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Methadone hapten, preparation method of methadone hapten, methadone antigen, methadone monoclonal antibody and application of methadone monoclonal antibody

A monoclonal antibody and methadone technology, applied in chemical instruments and methods, preparation of organic compounds, preparation of aminohydroxyl compounds, etc., can solve the problems of time-consuming and cumbersome operations, and achieve simple preparation methods, easy-to-obtain raw materials, and high sensitivity Effect

Active Publication Date: 2013-10-23
GUANGZHOU WONDFO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These chromatographic analysis methods have high sensitivity and specificity, but the operation is cumbersome, requires sophisticated instruments and professionally trained operators, and takes a long time

Method used

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  • Methadone hapten, preparation method of methadone hapten, methadone antigen, methadone monoclonal antibody and application of methadone monoclonal antibody
  • Methadone hapten, preparation method of methadone hapten, methadone antigen, methadone monoclonal antibody and application of methadone monoclonal antibody
  • Methadone hapten, preparation method of methadone hapten, methadone antigen, methadone monoclonal antibody and application of methadone monoclonal antibody

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preparation example Construction

[0065] Preparation of Methadone Monoclonal Antibody

[0066] Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, a complete antigen of the invention can be administered to an animal to induce the production of monoclonal antibodies. Monoclonal antibodies can be produced using hybridoma technology or can be produced using recombinant DNA methods.

[0067] The invention provides an anti-methadone monoclonal antibody MTD-5, which is identified as IgG1 type. In a preferred solution of the present invention, the monoclonal antibody MTD-5 is prepared by culturing hybridoma cells. The supernatant of the hybridoma cell culture was taken, the IgG was crudely extracted by saturated ammonium sulfate precipitation, and then the crudely extracted antibody was purified by an affinity chromatography column.

[0068] In a preferred embodiment of the present invention, the monoclonal antibody MTD-5 is prepared by a method...

Embodiment 1

[0102] Example 1 Preparation of methadone hapten

[0103] A methadone hapten, the methadone hapten is prepared by the following method, the specific steps are as follows:

[0104] (1) To reduce methadone to hydroxymethadone, the specific steps are: put methadone hydrochloride (2.4mmol) in a 50ml round bottom flask, add 10ml of anhydrous methanol and stir to dissolve, add sodium borohydride (4.8mmol) under ice bath, The temperature was controlled at 5°C for reaction. After completion of the reaction, add 30ml of ice water and stir to dissolve, extract 3 times with 15ml of ethyl acetate, combine the organic phases, wash twice with saturated aqueous sodium chloride solution, collect the organic phases, and dry over anhydrous sodium sulfate for 1 hour. The mother liquor was collected by filtration, and the solvent was evaporated to dryness.

[0105] (2) Dissolve the hydroxymethadone obtained in step (1) in 5ml of toluene, add succinic anhydride, and react at 110°C for 20 hours. ...

Embodiment 2

[0106]Example 2 Preparation of methadone-OVA complete antigen

[0107] A kind of methadone-OVA complete antigen, described methadone-OVA complete antigen is to use carbodiimide method (EDC) method to make methadone hapten and protein carrier connection, concrete steps are as follows:

[0108] Dissolve 9.0mg (0.029mmol) of methadone hapten in 500μl N,N-dimethylformamide, shake and mix well, add EDC0.04mmol, NHS0.04mmol and stir at 4°C for 4 hours;

[0109] Dissolve the carrier protein OVA0.003mmol in 2.0ml PBS (0.05M, pH7.0), slowly add to the above solution, after the addition, stir and react at room temperature for 24 hours, then dialyze in distilled water for 2 days, change the solution 3 times, Centrifuge in a high-speed refrigerated centrifuge, collect the supernatant, and obtain the methadone-OVA immunogen.

[0110] Visible by UV scanning (see figure 2 ), the maximum absorption peaks of the three in the range of 260-310nm are obviously different, that is, the coupling ...

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Abstract

The invention discloses a methadone hapten, a preparation method of the methadone hapten, a methadone antigen, a methadone monoclonal antibody and application of the methadone monoclonal antibody. The methadone hapten has a structure represented by a formula I shown in a drawing; according to the preparation method of the methadone hapten, methadone is reduced into hydroxyl methadone, the hydroxyl methadone is subjected to esterification reaction with succinic anhydride, and then, the methadone hapten is prepared; the methadone antigen is prepared through connecting the methadone hapten with a protein carrier; the methadone monoclonal antibody is prepared through applying the methadone antigen to an animal for immunization and then using a hybridoma technique or DNA (Deoxyribonucleic Acid) recombination method; and the methadone monoclonal antibody is applied to the preparation of reagents, reagent strips or kits for detecting samples. The methadone hapten, the methadone antigen and the methadone monoclonal antibody have the advantages that the antigen disclosed by the invention has high immunogenicity, and the immunoreactivity of methadone is preserved; the methadone monoclonal antibody is excellent in specificity and high in potency; and a colloidal gold-labeled methadone monoclonal antibody immunological test strip is high in sensitivity and strong in specificity, is simple, convenient and quick, and can be applied to site detection, and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to methadone hapten, antigen, monoclonal antibody and application thereof. Background technique [0002] Methadone hydrochloride (Methadone, MTD) is a synthetic opioid drug that is similar in efficacy to morphine, has analgesic effects, and is accompanied by side effects such as respiratory depression, miosis, and sedation. Because of its strong analgesic effect and low addiction, methadone is often used in the substitution treatment of opioid substance abuse, and has become the main detoxification drug in many countries in the world. Methadone itself has physiological and psychological dependence and tolerance, and its abuse may lead to the same consequences as opioid drugs. It is one of the targets pursued by drug addicts, so it must be strictly managed. At present, it has been included in the norms of narcotic drugs by national and international drug control conventions for strict c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07C219/22C07C213/06C07K14/765C07K14/77C07K14/795C07K16/44G01N33/558G01N33/531
Inventor 邓发亮唐时幸王继华
Owner GUANGZHOU WONDFO BIOTECH
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