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Preparation method and application of safrole hapten and safrole antigen

A technology of safrole and hapten, which is applied in the field of immunochemistry to achieve the effect of high antibody specificity, rapid detection and high sensitivity

Active Publication Date: 2018-10-23
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is no report about the preparation method of safrole hapten and antigen at present

Method used

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  • Preparation method and application of safrole hapten and safrole antigen
  • Preparation method and application of safrole hapten and safrole antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] The preparation of embodiment 1 safrole hapten

[0020] 1. Synthesis of safrole hapten

[0021] Take 1.0 g of 5-(2-bromo-2-propen-1-yl)-1,3-benzodioxolane, add 50 mL of anhydrous DMSO to dissolve, stir to clarify, add 0.48 g of potassium hydroxide, add Add 0.20 g of sodium iodide in water, stir vigorously, add 0.41 g of allanine, and react at 100°C for 4 hours. Stop the reaction, add 50 mL of water, add 1mol / L hydrochloric acid to adjust the pH value to 6, add 80 mL of ethyl acetate, extract three times, combine the organic phases, evaporate to dryness by rotary evaporation, put on a silica gel column, ethyl acetate / petroleum ether (v / v, 1 / 5) was eluted and separated to obtain 0.91 g of the hapten product safrole propionate, with a yield of 87.37%.

[0022] 2. Identification of safrole hapten

[0023] The above haptens were identified by H NMR spectroscopy, 1 H-NMR (CDCl 3 , 300MHz) δ: 11.0 (1H, -COOH), 6.90 (1H, ArH, J=6.864), 6.68 (1H, ArH), 6.76 (1H, ArH), 6.07...

Embodiment 2

[0025] Example 2 Preparation of safrole antigen

[0026] 1. Synthesis of safrole immunogen

[0027] Safrole hapten was coupled with bovine serum albumin (BSA) to obtain immune antigen.

[0028] Take 9 mg of safrole hapten, add 0.3 mL of N,N-dimethylformamide to dissolve, clarify, add 8.3 mg of carbodiimide (EDC), stir for 20 min, add 7 mg of N-succinimide, and continue stirring After 30 min, liquid A of the hapten activation solution was obtained; 50 mg of bovine serum albumin (BSA) was dissolved in 4 mL of 0.8% sodium chloride aqueous solution to obtain liquid B; liquid A was slowly added dropwise to liquid B at 4°C After stirring for 4 h, dialysis and purification in 0.02 mol / L PB buffer for 3 days, the safrole-BSA immunogen was obtained, which was aliquoted and stored at -20°C for future use.

[0029] 2. Synthesis of safrole coating original

[0030] The safrole hapten was conjugated with ovalbumin (OVA) to obtain the coating antigen.

[0031] Take 6 mg of safrole hapte...

Embodiment 3

[0034] Example 3 Preparation of Safrole Monoclonal Antibody

[0035] 1. Animal immunity

[0036] Inject the immunogen obtained in step 1 into Balb / c mice at an immunization dose of 150 μg / mouse to make them produce antiserum.

[0037] 2. Cell fusion and cloning

[0038] The splenocytes of Balb / c mice producing specific antibodies were fused with myeloma cells SP20, the cell supernatant was measured by indirect competitive enzyme-linked immunoassay method, and positive wells were screened. Positive wells were cloned by limiting dilution method to obtain and establish monoclonal antibody-producing hybridoma cell lines.

[0039] 3. Cell cryopreservation and recovery

[0040] The hybridoma cells in the logarithmic growth phase were taken to make a cell suspension with the cryopreservation medium, distributed into cryopreservation tubes, and stored in liquid nitrogen for a long time. When recovering, take out the cryopreservation tube, put it into a 37°C water bath to thaw quic...

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Abstract

The invention relates to a preparation method and application of a safrole hapten and a safrole antigen. The preparation method is characterized in that the safrole hapten is obtained by carrying outreaction on 5-(2-bromo-2-propene-1-yl)-1,3-benzodipentene and aminopropionic acid; and the safrole antigen is obtained by coupling the safrole hapten and carrier protein. The antigen prepared throughthe preparation method shows a specific safrole antigenic determinant, and a safrole monoclonal antibody can be prepared through immune animals; and the generated antibody is high in specificity and high in sensitivity and can be used for preparing an enzyme-linked immunosorbent assay kit for detecting safrole, a colloidal gold test strip and a time-resolved fluorescence immunochromatographic teststrip, so that the safrole in tobacco flavors and essences, tobacco, tobacco products and food can be rapidly detected. The formula of the safrole hapten is shown in the description.

Description

technical field [0001] The invention relates to a preparation method and application of safrole hapten and antigen. It belongs to the technical field of immunochemistry. Background technique [0002] Safrole, also known as safrole, safrole, is a colorless or slightly yellow liquid, insoluble in water, easily soluble in chloroform, ether and other non-polar organic solvents, with camphor wood smell, naturally occurring in cinnamon, nutmeg, black Plants such as pepper and perilla are the main constituents of many edible natural essential oils such as sassafras, star anise and camphor. Safrole compounds mainly include safrole, isosafrole, dihydrosafrole and so on. Because of its pleasant smell, sassafras oil was once used as a flavoring and fragrance in food, detergents, and cosmetics. However, studies in recent years have shown that safrole can form safrole-deoxyribonucleic acid adducts in liver tissue, which has certain toxic and side effects on the human body, and is easy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D317/58C07K14/795C07K14/765C07K14/77C07K16/44G01N33/577
CPCC07D317/58C07K14/765C07K14/77C07K14/795C07K16/44G01N33/577
Inventor 陈黎刘惠民赵乐崔华鹏樊美娟潘立宁蔡君兰余晶晶王洪波
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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