Ractopamine immunogen, coating antigen and use thereof in colloid-gold test paper

A technology of ractopamine and immunogen, which is applied in the field of immunology, can solve the problem that the detection limit cannot meet the national limit requirements, and achieve the effect of improving immunogenicity

Active Publication Date: 2011-03-30
深圳市三方圆生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the current ractopamine colloidal gold rapid test products on the ma

Method used

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  • Ractopamine immunogen, coating antigen and use thereof in colloid-gold test paper
  • Ractopamine immunogen, coating antigen and use thereof in colloid-gold test paper
  • Ractopamine immunogen, coating antigen and use thereof in colloid-gold test paper

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Experimental program
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Embodiment 1

[0037] Synthesis of embodiment 1 ractopamine hapten

[0038] 10mmol of 4-iodobutyric acid, 10mL of concentrated sulfuric acid, reflux in 100mL of ethanol for 48h, evaporate to the remaining 10mL of liquid, add 50mL of distilled water, use sodium carbonate to neutralize the excess acid, and adjust to pH = 10. 100mL of ethyl acetate is divided into 3 After extraction, the solvent was evaporated under reduced pressure to obtain ethyl 4-iodobutyrate.

[0039] Weigh 5 mmol of ractopamine and 10 mmol of sodium hydroxide, reflux in 20 mL of anhydrous methanol, slowly add 5 mmol of ethyl 4-iodobutyrate dropwise, and react for 48 hours. The reaction system was cooled to room temperature, the pH was adjusted to neutral, and the components were evaporated to dryness by rotary evaporation, and the components connected with ethyl 4-iodobutyrate at the hydroxyl end away from the nitrogen atom of ractopamine were separated using a silica gel column.

[0040] The above-mentioned intermediate...

Embodiment 2

[0043] Synthesis of embodiment 2 ractopamine immunogen

[0044] 0.1 mmol of ractopamine hapten was dissolved in 2 mL of DMF, and 27.5 mg of DCC and 14.4 mg of NHS were added with stirring. Magnetic stirring was carried out overnight at 4°C. After centrifugation, the supernatant was liquid A, and 140 mg of hemocyanin (KLH) was weighed and dissolved in 10 mL of PBS (pH 8.0) with a concentration of 0.1 mol / L. Add 1 mL of DMF, stir and dissolve to prepare liquid B. Under magnetic stirring, liquid A is gradually dropped into liquid B, and react at 4°C for 12 hours. After centrifugation, the supernatant was taken, dialyzed with normal saline at 4°C for 3 days, and the dialysate was changed 3 times a day. The obtained whole antigen was dispensed into 0.5 mL centrifuge tubes at a concentration of 1 mg / mL. Store in -20°C freezer.

[0045] The prepared immune antigen structure is:

[0046]

Embodiment 3

[0047] Embodiment 3 experimental animal immunization

[0048] The hapten ractopamine immunization antigen was used to immunize female balb / c mice for 6 weeks respectively, 3 mice in each group. For the first immunization injection, 100 μL of 100 μg / mL immune antigen was fully emulsified with the same amount of complete Freund’s adjuvant, and injected directly into the intraperitoneal cavity. After an interval of two weeks, take the sampled antigen, emulsify it with 100 μL of incomplete adjuvant, and inject it in the same way.

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Abstract

The invention relates to a ractopamine immunogen, a ractopamine coating antigen and use thereof in colloid-gold test paper and belongs to the technical field of immunology. The immunogen and coating antigen are products of the coupling of the hapten of the connecting arm of a hydroxyl terminal far away from a nitrogen atom of the ractopamine with a carrier protein. The hapten is similar to a ractopamine counterpart in terms of molecular structure, stereochemistry and electronic distribution. The molecule of the hapten has an active group for coupling with a protein carrier, and the existence of the active group exerts on influences on the electronic distribution of the molecules of a material to be tested. When coupled with hemocyanin, the hapten enables an organism to produce a high-potency and high-specificity antibody against ractopamine and be absorbed onto a glass cellulose membrane; and the coating antigen constructed by coupling with human haemocyanin can be absorbed onto a nitrocellulose membrane for constructing the colloid-gold test paper.

Description

technical field [0001] The invention relates to a ractopamine immunogen and a coater, belonging to the technical field of immunology. Background technique [0002] Ractopamine hydrochloride is a pharmaceutical raw material, a cardiotonic drug that can be used to treat congestive heart failure. It can also be used to treat muscular dystrophy, increase muscle and reduce fat accumulation. At the same time, it can increase the daily weight gain of animals, improve the utilization rate of feed, and increase the protein content of animals. Ractopamine is a beta stimulant, which can improve the growth rate and efficiency of lean tissue when added to pig feed, and the skin is smooth and smooth, and looks good. The poisoning phenomenon of ractopamine is similar to the poisoning phenomenon of "clenbuterol" when people consume ractopamine. Symptoms such as rapid heartbeat, tremor, and palpitations may occur, and more serious ones may cause human teratogenicity and carcinogenesis. Th...

Claims

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Application Information

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IPC IPC(8): C07K14/795C07K14/765G01N33/558
Inventor 钟松清谭攀李细清张世伟
Owner 深圳市三方圆生物科技股份有限公司
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