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Monoclonal antibody, ELISA method and kit for detecting methyltestosterone

An enzyme-linked immunosorbent reagent and monoclonal antibody technology, applied in the field of veterinary drug residue analysis and immunology, can solve the problems of high cost, cumbersome pretreatment, expensive instruments, etc., and achieve high accuracy, high sensitivity, specificity, and precision. Good results

Inactive Publication Date: 2017-07-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the instrumental analysis method is sensitive, accurate, high-resolution and can carry out qualitative and quantitative research on multi-residue detection, it requires expensive instruments, tedious pre-treatment, and skilled professional operators
If instrumental analysis is used to test a large number of samples, the cost will be very high; and most of the current national testing institutions are only equipped with sophisticated analytical instruments at the provincial and municipal levels

Method used

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  • Monoclonal antibody, ELISA method and kit for detecting methyltestosterone
  • Monoclonal antibody, ELISA method and kit for detecting methyltestosterone
  • Monoclonal antibody, ELISA method and kit for detecting methyltestosterone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The preparation of embodiment 1 hapten

[0028] Synthesis of methyltestosterone 3-p-hydrazinobenzoic acid (MT-CPD) Weigh 60 mg of methyltestosterone and dissolve it in 10 ml of methanol, add 30 mg of p-hydrazinobenzoic acid, add 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

[0029] The preparation of embodiment 2 immunogen and coating former

[0030] 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 solution A. Weigh 25 mg of MT-CPD and dissolve it in 200 μL of N,N-dimethylformamide to form solution B. Weigh 18 mg of DCC and 12 mg of NHS and dissolve in 200 μL of N,N-dimethylformamide to form 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:

[0031] 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.

[0032]

Embodiment 3

[0033] The preparation of embodiment 3 monoclonal antibody

[0034] 3.1 Animal immunity

[0035] 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.

[0036] 3.2 Cell fusion and cloning

[0037] 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 specificity monoclonal antibody capable of distinguishing methyltestosterone. The monoclonal antibody is secreted by a hybridoma cell strain MT9C10 of which the preservation number is CCTCC NO: C201493. The invention further discloses an enzyme linked immunosorbent assay method and kit for detecting the methyltestosterone. Compared with the prior art, the monoclonal antibody disclosed by the invention can be used for distinguishing the methyltestosterone, is high in distinguishing sensitivity and is 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 in particular relates to a monoclonal antibody capable of recognizing methyltestosterone, an enzyme-linked immunosorbent method (ELISA) and a kit for detecting methyltestosterone. Background technique [0002] Methyltestosterone is a synthetic steroidal androgen with the basic structure of cyclopentane polyhydrophenanthrene. In veterinary clinics, methyltestosterone is widely used in animal husbandry to promote protein synthesis and skeletal muscle Growth, by inhibiting the growth of fat, so as to make the muscles more developed, and can stimulate the animal's desire for food, so that the daily weight gain of the animal can be increased, and at the same time, the utilization efficiency of feed can be greatly improved. [0003] Methyltestosterone is usually relatively stable, stays in the animal body for a long time, and remains in the animal body. Long-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N5/20G01N33/74G01N33/577C12R1/91
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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