Ractopamine semiantigen, artificial antigen and monoclonal antibody, and preparation methods and applications thereof

A technology of ractopamine mono- and ractopamine, which is applied in the field of ractopamine hapten, monoclonal antibody and its preparation, and artificial antigen, can solve the problems of high production cost, low sensitivity of kit, low sensitivity of monoclonal antibody, etc., and achieve Improve sensitivity and potency, reduce cost, and achieve high specificity

Inactive Publication Date: 2015-01-28
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the chromatographic analysis method is relatively sensitive and has high accuracy, it needs the support of large instruments, cumbersome sample pretreatment and professional personnel to operate, and cannot meet the market's requirements for fast and simple analysis methods.
Although the immunoassay method is fast, simple, specific, high-sensitivity, does not require complicated sample pretreatment, and is suitable for on-site batch sample detection, etc., the enzyme-linked immunoassay kit is not ideal for the detection of ractopamine. Therefore, the preparation of ractopamine antibodies with high sensitivity and specificity is the key, and the key to solving the problem depends on the molecular structure of the immune hapten and the cell line that secretes the antibody
[0007] Patent 201210177057.X provides a preparation method of ractopamine hapten and artificial antigen. Although the synthesis method of ractopamine is simple, the purity and yield are high, but the hapten is synthesized based on ractopamine raw material, which will increase Cost of Ractopamine Testing
[0008] Patent CN 102924601B provides a preparation method of ractopamine monoclonal antibody, the hapten is also synthesized based on the structure of ractopamine, not only the production cost is relatively high, but also the sensitivity of preparing monoclonal antibody is low

Method used

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  • Ractopamine semiantigen, artificial antigen and monoclonal antibody, and preparation methods and applications thereof
  • Ractopamine semiantigen, artificial antigen and monoclonal antibody, and preparation methods and applications thereof
  • Ractopamine semiantigen, artificial antigen and monoclonal antibody, and preparation methods and applications thereof

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Comparison scheme
Effect test

Embodiment 1

[0048] 1. Preparation of ractopamine hapten

[0049] The specific experimental steps are:

[0050] (1) Dissolve compound 1 (164mg, 1.0mmol, 1.0eq) in 4mL acetonitrile, add NBS (N-bromosuccinimide) (200mg, 1.1mmol, 1.1eq) under stirring, and stir at room temperature for reaction 3h. Add 25mL H after the reaction 2 O, EA (ethyl acetate) (3×10 mL) extraction, combined organic phases, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, dry column chromatography, DCM (dichloromethane) as eluent, atmospheric pressure After elution, 209 mg of yellow oily substance, intermediate 2, was obtained. Yield: 86%. 1H NMR (400MHz, CDCl 3 )δ7.28(d,J=1.5Hz,1H), 7.02(dd,J=8.3,1.6Hz,1H), 6.91(d,J=8.3Hz,1H), 5.62(s,1H), 2.79( d, J=6.9 Hz, 2H), 2.73 (d, J=7.0 Hz, 2H), 2.14 (s, 3H).

[0051] (2) Intermediate 2 (1.75g, 7.29mmol, 1.0eq) was added to a dry two-necked flask, and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (970mg, 1.46mmol, 0.2eq), swap N 2 R...

Embodiment 2

[0149] Preparation of ractopamine hapten

[0150] The specific experimental steps are:

[0151] (1) Dissolve compound 1 (164mg, 1.0mmol, 1.0eq) in 4mL acetonitrile, add NBS (N-bromosuccinimide) (240mg, 1.5mmol, 1.5eq) under stirring, and stir at room temperature for reaction 4h. Add 25mL H after the reaction 2 O, EA (ethyl acetate) (3×10 mL) extraction, combined organic phases, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, dry column chromatography, DCM (dichloromethane) as eluent, atmospheric pressure After elution, 345 mg of yellow oily substance, intermediate 2, was obtained. Yield: 94.6%. 1H NMR (400MHz, CDCl 3 )δ7.28(d,J=1.5Hz,1H), 7.02(dd,J=8.3,1.6Hz,1H), 6.91(d,J=8.3Hz,1H), 5.62(s,1H), 2.79( d, J=6.9 Hz, 2H), 2.73 (d, J=7.0 Hz, 2H), 2.14 (s, 3H).

[0152] (2) Intermediate 2 (0.88g, 3.6mmol, 1.0eq) was added to a dry two-necked flask, and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (485mg, 0.73mmol, 0.2eq), swap N 2 Rep...

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Abstract

The invention provides an immunodetection technology, and concretely relates to a ractopamine semiantigen and an artificial antigen, preparation methods thereof, and a monoclonal antibody prepared through immunizing mice by using the artificial antigen. The monoclonal antibody is secreted by a hybridoma cell strain 12B2, and the hybridoma cell is preserved China Center for Type Culture Collection with the preservation number of (CCTCC NO):C2014127. The ractopamine monoclonal antibody provided by the invention has the advantages of high titer, high sensitivity and strong specificity, and can be applied in the detection of the residual of ractopamine medicines.

Description

Technical field [0001] The invention relates to the technical field of immunoassays, in particular to a ractopamine hapten, an artificial antigen, a monoclonal antibody, and a preparation method and application thereof. Background technique [0002] Ractopamine (RAC, structural formula such as figure 1 ), also known as clumbaan, is a β-adrenergic receptor agonist with diphenylalkaneamine structure. It is a synthetic synthetic that can produce corresponding physiological effects by binding to β receptors on the cell membrane Natural catecholamine analogs have similar effects to those of epinephrine and norepinephrine. They are generally used clinically to treat diseases such as bronchial asthma and bronchospasm. They can also be used to treat muscular dystrophy, increase muscles, reduce fat accumulation, and The growth of the fetus and newborn is beneficial. [0003] When ractopamine is used to feed animals, it can redistribute nutrients in the animal's body, change the animal's nu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07C229/34C07C227/18C07K14/765C07K16/44G01N33/577G01N33/531
Inventor 乔春华吴康许芸芸
Owner SUZHOU UNIV
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