Monoclonal antibody, ELISA method and kit for detecting benzodiazepines

An enzyme-linked immunization and drug technology, applied in the field of drug residue analysis and immunology, can solve problems such as inability to recognize norzepam, and achieve the effects of simple sample pretreatment method, good precision and high recognition sensitivity

Active Publication Date: 2017-10-20
HUAZHONG AGRI UNIV
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CN102565409A discloses a detection kit for benzodiazepines, which uses an anti-benzodiazepine monoclonal antibody purchased from American Medi Biotechnology Co., Ltd., which can recognize oxazepam, diazepam, Prazolam and other benzodiazepines, but can not recognize norazepam, temazepam, estazolam

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Monoclonal antibody, ELISA method and kit for detecting benzodiazepines
  • Monoclonal antibody, ELISA method and kit for detecting benzodiazepines
  • Monoclonal antibody, ELISA method and kit for detecting benzodiazepines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The preparation of embodiment 1 immunogen and coating former

[0028] 1.1 Preparation of immunogen

[0029] Weigh 28 mg of nitrazepam in a round-bottomed flask, dissolve in 10 mL of methanol, add 2.8 mg of palladium carbon, feed in hydrogen for 30 mM of reaction, filter off the palladium carbon, and evaporate the reaction solution to dryness to obtain the hapten 7-aminonitrazepam. Add 1 mL of N,N-dimethylformamide to redissolve.

[0030] Weigh 70 mg of bovine serum albumin (BSA) and dissolve it in 10 ml of borate buffer, and add the above-mentioned hapten to the carrier protein dropwise.

[0031] After drawing 0.2ml of 25% glutaraldehyde solution and diluting it 10 times with 1.8ml of PBS, slowly add dropwise to the mixture of hapten and carrier protein, and react overnight at 4°C.

[0032]

[0033] 1.2 Preparation of coating agent

[0034] Weigh 28 mg of nitrazepam in a round-bottomed flask, dissolve in 10 mL of methanol, add 2.8 mg of palladium carbon, feed in h...

Embodiment 2

[0038] The preparation of embodiment 2 monoclonal antibody

[0039] Preparation of hybridoma cells: refer to "Veterinary Immunology" by Du Nianxing.

[0040] The immunogen prepared in Example 1 was used to immunize Balb / C mice (purchased from the Experimental Animal Center of Hubei Academy of Medical Sciences). The immunization procedure is as follows: for basic immunization, a protein emulsion containing 100 μg of immunogen emulsified with an equal volume of Freund’s complete adjuvant is injected subcutaneously at the back of the neck of the mouse; 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 detected by indirect ELISA method. Immunization qualified mice (high titer, good sensitivity) stop immunization in preparation for fusion.

[0041] At the time of fusion, one Balb / C mouse that had undergone the final boo...

Embodiment 3

[0046] The establishment of embodiment 3 indirect competition ELISA detection method

[0047] 3.1 Preparation of reagents (the reagents used in this example were prepared by the following methods unless otherwise specified)

[0048] Phosphate buffer: NaCl 8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, KCl 0.2g, add double distilled water to 1000mL, adjust pH to 7.4;

[0049] Coating solution: Take Na 2 CO 3 1.59g, NaHCO 3 2.93g, add triple distilled water to 1000mL, adjust the pH value to 9.6;

[0050] 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 double distilled water to 1000mL, adjust pH to 7.4;

[0051] Blocking solution: Ovalbumin 1g dissolved in 100mL phosphate buffer;

[0052] Substrate solution A: 3,3',5',5-tetramethylbenzidinediamine (TMB) 200mg, absolute ethanol 100mL, add double distilled water to 1000mL;

[0053] Substrate B: Na 2 HPO 4 14.6g, citric acid 9.3g, 0.75% urea hydrogen peroxide 6....

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a specific monoclonal antibody capable of recognizing benzodiazepines such as diazepam and nitrazepam and an enzyme for detecting benzodiazepines such as diazepam and nitrazepam Immunization methods and kits. The monoclonal antibody of the present invention is secreted by the hybridoma cell 3D7 whose deposit number is CCTCC NO: C201352. Compared with the prior art, the monoclonal antibody prepared by the invention can simultaneously recognize various benzodiazepines such as diazepam and nitrazepam. The ELISA method and the kit of the invention have the advantages of high detection efficiency and sensitivity, good precision and accuracy, and the like.

Description

technical field [0001] The invention belongs to the technical field of drug residue analysis and immunology, and specifically relates to a monoclonal antibody for detecting benzodiazepines, and the invention also relates to an enzyme-linked immunosorbent method and reagents for detecting benzodiazepines box. Background technique [0002] Benzodiazepines are a class of sedatives that act on the central nervous system, which can cause animals to be sedated, lethargic, or indifferent to the surrounding environment. There are many kinds of drugs, most of which are derivatives of 1,4-benzodiazepine, whose basic structure is composed of two benzene rings and a heterocyclic ring containing 7 atoms. It is usually used in the process of transporting food-producing animals to slaughterhouses to reduce stress and reduce animal mortality. In addition, since it makes the animal lethargic, it plays an indirect role in promoting the growth of the animal. Therefore, the use of such drugs...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/44C12N5/20G01N33/577C12R1/91
Inventor 袁宗辉彭大鹏王涓潘源虎王玉莲冯亮刘振利
Owner HUAZHONG AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap