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

Method for detecting Listeria monocytogenes and monoclonal antibodies

A technology for mononucleosis and Listeria, applied in the field of microbial detection, can solve the problems of relying on imports of detection products, inability to adapt to a large number of sample screening, and long detection cycles

Active Publication Date: 2014-07-23
UNIV OF SHANGHAI FOR SCI & TECH
View PDF3 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the detection of Listeria monocytogenes mainly relies on the biochemical identification stipulated in the national standard. The disadvantage is that the operation is cumbersome, the detection cycle is long, and it cannot adapt to the screening of a large number of samples.
Immunological detection is the fastest, accurate and stable rapid detection method that has emerged in recent years, but all detection products rely on imports. At present, there is no rapid detection product with completely independent intellectual property rights in my country that can be used in detection practice. The patent is based on Listeria monocytogenes is the detection target, and the claimed technology involves rapid detection products for Listeria monocytogenes

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
  • Method for detecting Listeria monocytogenes and monoclonal antibodies
  • Method for detecting Listeria monocytogenes and monoclonal antibodies
  • Method for detecting Listeria monocytogenes and monoclonal antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1. Preparation of anti-Listeria monocytogenes monoclonal antibodies 6D4H7C8B6 and 1B4E7A6C9

[0077] 1. Preparation of immunogen and positive standard

[0078] Listeria monocytogenes (ATCC No.43251) was inoculated in Listeria broth, cultured at 37°C for 24h, picked a single colony in Listeria broth, cultured at 37°C, 150r / min shaking for 17h, counted , adding 0.3% formaldehyde solution to inactivate at room temperature for 1 day. Adjust the concentration of Listeria monocytogenes (ATCC No.43251) to 5×10 with normal saline 9 CFU / ml was used as immunogen; the concentration was adjusted to 10 with normal saline 8 cfu / ml Listeria monocytogenes liquid as a positive control standard, Listeria broth as a negative control standard.

[0079] 2. Preparation of monoclonal antibodies

[0080] 1) Experimental animals: Three 8-week-old, weighing about 20 g female Balb / c mice were selected as experimental animals.

[0081] 2) Immunization method: each mouse was intraperit...

Embodiment 2

[0106] Example 2. Characterization of monoclonal antibodies 6D4H7C8B6 and 1B4E7A6C9

[0107] 1. Monoclonal Antibody Subclass Identification

[0108] 1. Antigen coating: Coat goat anti-mouse secondary antibody IgG+A+M with 0.01M PBS, 50 μl per well, coat overnight at 4°C, discard the liquid in the well the next day, and wash the plate 3 times.

[0109] 2. Blocking: add 200 μl of 1% BSA to each well, and block overnight at 4°C. Pat the board dry the next day without washing it.

[0110] 3. Add monoclonal antibody hybridoma cell supernatant, 8 microwells for each sample, 50 μl per well. Incubate for 1 hour at 37°C.

[0111] 4. After washing the plate 4 times, add specific binding rabbit anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, κ, λ, and incubate at 37°C for 1 hour.

[0112] 5. After washing the plate 4 times, add diluted horseradish peroxidase-labeled anti-rabbit secondary antibody IgG (H+L) to each well, and incubate at 37°C for 30 minutes.

[0113] After washing the ...

Embodiment 3

[0127] Example 3. Composition, preparation and application of an enzyme-linked immunosorbent assay kit for detecting Listeria monocytogenes

[0128] 1. The enzyme-linked immunosorbent assay kit consists of the following materials:

[0129] (1) ELISA plate with pre-coated antibody: Dilute with 0.02M acetate buffer (pH 2.0) solution, coat 96-well ELISA plate with anti-Listeria monoclonal antibody 1B4E7A6C9, 100 μl per well. Incubate overnight at 4°C, block and wash according to conventional ELISA methods.

[0130] (2) Listeria positive control standard and negative control standard.

[0131] (3) Anti-Listeria monoclonal antibody 6D4H7C8B6 labeled with horseradish peroxidase.

[0132] (4) Enzyme-labeled antibody diluent: 0.01M PBS, pH7.6.

[0133] (5) 10× concentrated lotion: 0.1M phosphate buffer containing 0.5% Tween-20 and 0.2% sodium azide, pH 7.4, just dilute the concentrated lotion 10 times before use.

[0134] (6) Chromogenic solution A and chromogenic solution B. Mix...

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 belongs to the field of microbiological detection and in particular relates to a method for detecting Listeria monocytogenes and two Listeria monocytogenes resisting monoclonal antibodies generated in the same monoclonal antibody preparation process for the method. The monoclonal antibodies can be specifically bonded with the Listeria monocytogenes and is generated by mouse hybridoma cell line 6D4H7C8B6, CGMCC No.8002 or 1B4E7A7C9, CGMCC No.8765. The invention also provides application of the Listeria monocytogenes resisting monoclonal antibodies.

Description

technical field [0001] The invention belongs to the field of microorganism detection. Specifically, the present invention relates to a method for detecting Listeria monocytogenes, two strains of monoclonal antibodies against Listeria monocytogenes used in the method, and hybridoma cells producing the monoclonal antibodies . Background technique [0002] Listeria monocytogenes (Listeria monocytogenes) is a short Gram-positive non-spore facultative anaerobic bacilli, the most pathogenic bacteria in the Listeria genus, and the only one that is pathogenic to humans. Typical intracellular parasites that can cause severe zoonotic diseases such as meningitis, sepsis, miscarriage, and mononucleosis. In 1988, the World Health Organization (WHO) published the "Foodborne Listeriosis Advice" to guide countries around the world on how to prevent Listeria contamination and poisoning. Since then, LM has become a new important food-borne disease pathogen, and WHO listed it as the four ma...

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 Applications(China)
IPC IPC(8): G01N33/577C07K16/12C12N5/20C12R1/91
CPCC07K16/1296G01N33/56911
Inventor 刘箐李森曾海娟
Owner UNIV OF SHANGHAI FOR SCI & TECH
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