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Preparation and application of highly specific amantadine artificial antigen

A technology of amantadine and artificial antigen, which is applied in the preparation of organic compounds, cyanide reaction preparation, chemical instruments and methods, etc., can solve the problems of expensive instruments, time-consuming detection, failure to meet the requirements of fast and accurate, and achieve specificity High performance and high sensitivity

Inactive Publication Date: 2015-09-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection of amantadine mainly relies on high-performance liquid chromatography (HPLC), gas chromatography (GC), thin-layer chromatography (TLC), mass spectrometry (MS), etc., but there are defects such as expensive instruments and time-consuming detection , and requires professional and technical personnel to operate, which cannot meet the fast and accurate requirements of modern detection

Method used

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  • Preparation and application of highly specific amantadine artificial antigen
  • Preparation and application of highly specific amantadine artificial antigen
  • Preparation and application of highly specific amantadine artificial antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of a small molecule compound (i.e. amantadine hapten)

[0033] Amantadine (100mg, 0.66mmol) and 6-bromohexanoic acid (130mg, 0.8mmol) were added to toluene, and the temperature was raised to 130°C under nitrogen protection to reflux for 48h. Concentrate under reduced pressure, add water (10 mL), adjust the pH of the aqueous phase to 2-3 with hydrochloric acid, add ethyl acetate for extraction, wash with saturated NaCl solution, dry and concentrate to obtain the amantadine hapten product.

Embodiment 2

[0034] Example 2 Preparation of amantadine artificial antigen (synthesized immunogen by EDC method)

[0035] Take 10mg of amantadine hapten, add 2mL of ultrapure water to dissolve, then add NHS and EDC respectively (the molar ratio of hapten, NHS, and EDC is 1: 2: 2), mix well at room temperature, and react at room temperature at 25°C 12h. Take 25mg bovine serum albumin (the molar ratio of hapten and bovine serum albumin is 80:1), add 5mL 0.1M pH9.6 carbonate buffer. The activated hapten solution was slowly added dropwise to the protein solution, and reacted at room temperature for 24 hours. The amantadine artificial antigen was obtained by dialysis with PBS buffer solution for 2 days, during which the water was changed 4 times.

Embodiment 3

[0036] Example 3 Preparation of amantadine artificial antigen (synthesis of immunogen by mixed anhydride method)

[0037] Take 9 mg of amantadine hapten and add 2 mL of ultrapure water to dissolve it. At 0°C, add tri-n-butylamine and isobutyl chloroformate (the molar ratio of hapten, tri-n-butylamine, and isobutyl chloroformate is 1: 1.5: 1.5), and react at 0°C for 1 hour. Take 30mg of bovine serum albumin (the molar ratio of hapten to bovine serum albumin is 60:1), add 5mL of 0.1M pH9.6 carbonate buffer, and pre-cool at 0°C for 30min. At 0°C, the activated hapten solution was slowly added dropwise to the protein solution, reacted at 0°C for 1 hour, and then reacted at room temperature for 24 hours. The amantadine artificial antigen was obtained by dialysis with PBS buffer solution for 2 days, during which the water was changed 4 times.

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Abstract

Preparation and application of a highly specific amantadine artificial antigen belong to the technical field of biochemistry engineering. The method is as below: adding amantadine, 6-bromo-hexanoic acid to toluene, heating to 130 DEG C under nitrogen protection, and conducting reflux for 48 h; concentrating under reduced pressure, adding water, and adjusting the pH value of the aqueous phase with hydrochloric acid; adding ethylacetate for extraction, washing with saturated NaCl solution, drying and concentrating to obtain a product semiantigen; and coupling the carboxyl on the semiantigen with amino on a carrier protein to obtain an artificial antigen. Experimental results show that an antiserum obtained by immunizing animals with the antigen obtained by the invention has titer up to 81000, detection limit of 0.1ng / mL, and half inhibitory concentration of 1ng / mL; and the produced antibody has high specificity and high sensitivity. The antigen or antibody provided by the invention has wide application prospects.

Description

technical field [0001] The invention relates to a preparation method and application of a highly specific amantadine artificial antigen, belonging to the technical field of biochemical engineering. Background technique [0002] Amantadine can inhibit virus reproduction and is clinically used to prevent and treat various type A influenza virus infections. However, due to economic interests and the pressure of disease prevention and control, amantadine is abused in poultry breeding in my country. The residual accumulation of amantadine will cause drowsiness, insomnia, dizziness, depression, nausea and other symptoms to the eaters; in December 2012, a CCTV survey stated that in order to further shorten the breeding cycle, some farmers let the white feather, which was already a fast-growing variety, The chickens grow faster and are secretly fed illicit drugs, which include antivirals such as amantadine. The outbreak of the "quick chicken" incident has aroused widespread concer...

Claims

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Application Information

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IPC IPC(8): C07C229/46C07C227/10C07K14/47C07K16/44G01N33/577
Inventor 匡华彭双胥传来徐丽广马伟刘丽强宋珊珊胡拥明
Owner JIANGNAN UNIV
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