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Ractopamine immunomagnetic bead separation enrichment kit and application

A technology of ractopamine coupled with ractopamine monolayer, applied in the field of immunology, can solve the problems of high binding strength, low binding probability of antibodies and detection target substances, etc., and achieves time saving, environmental protection in rinsing and elution processes, and separation processes simple effect

Active Publication Date: 2016-01-27
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The covalent bond between the antibody and the carrier is a physical binding force such as hydrophobic force and ion exchange force, and its binding force is not as strong as that of the chemical bond of immunomagnetic beads
Moreover, the carrier of the immunoaffinity column is a solid phase, and the binding probability of the antibody to the detection target substance will be lower than that of the immunomagnetic beads.

Method used

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  • Ractopamine immunomagnetic bead separation enrichment kit and application
  • Ractopamine immunomagnetic bead separation enrichment kit and application
  • Ractopamine immunomagnetic bead separation enrichment kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Preparation of specific components of the kit

[0021] 1. Ractopamine Hapten Synthesis

[0022] The ractopamine hapten is oxidized by pyridinium dichromate from ractopamine, and then undergoes condensation reaction with p-hydroxyphenylhydrazine, and a carboxyl spacer arm is introduced at the ortho position of the benzene ring to obtain p-hydroxyphenylhydrazine ractopamine hapten.

[0023] Take 0.1g of ractopamine and add 1.0g of ractopamine to dissolve in acetonitrile, add 0.78g of pyridinium chromate, stir, then add 500μL of acetic acid, stir at 80°C for 10h, after stopping the reaction, use a rotary evaporator to remove acetonitrile, then add water and ethyl acetate Ester extraction, after layering, remove the water phase, dry the organic phase with anhydrous sodium sulfate and evaporate to dryness, put it on a silica gel column, the volume ratio of mobile phase chloroform and methanol is chloroform:methanol=10:1, carry out elution and separation, and ob...

Embodiment 2

[0036] Embodiment 2: the formation of kit

[0037] Set up the detection ractopamine magnetic immunomagnetic bead separation and enrichment kit, so that it contains the following components:

[0038] Magnetic Beads Conjugated to Ractopamine Monoclonal Antibody

[0039] Reconstitution solution

[0040] magnet

[0041] Add the magnetic beads coupled with ractopamine monoclonal antibody to the reconstitution solution to a final concentration of 10 mg / mL.

Embodiment 3

[0042] Example 3: Kit for separation and enrichment of ractopamine in samples

[0043] 1) Take 0.1 mL of magnetic bead reconstitution solution dissolved with conjugated ractopamine monoclonal antibody in a 10 mL centrifuge tube, wash the magnetic beads with 5 mL of deionized water for 1-2 times, and place the centrifuge tube on the magnet for 2-3 minutes , to ensure that all the magnetic beads are adsorbed on the magnet, and separate the magnetic beads and washing solution with a magnet every time;

[0044] 2) Add 5 mL of urine sample into a 10 mL centrifuge tube filled with rinsed magnetic beads coupled with ractopamine monoclonal antibody, mix well, and react at room temperature for 20 min; or use a homogenizer for animal tissue and feed samples After homogenization, weigh 5.0±0.05g sample into the sample bottle, add 1.5±0.05g sodium chloride and 20mL50% methanol solution respectively, vortex with vortex for 5min, centrifuge at 3000r / min for 5min at room temperature, or let ...

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PUM

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Abstract

The present invention relates to a ractopamine immunomagnetic bead separation enrichment kit comprising ractopamine monoclonal antibody coupled magnetic beads, a reconstitution fluid and a magnet, the ractopamine monoclonal antibody coupled magnetic beads are prepared by mixing ractopamine monoclonal antibody and magnetic beads in the mass ratio of 1: 500 to 800 and dissolving in 2-(N-morpholino) ethanesulfonic acid monohydrate for coupling, and the ractopamine monoclonal antibody is prepared by using a conjugate obtained from ractopamine hapten and bovine serum albumin as an immunogen to immunize Balb / c mice. The present invention also relates to a sample pre-processing method of the ractopamine immunomagnetic bead separation enrichment kit for separating ractopamine, the method has high ractopamine specificity, recovery rate and accuracy, simplifies sample preprocessing steps, and avoids use of a variety and a large number of organic solvents in the sample preprocessing process.

Description

technical field [0001] The invention relates to a ractopamine immunomagnetic bead kit and a method for separating and enriching ractopamine in a sample. in the field of immunology. Background technique [0002] Ractopamine (RAC), the chemical name is 1-(4-hydroxyphenyl)-2[1-methyl-3-(4-hydroxyphenyl)-propylamino]-ethanol hydrochloride, and Clen Tero (Clenbuterol) is a beta-receptor stimulant and is commonly used clinically to treat bronchitis and asthma. Studies in recent years have shown that RAC has the functions of controlling animal nutrition metabolic pathway and enhancing feed conversion rate, so it is used as a new growth-promoting additive. However, because ractopamine has residues in pigs and other animals and can enter the human body through the biological chain, when the accumulation exceeds a certain amount, it will show toxic and side effects, and symptoms such as tachycardia, arrhythmia, and muscle pain will appear. In recent years, as the government has str...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/40G01N1/34
Inventor 徐念琴聂雯莹马腊腊曹东山杨昌松何方洋罗晓琴万宇平
Owner BEIJING KWINBON BIOTECH
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