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Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting androgens

An enzyme-linked immunosorbent assay and monoclonal antibody technology, which is applied in the field of veterinary drug residue analysis and immunology, can solve the problems of inability to detect multiple drug components, and no enzyme-linked immunosorbent kit that can detect androgen drugs at the same time. High accuracy, good specificity, good precision

Active Publication Date: 2015-04-29
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing enzyme-linked immunosorbent methods and kits can only identify 1-2 kinds of androgen drugs, and cannot detect multiple drug components at the same time, let alone detect nandrolone, methyltestosterone, testosterone, and trenbolone at the same time ELISA Kit for Androgen Drugs

Method used

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  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting androgens
  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting androgens
  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting androgens

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Experimental program
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Effect test

Embodiment 1

[0031] The preparation of embodiment 1 hapten

[0032] Synthesis of the hapten methyltestosterone 3-p-hydrazinobenzoic acid (MT-CPD): Weigh 60 mg methyltestosterone and dissolve it in 10 ml methanol, add 30 mg p-hydrazinobenzoic acid, add an appropriate amount of Na 2 CO 3 , reacted at room temperature, TLC monitored the reaction progress, and the reaction was complete in about 2 hours. Blow dry with nitrogen, adjust the acidity of the reaction solution with dilute hydrochloric acid, add ethyl acetate for extraction, collect the organic layer, and blow dry with nitrogen to obtain the final product MT-CPD.

Embodiment 2

[0033] The preparation of embodiment 2 immunogen and coating former

[0034] Synthesize the complete antigen MT-CPD-KLH (OVA) by DCC method: take 2ml / 10mg of KLH or 20mg of OVA and dissolve it in 15mL of PBS to make liquid A. Weigh 25 mg of MT-CPD and dissolve it in 200 μL N,N-dimethylformamide to form solution B. Weigh 18mg DCC and 12mg NHS respectively and dissolve in 200μL N,N-dimethylformamide as solution C. Under room temperature, liquid B and liquid C were mixed and reacted for 12 hours to obtain liquid D. Slowly drop solution D into solution A, and react overnight in ice bath. The reaction process is as follows:

[0035] The supernatant was dialyzed against PBS at 4°C for 7 days, and the dialysate was changed twice a day to remove unreacted small molecular substances. The MT-CPD-KLH or MT-CPD-OVA solution was lyophilized and stored in a -20°C refrigerator, which were immunogens and coatings, respectively.

[0036]

Embodiment 3

[0037] The preparation of embodiment 3 monoclonal antibody

[0038] 3.1 Animal immunity

[0039] With reference to Yang Hanchun's "Animal Immunology", the MT-CPD-KLH immunogen prepared by the inventor was used to immunize Balb / C mice (purchased from the Experimental Animal Center of the Center for Disease Control and Prevention of Hubei Province). Equal volume of complete Freund's adjuvant was emulsified, and injected subcutaneously at multiple points on the back of the mice, and then boosted immunization once every 2 weeks, and emulsified with incomplete adjuvant. Finally, intraperitoneal injection three days before fusion (preferably resting for one month after the end of immunization) for booster immunization, doubling the amount of antigen without adding adjuvant.

[0040] 3.2 Cell fusion and cloning

[0041] At the time of fusion, take a Balb / C mouse that has undergone the final booster immunization, sacrifice it by bleeding from the eye socket (collect the serum, it is...

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Abstract

The invention discloses a specific monoclonal antibody capable of distinguishing nandrolone, methyltestosterone, testosterone and trenbolone at the same time. The monoclonal antibody is secreted by a hybridoma cell strain NT4D12 of which the preservation number is CCTCC No.C201494. The invention further discloses an enzyme-linked immunosorbent assay method and kit for detecting the androgens. Compared with the prior art, the monoclonal antibody prepared by the invention can be used for distinguishing four androgens namely the nandrolone, the methyltestosterone, the testosterone and the trenbolone, is high in detection sensitivity and good in specificity. The ELISA method and the kit, disclosed by the invention, have the advantages of high detection sensitivity, high accuracy and high precision.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue analysis and immunology, and specifically relates to a monoclonal antibody capable of recognizing androgen drugs, an enzyme-linked immunosorbent method (ELISA) and a kit for detecting androgen drug residues. Background technique [0002] Anabolic androgen is a class of synthetic steroidal sex hormones with the basic structure of cyclopentane polyhydrophenanthrene. This type of drug plays an important role in veterinary medicine. It can promote protein synthesis and skeletal muscle growth. By inhibiting The growth of fat makes the muscles more developed, and can stimulate the animal's desire for food, increase the daily weight gain of the animal, and greatly improve the utilization efficiency of feed. If a large dose of androgen is taken, it will stimulate the hematopoietic function of the bone marrow, especially to promote the production of red blood cells and stimulate the growth ...

Claims

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Application Information

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IPC IPC(8): C07K16/26C12N5/20G01N33/72G01N33/577C12R1/91
Inventor 袁宗辉王惠彭大鹏潘源虎王玉莲陈冬梅冯亮朱永利刘振利
Owner HUAZHONG AGRI UNIV
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