Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Preparation method and application of ractopamine monoclonal antibody

A kind of technology of ractopamine mono and ractopamine hydrochloride

Inactive Publication Date: 2011-12-21
安徽缘远博爱生物技术有限公司
View PDF4 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The applicant found through searching that the specificity of the ractopamine monoclonal antibody prepared in domestic reports is not strong, and in the application of the colloidal gold test strip prepared with the monoclonal antibody as the core, there is no treatment method for different detection samples And the report of the lowest detection concentration, and the false positive rate of test strips currently on the market is relatively high
Therefore, the detection of ractopamine is far from meeting the requirements of rapidity, cheapness, stability and accuracy.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation method and application of ractopamine monoclonal antibody
  • Preparation method and application of ractopamine monoclonal antibody
  • Preparation method and application of ractopamine monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Synthesis of embodiment 1 artificial immunization antigen and coating antigen

[0022] Weigh 34 mg of ractopamine hydrochloride, add it to a solution containing 10 mg of succinic anhydride dissolved in 2 mL of pyridine, place it in a fume hood and stir for 24 hours at room temperature, and blow dry the pyridine with nitrogen to obtain a ractopamine-semisuccinic anhydride compound. Dissolve ractopamine-semisuccinic anhydride with 4 mL of dissolving solution (N, N-dimethylformamide and 1,4-dioxane in equal amounts), add 26.2 μL of tri-n-butylamine, and stir in ice bath for 10 min Afterwards, add 15 μL of isobutyl chloroformate, stir and react at room temperature for 1 h, place the mixture in an ice bath, and add the pre-frozen protein solution dropwise (100 mg protein dissolved in 0.1 M sodium borate solution, pH 8.5 ), put it at room temperature to react overnight, put the above reaction product into a dialysis bag (molecular weight cut-off 3500) and dialyze in PBS for m...

Embodiment 2

[0023] Example 2 Preparation of Monoclonal Antibody

[0024] 500μg·mL -1 The PBS solution of Rac-KLH (0.22μm filter membrane filtration sterilization) was mixed with an equal volume of Freund's complete adjuvant to form an emulsion, and the healthy 6-week-old female BALB / C mice were subcutaneously injected, each 0.4mL (containing Rac- KLH 100 μg), the same dose was boosted 3 weeks after the first immunization, and the adjuvant was Freund’s adjuvant, and a total of 3 times were immunized with an interval of 2 weeks. The serum antibody titer was detected by indirect ELISA method, and the small antibody with the highest titer was selected The mice were subjected to cell fusion, and the mice were boosted with Rac-KLH three days before the fusion. Take mouse splenocytes and myeloma cells SP2 / 0 for fusion, add HAT medium, and culture in a 37°C, 6% CO2 incubator. Half of the medium was replaced with fresh HAT medium after 5 days, and the HAT medium was replaced with HT medium after...

Embodiment 3

[0027] Example 3 Purification and Identification of Monoclonal Antibody

[0028] 1. Antibody Purification

[0029] The ascites was purified by octanoic acid-ammonium sulfate salting-out method and Protein A immunochromatography, and the purified product was identified by SDS-PAGE electrophoresis. A 51KD antibody heavy chain band and a 26KD antibody light chain band appeared, and the purity was determined to be 98.2% ( figure 1 ).

[0030] 2. Determination of Antibody Sensitivity

[0031] The blocking efficiency of the purified antibodies was determined by indirect competition ELISA. Ractopamine hydrochloride standard was dissolved in PBS, and then diluted into a series of concentration gradients (20ng·mL -1 , 10ng·mL -1 , 5ng·mL -1 , 2.5ng·mL -1 , 1ng·mL -1 , 0.5ng·mL -1 , 0ng·mL -1 ), add 50 μL of working concentration 6E5 antibody to 50 μL of each concentration standard, set 0 standard wells and blank control wells at the same time, act in a water bath at 37°C for 1...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a method for preparing a specific monoclonal antibody and its application, and relates to the technical field of detection. By modifying the structure of ractopamine hydrochloride and synthesizing an artificial antigen of ractopamine, a specific monoclonal antibody of ractopamine is prepared. Ractopamine monoclonal antibody is used as the core reagent to prepare colloidal gold immunological test strips for detecting ractopamine. The ractopamine antibody prepared by the present invention is a highly specific monoclonal antibody, which has the characteristics of strong specificity, high sensitivity, and high accuracy; the present invention utilizes the test strip prepared on the basis of the highly specific monoclonal antibody for detecting Residual ractopamine has the characteristics of simplicity, rapidity, sensitivity, accuracy and low cost, and its application has broad prospects.

Description

Technical field: [0001] The invention relates to the preparation of a specific monoclonal antibody of ractopamine, which is used for the rapid determination of clenbuterol in feed and animal food, and belongs to the technical field of detection. Background technique: [0002] Ractopamine (Rac) belongs to the class of β-adrenoceptor agonists (abbreviated as β-agonists) drugs, which can promote animal muscle growth, reduce carcass fat content, improve feed utilization, and significantly increase lean meat percentage. It is one of the most practical β-agonists. However, the cumulative residue of ractopamine in animals poses a potential threat to the safety of consumers, often causing clinical symptoms such as heart palpitations, muscle tremors, nausea and vomiting, fever and chills, especially for patients with heart disease, diabetes, hypertension, etc. more harmful. As early as 1986, Europe issued a ban on beta-agonists. my country's Ministry of Agriculture and the General...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K16/44G01N33/577
Inventor 李柏林左伟勇洪伟鸣吴双孟婷王永娟孙勇王帅兵
Owner 安徽缘远博爱生物技术有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com