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A kind of histamine hapten, artificial antigen, antibody and its preparation method and application

An artificial antigen and hapten technology, applied in the preparation methods of peptides, chemical instruments and methods, animal/human proteins, etc., can solve the problems of low antibody preparation titer and poor repeatability, and achieve enhanced specificity, short time, specific effect

Active Publication Date: 2015-09-30
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to overcome the defects of low titer and poor repeatability of antibody preparation in traditional histamine detection technology, and provide a histamine hapten, artificial antigen and specific antibody

Method used

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  • A kind of histamine hapten, artificial antigen, antibody and its preparation method and application
  • A kind of histamine hapten, artificial antigen, antibody and its preparation method and application
  • A kind of histamine hapten, artificial antigen, antibody and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0044] The preparation method of hapten BIA (n=0, Y=COOH

[0045] S1. Take 1.8 g (10 mmoL) of monomethyl terephthalate, put it into a 50 mL round bottom flask, add 10 mL (135 mmoL) of thionyl chloride, reflux at 70°C for 1 h, and distill at atmospheric pressure , remove too much thionyl chloride, then add 10 ml of dichloromethane to the residue, rotary evaporation, repeat three times, remove too much thionyl chloride, obtain 1.7g white solid, about 85% yield; Dissolve 1.38 g (7.5 mmoL) of histamine dihydrochloride in 10 mL of 15% NaOH solution, dissolve 1.7 g (8.5 mmoL) of the white solid obtained above in 10 mL of acetonitrile, and add dropwise to histamine dihydrochloride (the NaOH solution of histamine dihydrochloride is prepared as follows: weigh 0.92 g of histamine dihydrochloride (5 mmoL) and dissolve it in 10 mL of 15% NaOH solution, stir at room temperature for 1 h, filter the precipitate After washing with NaOH solution and drying, 1.64 g (6.6 mmoL) of a ligh...

Embodiment 2

[0049] Example 2 Preparation method of hapten BII (n=0)

[0050] Add 0.9 g (6 mmoL) of p-aldehyde benzoic acid to a 50 mL round bottom flask, slowly add methanol until dissolved, add 0.55 g (5 mmoL) histamine while stirring, stir overnight at room temperature, filter the reaction, wash with methanol twice , to obtain 1.1 g of the target product, yield (89%). ESI-MS analysis (positive) m / z 244[M+H]+; 1H NMR (600 MHz, ) δ 8.37 , 7.98 (d, J = 8.0 Hz), 7.80 (d, J = 8.0 Hz), 7.51 , 6.76 , 3.87–3.81 (m), 2.85 (t, J = 7.3 Hz).

Embodiment 3

[0051] Example 3 Preparation of Immunogen / Coating Gen

[0052] The difference between the preparation of the immunogen and the coating source lies in the carrier protein, the immunogen carrier protein uses bovine serum albumin (BSA), and the coating original carrier protein uses ovalbumin (OVA). The preparation method of the immunogen is used as an example in the following.

[0053] Active ester method: Dissolve 0.1 mmoL of hapten BIA (n=0, Y=COOH) or BII (n=0) in 0.5 mL DMF, stir and add 0.0512 g (0.2 mmoL) DCC and 0.023 g (0.2 mmoL) NHS , and reacted overnight with magnetic stirring at 4°C, and the supernatant after centrifugation was liquid A. Weigh 0.02 g of BSA or OVA and dissolve in 2 mL of PBS (pH8.0) with a concentration of 0.1 mol / L, stir and dissolve to prepare solution B. Under magnetic stirring, liquid A was gradually dropped into liquid B, and reacted at 4 °C for 12 h. After centrifugation, the supernatant was taken and dialyzed with normal saline at 4 °C f...

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Abstract

The invention discloses a histamine hapten, an artificial antigen, an antibody, as well as preparation methods and applications thereof. The histamine hapten has a molecular structure as shown in formula (I) or (II), wherein n=0-6, Y=COOH and NO2. The histamine artificial antigen has a molecular structure as shown in the formula (III) or (IV), wherein n=0-6. The titer of antiserum obtained through adopting the antigen can reach 1*10<7>, the minimum detection limit is 0.13ng / mL, the 50% inhibiting concentration is 7.6ng / mL, and the produced antigen has high specificity, high sensitivity and high accuracy. The histamine derivative preparation methods are simple and efficient, have high repeatability and lay foundations for building a quick accurate and flexible histamine immunodetection method.

Description

technical field [0001] The invention belongs to the technical field of food safety immunoassay, and more specifically relates to a histamine hapten, an artificial antigen, an antibody and a preparation method and application thereof. Background technique [0002] Biogenic amines are a class of nitrogen-containing low-molecular organic bases, which are mainly formed by the decarboxylation of amino acids, and are widely found in meat products, aquatic products, and fermented products rich in amino acids and proteins. An appropriate amount of biogenic amines is necessary to maintain the normal physiological functions of the human body, and excessive amounts can cause allergic reactions, and even endanger life in severe cases. In addition, the content of biogenic amines in food has a certain correlation with food quality. Therefore, it is of great significance to strengthen the monitoring of biogenic amines in food to improve food quality and safety. Among biogenic amines, his...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D233/64C07K14/77C07K14/765C07K1/107C07K16/44G01N33/68
Inventor 徐振林孙远明罗林沈玉栋肖治理杨金易雷红涛王弘
Owner SOUTH CHINA AGRI UNIV
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