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Preparation method and application of a kind of coumarin hapten and antigen

A coumarin and hapten technology applied in the field of immunochemistry to achieve the effects of high antibody specificity, rapid detection, and high sensitivity

Active Publication Date: 2021-11-23
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The preparation method of coumarin hapten and antigen has not been reported yet

Method used

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  • Preparation method and application of a kind of coumarin hapten and antigen
  • Preparation method and application of a kind of coumarin hapten and antigen
  • Preparation method and application of a kind of coumarin hapten and antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Preparation of Coumarin Hapten

[0021] 1. Synthesis of coumarin hapten

[0022] Take 1.00 g of coumarin, add 80 mL of chlorobenzene and stir to dissolve, add 2.50 g of tert-butanol hydroperoxide, stir and mix well, add 3-(1,3-dioxa-2-yl) propanal 0.89 g, stirred at 100°C for 24 h. After stopping the reaction, add 100 mL of cold water, extract 3 times with 100 mL of ethyl acetate, wash with 50 mL of water, dry the organic phase over anhydrous sodium sulfate and evaporate to dryness, and 60 mL of petroleum ether / dichloromethane (v / v, 1 / 1 ) recrystallized to obtain 1.70 g of acetal coumarin compound with a yield of 94.00%.

[0023] Take 1.70 g of acetal coumarin, add 20 mL of acetonitrile to dissolve, add 10 mL of 1 mol / L dilute hydrochloric acid, stir vigorously at room temperature for 4 hours, stop the reaction, rotary evaporate, remove acetonitrile, add 50 mL of water, add 1 mol / L NaOH to adjust When the pH value reaches 6, add 70 mL of 1,2-dichloroethane ...

Embodiment 2

[0027] Example 2 Preparation of Coumarin Antigen

[0028] 1. Synthesis of coumarin immune antigen

[0029] The coumarin hapten is conjugated with bovine serum albumin (BSA) to obtain the immune antigen.

[0030] Take 16 mg of coumarin hapten, add ethanol to dissolve to obtain liquid A, take 0.1 g of bovine serum albumin, add acetate buffer solution with a pH value of 5.6 to dissolve to obtain liquid B, add liquid A to liquid B dropwise Stir at 4°C for 20 h, add 1 mg sodium borohydride, continue to stir for 2 h, dialyze and purify with 0.02 mol / L PB for three days, change the medium three times a day, aliquot the immunogen, and store it at -20°C for future use.

[0031] 2. Synthesis of coumarin-coated antigen

[0032] The coumarin hapten was conjugated with ovalbumin (OVA) to obtain the coating antigen.

[0033] Take 5 mg of coumarin hapten, add ethanol to dissolve to obtain liquid A, take 50 mg of ovalbumin, add carbonate buffer solution with a pH value of 9.1 to dissolve to ...

Embodiment 3

[0036] Example 3 Preparation of coumarin monoclonal antibody

[0037] 1. Obtaining hybridoma cells

[0038] 1) Animal immunization: Fully emulsify the coumarin hapten-BSA conjugate (immunogen) with an equal amount of Freund's complete adjuvant, and inject 6-week-old Balb / c mice subcutaneously, 0.2 mL each; thereafter , a booster immunization every two weeks, with Freund's incomplete adjuvant instead of Freund's complete adjuvant, the method and dosage are the same as the first immunization;

[0039] 2) One week after the last booster immunization, the fundus vein blood was collected to measure the titer and inhibitory effect of coumarin antibody. When there was inhibition and the titer reached 1:10000 or more, the following final immunization was performed: intraperitoneal injection of immunogen solution without any adjuvant 0.1 mL, the mice were sacrificed three days later, and the spleen lymphatic B cells were fused with myeloma cells;

[0040] 3) After 7 days of cell fusi...

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Abstract

A preparation method and application of coumarin hapten and antigen, characterized in that: the coumarin hapten is obtained by acetal reaction of coumarin; the coumarin antigen is obtained by coumarin hapten and Carrier protein coupling is obtained. The antigen prepared by the invention presents specific coumarin antigenic determinants, and the coumarin monoclonal antibody can be prepared by immunizing animals. The antibody produced has high specificity and high sensitivity, and can be used to prepare enzyme-linked immunosorbent assay kits, colloidal gold test strips and time-resolved fluorescent immunochromatographic test strips for the detection of coumarin, thereby realizing the detection of flavors in tobacco, flavors and fragrances and food. Rapid detection of soylin.

Description

technical field [0001] The invention relates to a preparation method and application of a coumarin hapten and an antigen. It belongs to the technical field of immunochemistry. Background technique [0002] Coumarin-based compounds are a class of natural products with an aromatic odor and a benzoα-pyrone structure as the core. They are widely distributed in the plant kingdom and can be divided into simple coumarin-based compounds and furanocoumarin-based compounds according to their structures. Compounds, pyranocoumarin-based compounds and other coumarin-based compounds, etc. Coumarin, also known as o-hydroxycinnamic acid lactone and 1,2-benzopyrone, is a natural plant secondary product and a lactone compound widely present in nature. It was discovered in 1820 Found in tonka bean, mainly exists in plants in the form of free state and glycosides, most in Rutaceae and Umbelliferae plants, followed by Leguminosae, Orchidaceae, Oleaceae, Solanaceae and Compositae plants , a fe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D311/12G01N33/577C07K14/765C07K14/77C07K14/795
CPCC07D311/12C07K14/765C07K14/77C07K14/795C07K19/00G01N33/577
Inventor 陈黎刘惠民崔华鹏樊美娟赵乐潘立宁王洪波郭军伟秦亚琼
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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