Listeria monocytogenes enzyme-linked immunosorbent assay kit

A mononuclear cell hyperplasia and enzyme-linked immunological reagent technology, applied in the fields of biotechnology and immunology, can solve the problems of inability to adapt to a large number of sample screening, complicated operations, and dependence on imports of detection products.

Active Publication Date: 2014-07-23
UNIV OF SHANGHAI FOR SCI & TECH
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  • 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

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  • Listeria monocytogenes enzyme-linked immunosorbent assay kit
  • Listeria monocytogenes enzyme-linked immunosorbent assay kit
  • Listeria monocytogenes enzyme-linked immunosorbent assay kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Preparation of anti-Listeria monocytogenes monoclonal antibodies 1B4E7A6C9 and 6D4H7C8B6

[0057] 1. Preparation of immunogen and positive standard

[0058] Listeria monocytogenes (ATCC No.43251) was inoculated in Listeria broth, cultured at 37°C and shaken at 150r / min for 17 hours, counted, and inactivated by adding 0.3% formaldehyde solution 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 was used as a positive control standard, and Listeria broth was used as a negative control standard.

[0059] 2. Preparation of monoclonal antibodies

[0060] 1) Experimental animals: Three 8-week-old, weighing about 20 g female Balb / c mice were selected as experimental animals.

[0061] 2) Immunization method: each mouse was intraperitoneally injected with 0.2ml of immunogen, and the same dose was...

Embodiment 2

[0083] Example 2. Characterization of monoclonal antibodies 1B4E7A6C9 and 6D4H7C8B6

[0084] 1. Monoclonal Antibody Subclass Identification

[0085] 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.

[0086] 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.

[0087] 3. Add monoclonal antibody hybridoma cell supernatant, 8 microwells for each sample, 50 μl per well. Incubate for 1 hour at 37°C.

[0088] 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.

[0089] 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.

[0090] 6. After washing t...

Embodiment 3

[0104] Example 3. The composition, preparation and application of the ELISA kit for detecting Listeria

[0105] 1. The enzyme-linked immunosorbent assay kit consists of the following materials:

[0106] (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.

[0107] (2) Listeria positive control standard and negative control standard.

[0108] (3) Anti-Listeria monoclonal antibody 6D4H7C8B6 labeled with horseradish peroxidase.

[0109] (4) Enzyme-labeled antibody diluent: 0.01M PBS, pH7.6.

[0110] (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.

[0111] (6) Chromogenic solution A and chromogenic solution B. Mix equal volumes of liquid A and liquid...

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Abstract

The invention discloses a Listeria monocytogenes enzyme-linked immunosorbent assay kit. The kit contains two monoclonal antibodies which can be specifically bonded to the Listeria monocytogenes, wherein one monoclonal antibody is 1B4E7A6C9 specially used for capturing the antibody, and other monoclonal antibody is 6D4H7C8B6 used for detecting the antibody. The kit is proved to be capable of detecting the Listeria monocytogenes specifically in a high-efficiency manner and having no cross reaction with other 68 common pathogenic bacteria through substantive tests, and therefore, the kit has good performances on detecting the pathogenic bacteria.

Description

technical field [0001] The invention belongs to the fields of biotechnology and immunology, in particular to an enzyme-linked immunoassay kit for detecting Listeria monocytogenes. 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 major food-borne pathogens in the 1990s along with E.coli O157, Salmonella and Staphylococcus aureus. The bacteria mainly contaminate milk and dairy ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56911G01N2333/195
Inventor 刘箐郭慧琴吴淑燕
Owner UNIV OF SHANGHAI FOR SCI & TECH
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