Semi-antigen, artificial antigen, and antibody aiming directly at histamine, preparation method and applications thereof

A technology of artificial antigen and hapten, which is applied in the field of hapten directly targeting histamine, antibody and its preparation, and artificial antigen, to achieve the effect of strengthening the immunogen stress response, eliminating the derivation step, and improving the detection sensitivity and specificity

Active Publication Date: 2014-10-29
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 the traditional histamine detection technology that the antibody is only aimed at histamine derivatives, and the histamine detection needs to be derivatized first, and provide a hapten, artificial antigen, Antibody

Method used

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  • Semi-antigen, artificial antigen, and antibody aiming directly at histamine, preparation method and applications thereof
  • Semi-antigen, artificial antigen, and antibody aiming directly at histamine, preparation method and applications thereof
  • Semi-antigen, artificial antigen, and antibody aiming directly at histamine, preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The preparation method of embodiment 1 hapten HI (n=0)

[0050] Add 0.92 g (5 mmoL) histamine dihydrochloride to a 50 mL round bottom flask, and slowly add methanol until dissolved, then add 0.54 g (10 mmoL) sodium methoxide, and add 0.75 g p-formylbenzoic acid ( 5 mmoL), stirred at room temperature for 1 hour, added 0.37 g (10 mmoL) of sodium borohydride, and continued to react for 0.5 hours. The product was dissolved in ethanol and kept overnight in a refrigerator at 4°C. Crystals were precipitated, filtered, washed twice with cold ethanol, and dried to obtain 0.84 g of white crystals, which were haptens, with a yield of (69.4%). ESI-MS analysis (positive) m / z 246 [M+H]+; 1 H NMR (600 MHz, D 2 O) δ = 8.48 (d, J=1.2, 1H), 7.72 (d, J=8.1, 2H), 7.36 (d, J=8.2, 2H), 7.22 (s, 1H), 4.20 (s, 2H) , 3.32 (t, J=7.6, 2H), 3.08 (t, J=7.6, 2H).

Embodiment 2

[0051] Example 2 Preparation of Immunogen / Coating Gen

[0052] In the preparation method of the immunogen and the coating original, the difference lies in the carrier protein used, the immunogen carrier protein adopts bovine serum albumin (BSA), and the coating original carrier protein adopts ovalbumin (OVA ), the coupling method used was the carbodiimide method with certain modifications. The preparation method of the immunogen is used as an example in the following.

[0053] Carbodiimide method: Dissolve 24.6 mg (0.1 mmoL) of the hapten HI (n=0) prepared in Example 1 in 4 mL of boric acid solution (0.1M, pH4.5), stir and add 38.4 mg (0.2 mmoL) 1 -Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), then add 68mg (0.001mmoL) bovine serum albumin, and react overnight with magnetic stirring at 4°C, keeping the pH at 4.5-5.0 . The mixture after the coupling reaction was dialyzed against normal saline at 4°C for 3 days, and the dialysate was changed 4 times a d...

Embodiment 3

[0055] Example 3 Preparation and identification of antibodies

[0056] Three immunogens with different binding ratios were emulsified with an equal dose of immune adjuvant (Freund's complete adjuvant for the first immunization, and Freund's incomplete adjuvant for subsequent booster immunizations) with a stirrer. After complete emulsification, a 7-week-old Balb / c female mouse was immunized with various immunogens of each combination ratio by subcutaneous injection in the back, subcutaneous injection in various parts, leg muscle, and ear vein. A booster immunization was given 4 weeks after the first immunization, and a booster immunization was given 3 weeks later. One week after the fourth booster immunization, blood was collected from the ear vein, and the serum titer was determined by indirect ELISA. When the titer no longer rises, the ear vein is used to boost the immunization. One week later, the eyeballs were picked for blood collection, and the blood was collected...

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Abstract

The invention belongs to the technical field of food safety immunity detection, and specifically discloses a semi-antigen, artificial antigen, and antibody aiming directly at histamine, a preparation method and applications thereof. The molecular structure of the semi-antigen is represented by the formula (I), which is shown in the description, wherein the n=0-6. The semi-antigen can combine with a carrier protein to obtain the artificial antigen, and the antibody obtained from animals immunized by the artificial antigen can directly recognize histamine, while no obvious cross reaction exists between the antibody and other substances with a similar structure as the histamine. Compared to the indirect derivation method, which has been reported before, the histamine immunoanalysis method, which is built on the basis of the antibody, saves the derived sample pretreatment step, so the organic reagent consumption and analysis time are saved, and the detection cost is reduce. The titer of the polyclone antibody is 1:64000, the semi-inhibition concentration is 56.3 mg / L, and the lowest detection limit of histamine can reach 25.8 mg / L lower than the prescribed limit standards of every country.

Description

technical field [0001] The invention belongs to the technical field of food safety immunoassay, and more specifically relates to a hapten, an artificial antigen, an antibody directly targeting histamine, a preparation method and application thereof. Background technique [0002] Histamine, also known as histamine, has a chemical name of 4-(5)-(2-aminoethyl)imidazole, and is an amine substance widely present in various biological tissues. Histidine in organisms forms histamine through the action of histidine decarboxylase, and histamine is the substance that has the greatest impact on human health among biogenic amines. Histamine is generally easy to metabolize, but if the level of histamine in food due to bacterial contamination exceeds the body's ability to metabolize it, it will cause harm to the body. Histamine poisoning is one of the main safety problems in aquatic food, and incidents of histamine poisoning occur frequently all over the world, especially in coastal area...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D233/64C07K1/107C07K14/765C07K14/77C07K16/06C07K16/18G01N33/535
CPCC07D233/64C07K1/1077C07K14/765C07K14/77C07K16/18C07K19/00G01N33/554
Inventor 孙远明罗林徐振林沈玉栋王弘雷红涛杨金易肖治理
Owner SOUTH CHINA AGRI UNIV
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