Hybridoma cell strain capable of secreting fluazinam monoclonal antibody and application thereof

A hybridoma cell line, fluazinamide single technology, applied in analytical materials, preparation methods of peptides, animal/human proteins, etc., can solve the problems of high solvent, expensive equipment, consumption, etc., and achieve the effect of good detection sensitivity

Active Publication Date: 2021-10-15
JIANGNAN UNIV
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite the high sensitivity and specificity of these chromatography-based methods, there are some disadvantages such as the need for thorough sample cleanup, high solvent consumption, expensive equipment, and skilled technicians

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Hybridoma cell strain capable of secreting fluazinam monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Synthesis of fluazinam hapten

[0051] Since the small molecule of fluazinam is not immunogenic and cannot stimulate the mice to produce an immune response and then produce antibodies, it is necessary to couple fluazinam to the protein by protein linking technology to obtain immunogenicity; protein coupling Active groups commonly used in the technology include amino, carboxyl, hydroxyl, mercapto, etc. Since the molecular structure of fluazinam does not contain these active groups, derivatization is required.

[0052] Weigh 9.30 g (20 mmol) of fluazinam into a 100 ml three-necked flask, dissolve it in 20 mL DMSO, and then add 2.25 g (40 mmol) of KOH. Another 2.10 g (20 mmol) of β-mercaptopropionic acid was weighed, dissolved in 10 mL DMSO and transferred to a 50 ml constant pressure dropping funnel. Slowly add the β-mercaptopropionic acid solution dropwise into the three-necked flask with stirring, and slowly raise the temperature to 100°C on the oil bath, ke...

Embodiment 2

[0053] Example 2: Synthesis of complete antigen of fluazinam

[0054] Weigh 8.5 mg fluazinamide hapten, 4.2 mg N-hydroxysuccinimide (NHS), dissolve in 300 μL N,N-dimethylformamide (DMF), stir at room temperature for 10 min; then weigh 6.9 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), fully dissolved with 100 μL DMF, added to the fluazinam hapten solution, stirred at room temperature to react 6- 8h (called A liquid). Take 8 mg of BSA, dilute it to 4 mg / mL with 0.01 M carbonate buffer (CBS) (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 Dialyzed to remove unreacted small molecular hapten to obtain the complete antigen, which was identified by UV absorption scanning method.

Embodiment 3

[0055] Embodiment 3: the synthesis of fluazinam coating original

[0056] Dissolve 5.0 mg of fluazinamide hapten and 2.3 mg of N-hydroxysuccinimide (NHS) in 300 μL of anhydrous N,N-dimethylformamide (DMF), and stir at room temperature for 10 min to obtain fluazinamide semi- Antigen solution; 3.8 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in 100 μL anhydrous DMF, and added to the fluazinam hapten 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 carbonate buffer solution (CBS) with a concentration of 0.01 mmol / L to obtain liquid B; Adding it into solution B for reaction to obtain a reaction solution; dialyzing the reaction solution with PBS solution to remove unreacted small molecule hapten to obtain the coating original.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Sensitivityaaaaaaaaaa
Sensitivityaaaaaaaaaa
Login to view more

Abstract

The invention discloses a hybridoma cell strain capable of secreting a fluazinam monoclonal antibody and application of the hybridoma cell strain, and belongs to the field of food safety immunodetection. The invention provides the hybridoma cell strain CKG secreting a fluazinam monoclonal antibody, the hybridoma cell strain CKG is classified and named as a monoclonal cell strain, the preservation date is September 27, 2020, and the accession number is CGMCC No.20790. A complete antigen of fluazinam and an equal amount of a Freund's adjuvant are mixed and emulsified, and BALB/c mice are immunized through neck and back subcutaneous multipoint injection. Splenocytes of a high-titer and low-IC50 mouse are fused with myeloma cells of the mouse through a PEG method, and hybrid cells after fusing the two cells are screened out by adopting a selective culture medium; and cells are screened by an indirect competitive enzyme-linked immunosorbent assay and subcloning is performed for three times to finally obtain the monoclonal antibody hybridoma cell strain CKG. The monoclonal antibody secreted by the cell strain has good detection sensitivity (IC50 value being 2.68 ng/mL) to fluazinam, and can be used for residue detection of fluazinam in food.

Description

technical field [0001] The invention relates to a hybridoma cell strain secreting fluazinam monoclonal antibody and application thereof, belonging to the field of food safety immunoassay. Background technique [0002] Fluazinam is a 2,6-dinitroaniline fungicide developed by Ishihara Co., Ltd. in Japan. It is commonly used to control tomato late blight, citrus gray mold, soybean sclerotinia, pepper blight and clubroot of cruciferous plants. illness etc. Fluazinam is not systemic and has no therapeutic effect, but it is extremely resistant to rain erosion and has a long residual effect. It is a good foliar spray and can also treat spider mites. However, the environmental toxicity of fluazinam is worrying. Studies have shown that fluazinam can cause asthma and dermatitis and other diseases that damage the human immune system. Therefore, establishing an effective detection method for fluazinam residues has far-reaching significance for effectively promoting environmental prote...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44C07K14/765C07K14/77C07K1/107G01N33/577C07D213/74
CPCC07K16/44C07K14/765C07K14/77G01N33/577G01N33/5308C07D213/74Y02A50/30
Inventor 胥传来刘杰匡华刘丽强宋珊珊胡拥明徐丽广胥欣欣吴爱红
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap