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Hybridoma cell strain capable of generating anti-kratom monoclonal antibody, and preparation method and application of hybridoma cell strain

A hybridoma cell line and a technology for cloning antibodies, applied in the field of hybridoma cell lines and their preparation, can solve problems such as doping or pollution, and achieve the effects of high sensitivity, high affinity, and shortening the preparation time.

Active Publication Date: 2021-07-16
HANGZHOU CLONGENE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Adulteration or contamination with any co-swallowed substances is also a risk

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  • Hybridoma cell strain capable of generating anti-kratom monoclonal antibody, and preparation method and application of hybridoma cell strain
  • Hybridoma cell strain capable of generating anti-kratom monoclonal antibody, and preparation method and application of hybridoma cell strain
  • Hybridoma cell strain capable of generating anti-kratom monoclonal antibody, and preparation method and application of hybridoma cell strain

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preparation example Construction

[0073] A preparation method of kratom antigen, comprising the following steps:

[0074] Preparation of mitragynine complete antigen:

[0075] a) Protected amino groups: dissolve mitragynine in an organic solvent, stir and dissolve at room temperature, and slowly add trifluoroacetic anhydride which is 1-3 times the molar amount of mitragynine in an ice-water bath under the protection of an inert gas, and remove the ice Water bath, stirring at room temperature for 2-4 hours; after the reaction, evaporate the solvent under reduced pressure in a 40-50°C water bath to obtain a light yellow oily liquid, add 5-15ml of n-hexane to dissolve, and crystallize into a white solid in an ice-water bath. Wash the white solid with n-hexane;

[0076]

[0077] mitragynine-COCF 3

[0078] b) Hydrolysis of the ester group: Add 5-15mL of methanol and 1-3ml of water to the obtained white solid, then add 5-10 times the molar amount of lithium hydroxide, stir and dissolve and continue to react f...

Embodiment 1

[0112] Example 1: Preparation of Kratom Antigen

[0113] Preparation of complete antigen of mitragynine (Antigen1):

[0114] a) Protected amino group

[0115] Weigh 398.5mg (1mmol) of mitragynine powder in a 150ml three-necked flask, add 60ml of dichloromethane, stir and dissolve at room temperature, and slowly add 420mg (2mmol) of trifluoroacetic anhydride dropwise under the condition of ice-water bath and nitrogen protection, The ice-water bath was removed, and stirred at room temperature for 3 hours. After the reaction, the solvent was evaporated to dryness under reduced pressure in a water bath at 45° C. to obtain a light yellow oily liquid, which was dissolved by adding 10 ml of n-hexane. It can crystallize into a white solid in an ice-water bath. 420 mg (0.86 mmol) of the white solid were washed with n-hexane.

[0116] b) Hydrolysis of the ester group Add 10 mL of methanol and 2 mL of water to the above white solid, then add 158 mg (6.88 mmol) of lithium hydroxide, s...

Embodiment 2

[0136] Example 2: Preparation of Kratom Antigen

[0137] Preparation of complete antigen of mitragynine (Antigen1):

[0138] a) Protected amino group

[0139] Weigh 398.5mg (1mmol) of mitragynine powder in a 150ml three-necked flask, add 60ml of dichloromethane, stir and dissolve at room temperature, and slowly add 210mg (1mmol) of trifluoroacetic anhydride dropwise under the condition of an ice-water bath under nitrogen protection, The ice-water bath was removed, and stirred at room temperature for 3 hours. After the reaction, the solvent was evaporated under reduced pressure in a water bath at 405° C. to obtain a light yellow oily liquid, which was dissolved by adding 10 ml of n-hexane. It can crystallize into a white solid in an ice-water bath. 239 mg (0.6 mmol) of the white solid were washed with n-hexane.

[0140] b) Hydrolysis of ester groups

[0141] Add 10 mL of methanol and 2 ml of water to the above white solid, then add 110 mg (4.8 mmol) of lithium hydroxide, s...

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Abstract

The invention relates to the field of biological detection, and discloses a hybridoma cell strain capable of generating an anti-kratom monoclonal antibody, and a preparation method and application of the hybridoma cell strain. The hybridoma cell strain is named as Kratom11B7, and has been preserved in China Center for Type Culture Collection on January 31, 2021, with the preservation number of CCTCCNO: C202146. The preparation method comprises the following steps: step 1, preparing a mitragynine complete antigen; step 2, preparing a 7-hydroxy mitragynine complete antigen; and step 3, preparing the hybridoma cell strain, specifically, immunizing a mouse with the mitragynine complete antigen and 7-hydroxy mitragynine complete antigen together. The hybridoma cell strain Kratom11B7 has high secretion yield, and the anti-kratom monoclonal antibody obtained by secretion has the characteristics of high affinity, high specificity and high sensitivity. By adopting the continuous immunization method, the using amount of the antigen can be effectively reduced, and the immunization time is shortened.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a hybridoma cell strain capable of producing anti-kratom monoclonal antibody, a preparation method and application thereof. Background technique [0002] Kratom, of the Rubiaceae family, is a psychoactive plant that grows in Southeast Asia, especially in Thailand and Malaysia. .Fresh or dried kratom leaves can be smoked, chewed or consumed as a tea. Low doses of kratom have a stimulating effect and have long been used by manual laborers in Southeast Asia as a stimulant to offset work fatigue; high doses of kratom have a sedative-anesthetic effect, so kratom leaves are used as a substitute for traditional drugs and opium. [0003] The pharmacological function of kratom is mainly realized by the alkaloids 7-hydroxy mitragynine (formula II) and mitragynine (formula I). Although the molecular structure of these alkaloids is similar to that of hallucinogens, these substances do n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/16C07K14/765C07K1/107G01N33/577G01N33/58G01N33/532G01N33/558C07D471/14
CPCC07K16/16C07K14/765G01N33/577G01N33/587G01N33/532G01N33/558G01N33/94C07D471/14C07K2317/92C07K2317/94Y02P20/55
Inventor 郑智彪郑曙剑
Owner HANGZHOU CLONGENE BIOTECH
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