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Multi-cluster antigen and wide-spectrum specific antibody of beta-adrenoceptor agonists and application of multi-cluster antigen and wide-spectrum specific antibody

A receptor agonist and epinephrine technology, applied in the fields of immunochemistry and analysis, can solve the problems of inability to detect similar veterinary drug residues, high cost of chromatographic detection, and easy missed detection.

Inactive Publication Date: 2015-01-28
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Chromatography is the main method for the detection of β-agonist veterinary drug residues, but the detection cost of chromatography is high, the operation is complicated, and a large number of negative samples are repeatedly detected; while the enzyme-linked immunoassay has the advantages of strong specificity and simple sample pretreatment, but it is specific The strong characteristic also determines its fatal shortcoming: an enzyme-linked immunosorbent assay kit or test strip can only detect one kind of veterinary drug residue, but cannot detect other similar veterinary drug residues, which is the reason for missed detection ( Such as the "Shuanghui Clenbuterol" event that caused a sensation in the country in 2011); in addition, with the emergence of multiple alternatives to β-agonists, if a complete test is performed, a sample needs to use multiple test products, which greatly increases the test cost and cost. Working hours, lost the meaning of quick screening

Method used

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  • Multi-cluster antigen and wide-spectrum specific antibody of beta-adrenoceptor agonists and application of multi-cluster antigen and wide-spectrum specific antibody
  • Multi-cluster antigen and wide-spectrum specific antibody of beta-adrenoceptor agonists and application of multi-cluster antigen and wide-spectrum specific antibody
  • Multi-cluster antigen and wide-spectrum specific antibody of beta-adrenoceptor agonists and application of multi-cluster antigen and wide-spectrum specific antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Preparation of β-agonist multi-cluster antigen SAL-RAC-BSA and SAL-RAC-OVA conjugates

[0046] 1) Coupling the SAL hapten on BSA by the mixed anhydride method, and then purifying:

[0047] Dissolve 0.2 mmol of salbutamol sulfate in 5-10 ml of absolute ethanol, add 0.2 mmol of glutaric anhydride, react at room temperature for 3-5 hours, and centrifuge. Dissolve the solid in the lower layer in 5-10ml of N,N-dimethylformamide, add 0.2mmol of tri-n-butylamine and 0.2mmol of isobutyl chloroformate, and continue the reaction for 1-3h. The above reaction solution was slowly added to 5-10 ml of phosphate buffer solution containing 5-10 mg / ml BSA, and reacted overnight at room temperature. The above reaction solution is dialyzed with phosphate buffer solution for 3 to 6 times, and then freeze-dried to obtain the freeze-dried powder of the SAL-BSA conjugate. According to the ultraviolet-visible absorption spectrogram of SAL, BSA and SAL-BSA conjugate (see attached fi...

Embodiment 2

[0051] Example 2 Preparation of β-agonist broad-spectrum specific antibody SAL-RAC-BSAAb

[0052] Healthy white rabbits weighing about 2 kg were immunized with the SAL-RAC-BSA conjugate described in Example 1 as an immunogen. For the first immunization, 0.25 mg of SAL-RAC-BSA conjugate was mixed with an equal amount of Freund's complete adjuvant and fully emulsified, and injected subcutaneously at multiple points on the back. Two weeks later, the same dose of immunogen and the same amount of Freund's incomplete adjuvant were used for emulsification and booster immunization, and booster immunization was performed every two weeks, for a total of three times. 10 days after the last immunization, blood was collected from the jugular vein of the white rabbits, placed in a 4°C environment for 30 minutes, and then purified by ammonium sulfate multi-stage precipitation method to obtain the β-agonist broad-spectrum specific antibody SAL-RAC- BSAAb.

Embodiment 3

[0053] Example 3 Preparation and Application of a Kit Using SAL-RAC-OVA Conjugate as Coating Source and Enzyme-labeled Secondary Antibody as Enzyme Label

[0054] 1. ELISA kit, including:

[0055] 1) For the enzyme plate coated with SAL-RAC-OVA conjugate, the concentration of the coating source can be 0.15μg / ml~0.25μg / ml;

[0056] 2) β-agonist broad-spectrum specific antibody SAL-RAC-BSAAb;

[0057] 3) horseradish peroxidase-labeled goat anti-rabbit IgG;

[0058] 4) β-agonist standard solution;

[0059] 5) Substrate chromogenic solution: composed of liquid A and liquid B, liquid A is a 2% aqueous solution of carbamide peroxide, and liquid B is an aqueous solution of 1% tetramethylbenzidine;

[0060] 6) Termination solution: 1-2mol / L hydrochloric acid or sulfuric acid solution;

[0061] 7) Washing solution: 0.01 mol / L phosphate buffer containing 0.8% to 1.2% Tween 20 and 0.5% sodium azide with a pH value of 7.4;

[0062] 8) Diluent: 0.01-0.1mol / L phosphate buffer;

[0063...

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Abstract

The invention relates to a multi-cluster antigen and an antibody of beta-adrenoceptor agonists and application of the multi-cluster antigen and the antibody to detection on multi-residue of the beta-adrenoceptor agonists. The application is implemented by a detection kit and test paper. The multi-cluster antigen of the beta-agonists is salbutamol-ractopamine-bovine serum albumin (SAL-RAC-BSA) or salbutamol-ractopamine-ovalbumin (SAL-RAC-OVA), and the salbutamol-ractopamine-bovine serum albumin antibody (SAL-RAC-BSAAb) is prepared from SAL-RAC-BSA through animal immunization. The invention also discloses the kit and the test paper, which are used for detecting the multi-residue of the beta-agonists and take the SAL-RAC-BSAAb as a core agent. Based on a cross immunization reaction of the SAL-RAC-BSAAb on the beta-agonists, three beta-agonists, namely clenbuterol, SAL and RAC, can be detected.

Description

technical field [0001] The invention belongs to the technical field of immunochemistry and analysis, and relates to a β-adrenoceptor agonist (hereinafter referred to as β-agonist) multi-cluster antigen and broad-spectrum specific antibody and its preparation and application, in particular to a The broad-spectrum specific antibody is the core kit and colloidal gold test paper. Background technique [0002] β-adrenergic receptor agonists, referred to as β-agonists, are the chemical residue hazards with the highest detection rate and the most serious hazards in animal-derived foods, the most famous of which is clenbuterol (CL) , commonly known as "clenbuterol". In the 1980s, researchers from the U.S. fatty amine company accidentally discovered that "lean meat powder" can enhance the decomposition of animal fat, promote protein synthesis, greatly shorten the time to market of meat products, and significantly improve carcass lean meat rate, feed return rate and economic benefits...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K14/77C07K1/107C07K16/44G01N33/558G01N33/543
CPCC07K14/765C07K14/77C07K16/44G01N33/535G01N33/558
Inventor 邵科峰赵波张红琳任鹏飞常其沛
Owner NANJING NORMAL UNIVERSITY
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