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Yohimbine hapten YHA, artificial antigen and antibody thereof, and preparation and application thereof

An artificial antigen, yohimbine technology, applied in the field of artificial antigen and its antibody and its preparation and application, yohimbine hapten YHA, to achieve high sensitivity, low cost, and strong specificity

Active Publication Date: 2020-08-07
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved in the present invention is to overcome the blank of the existing method for immunological detection of yohimbine, to provide a yohimbe hapten YHA, artificial antigen and its antibody and its preparation and application

Method used

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  • Yohimbine hapten YHA, artificial antigen and antibody thereof, and preparation and application thereof
  • Yohimbine hapten YHA, artificial antigen and antibody thereof, and preparation and application thereof
  • Yohimbine hapten YHA, artificial antigen and antibody thereof, and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Synthesis and identification of yohimbine hapten YHA

[0041] 1. Experimental method

[0042] (1) Yohimbine hydrochloride (YHB HCl) is dissolved in methanol, and the obtained concentration is that the yohimbine hydrochloride-methanol solution of 0.128mol / L carries out hydrolysis reaction with the sodium hydroxide aqueous solution of 1mol / L (volume ratio is 1:1), after the reaction product was acidified with hydrochloric acid, the solvent was removed by rotary evaporation, reconstituted with anhydrous methanol solution and filtered, and the obtained filtrate was dried with anhydrous sodium sulfate;

[0043] (2) After the liquid was evaporated to dryness in vacuum, the product was purified by liquid phase, the mobile phase was 0.1% formic acid aqueous solution, the column temperature was 40°C, and the flow rate was 300 μL / min to collect the target product;

[0044] (3) Carry out nuclear magnetic resonance identification with a standard product of yohimbine, and...

Embodiment 2

[0054] Example 2 Synthesis and identification of yohimbine artificial antigen

[0055] The present invention has successfully synthesized yohimbine-EDC-cBSA artificial antigen and yohimbine-EDC-cOVA artificial antigen, and the specific experimental methods and results are as follows:

[0056] 1. Cationicization of carrier protein

[0057] (1) Dissolve 0.675g bovine serum albumin (BSA) (0.015mmol) and 56mg ethyl-[3-(dimethylamino)propyl]carbodiimide (EDC) (0.3mmol) in 20mL PBS buffer (0.1M, pH7.4), placed on a magnetic stirrer and stirred at room temperature for 30min to fully dissolve;

[0058] (2) Slowly add the buffer solution into 20 mL of PBS buffer solution (0.1 M, pH 7.4) dissolved with 18 mg of ethylenediamine (0.3 mmol), place on a magnetic stirrer and stir at room temperature for 2 h;

[0059] (3) Transfer the mixed solution to a dialysis bag, use PBS buffer (0.1M, pH 7.4) as the dialysate, place it at 4°C for 3 days, and change the dialysate every 6 hours to remove...

Embodiment 3

[0073] Example 3 Preparation of Yohimbine Antibody YHA-IgG

[0074] The preparation method of Yohimbine antibody YHA-IgG comprises the following steps:

[0075] (1) Using 300 μL yohimbine-EDC-cBSA artificial antigen (1mg / mL) as the immunogen, immunize two 5-week-old New Zealand white rabbits (female and male), and YHA-cBSA by volume for the first immunization Emulsify with Freund's complete adjuvant at a ratio of 1:1, and immunize female New Zealand white rabbits and male New Zealand white rabbits respectively; then emulsify YHA-cBSA with Freund's incomplete adjuvant at a volume ratio of 1:1, and perform 1 week every three weeks A booster immunization, a total of 5 booster immunizations;

[0076] (2) One week after the third immunization, blood was collected from the ear vein of the rabbit, and the rabbit blood was collected. After labeling, it was centrifuged at 5000r / min for 20min at 4°C to obtain the supernatant, which was used to detect the immune effect by ELISA;

[007...

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Abstract

The invention discloses a yohimbine hapten YHA, an artificial antigen and an antibody thereof as well as preparation and application thereof. The preparation method comprises preparing a yohimbine hapten YHA, wherein the structural formula of the yohimbine hapten YHA is shown as a formula (I); preparing a yohimbine artificial antigen and a yohimbine antibody YHA-IgG by using the hapten. The antibody YHA-IgG has high detection sensitivity and strong specificity for yohimbine, the half inhibition concentration is 0.50 mu g / kg, the detection range for yohimbine is 0.12-2.18 mu g / kg, the lowest detection limit is 1.13 mu g / kg, and the detection requirements are met; besides, the preparation methods of the yohimbine hapten YHA, the yohimbine artificial antigen and the yohimbine antibody YHA-IgGare simple and low in cost, and a core raw material is provided for establishing an immunological detection method of specific yohimbine; therefore, the yohimbine antibody YHA-IgG has a wide application prospect in detection of yohimbine.

Description

technical field [0001] The invention belongs to the technical field of food safety detection. More specifically, it relates to a yohimbe hapten YHA, an artificial antigen and its antibody, as well as its preparation and application. Background technique [0002] Yohimbine is a natural alkaloid extracted from the bark of the Yohimbine tree and the root of the Rauwolfia tree. Yohimbine is mainly used to treat male sexual dysfunction, arteriosclerosis, rheumatism, etc. It also has the effects of clearing wind heat, reducing liver fire, and reducing swelling and poison. In addition, yohimbine also has the effect of accelerating the body's energy metabolism and increasing energy supply. Therefore, yohimbine is easily illegally added to food or health products by unscrupulous merchants. [0003] When yohimbine is overdose, it usually causes agitation, anxiety, high blood pressure and rapid heartbeat. In severe cases, it can cause acute neurotoxicity and even death; for example: ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D471/14C07K14/765C07K14/77C07K1/107C07K16/16G01N33/53
CPCC07D471/14C07K14/765C07K14/77C07K16/16G01N33/5308
Inventor 雷红涛林建浩李向梅沈兴陈佳虹沈玉栋徐振林孙远明
Owner SOUTH CHINA AGRI UNIV
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