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Albendazole-2-amino sulfone residue detection monoclonal antibody as well as preparation method and application thereof

A monoclonal antibody and albendazole technology, applied in the field of veterinary drug residue analysis and immunology, to achieve the effect of simple sample processing method, good accuracy and precision, and easy operation

Inactive Publication Date: 2013-09-11
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the defect that the prior art cannot recognize albendazole residue marker albenda-2-aminosulfone, and provide a kind of specific recognition albendazole residue marker albenda-2- monoclonal antibody to aminosulfone

Method used

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  • Albendazole-2-amino sulfone residue detection monoclonal antibody as well as preparation method and application thereof
  • Albendazole-2-amino sulfone residue detection monoclonal antibody as well as preparation method and application thereof
  • Albendazole-2-amino sulfone residue detection monoclonal antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Preparation of Immunogen and Coating Original

[0055] Accurately weigh ABZSO 2 NH 2 18.00mg, completely dissolved in 5mL N,N-dimethylformamide as liquid A. Accurately weigh 90.00 mg of BSA and fully dissolve it in 40 mL of phosphate buffer (pH 7.2) to form B solution. At room temperature (20-25°C, the same below), add liquid A to liquid B dropwise under magnetic stirring. After the two are evenly mixed, slowly add 1.5 mL of glutaraldehyde solution (GA) with a volume ratio of 15%. . Magnetic stirring was carried out at room temperature for 2 h. After the reaction was completed, the reaction solution was transferred into a dialysis bag, and dialyzed in normal saline at 4°C for 3 days, and the dialysate was changed 3 times a day. Centrifuge at 4000r / min for 5min, take the supernatant and freeze-dry to obtain the conjugate ABZSO 2 NH 2 -GA-BSA, stored at -20°C, used as an immunogen.

[0056] In the above steps, BSA (purchased from sigma company) was rep...

Embodiment 2

[0057] Embodiment 2: Preparation of monoclonal antibody

[0058] Preparation of hybridoma cell lines: refer to the method in Xue Qingshan's "Principles and Techniques of In Vitro Culture" Science Press, 2001 edition: ABZO prepared in Example 1 2 NH 2 -GA-BSA conjugate is the immunogen, and Balb / C female mice are immunized. One basic immunization and three to five booster immunizations were used in the immunization program. For the first immunization, a protein emulsion containing 100 μg of immunogen emulsified with an equal volume of Freund’s complete adjuvant was injected subcutaneously on the back of the neck of the mouse for basic immunization, and then every 15 days with protein emulsion containing 100 μg of immunogen emulsified with Freund’s incomplete adjuvant Protein emulsion of immunogen for booster immunization. From the third immunization, the tail blood was collected on the 8th day after each immunization, the serum was separated, and the serum antibody titer was...

Embodiment 3

[0060] Embodiment 3: the establishment of indirect competitive ELISA detection method

[0061] 1 reagent preparation

[0062] Carbonate buffer (pH 9.6): Accurately weigh Na 2 CO 3 1.59g, NaHCO 3 2.93g, dissolved in a small amount of ultrapure water, and adjusted to 1000mL.

[0063] Washing solution (pH 7.4): Accurately weigh 8.00g of NaCl, KH 2 PO 4 0.20g, Na 2 HPO 4 12H 2 O 2.90g, KCl 0.20g, dissolved in a small amount of ultrapure water, Tween 20 0.50mL was added, and the volume was adjusted to 1000mL.

[0064] Phosphate buffer (pH 7.4): Accurately weigh 8.00g of NaCl, KH 2 PO4 0.20g, Na 2 HPO 4 12H 2 Dissolve 2.90g of O, 0.20g of KCl in a small amount of ultrapure water, and dilute to 1000mL.

[0065] Blocking solution: Accurately weigh 10.00 g of ovalbumin, add 1000 mL of phosphate buffer, stir and mix until the protein is completely dissolved.

[0066] Substrate mixture: Accurately absorb 10 mL of Substrate B solution (purchased from Wuhan Feiyuan Techno...

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Abstract

The invention discloses an albendazole-2-amino sulfone residue detection monoclonal antibody as well as a preparation method and an application thereof, and belongs to a hybridoma cell GA / 2A11 with the collection number of CCTCCNO:C201142. The preparation method comprises the following steps: a, coupling hapten albendazole-2-amino sulfone with bovine serum albumin, thereby obtaining an immunogen; b, coupling the hapten albendazole-2-amino sulfone with ovalbumin, thereby obtaining a coating antigen; c, preparing a hybridoma cell strain by using the immunogen; d, coating a solid phase carrier with the coating antigen; e, extracting a sample to be detected by using ethyl acetate under the action of potassium hydroxide, thereby obtaining an object to be detected; f, performing enzyme-linked immunity detection on the object to be detected. A kit comprises: (1) an elisa plate coated with the coating antigen ABZSO2NH2-GA-OVA, (2) an ABZSO2NH2 standard substance solution, (4) a Goat anti Mouse IgG antibody working solution marked by a horse radish peroxidase, (5) a concentrated phosphate buffering solution, (6) a concentrated washing solution, (7) a substrate mixing solution and (9) a stop solution. The invention further discloses the application of the kit in the detection of albendazole-2-amino sulfone residue detection. The kit has the characteristics of being simple, convenient, rapid, sensitive and accurate.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue analysis and immunology. It specifically relates to a monoclonal antibody that can recognize the albendazole residue marker albendazole-2-aminosulfone, and also relates to a kind of albendazole residue marker that can recognize albendazole-2-aminosulfone The preparation method of the monoclonal antibody also relates to the use of a monoclonal antibody capable of recognizing albendazole residue marker albendazole-2-aminosulfone. Background technique [0002] Albendazole is a benzimidazole anthelmintic widely used in veterinary clinics. It has the characteristics of broad spectrum, high efficiency and low toxicity. It is widely used in ruminants such as cattle and sheep, and has repellent effect on common nematodes, trematodes and tapeworms. After albendazole is administered, it is rapidly metabolized into albendazole sulfoxide and albendazole sulfone through the first-pass effect ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44C12N5/20G01N33/577G01N33/543C12R1/91
Inventor 袁宗辉蒋能辉彭大鹏王玉莲黄玲利陈冬梅陶燕飞戴梦红刘振利
Owner HUAZHONG AGRI UNIV
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