Anti-cocaine monoclonal antibody, cell line capable of secreting same and preparation method

A monoclonal antibody, anti-cocaine technology, applied in biochemical equipment and methods, chemical instruments and methods, analytical materials, etc., can solve the problems of large sample consumption, inability to realize on-site instant detection, low detection sensitivity and precision, etc. Achieve high secretion yield, shorten preparation time, and shorten immunization time

Inactive Publication Date: 2018-05-04
HANGZHOU CLONGENE BIOTECH
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  • Claims
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AI Technical Summary

Problems solved by technology

[0006] Currently, urine detection of cocaine mainly relies on high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS). These methods have extremely high sensitivity. and precision, but requires expensive instruments

Method used

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  • Anti-cocaine monoclonal antibody, cell line capable of secreting same and preparation method
  • Anti-cocaine monoclonal antibody, cell line capable of secreting same and preparation method
  • Anti-cocaine monoclonal antibody, cell line capable of secreting same and preparation method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Anti-cocaine hybridoma cell line COC 2B1 capable of producing anti-cocaine monoclonal antibody. The cell line was preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO.C201727, and it was preserved in the China Center for Type Culture Collection (CCTCC) on February 21, 2017. The address of the object collection center is: Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province.

[0051] Specifically, the anti-cocaine hybridoma cell line COC 2B1 is obtained by fusion of mouse lymphocytes and mouse myeloma cells, and the anti-cocaine monoclonal antibody produced by it has highly specific reactivity with cocaine antigen.

[0052] More specifically, the anti-cocaine hybridoma cell line COC 2B1 is produced by cocaine artificial antigen-induced mice. In the process of preparing the cocaine artificial antigen, the first method is to use potassium permanganate to oxidize demethyl cocaine, and then use dibutyl Co...

Embodiment 2

[0055] In the following examples, the PBS buffer solution is an aqueous solution containing 0.008M disodium hydrogen phosphate dodecahydrate, 0.15M sodium chloride, and 0.002M sodium dihydrogen phosphate dihydrate, and the pH is 7.4; Forearm or neck; 1×HAT DMEM culture solution contains 15% FBS; 1×HT DMEM culture solution contains 15% FBS.

[0056] The preparation method of the above-mentioned anti-cocaine hybridoma cell line COC 2B1, the method comprises the following steps:

[0057] (1), preparation of cocaine artificial antigen;

[0058] (2) Stimulate mouse splenocytes to produce specific B lymphocytes through continuous short-term rapid immunization;

[0059] (3) The above-mentioned mouse B lymphocytes and mouse myeloma cells were fused to obtain an anti-cocaine hybridoma cell line COC2B1, and the anti-cocaine monoclonal antibody produced by it had highly specific reactivity with cocaine antigen.

[0060] The above three steps are described in detail below.

[0061] In ...

Embodiment approach

[0064] Method 1: use cocaine hydrochloride as raw material, after freeing, use potassium permanganate to oxidize to obtain N-desmethyl cocaine, and then use succinic anhydride to condense N-desmethyl cocaine to obtain carboxyl-containing cocaine hapten Hapten1, which can be It is called hapten No. 1. Cocaine artificial antigen (COCAg1) is prepared by combining cocaine hapten with bovine serum albumin (BSA) by carbodiimide method, which can be called cocaine artificial antigen No. 1.

[0065] Specifically: 1000 mg of cocaine hydrochloride is dissolved in 20 ml of water, and the pH is adjusted to 9-10 with concentrated ammonia water to produce a large amount of white precipitate. Add 20 ml of dichloromethane and transfer it to a 125 ml separatory funnel for extraction and separation. Extracted twice with methyl chloride, combined the organic phases, dried and filtered with anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to obtain 925 mg of white crystal...

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Abstract

The invention provides a hybridoma cell line COC 2B1 which is capable of producing an anti-cocaine monoclonal antibody and preserved in CCTCC (China Center for Type Culture Collection) with the preservation number being NO.C201727. The invention further provides the anti-cocaine monoclonal antibody secreted by the hybridoma cell line COC 2B1, a kit for detecting cocaine in urine as well as a detection method and a production method of the cocaine detection kit. The invention has the following beneficial effects: ethyl bromoacetate as a crosslinking agent is used for activating cocaine, and anobtained cocaine artificial antigen keeps structural specificity of cocaine. A continuous immunization method is adopted, antigen dosage is saved effectively, immunization time is shortened, secretionyield of the hybridoma cell line COC 2B1 is high, and the cocaine monoclonal antibody secreted by the hybridoma cell line COC 2B1 has the characteristics of high affinity, high specificity and high sensitivity. The detection product has more advantages in aspects of sensitivity, specificity, detection limit and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an anti-cocaine monoclonal antibody and a hybridoma cell line secreting the antibody [0002] The invention also relates to a cocaine detection kit and a production method thereof. Background technique [0003] Cocaine is a tropane-type alkaloid, which is extracted from coca leaves. It is a natural central nervous system stimulant. In medicine, it is used as a local anesthetic or vasoconstrictor because of its good anesthetic effect and strong penetrating power. , mainly used for topical anesthesia, but due to its strong toxicity, it is not suitable for injection, and it is clinically available in the form of hydrochloride and sulfate. [0004] However, cocaine has the function of depressing the central nervous system, so it is abused. Cocaine has damaging effects on digestive system, immune system, cardiovascular system and genitourinary system, especially as a dose-dependent hepa...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44G01N33/577
CPCC07K16/44C07K2317/35C07K2317/92G01N33/577G01N33/946
Inventor 郑曙剑郑智彪刘静
Owner HANGZHOU CLONGENE BIOTECH
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