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Beta-agonist semi-antigen, and preparation method and application thereof

A stimulant and hapten technology, applied in the field of hapten and its preparation, can solve the problems of high detection cost, inability to operate on-site, complicated pretreatment, etc., and achieve low detection cost, good affinity and simple synthesis method

Active Publication Date: 2014-01-15
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chromatographic method is sensitive, accurate, strong specificity, good separation, and can determine multiple drugs at the same time, but requires expensive instruments, complicated sample pretreatment, tedious and time-consuming, high cost of detection, cannot be operated on site, and requires professionals operation, so its application is limited; in contrast, ELISA has the advantages of good specificity, high sensitivity, easy operation, low detection cost, suitable for screening and detection of batch samples, etc., and requires less equipment and is simple to operate Easy to learn and low cost, it can better meet the needs of my country's livestock and poultry farmers, slaughterhouses, food companies, government functional supervision departments, etc.

Method used

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  • Beta-agonist semi-antigen, and preparation method and application thereof
  • Beta-agonist semi-antigen, and preparation method and application thereof
  • Beta-agonist semi-antigen, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Synthesis and identification of β-agonist hapten

[0019] 1. Synthesis of β-agonist hapten (synthesis route such as figure 1 )

[0020] 1.0g salbutamol, dissolved in 20ml N,N-dimethylformamide, then add 10% molar equivalent of tetramethylpiperidine oxide, 10% molar equivalent of tetrabutylammonium chloride and 20ml 0.5mol / L NaHCO 3 After stirring the aqueous solution at room temperature for 30 minutes, add 1.1 molar equivalent of N-chlorosuccinimide, and after reacting at room temperature for 2 to 5 hours, remove most of the solvent, extract with ethyl acetate, wash, dry, remove the solvent and purify by column chromatography , Get white solid salbutamol monooxide, which is β-agonist hapten.

[0021] 2. Identification of β-stimulant haptens

[0022] Take the synthesized β-agonist hapten and measure it by proton NMR spectroscopy, such as figure 2 As shown, the aldehyde group signal peak increased by about 10.2 ppm in the spectrum indicates that the hapten synthesis i...

Embodiment 2

[0023] Example 2 β-Agonist Antigen

[0024] The β-agonist hapten is coupled with the carrier protein to obtain the β-agonist antigen.

[0025] 1. The preparation of immunogen-synthesis of β-agonist hapten-bovine serum albumin conjugate

[0026] Take 7mg hapten and dissolve it in 1ml N,N-dimethylformamide, take 30mg each of carbodiimide and N-hydroxysuccinimide, dissolve it in 0.2ml water, add to the hapten solution, stir at room temperature 24h, you can get reaction solution A; weigh 50mg bovine serum albumin, make it fully dissolved in 3.8ml PBS (pH 7.2), slowly drop reaction solution A into the protein solution, and stir at room temperature After 24 hours, dialyzed with 0.01mol / L PBS at 4°C for 3 days, and change the dialysate 3 times a day to remove unreacted small molecules to obtain β-agonist immunogens; subpackage and store at -20°C for later use.

[0027] 2. Preparation of Coating Agent——Synthesis of β-agonist hapten-ovalbumin conjugate

[0028] Take 7mg of hapten and dissolve ...

Embodiment 3

[0031] Example 3 β-agonist monoclonal antibody

[0032] 1. Preparation of β-agonist monoclonal antibodies

[0033] Animal immunization: Inject the immunogen into Balb / c mice at a dose of 150μg / mouse to produce antiserum.

[0034] Cell fusion and cloning: After the mouse serum determination result is high, take its spleen cells and fuse with SP2 / 0 myeloma cells at a ratio of 8:1 (quantity ratio), and use indirect competitive ELISA to determine the cell supernatant for screening Positive hole. Use the limiting dilution method to clone the positive wells until a hybridoma cell line secreting monoclonal antibodies is obtained.

[0035] Cell cryopreservation and resuscitation: The β-agonist monoclonal antibody hybridoma cell line is made into 1×10 cryopreservation solution 6 Cells / ml cell suspension, long-term storage in liquid nitrogen. Take out the cryopreservation tube during resuscitation, immediately put it into a 37℃ water bath for rapid thawing, centrifuge to remove the cryopreser...

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Abstract

The invention discloses a semi-antigen and a preparation method and application thereof and specifically relates to a beta-agonist semi-antigen. The invention further discloses a preparation method and application of the beta-agonist semi-antigen. A kit rapid detection product established on the basis of the beta-agonist semi-antigen has the advantages of convenient usage, low detection cost, usage of highly efficient, accurate and rapid detection methods, capacity of simultaneous detection of large batch samples and applicability to on-site monitoring of residue of beta-agonist drugs in animal-origin samples and screening of considerable samples.

Description

Technical field [0001] The invention relates to a hapten and a preparation method and application thereof, in particular to a beta-agonist hapten and a preparation method and application thereof. technical background [0002] β-agonist (β-agonist) full name β-adrenoceptor agonist (β-adrenoceptor agonist), also known as β-agonist, is a type of benzene with chemical structure and physiological function similar to adrenaline and norepinephrine Ethylamine drugs. Common β-stimulants are clenbuterol, ractopamine, salbutamol, cimaterol, mabuterol, fenoterol, terbutaline and so on. β-agonists can selectively bind to β-adrenergic receptors on cell membranes to regulate sympathetic nerve excitement and relax tracheal smooth muscle. Therefore, they are often used clinically for the treatment of human and animal asthma. Studies in the 1980s showed that β-stimulants affected the flow and redistribution of nutrients in animals, enhanced fat catabolism and protein synthesis in the body, and s...

Claims

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Application Information

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IPC IPC(8): C07C223/02C07C221/00C07K14/765C07K14/77C07K14/795C07K14/75C07K14/47C07K1/113C07K16/44G01N33/577
Inventor 何方洋陶光灿冯才伟吴鹏赵正苗崔廷婷李勇张禹
Owner BEIJING KWINBON BIOTECH
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