Clenbuterol complete antigen and method for preparing monoclonal antibody thereof

A clenbuterol hydrochloride and monoclonal antibody technology, applied in biochemical equipment and methods, anti-animal/human immunoglobulin, specific peptides, etc., can solve the problem of low antibody specificity and affinity, cumbersome operation steps, and dangerous Reagents and other issues, to achieve the effect of improving sensitivity and specificity, simple preparation steps, and low potential harm to human body

Inactive Publication Date: 2008-05-21
UNIV OF SHANGHAI FOR SCI & TECH
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0004] The invention provides a preparation method of clenbuterol hydrochloride complete antigen and anti-clenbuterol hydrochloride monoclonal antibody, which overcomes the cumbersome operation steps and dangerous reagents in the prior art, and it is easy to produce antibodies against other antigenic determinants and produce anti-clenbuterol monoclonal antibodies. The disadvantages of antibody specificity and low affinity

Method used

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  • Clenbuterol complete antigen and method for preparing monoclonal antibody thereof
  • Clenbuterol complete antigen and method for preparing monoclonal antibody thereof
  • Clenbuterol complete antigen and method for preparing monoclonal antibody thereof

Examples

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Embodiment 1

[0029] Example 1: Synthesis and purification of complete antigen

[0030] Prepare a 1mol / L hydrochloric acid solution and pre-cool it in a refrigerator at 4°C. Weigh 80.00 mg of clenbuterol hydrochloride (CLB) into a beaker, slowly add the pre-cooled hydrochloric acid solution dropwise, and stir with a glass rod until the CLB is dissolved, then put the dissolved solution into an ice bath, and add rotor. Place the ice bath on the magnetic stirrer, turn on the switch, stir slowly, and gradually add 30% NaNO 2 solution. Add NaNO dropwise 2 Use a glass rod to dip a little droplet of the reaction liquid into the solution, check it with starch KI test paper, and stop adding 30% NaNO when it changes to dark purple.2 solution. The reaction solution was continued, and after 45 min, 1 ml of 5% ammonium sulfamate was added dropwise to terminate the reaction. Weigh 300 mg of BSA into a beaker, slowly add 0.01 mol / L pH=7.2-7.4 phosphate buffer saline (PBS) dropwise until completely di...

Embodiment 2

[0044] Example 2: Preparation of anti-clenbuterol monoclonal antibody by animal immunization

[0045] 1) Female pure-line Balb / c mice aged 6-8 weeks and weighing 18-22 g are selected. The dose of immunization was 50 μg / monkey for basic immunization, and 100 μg / bird for booster immunization. The quantitative artificial antigen complexes were diluted with PBS buffer respectively, and an equal volume of Freund's complete adjuvant was added to the initial immunization, and fully emulsified until the milk droplets did not spread when dripped into water. Intraperitoneal injection method was used. After 2-3 weeks, booster immunization was carried out, and then booster immunization was carried out every two weeks, and Freund's incomplete adjuvant was used for booster immunization. From the third immunization, orbital blood was collected on the third day of each immunization to determine the titer and specificity. After the immune serum titer is qualified, prepare for cell fusion. ...

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Abstract

The invention discloses the preparation methods of a complete antigen of clenbuterol hydrochloride and a monoclonal antibody of the clenbuterol hydrochloride. Firstly, the clenbuterol hydrochloride reacts with sodium nitrite under the acid condition to obtain azo clenbuterol hydrochloride; then the azo clenbuterol hydrochloride coupled with bovine serum albumin under the alkaline condition to prepare for the complete antigen of the clenbuterol hydrochloride. A balb / c pure line rat is immunized, after IELISA test shows that the serum of the rat after immunity is eligible, the cell fusion is processed for preparing for the monoclonal antibody of the clenbuterol hydrochloride. The complete antigen of the clenbuterol hydrochloride prepared by the invention can be used for immunizing the animal. The prepared monoclonal antibody can be used for testing the residual quantity of the clenbuterol hydrochloride in meat, meat products, livestock feed and animal body before the animal is slaughtered. The coupling rate of the clenbuterol hydrochloride in the complete antigen of the clenbuterol hydrochloride obtained by the method of the invention with the bovine serum albumin is 17, and a molecular structure formula thereof is as above.

Description

technical field [0001] The invention relates to a preparation method of clenbuterol hydrochloride complete antigen and anti-clenbuterol hydrochloride monoclonal antibody, and the antibody can be used to detect the residual amount of clenbuterol hydrochloride in meat products and livestock feed. Background technique [0002] Clenbuterol hydrochloride (CLB), also known as clenbuterol, is a beta 2 Adrenergic agonists. Animals can accelerate the conversion and decomposition of body fat after eating, and promote protein synthesis, so it is used by illegal traders to raise pigs and cattle to increase the lean meat rate for huge profits. However, after people eat meat products containing CLB, they will have symptoms such as palpitations, muscle tremors in the face, neck and limbs, dizziness, fatigue, and arrhythmia, which seriously affect people's lives and social stability. Therefore, in 1997, my country has banned CLB as a feed additive for poultry meat production, and formulat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K16/18C12N5/18
Inventor 李红梅李琳徐斐胥义陈佳
Owner UNIV OF SHANGHAI FOR SCI & TECH
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