Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

ScFv (single chain variable fragment) antibody used for detecting norfloxacin, and encoding gene and application thereof

A norfloxacin and antibody technology, which is applied in the field of immunological detection, can solve the problems of being unsuitable for on-site monitoring and detection, cumbersome preparation and screening processes, and high technical requirements for operation, and achieves convenient and easy-to-implement detection methods, high-efficiency methods, and advanced technology. Simple processing effect

Active Publication Date: 2011-10-12
商务部流通产业促进中心
View PDF4 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chemical analysis methods are not suitable for on-site monitoring and testing due to the need for expensive instruments and equipment, high requirements for sample pretreatment, and the need for specialized operators.
Immunological methods are currently mainly Elisa (enzyme-linked immunosorbent) method, but the core reagent of this method-monoclonal antibody, is not conducive to wide application because of its cumbersome preparation and screening process and high requirements for operating techniques

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
  • ScFv (single chain variable fragment) antibody used for detecting norfloxacin, and encoding gene and application thereof
  • ScFv (single chain variable fragment) antibody used for detecting norfloxacin, and encoding gene and application thereof
  • ScFv (single chain variable fragment) antibody used for detecting norfloxacin, and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Coupling Preparation of Immunogen and Detector

[0022] 1. Materials

[0023] Bovine serum albumin (BSA), ovalbumin (OVA), N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylpropyl)carbodiimide (EDC), N, N-dimethylformamide (DMF), norfloxacin (NFLX) standard, dialysis membrane, dialysis membrane treatment fluid, magnetic stirrer, UV spectrophotometer.

[0024] 2. Method

[0025] The mixed acid anhydride method is used to couple norfloxacin to protein macromolecules (BSA, OVA) to prepare immunogens and coating agents.

[0026] 2.1 Synthesis and purification of immunogen and coater

[0027] A. Intermediate Product Preparation

[0028] a. Take 20mg of the drug and put it in a beaker, add 2ml of DMF, if the dissolution is not enough, it can be dissolved in ultrasonic wave;

[0029] b. After fully dissolving, add 25mg EDC and 20mg NHS, and react with magnetic stirring;

[0030] c. Wrap the above beaker with tinfoil, and stir with magnetic force at room temperatur...

Embodiment 2

[0038] Embodiment 2 mouse immunization

[0039] 1. Materials

[0040] Balb / c mice (male, 6 weeks old), complete Freund's adjuvant (Sigma), incomplete Freund's adjuvant (Sigma), 96-well microtiter plate, immunogen NFLX-BSA, coating original NFLX -OVA, coating buffer (Na 2 CO 3 buffer (0.05N, pH9.6), blocking solution (2% skim milk PBS), washing buffer (PBS, 0.1% Tween20PBST), stop solution, mouse anti-norfloxacin monoclonal antibody, HRP-labeled goat anti-mouse IgG antibody (sigma company), TMB chromogenic solution (sigma company).

[0041] 2. Method

[0042] 2.1 Immunization of mice

[0043] a. Take 100 μg of immunogen NFLX-BSA and add an equal amount of complete Freund's adjuvant to make an emulsifier;

[0044] b. First immunization: Take 8 6-week-old Balb / c mice and immunize them by multi-point injection on the back, 0.2ml / mouse, and set a normal saline control group at the same time;

[0045] c. Second immunization and third immunization: Immunization method: Take 10...

Embodiment 3

[0060] Example 3 Extraction of immune mouse splenocyte RNA and obtaining cDNA by RT-PCR

[0061] 1. Materials

[0062] Immune mouse spleen, cell screen, DEPC-treated sterilized PBS, DEPC-treated deionized water, total RNA extraction kit RNA iso Plus (Takara Company), reverse transcription kit (Takara Company).

[0063] 2. Method

[0064] 2.1 Extraction of total RNA from splenocytes

[0065] The total RNA extraction kit RNAiso Plus was used according to the instructions.

[0066] After the RNA was completely dissolved, the quality and quantity of the obtained RNA were measured on a UV spectrophotometer. RNA was frozen at -70°C.

[0067] 2.2 Synthesis of cDNA by reverse transcription

[0068] The obtained mouse spleen RNA was taken out from the -70°C low-temperature refrigerator, and operated in a low-temperature environment. A commercial total RNA extraction kit (Takara) was used according to the instructions.

[0069] The first strand of cDNA was amplified and stored at...

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 provides an scFv (single chain variable fragment) antibody used for detecting norfloxacin, which comprises a heavy chain and a light chain, wherein the heavy chain and the light chain are connected by a flexible linking peptide (Gly4Ser)3, and the amino acid sequences of the heavy chain and the light chain are separately shown in SEQ ID No. 1 and 2. The invention also provides a gene encoding the scFv antibody. The scFv (single chain variable fragment) antibody used for detecting norfloxacin provided by the invention mainly utilizes a competitive Elisa method to quantitatively or qualitatively detect the residue of norfloxacin in a sample, has low pre-treatment requirement on the sample and simple pre-treatment process, and can simultaneously and rapidly detect large quantity of samples; and after first library building, the scFv antibody is simple and convenient to prepare. A main reagent is provided in a working liquid form for using high-specificity anti-norfloxacin scFv antibody, and the detection method is convenient and feasible and has the characteristics of high specificity, high sensitivity, high precision, high accuracy and the like.

Description

technical field [0001] The invention relates to the field of immunological detection, in particular to a scFv recombinant antibody for detecting norfloxacin, its encoding gene and application. Background technique [0002] Norfloxacin (Norfloxacin, NFLX), the chemical name is 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid , is a fluoroquinolone antibacterial drug with a broad-spectrum antibacterial effect, especially high antibacterial activity against aerobic Gram-negative bacilli, and has a good antibacterial effect on the following bacteria in vitro: most bacteria of the Enterobacteriaceae family, including Citrobacter Enterobacter cloacae, Enterobacter aerogenes, Escherichia coli, Klebsiella, Proteus, Salmonella, Shigella, Vibrio, Yersinia, etc. Norfloxacin also has antibacterial activity against multidrug-resistant bacteria in vitro. It also has good antibacterial effect on penicillin-resistant Neisseria gonorrhoeae, Haemophilus influe...

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
IPC IPC(8): C07K16/44C12N15/13C12N15/63C12N7/01G01N33/543C12R1/92
Inventor 郑志明侯晓林金社胜刘文娟赵扬
Owner 商务部流通产业促进中心
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com