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Monoclonal antibody, ELISA method and kit for detecting nitroimidazole drugs

A nitroimidazole and monoclonal antibody technology is applied in the field of veterinary drug residue analysis and immunology. The effect of good economic value

Active Publication Date: 2017-09-01
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method can recognize a variety of drugs, polyclonal antibodies are highly variable, not stable enough, and cannot be mass-produced, and the sensitivity of other drugs is low except for dimetidazole

Method used

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  • Monoclonal antibody, ELISA method and kit for detecting nitroimidazole drugs
  • Monoclonal antibody, ELISA method and kit for detecting nitroimidazole drugs
  • Monoclonal antibody, ELISA method and kit for detecting nitroimidazole drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of Immunogen and Coating Original

[0028] 1.1 Preparation of immunogen 3-metronidazole mercaptopropionate and hemocyanin conjugate (MNZ-MPA-DCC-KLH)

[0029] Weigh 17g (0.1mol) of MNZ into a 500mL round bottom flask, and dissolve it with acetone and a small amount of N,N'-dimethylformamide. Add 10g of potassium carbonate and 16g of acetic anhydride, and react in an oil bath at 40°C for 1d. Suction filtration, the solvent was evaporated under reduced pressure to obtain the reaction product of the first step. Dissolve 10 g of the first step product in dichloromethane, add an equal amount of N-bromosuccinimide, light for 3-5 days, filter with suction, and evaporate the filtrate to dryness. The residue was purified by silica gel column, the eluent was ethyl acetate:petroleum ether=1:4, the organic phases were combined, and the solvent was evaporated under reduced pressure to obtain a reddish-brown solid, which was the reaction product of the second ...

Embodiment 2

[0036] Example 2 Preparation of Monoclonal Antibody

[0037] 2.1 Animal immunity

[0038] Balb / C female mice (purchased from Hubei Province CDC Center for Laboratory Animals). The immunization procedure is to take a protein solution containing 50-100 μg of MNZ-MPA-DCC-KLH conjugate, mix it with an equal amount of adjuvant, and then inject it into the mouse body to make it produce specific serum.

[0039] 2.2 Cell fusion and cloning

[0040] 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 positive serum), and immerse it in 75% alcohol for 5 minutes for disinfection. Remove the mouse spleen aseptically, separate the splenocytes, and mix with freshly prepared SP2 / 0 myeloma cells (SP2 / 0 myeloma cells come from our laboratory) by 1~2×10 7 SP2 / 0 with 10 8The ratio of immune spleen cells (1:10 to 1:15) was placed in a 50mL centrifuge tube, the cells were resuspend...

Embodiment 3

[0044] Example 3 Establishment of dimetidazole indirect competition ELISA detection method

[0045] 3.1 Preparation of reagents (the reagents used in this example were prepared by the following methods unless otherwise specified)

[0046] Phosphate buffer: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, KCl 0.2g, add deionized water to 1000mL, adjust the pH to 7.4;

[0047] Coating solution: Take Na 2 CO 3 1.59g, NaHCO 3 2.93g, add deionized water to 1000mL, adjust the pH value to 9.6;

[0048] Washing liquid: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, KCl 0.2g, Tween 200.5mL, add deionized water to 1000mL, adjust the pH to 7.4;

[0049] Blocking solution: Ovalbumin 1g dissolved in 100mL phosphate buffer;

[0050] Substrate solution A: 3,3',5',5-tetramethylbenzidinediamine (TMB) 200mg, absolute ethanol 100mL, add deionized water to 1000mL;

[0051] Substrate B: Na 2 HPO 4 14.6g, citric acid 9.3g, 0.75% urea hydrogen peroxide 6.4mL, add deion...

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PUM

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Abstract

The invention discloses a specific monoclonal antibody capable of recognizing nitroimidazole drugs, which is secreted by the hybridoma cell line DMZ1D5 with the preservation number CCTCC NO: C201499. The invention also discloses an ELISA method and a kit for detecting nitroimidazole drugs. Compared with the prior art, the monoclonal antibody prepared by the present invention can simultaneously recognize five nitro groups of dimethylmetronidazole, isoprenidazole, ronidazole, hydroxypronidazole and hydroxymethylmetronidazole. For imidazole drugs, the enzyme-linked immunosorbent method and kit of the present invention can detect nitroimidazole drug residues in animal feed or edible animal tissues in one measurement, and have the advantages of simplicity, speed, sensitivity, accuracy and the like.

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 nitroimidazole drugs, an enzyme-linked immunosorbent method (ELISA) and a kit for detecting nitroimidazole drugs. Background technique [0002] Nitroimidazoles are synthetic antiprotozoal and antianaerobic drugs. The basic structure is 5-nitroimidazole ring. It has strong antiprotozoal effect and can prevent protozoan infection in various parts; it has a wide antibacterial spectrum and can be used in veterinary clinics in combination with various antibiotics. Because it can promote animal growth and improve feed conversion rate, it is widely used as feed additive in livestock and poultry production. [0003] The irrational use of such drugs has caused residues in animal foods, and then enters the human body through the food chain. Studies have shown that nitroimidazole heterocycles ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/44C12N5/20G01N33/577C12R1/91
Inventor 袁宗辉彭大鹏韩玮王玉莲潘源虎陈冬梅周琪冯亮
Owner HUAZHONG AGRI UNIV
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