Forchlorfenuron monoclonal antibody hybridoma cell strain and application thereof

A technology of forchlorfenuron monoclonal antibody, applied in animal/human protein, serum albumin, biochemical equipment and methods, etc., can solve the problems of high solvent, expensive equipment, consumption, etc., and achieve strong specificity and detection sensitivity The effect of high and good practical application value

Active Publication Date: 2021-03-16
JIANGNAN UNIV
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  • 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

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  • Forchlorfenuron monoclonal antibody hybridoma cell strain and application thereof
  • Forchlorfenuron monoclonal antibody hybridoma cell strain and application thereof
  • Forchlorfenuron monoclonal antibody hybridoma cell strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: the synthesis of forchlorfenuron hapten

[0053] Since the small molecule of forchlorfenuron is not immunogenic and cannot stimulate the immune response of mice to produce antibodies, it is necessary to couple forchlorfenuron to the protein through 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 forchlorfenuron does not contain these active groups, it needs to be derivatized.

[0054] Weigh forchlorfenuron 4.95g (20mmol) in a 100ml three-necked flask, dissolve it with 20ml DMSO, and then add KOH 2.25g (40mmol). 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 drop the β-mercaptopropionic acid solution into the three-necked flask under stirring, and slowly raise the temperature to 100°C on the oil ba...

Embodiment 2

[0055] Embodiment 2: the synthesis of complete antigen of forchlorfenuron

[0056] Weigh 5.5mg forchlorfenuron hapten, 4.2mg N-hydroxysuccinimide (NHS), dissolve in 300μL N,N-dimethylformamide (DMF), stir at room temperature for 10min; then weigh 6.9mg 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), fully dissolved with 100μL DMF, added to the forchlorfenuron hapten solution, stirred at room temperature for 6-8h (called liquid A). Take 8 mg of BSA, dilute it to 4 mg / mL with 0.01M 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

[0057] Embodiment 3: the synthesis of forchlorfenuron coating former

[0058] Dissolve 3.0 mg of forchlorfenuron 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 minutes to obtain forchlorfenuron hapten Solution: Dissolve 3.8mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in 100 μL of anhydrous DMF, add to the forchlorfenuron hapten solution, room temperature Stir and react for 6-8 hours to obtain liquid C; 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 D; then slowly add liquid A drop by drop React in solution B to obtain reaction solution E; dialyze reaction solution C with PBS solution to remove unreacted small-molecule haptens to obtain the coating original.

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Abstract

The invention discloses a forchlorfenuron monoclonal antibody hybridoma cell strain and an application thereof, and belongs to the technical field of food safety immunodetection. The preservation number of the hybridoma cell strain is CGMCC No. 19179. Forchlorfenuron hapten and complete antigen are firstly synthesized, mixing and emulsifying are performed by using a Freund's adjuvant, and injecting is performed to immunize a BALB / c mouse. High-titer and low-IC50 mouse splenocytes are screened, and are fused with mouse myeloma cells by a PEG method, a culture medium is selected, and hybrid cells obtained by fusing the two cells are screened. The cells are screened by an indirect competitive enzyme-linked immunosorbent assay and subcloning is performed for five times to finally obtain the monoclonal antibody hybridoma cell strain. The monoclonal antibody secreted by the cell strain has good detection sensitivity to the forchlorfenuron, can be used for preparing forchlorfenuron immunoassay kits and colloidal gold test strips, and is used for residue detection of the forchlorfenuron in foods.

Description

technical field [0001] The invention belongs to the technical field of food safety immunoassay, and in particular relates to a forchlorfenuron monoclonal antibody hybridoma cell line and application thereof. Background technique [0002] The English common name of forchlorfenuron is: Forchlorfenuron, chemical name: 1-(2-chloro-4-pyridine)-3-phenylurea; forchlorfenuron belongs to phenylurea cytokinin, and its physiological effects mainly include: 1 ) Promote cell division and expand cell volume; 2) Promote organ formation and protein synthesis; 3) Enhance stress resistance and delay aging; 4) Break the dominance of terminal buds; 5) Induce the formation of dormant buds; 7) Improve photosynthetic efficiency; 8) Induce parthenocarpy. Its physiological effect is similar to that of general purine-type cytokinins, but its concentration is lower and its activity is higher. It can also be used as a herbicide in high concentration. [0003] Forchlorfenuron, as a new type of high-e...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K14/765C07K16/44G01N33/53C07D213/75
CPCC07K16/44C07K14/765G01N33/577G01N33/5308C07D213/75G01N2430/20C07K2317/92
Inventor 胥传来刘杰匡华刘丽强宋珊珊胡拥明
Owner JIANGNAN UNIV
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