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
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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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