Dihydrocapsaicin artificial hapten and artificial antigen as well as preparation methods thereof

A technology of dihydrocapsaicin and artificial hapten, applied in the field of immunochemistry, can solve the problems of high detection cost, complicated operation, long cycle and the like, and achieve the effects of high sensitivity, strong specificity and high affinity

Active Publication Date: 2015-03-25
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Dihydrocapsaicin has the properties of fat solubility, good stability, and high boiling point, and the swill oil in the catering industry is one of the main sources of waste oil. It is difficult to completely remove compounds with the above properties in the current waste oil processing methods
The existing detection methods for dihydrocapsaicin are mainly high performance liquid chromato

Method used

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  • Dihydrocapsaicin artificial hapten and artificial antigen as well as preparation methods thereof
  • Dihydrocapsaicin artificial hapten and artificial antigen as well as preparation methods thereof
  • Dihydrocapsaicin artificial hapten and artificial antigen as well as preparation methods thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0034] Example 1

[0035] Preparation of dihydrocapsaicin artificial hapten

[0036] Weigh 5.86g of N-(4-hydroxy-3-methoxybenzyl)nonanamide, 2.36g of maleic anhydride and 0.49g of N,N'-dicyclohexylcarbodiimide, 25mL of dichloromethane in React for 0.5h at room temperature in the reaction flask, then place the above reaction solution in an ice bath to control the temperature within the range of 0~5℃, take 0.30g of 4-dimethylaminopyridine and 4mL of dichloromethane and place it in a constant pressure drop Mix thoroughly in the funnel, slowly add dropwise to the reaction flask, 0.5h dripping is completed, after the dripping is completed, keep the room temperature and 25℃ and continue to stir for 6 hours, filter the reaction solution, and distill under reduced pressure to remove the solvent to obtain a pale yellow oil Then, the crude product was passed through silica gel column chromatography, column chromatography reagent: a mixed solution of petroleum ether and ethyl acetate with a ...

Example Embodiment

[0039] Example 2

[0040] Preparation of dihydrocapsaicin artificial antigen

[0041] Weigh 6 mg of dihydrocapsaicin hapten (E)-4-[2-methoxy-4-(nonamidomethyl)phenoxy]-4-oxo-2-butenoic acid, and then weigh 10mg NHS, Dissolve 0.2mL DMF in the reaction device, and carry out the reaction at room temperature (25-27°C) under magnetic stirring for 1 hour; weigh 20 mg of DCC and dissolve it in 200uL DMF, add dropwise to the above reaction device, react at room temperature for 4 hours, and a white precipitate is formed , Let stand overnight, centrifuge at 8000r / min, 5min, and take the supernatant;

[0042] The supernatant was slowly added dropwise to 10ml of 2mg / mL bovine serum albumin phosphate buffer solution and stirred at room temperature for 12h; the bovine serum albumin phosphate buffer solution was bovine serum albumin with 0.2M pH 8 is obtained by dissolving in phosphate buffer. Then it was dialyzed with 0.01M PBS buffer for 72 hours, and the dialysate was replaced once in 4-8 hou...

Example Embodiment

[0043] Example 3

[0044] Preparation of Dihydrocapsaicin Antiserum

[0045] Using 400 uL of the dihydrocapsaicin artificial antigen prepared in Example 2 as the immunogen, it was mixed with an equal volume of Freund's complete adjuvant to emulsify completely, and 4 Balb / c mice were immunized and injected subcutaneously on the back. The first immunization was emulsified with Freund's complete adjuvant, and the subsequent emulsification with incomplete adjuvant, each 100ug / time, the interval between the first immunization and the second immunization was 4 weeks, and the interval between each immunization was 3 weeks, and a total of 4 immunizations. Starting from the second immunization, collect blood from the tail of the mice 7-10 days after each immunization, about 25μL per mouse, place the blood sample at 37°C for half an hour, centrifuge at 5,000r / min for 1min, take the supernatant and mix with equal volume of glycerol ,Store at -20℃ for later use for potency testing.

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Abstract

The invention relates to a dihydrocapsaicin artificial hapten and artificial antigen and as well as preparation methods thereof. The molecular structural formula of the dihydrocapsaicin artificial hapten is shown in the formula I; the molecular structural formula of the artificial antigen is shown in the formula II. According to the invention, the phenolic hydroxyl group of N-(4-hydroxy-3-methoxyl benzyl) is modified; the dihydrocapsaicin artificial hapten retains characteristics and structure of dihydrocapsaicin matters to the maximum extent, can be used as antigenic determinant, and has reactive groups which can be coupled with carrier protein; the obtained specific dihydrocapsaicin artificial antigen can acquire a dihydrocapsaicin antibody which is high in appetency, sensitivity and specificity in an immunizing manner, and can be used for immunity detection for dihydrocapsaicin in edible vegetable oil.

Description

technical field [0001] The invention relates to a dihydrocapsaicin artificial hapten, an artificial antigen and a preparation method thereof, belonging to the technical field of immunochemistry. Background technique [0002] In recent years, the act of mixing abnormal cooking oil (commonly known as gutter oil) into qualified cooking oil for profit has been repeatedly banned, which seriously threatens the health of consumers and has become an urgent problem in the field of food safety in my country. [0003] The Ministry of Science and Technology, the State Administration for Industry and Commerce, the General Administration of Quality Supervision, Inspection and Quarantine, the Food and Drug Administration, the Grain Bureau, and the China Center for Disease Control and Prevention jointly tackle key problems in gutter oil identification methods, mainly including: heavy metal content, polycyclic aromatic hydrocarbons, cholesterol, 3-chloropropanediol and Its ester, viscosity, ...

Claims

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Application Information

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IPC IPC(8): C07C233/18C07C231/12C07K14/765C07K14/77C07K14/795C07K1/107C07K16/16G01N33/566
Inventor 李培武马飞张奇杨青青丁小霞张良晓
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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