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Safrole hapten, artificial antigen, antibody as well as preparation method and application of safrole hapten and artificial antigen

An artificial antigen, safrole technology, applied in chemical instruments and methods, animal/human proteins, resistance to vector-borne diseases, etc., to achieve the effect of strong specificity, low detection limit, and wide linear range

Active Publication Date: 2022-05-27
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 defect and deficiency that safrole immunodetection hapten and antibody exist in the prior art, and provide a kind of safrole hapten, artificial antigen , Antibody and its preparation method and application

Method used

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  • Safrole hapten, artificial antigen, antibody as well as preparation method and application of safrole hapten and artificial antigen
  • Safrole hapten, artificial antigen, antibody as well as preparation method and application of safrole hapten and artificial antigen
  • Safrole hapten, artificial antigen, antibody as well as preparation method and application of safrole hapten and artificial antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1 Synthesis and identification of safrole hapten

[0083] 1. Synthesis and identification of safrole hapten H-SG

[0084] (1) Synthesis of safrole hapten H-SG

[0085] 3,4-methylenedioxyphenethylamine (1 mol) and succinic anhydride (1.2 mol) were dissolved in pyridine, 4-dimethylaminopyridine (DMAP) (0.4 mol) was added and the reaction was refluxed overnight. After separation and purification, the hapten H-SG is obtained by drying. The synthetic route of the hapten H-SG is as follows figure 1 shown.

[0086] (2) Identification of safrole hapten H-SG

[0087] NMR identification results of the hapten H-SG: 1 HNMR(600MHz, Methanol-d4)δ12.18(s, 1H), 6.66–6.59(m, 2H), 6.56(dd, J=7.9, 1.6Hz, 1H), 5.78(s, 2H), 3.29–3.17 (m, 3H), 2.59(t, J=7.3Hz, 2H), 2.46(t, J=7.0Hz, 2H), 2.33(t, J=7.0Hz, 2H).

[0088] The mass spectrometry result of the hapten H-SG is: MS:C 13 H 15 NO 5 : 265.27, ESI-[M-H]+: 266.3.

[0089] The structural formula of hapten H-SG is shown in ...

Embodiment 2

[0102] The synthesis and identification of embodiment 2 safrole artificial antigen

[0103] 1. Synthesis of safrole artificial antigen

[0104]The hapten H-SG and hapten H-2C prepared in Example 1 were coupled to bovine serum albumin (BSA) and chicken ovalbumin (OVA) by the active ester method.

[0105] Weigh the hapten H-SG (1moL) prepared in Example 1, mix it with N-hydroxysuccinimide (NHS) (0.8moL) and 1-(3-dimethylaminopropyl)-3-ethyl carbon Diimide hydrochloride (EDC) (1.9 moL) was dissolved in 50-200 μL N,N-dimethylformamide (DMF), and stirred at room temperature in the dark for 2-4 h to obtain hapten H-SG activation solution; BSA (10 mg) was added to 1 mL of PBS buffer (0.01moL / L, pH=7.4); hapten H-SG activation solution was slowly added dropwise to BSA in PBS buffer solution, and reacted at 4°C for 12 h; The buffer solution was dialyzed for 3 days, 3 times a day. After the dialysis, the safrole artificial antigen H-SG-BSA was obtained, which was divided into centrifu...

Embodiment 3

[0113] Example 3 Preparation of Antibodies

[0114] 1. Preparation of Polyclonal Antibodies

[0115] H-SG-BSA prepared in Example 2 was used as immunogen and immune adjuvant (incomplete Freund's adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent booster immunizations) by emulsification in a volume ratio of 1:1. Evenly, the immune body weight was 2.5 to 3 kg of New Zealand white rabbits. Multiple subcutaneous injections on the neck and back were used, the second immunization was performed after 4 weeks, and the booster immunization was performed every 3 weeks thereafter. One week after the third booster immunization, blood was collected from the marginal ear vein, and the serum titer was determined by indirect competitive ELISA. When the titer no longer rises, boost the immunization with the ear vein. One week later, blood was collected from the heart, and the collected blood was obtained by incubating the blood for 0.5 to ...

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Abstract

The invention provides a safrole hapten, an artificial antigen, an antibody as well as a preparation method and application of the safrole hapten, the artificial antigen and the antibody, two haptens, namely a hapten H-SG and a hapten H-2C are prepared, the hapten H-SG is coupled with carrier protein BSA to obtain the artificial antigen H-SG-BSA, and the specific antibody for detecting safrole is further prepared. An artificial antigen H-2C-OVA is used as a coating antigen; the lowest detection limit of the antibody to safrole is 0.088 ng / mL, the half inhibitory concentration is 2.79 ng / mL, the quantitative detection range is 0.31-24.74 ng / mL, the antibody has good sensitivity and specificity, has no cross reaction to safrole analogues, and can specifically detect safrole; the invention provides a better immunodetection raw material of safrole, and provides a better choice for rapid and accurate detection of safrole.

Description

technical field [0001] The invention relates to the technical field of food detection, in particular to a safrole hapten, an artificial antigen, an antibody and a preparation method and application thereof. Background technique [0002] Safrole, also known as safrole, safrole, colorless or slightly yellow liquid, insoluble in water, soluble in chloroform, ether and other non-polar organic solvents, has the smell of camphor wood, naturally exists in cinnamon, meat Plants such as cardamom, black pepper, and perilla are the main constituents of many edible natural essential oils such as sassafras, star anise, and camphor oils. Due to its pleasant smell, sassafras oil was once used as a flavoring and fragrance agent in food, detergents and cosmetics. Many common spices, including black pepper, cocoa, nutmeg, cinnamon, tarragon, star anise, fennel, coriander, parsley, bay, cumin, pimento, and cloves contain trace amounts of safrole. Safrole is used to synthesize piperonyl butox...

Claims

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Application Information

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IPC IPC(8): C07D317/58C07K14/765C07K14/77C07K14/79C07K14/795C07K16/44G01N33/53G01N33/543G01N33/58
CPCC07D317/58C07K14/765C07K14/77C07K14/795C07K14/79C07K16/44G01N33/54306G01N33/581G01N33/5308Y02A50/30
Inventor 雷红涛潘康亮沈兴李向梅韦晓群徐小艳
Owner SOUTH CHINA AGRI UNIV
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