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A clenbuterol immunomagnetic bead separation and enrichment kit and its application

A technology for separation and enrichment and immune magnetic beads, which is applied in the field of immunology, can solve the problems of high binding force and low binding probability between antibodies and detection target substances, and achieve time saving, environmental protection and high specificity in the washing and elution process and accuracy effects

Active Publication Date: 2017-04-12
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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  • A clenbuterol immunomagnetic bead separation and enrichment kit and its application
  • A clenbuterol immunomagnetic bead separation and enrichment kit and its application
  • A clenbuterol immunomagnetic bead separation and enrichment kit and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0021] 1. Clenbuterol Hapten Synthesis

[0022] The clenbuterol hapten is a clenbuterol hapten obtained by diazotizing clenbuterol hydrochloride and p-hydroxyphenylpropionic acid, and introducing a spacer arm with a carboxyl group into the amino group. Add 0.2-0.5g of clenbuterol hydrochloride into 2-4mL of 1mol / L dilute hydrochloric acid to dissolve, add 10mL of distilled water, stir, and cool the solution to 0°C, then add 0.1-0.2g of nitrite, stir for 2h to obtain Diazonium salt solution. Take 0.3-0.5g p-hydroxyphenylpropionic acid, add it into 5mL ethanol to dissolve, then add 0.2-0.35g potassium carbonate, stir to dissolve, cool the solution to 0°C, add diazonium salt solution, stir for 4h, stop the reaction, Add an appropriate amount of water to the reaction solution, extract with ethyl acetate, separate the organic phase, add hydrochloric acid to adjust the pH value to 5, add ethyl acetate for extract...

Embodiment 2

[0035] Embodiment 2: the formation of kit

[0036] Set up the Clenbuterol Magnetic Immunomagnetic Beads Separation and Enrichment Kit to contain the following components:

[0037] Magnetic Beads Conjugated to Clenbuterol Monoclonal Antibody

[0038] Reconstitution solution

[0039] magnet

[0040] Add the magnetic beads coupled with clenbuterol monoclonal antibody to the reconstitution solution to a final concentration of 10 mg / mL.

Embodiment 3

[0041] Example 3: Kit for separation and enrichment of clenbuterol in samples

[0042] 1) Take 0.1 mL of magnetic bead reconstitution solution containing conjugated clenbuterol 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 -3min, ensure that all the magnetic beads are adsorbed on the magnet, and separate the magnetic beads and washing solution with a magnet each time;

[0043] 2) Add 5 mL of urine sample into a 10 mL centrifuge tube filled with rinsed and coupled clenbuterol monoclonal antibody magnetic beads, mix well, and react at room temperature for 20 min; After the homogenizer is homogenized, weigh 5.0±0.05g of the sample into the sample bottle, add 1.5±0.05g of sodium chloride and 20mL of 50% methanol solution, vortex with a vortexer for 5min, and centrifuge at 3000r / min for 5min at room temperature. Or take 10mL of supernatant to filter instead of ce...

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PUM

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Abstract

The invention relates to a clenbuterol immunomagnetic bead separation and enrichment kit. The clenbuterol immunomagnetic bead separation and enrichment kit comprises a magnetic bead which is coupled to a clenbuterol monoclonal antibody, reconstitution fluid and a magnet, wherein the magnetic bead which is coupled to the clenbuterol monoclonal antibody is formed by mixing and dissolving the clenbuterol monoclonal antibody and the magnetic bead in 2-(N-morpholino) ethanesulfonic acid monohydrate in a coupling way according to a mass ratio of 1:(500 to 1000); the clenbuterol monoclonal antibody is obtained by taking a coupling substance obtained from clenbuterol haptin and bovine serum albumin as an immunogen to immune Balb / c mice; the clenbuterol haptin is obtained by carrying out diazo-reaction on clenbuterol and P-hydroxybenzene propanoic acid and bringing a carboxyl spacer arm on amino. The invention also relates to a sample preprocessing method for separating the clenbuterol by adopting the clenbuterol immunomagnetic bead separation and enrichment kit. According to the sample preprocessing method disclosed by the invention, higher specificity, higher recovery rate and higher accuracy on the clenbuterol are obtained, sample preprocessing steps are simplified, and various and a mass of organic solvents can be prevented from being used during a sample preprocessing procedure.

Description

technical field [0001] The invention relates to a clenbuterol immunomagnetic bead kit and a method for separating and enriching clenbuterol in a sample. in the field of immunology. Background technique [0002] Clenbuterol (Clenbuterol), the synonym of clenbuterol, ammonia clenbuterol, common name "leptin", chemical name α [(tert-butylamino) methyl] -4-amino-3,5-dichlorobenzyl alcohol Hydrochloride. Clenbuterol is a β-stimulant. In the early 1980s, a company in the United States began to add it to feed to increase lean meat percentage. After eating pigs, it can increase the growth rate, promote protein synthesis in the metabolic process, accelerate the transformation and decomposition of fat, and increase the lean meat percentage of pork, so it becomes clenbuterol; Good phase; after slaughter, the meat is bright red, the fat layer is extremely thin, and the skin is often lean against the lean meat, which is plump. However, fattened pigs will have toxic and side effects s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/543G01N33/577
CPCG01N33/54326G01N33/577
Inventor 聂雯莹崔海峰赵正苗宋灏徐念琴何方洋冯才伟尚朋朋
Owner BEIJING KWINBON BIOTECH
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