Artificial antigen and antibody capable of simultaneously detecting acetaminophen and phenacetin and preparation method and application of artificial antigen and antibody

An acetaminophen and artificial antigen technology, which is applied in the preparation methods of peptides, chemical instruments and methods, biological tests, etc. The effect of eliminating distractions

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

[0006] The purpose of the present invention is to overcome the defects and deficiencies in the prior art that paracetamol and phenacetin cannot be detected simultaneously,

Method used

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  • Artificial antigen and antibody capable of simultaneously detecting acetaminophen and phenacetin and preparation method and application of artificial antigen and antibody
  • Artificial antigen and antibody capable of simultaneously detecting acetaminophen and phenacetin and preparation method and application of artificial antigen and antibody
  • Artificial antigen and antibody capable of simultaneously detecting acetaminophen and phenacetin and preparation method and application of artificial antigen and antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Synthesis and Identification of Hapten ACE1

[0070] 1. Synthesis of Hapten ACE1

[0071] Take N-(4-hydroxyphenyl)acetamide (N-(4-hydroxyphenyl)acetamide) (1moL), potassium carbonate (1~4moL), use acetone as solvent, and ethyl bromoacetate (1~2moL) in React at room temperature for 3 to 8 hours, separate and purify to obtain 2-(4-acetamidophenoxy) ethyl acetate (ethyl2-(4-acetamidophenoxy) acetate). Dissolve ethyl 2-(4-acetamidophenoxy)acetate in methanol, the volume ratio of ethyl 2-(4-acetamidophenoxy)acetate to methanol is 1:1, add 1mol / L hydrogen The sodium aqueous solution was stirred and reacted at room temperature for 2-5 hours. After the reaction, the pH was adjusted to 5-6 with 1 mol / L hydrochloric acid to obtain the hapten ACE1.

[0072] 2. Identification of Hapten ACE1

[0073] The prepared hapten ACE1 was identified by H NMR spectrum and determined by mass spectrometry.

[0074] H NMR spectrum results of hapten ACE1: 1 H NMR (600MHz, Methanol-d...

Embodiment 2

[0079] Synthesis and identification of embodiment 2 artificial antigen

[0080] 1. Synthesis of artificial antigen

[0081] (1) Synthesis of artificial antigen ACE1-BSA / OVA

[0082] The hapten ACE1 was coupled to bovine serum albumin (BSA) and chicken ovalbumin (OVA) respectively by the active ester method.

[0083] Weigh 1moL hapten ACE1, 0.8moL NHS and 2.6moL EDC, dissolve them in 50-200μL N,N-dimethylformamide (DMF), stir at room temperature for 2-4 hours in the dark to obtain ACE1 hapten activation solution; Add 10mg of BSA or OVA to 1mL of PBS buffer (0.01moL / L, pH=7.4); slowly add ACE1 hapten activation solution to the BSA or OVA solution drop by drop, react at 4°C for 12h; dialyze with PBS buffer for 3 day, 3 times a day. After the dialysis, the artificial antigen ACE1-BSA / OVA was obtained, which was divided into centrifuge tubes and stored at -20°C for use.

[0084] (1) Synthesis of artificial antigen ACE2-BSA / OVA

[0085] The hapten ACE2 was coupled to bovine seru...

Embodiment 3

[0097] Example 3 Simultaneously detect the preparation of paracetamol and phenacetin bispecific antibody

[0098] 1. Preparation of polyclonal antibody, the specific steps are as follows:

[0099] The artificial antigen ACE1-BSA prepared in Example 2 and the immune adjuvant (complete Freund's adjuvant for the first immunization, and incomplete Freund's adjuvant for the booster immunization) were evenly emulsified at a volume ratio of 1:1, and the New Zealand white rabbit was immunized . The New Zealand white rabbits weigh 2.5-3 kg, and are injected subcutaneously at multiple points on the neck and back, and immunized for the second time after 4 weeks, and boosted immunization 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...

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Abstract

The invention discloses an artificial antigen and antibody capable of simultaneously detecting acetaminophen and phenacetin and a preparation method and application of the artificial antigen and antibody. An artificial antigen 1 and an artificial antigen 2 are prepared, the artificial antigen 1 is used for preparing a bispecific antibody for simultaneously detecting acetaminophen and phenacetin, the artificial antigen 2 is used as an artificial coating antigen, the antibody has high-sensitivity and high-specificity recognition capability on acetaminophen and phenacetin, the lowest detection limits are 2.8 ng/mL and 2.1 ng/mL respectively, the cross reaction rate to structural analogues is lower than 0.1%, an immunoassay method of the acetaminophen and the phenacetin is established, and the purpose of rapidly and accurately detecting the acetaminophen and the phenacetin in food is achieved.

Description

technical field [0001] The invention relates to the technical field of food safety detection, and more specifically relates to an artificial antigen capable of simultaneously detecting acetaminophen and phenacetin, an antibody and a preparation method and application thereof. Background technique [0002] Acetaminophen (Acetaminophen), which belongs to the acetanilide antipyretic and analgesic drugs, has slow and long-lasting antipyretic effect and is well tolerated. It is the main component of many anti-cold and flu drugs. Acetaminophen is used in therapeutic concentrations It is safe, but long-term or overdose can cause liver damage, acute liver failure and even death. Phenacetin is an acetanilide antipyretic and analgesic drug. Its pharmacological effect is similar to that of acetaminophen, but it is more toxic. Studies have shown that long-term or excessive use can damage the kidneys, lead to hemolytic anemia, and induce cancer. . [0003] In recent years, some unscrup...

Claims

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Application Information

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IPC IPC(8): C07K14/765C07K14/77C07K14/795C07K14/79C07K1/107C07K16/44C07K16/06G01N33/577G01N33/53G01N33/543
CPCC07K14/765C07K14/77C07K14/795C07K14/79C07K16/44C07K16/06G01N33/9486G01N33/577G01N33/5308G01N33/54393C07K2317/31C07K2317/33C07K2317/76
Inventor 雷红涛赖玮林建浩王锦
Owner SOUTH CHINA AGRI UNIV
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