Monoclonal antibody of ractopamine and preparation method and application thereof

A technology of ractopamine monoclonal antibody, which is applied in the field of immunochemistry, can solve the problem of low sensitivity of ractopamine monoclonal antibody, and achieve the effect of high sensitivity and convenient use

Active Publication Date: 2010-12-22
JIANGSU HUACHUANG MEDICINE RES & DEV PLATFORM MANAGEMENT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The technical problem to be solved in the present invention is: aiming at the current situation that the sensitivity of the existing monoclonal antibody to ractopamine is not high, a mon

Method used

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  • Monoclonal antibody of ractopamine and preparation method and application thereof
  • Monoclonal antibody of ractopamine and preparation method and application thereof
  • Monoclonal antibody of ractopamine and preparation method and application thereof

Examples

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Effect test

Embodiment 1

[0029] Reagent and material preparation

[0030] Ractopamine (sigma, 34198), glutaric anhydride (sigma, G3806), sodium borate (sigma, S9640), pyridine (Sinopharm Chemical Reagent Co., Ltd.), tri-n-butylamine (Sinopharm Chemical Reagent Co., Ltd.), chloroformic acid Isobutyl ester (Sinopharm Group Chemical Reagent Co., Ltd.), 1,4-dioxane (Sinopharm Group Chemical Reagent Co., Ltd.), bovine serum albumin (BSA) (Jackson, 001-000-173), ovalbumin ( OVA) (Sigma, A5503), human serum albumin (HSA) (Sigma, A9511), sodium chloride (Amresco, 0241), potassium chloride (Amresco, 0395), potassium dihydrogen phosphate (Sigma, P9791), phosphoric acid Sodium hydrogen disodium (Sigma, 71639), Freund's complete adjuvant (Sigma, F5881), Freund's incomplete adjuvant (Sigma, F5506), tetramethylbenzidine (TMB) (Amresco, 0759), HAT (Sigma, H0262) and HT (Sigma, H0137), goat anti-mouse secondary antibody IgG-HRP (Jackson, 115-035-044), dimethylsulfoxide (DMSO) (Applichem, 0231), polyethylene glycol 4...

Embodiment 2

[0115] drug cross reaction test

[0116] According to the monoclonal antibody screening indirect competition ELISA method established in Example 1, carry out the indirect competition ELISA of the monoclonal antibody of ractopamine and clenbuterol, albuterol, terbutaline, and draw the inhibition curve, such as Figure 8 . Calculate the IC of the competitor 50 Value and cross-reactivity rate, the cross-reactivity rate is less than 0.01%. Therefore, the anti-ractopamine monoclonal antibody prepared in the present invention has very good specificity.

Embodiment 3

[0118] 1. This example is an example of the application of the monoclonal antibody 5D8' in the establishment of an ELISA method for the detection of ractopamine residues in the present invention, which can be used for the detection of ractopamine residues in pig urine, pork, and pig viscera.

[0119] 2. The detection principle of the kit in the present invention is the indirect competitive ELISA method, and the operation steps are the same as the conventional indirect ELISA method. The original OVA-RAC is coated on the microwell plate, and 10, 5 , 2.5, 1.25, 0.616, the ractopamine standard substance of 0.313ng / ml, add the sample in all the other wells, then add the monoclonal antibody against ractopamine (the monoclonal antibody that chooses in this embodiment is 5D8 '), residual in the sample The ractopamine and the coated antigen compete with the anti-ractopamine antibody at the same time, then add the enzyme-labeled secondary antibody, TMB color development, and read the OD ...

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Abstract

The invention relates to a monoclonal antibody of ractopamine and a preparation method and application thereof, which belongs to the field of immunochemical technology. In the invention, the ractopamine and carrier proteins BSA, HSA and OVA are coupled by a mixed anhydride method to synthesize artificial immunogens RAC-BSA and RAC-HSA and coating antigen RAC-OVA; a Balb/c mouse is immunized by the synthesized artificial immunogens RAC-BSA and RAC-HSA; the spleen cell of the immune mouse is fused with the SP2/0 myeloma cell and the two are coated by the coating antigen RAC-OVA; AND an indirect ELISA method and an indirect competition ELISA method are established to screen out a hybridoma cell strain (5D8) capable of stably excreting specific antibody. The Balb/c mouse is immunized by the obtained cell strain to prepare ascites which is purified by an octanoic acid-ammonium sulfate method and an ion exchange method, and the titer of the purified antibody is more than 5.12*10<5>. The monoclonal antibody has strong specificity, is used for preparing a ractopamine residue detection kit and colloidal gold test paper strips and can sensitively and quickly detect the ractopamine residue.

Description

technical field [0001] The invention relates to a monoclonal antibody and its application, belonging to the technical field of immunochemistry. Background technique [0002] Ractopamine hydrochloride (RAC), is a phenamine β 2 An adrenergic agonist, commonly used clinically in the treatment of bronchial asthma. In addition, as one of the most practical β-stimulants at present, it will also be used in sports competitions to strengthen the muscles of animals or people to improve sports performance. The International Olympic Committee has listed it as a banned drug. A series of animal experiments in the 1980s showed that when the dosage was 5-10 times the therapeutic dose, ractopamine could enhance fat catabolism, promote protein synthesis, significantly increase ketone body lean meat rate, and improve feed conversion rate Therefore, ractopamine can be used as a growth-promoting additive in the production of animal products, but human consumption of animal products containing ...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44G01N33/577C12R1/91
Inventor 唐宏王晓艳崔迎利王文夏文静尹丽梅韩晓娟戴威林纪昀杨利倪同浩郁靓汪纪宗
Owner JIANGSU HUACHUANG MEDICINE RES & DEV PLATFORM MANAGEMENT CO LTD
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