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Hapten, artificial antigens and antibody for detecting phenacetin as well as preparation method and application of hapten, artificial antigens and antibody

A technology of artificial antigen and phenacetin, applied in the field of detection of phenacetin hapten, antibody and its preparation, and artificial antigen, to achieve the recognition ability of eliminating interference, high detection sensitivity, high sensitivity and high specificity Effect

Active Publication Date: 2021-08-13
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 defects and deficiencies of the phenacetin detection method in the prior art, and provide a hapten, artificial antigen, antibody and its preparation method and application for detecting phenacetin

Method used

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  • Hapten, artificial antigens and antibody for detecting phenacetin as well as preparation method and application of hapten, artificial antigens and antibody
  • Hapten, artificial antigens and antibody for detecting phenacetin as well as preparation method and application of hapten, artificial antigens and antibody
  • Hapten, artificial antigens and antibody for detecting phenacetin as well as preparation method and application of hapten, artificial antigens and antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Synthesis and identification of embodiment 1 phenacetin hapten

[0073] Using the structural characteristics of phenacetin and on the basis of experimental data, an artificial hapten was designed.

[0074] 1. Synthesis and identification of hapten PNT1

[0075] Take N-(4-hydroxyphenyl)acetamide (1moL), potassium carbonate (3moL), use acetone as solvent, and react with methyl 4-bromomethylbenzoate (1.2moL) under stirring at room temperature for 5-8h, Separation and purification yielded methyl 4-((4-acetamidophenoxy)methyl)benzoate. Dissolve methyl 4-((4-acetamidophenoxy)methyl)benzoate in methanol, the volume ratio of methyl 4-((4-acetamidophenoxy)methyl)benzoate to methanol is 1 : 1, add 1mol / L aqueous sodium hydroxide solution and stir at room temperature for 5-10 hours. After the reaction, adjust the pH to 5-7 with 1mol / L hydrochloric acid to obtain the hapten PNT1.

[0076] Hapten PNT1 NMR results: 1 H NMR (600MHz, Methanol-d4) δ8.07–7.98(m,11H),7.55(dp,J=7.6,0.8...

Embodiment 2

[0088] Synthesis and identification of embodiment 2 phenacetin artificial antigen

[0089] 1. Synthesis of phenacetin artificial antigen

[0090] The synthetic method of phenacetin artificial antigen comprises the following steps:

[0091] Using PNT1 and PNT2 in Example 1 as haptens, respectively coupled with bovine serum albumin (BSA) and chicken ovalbumin (OVA) by active ester method, respectively weighed 1moL of the above-mentioned phenacetin hapten, 1.1moL of NHS and Dissolve 1.4moL EDC in 50-200μL DMF, and stir in the dark for 2-4 hours at room temperature to obtain phenacetin hapten PNT1, PNT2 activation solution; add 10mg BSA or OVA to 1mL PBS buffer (0.01moL / L, pH=7.4); slowly add the phenacetin hapten PNT1, PNT2 activation solution dropwise to the BSA or OVA solution, react at 4°C for 12 hours; dialyze with PBS buffer for 2 days, change the dialysate 4 times a day, after the dialysis The obtained phenacetin artificial antigens PNT1-BSA, PNT1-OVA, PNT2-BSA, and PNT2-...

Embodiment 3

[0103] The preparation of the antibody of embodiment 3 phenacetin

[0104] 1. Preparation of polyclonal antibody, the specific steps are as follows:

[0105] Emulsify the prepared artificial antigens PNT1-BSA, PNT2-BSA and immune adjuvant (incomplete Freund's adjuvant for the first immunization, and incomplete Freund's adjuvant for subsequent booster immunizations) at a volume ratio of 1:1 , to immunize New Zealand white rabbits. The weight of the New Zealand white rabbit is 2.5-3.0 kg, and the neck and back are injected subcutaneously at multiple points, and the second immunization is performed 3 weeks later, and the immunization is boosted once every 3 weeks thereafter. One week after the third booster immunization, blood was collected from the ear vein, and the serum titer was determined by indirect competitive ELISA. When the titer no longer rises, the ear vein is used to boost the immunization. One week later, blood was collected from the heart, and the method of obtai...

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Abstract

The invention provides a hapten, artificial antigens and an antibody for detecting phenacetin as well as a preparation method and application of the hapten, the artificial antigens and the antibody. A hapten 1, an artificial antigen 1 and an artificial antigen 2 are prepared, the artificial antigen 2 is used for preparing the specific antibody for detecting phenacetin, and the artificial antigen 1 is used as a coating antigen. The antibody has high-sensitivity and high-specificity recognition capability to phenacetin. The detection limit is 2ng / mL, and the cross reaction rate to structural analogues is lower than 3%. An immunoassay method of phenacetin is established, and the purpose of rapidly and accurately detecting phenacetin in herbal tea and other food is achieved.

Description

technical field [0001] The invention relates to the technical field of food safety detection, more specifically, to a hapten, an artificial antigen, an antibody for detecting phenacetin, a preparation method and application thereof. Background technique [0002] After Guangdong herbal tea was successfully included in the first batch of national intangible cultural heritage protection in 2006, the herbal tea industry developed more rapidly, and even the herbal tea trend began to set off across the country. However, while the herbal tea industry is developing rapidly, many problems have also appeared. In order to enhance the curative effect, some unscrupulous merchants illegally add antipyretic and analgesic drugs, such as phenacetin, to herbal tea. This practice not only affects the whole herbal tea The reputation of the industry may also cause serious harm to the life safety of drinkers. For example, in 2019, the Market Supervision and Administration Bureau of Wuzhou City, ...

Claims

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Application Information

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IPC IPC(8): C07C233/25C07K14/765C07K14/77C07K1/107C07K16/44C07K16/06G01N33/53G01N33/577G01N33/558G01N33/58G01N33/543
CPCC07C233/25C07K14/765C07K14/77C07K16/44C07K16/06G01N33/9486G01N33/5308G01N33/577G01N33/558G01N33/587G01N33/54393C07K2317/33C07K2317/76
Inventor 雷红涛赖玮林建浩王锦
Owner SOUTH CHINA AGRI UNIV
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