Anti-ractopamine antibody and application thereof

A technology of ractopamine and single-chain antibody, which is applied in the application, use of vectors to introduce foreign genetic material, and cells modified by introducing foreign genetic material. Simple, sensitive, and easy-to-produce effects

Inactive Publication Date: 2011-01-26
北京维德维康生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation of monoclonal antibodies or polyclonal antibodies must be obtained through cell culture. The entire production process is complicated, time-consuming, expensive, and difficult to operate

Method used

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  • Anti-ractopamine antibody and application thereof
  • Anti-ractopamine antibody and application thereof
  • Anti-ractopamine antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Preparation and functional detection of antibodies

[0057] 1. Preparation of ractopamine single-chain antibody

[0058] (1) Screening of antibodies

[0059] Six-month-old male Balb / c mice were taken, and the total RNA of spleen cells was extracted by one-step Trizol method, purified to obtain mRNA, and then reverse transcribed to obtain cDNA. A full set of primers were used to amplify the heavy chain variable region (VH) and light chain variable region (VL) genes respectively with cDNA as the template. Splicing into a single-chain antibody gene (ScFv), then connecting the ScFv with the vector pCANTAB5E to obtain pCANTAB5E / ScFv, and transforming E. coli TG1 to obtain a mouse-derived non-immune single-chain antibody library. Phage particles with specific anti-multiple ractopamine single-chain antibodies were screened by ELISA, and their corresponding nucleotide sequences were obtained by sequencing.

[0060] The single-chain antibody is composed of a heavy ...

Embodiment 2

[0090] Example 2. ELISA kit for detecting ractopamine and preparation thereof

[0091] 1. The ELISA kit consists of the following substances:

[0092] 1. ELISA plate coated with ractopamine hydrochloride and carrier protein conjugate (RCT-BSA);

[0093] 2. Ractopamine antibody: the single-chain antibody described in Example 1. The concentration of the antibody working solution was 5.0 ng / mL, and the antibody working solution was obtained by diluting the purified antibody in Example 1 with the sample diluent;

[0094] 3. Ractopamine standard: Ractopamine standard is ractopamine hydrochloride, the concentration of standard solution is 0μg / L, 0.15μg / L, 0.45μg / L, 1.35μg / L, 4.05μg / L and 12.15μg / L respectively ; Ractopamine hydrochloride was purchased from Sigma-Aldrich company in the United States; product catalog number is 34198; diluted to the above concentrations with sample diluent;

[0095] 4. Enzyme-labeled secondary antibody: horseradish peroxidase (HRP)-labeled mouse ant...

Embodiment 3

[0148] Embodiment 3, the test paper for detecting ractopamine and its preparation and application

[0149] 1. The structure of the test paper

[0150] The test paper is composed of a sample absorption pad, a colloidal gold pad, a reaction film and a water absorption pad;

[0151] The sample absorption pad, the colloidal gold pad, the reaction membrane and the water absorption pad are connected in sequence, the end of the sample absorption pad is connected to the beginning of the colloidal gold pad, the end of the colloidal gold pad is connected to the beginning of the reaction membrane, and the end of the reaction membrane is connected to the beginning of the colloidal gold pad. The beginning ends of the absorbent pads are connected;

[0152] The colloidal gold pad is coated with colloidal gold-labeled ractopamine antibody (the protein shown in sequence 1 in the sequence listing);

[0153] The reaction membrane has a detection area and a quality control area, and the detecti...

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Abstract

The invention discloses an anti-ractopamine antibody and application thereof. The antibody consists of a heavy chain variable region, a short peptide for connecting the heavy chain variable region with a light chain variable region, and the light chain variable region connected in turn, wherein an amino acid sequence of the heavy chain variable region is shown as No.9-No.130 amino acid residues from an N terminal in a sequence 1; and an amino acid sequence of the light chain variable region is shown as No.146-No.250 amino acid residues from the N terminal in the sequence 1. A kit and a colloidal gold test paper card have the advantages of high sensitivity, high accuracy, high precision, low cost, simple operation, short detection time, suitability for various units, simple storage, long quality guarantee period, and can rapidly detect mass samples simultaneously and realize on-site high-throughput rapid detection. Therefore, the antibody, the kit, the colloidal gold test paper and the detection method have important significance for detecting ractopamine.

Description

technical field [0001] The present invention relates to a ractopamine antibody and its application. Background technique [0002] Ractopamine (RCT) is a phenethanolamine. For animal husbandry and fishery, it is a kind of "nutrient redistribution agent", which belongs to beta stimulant. As a substitute for "clenbuterol", ractopamine has good effect, high economic return, and is widely used; for medicine, it is a A cardiotonic drug used to treat obesity and muscular dystrophy. However, when the cumulative intake of a person exceeds a certain value, or when a person consumes high residual ractopamine in the internal organs, a toxic reaction occurs. The symptoms are increased skeletal muscle contraction, which destroys the fusion between fast-twitch muscle fibers and slow-twitch muscle fibers, causing muscle tremors. The muscles of the limbs and face are especially obvious. Other symptoms of poisoning include tachycardia, arrhythmia, abdominal pain, muscle pain, nausea and dizz...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44C12N15/13C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10G01N33/566G01N33/577G01N33/531
Inventor 沈建忠江海洋李杰超吴小平王战辉徐飞刘丹王照鹏程晶杨光王富伟李艳伟
Owner 北京维德维康生物技术有限公司
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