 14699. According to the invention, a complete Freund's adjuvant is used for primary immunization of a BALB/c mouse, then an incomplete Freund's adjuvant is used for booster immunization three times, and a thiamethoxam complete antigen containing no adjuvant is used for impact immunization once, so the BALB/c mouse is immunized; and then the high-titer low-IC50spleen cells of the immunized mouse are fused with mouse myeloma cells by using a PEG method, and then the cell strain is obtained through indirect competitive ELISA screening and subcloning three times. The monoclonal antibody secreted by the cell strain has good specificity and detection sensitivity (with an IC50 value of 0.81 ng/mL) to thiamethoxam and can be used for detection of thiamethoxamresidues in food.

Description

technical field [0001] The invention relates to a hybridoma cell strain secreting thiamethoxam monoclonal antibody and application thereof, belonging to the field of food safety immunological detection. Background technique [0002] Thiamethoxam (Thiamethoxam, TMX) is the second-generation neonicotinoid insecticide developed by Syngenta in 1991; structurally, it uses 2-chloro-5-thiazolyl and methyl as the pharmacophore. The most ideal modification and combination of oxadiazine lead substances; from the perspective of the target, it acts as an acetylcholine receptor inhibitor to interfere with insect nerve signal transduction in a novel way; from the perspective of insecticidal activity, it is more active than the first generation of neonicotinoid insecticides It is strong and can be used in a variety of ways; from the perspective of the scope of action, it has excellent control effects on almost all chewing mouthparts and most piercing and sucking mouthparts pests, especiall...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44C07K14/795G01N33/577C12R1/91
CPCC07K14/795C07K16/44C07K19/00G01N33/577
Inventor 匡华叶丽雅胥传来徐丽广马伟刘丽强吴晓玲宋珊珊胡拥明
Owner JIANGNAN UNIV
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