Hybridoma cell strain capable of secreting fluridone monoclonal antibody as well as preparation method and application of hybridoma cell strain

A hybridoma cell line and monoclonal antibody technology, applied in the field of immunodetection, can solve the problems of complex processing, unsuitable for rapid detection of a large number of samples, long detection time, etc., and achieve the effect of good sensitivity

Active Publication Date: 2021-11-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods have problems such as complicated sample pretreatment and long detection time, and are not suitable for rapid detection of a large number of samples. Therefore, it is necessary to establish an efficient and rapid detection method for chlorpyrifos

Method used

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  • Hybridoma cell strain capable of secreting fluridone monoclonal antibody as well as preparation method and application of hybridoma cell strain
  • Hybridoma cell strain capable of secreting fluridone monoclonal antibody as well as preparation method and application of hybridoma cell strain
  • Hybridoma cell strain capable of secreting fluridone monoclonal antibody as well as preparation method and application of hybridoma cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0051] Example 2 Synthesis of complete antigen of chlorpyrazone

[0052] Weigh 2.75mg of fluoxetalone hapten (FRD-COOH), 1.5mg of N-hydroxysuccinimide (NHS), dissolve in 300μL N,N-dimethylformamide (DMF), stir at room temperature for 10min ; Then weigh 2.76mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), fully dissolve it with 100 μL DMF, add it to the FRD-COOH solution, and stir at room temperature Reaction 4-6h (called A solution). Take 6mg of KLH, dilute it to 3mg / mL with 0.01M carbonate buffer (CBS) (referred to as solution B), then slowly add solution A to solution B drop by drop, react overnight at room temperature; then use 0.01M PBS solution Dialyze to remove the unreacted small molecule hapten to obtain the complete antigen of chlorpyridone (FRD-COOH-KLH), which is identified by the ultraviolet absorption scanning method.

Embodiment 3

[0053] Example 3 Synthesis of the Coating Progen of Chlorpyridone

[0054] Dissolve 2.17mg of fluoxetalone hapten (FRD-COOH) and 1.87mg of N-hydroxysuccinimide (NHS) in 300μL of anhydrous N,N-dimethylformamide (DMF), and stir at room temperature for 10min. To obtain the fluoxetal hapten (FRD-COOH) solution; after dissolving 3.16 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in 100 μL of anhydrous DMF , added to the FRD-COOH solution, stirred at room temperature to react for 4-6h to obtain liquid A; dilute 6 mg of chicken ovalbumin (OVA) with 1 mL of carbonate buffer solution (CBS) with a concentration of 0.01 mmol / L to obtain 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, and obtain chlorpyridone-coated original (FRD-COOH- OVA).

Embodiment 4

[0055] Example 4 Preparation of hybridoma cell lines secreting chlorpyrazone monoclonal antibody

[0056] 1. Acquisition of animal immunity: Mix and emulsify the complete fluridoxazone antigen with an equal amount of Freund's adjuvant, and then immunize BALB / c mice with multiple subcutaneous injections on the back of the neck (except for sprint immunity); the first immunization uses complete Freund's adjuvant Adjuvant, the dose is 100μg / 30g; Incomplete Freund's adjuvant is used for multiple booster immunizations and the dose is halved to 50μg / 30g; no adjuvant is used for sprint immunization, it is directly diluted with normal saline and injected intraperitoneally, and the dose is halved again That is 25μg / 30g; the interval between the first immunization and the second booster immunization is one month, the interval between multiple booster immunizations is 21 days, and the interval between sprint immunization and the last booster immunization is 20 days; through indirect compet...

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Abstract

The invention relates to a hybridoma cell strain secreting a fluridone monoclonal antibody. The hybridoma cell strain is named as a monoclonal cell strain LKS, and is preserved in China General Microbiological Culture Collection Center (CGMCC) on May 13, 2021, the preservation address is No. 3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 22324. The hybridoma cell strain disclosed by the invention can efficiently and stably secrete the fluridone monoclonal antibody and has relatively good sensitivity and specificity when being applied to immunoassay detection of fluridone, the IC50 value is 0.86 ng / mL, and the cross-over rate of fluridone analogues is less than 10%.

Description

technical field [0001] The invention relates to the technical field of immunoassay, in particular to a hybridoma cell line secreting chlorpyrizone monoclonal antibody and its preparation method and application. Background technique [0002] Fluridone belongs to pyrrolidone herbicides, which can be used in wheat, rice, corn, cotton, pasture and non-arable land, etc., to control annual gramineous weeds, broad-leaved weeds and some perennial weeds, as well as It is suitable for some weeds that are resistant to glyphosate. It is a systemic herbicide that can be absorbed by the roots of plants and transmitted to the leaves. Its mechanism of action is to inhibit lycopene dehydrogenase, causing plants to The reduction of carotenoid biosynthesis in the body leads to the depletion of chlorophyll, which eventually inhibits photosynthesis and causes plant death. At present, there are many studies on the residues of chlorpyrazone in animals and plants. The United States has stipulated...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44C07K14/765C07K14/795C07K14/77G01N33/53G01N33/577
CPCC07K16/44C07K14/765C07K14/795C07K14/77G01N33/5308G01N33/577G01N2033/184Y02A50/30
Inventor 胥传来路倩倩匡华徐丽广孙茂忠刘丽强宋珊珊吴晓玲郝昌龙胡拥明吴爱红马伟
Owner JIANGNAN UNIV
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