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Double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) method for detecting listeria in food on basis of monoclonal antibodies

A monoclonal antibody and double antibody sandwich technology, applied in the field of immunoassay, can solve the problems of human health threats from refrigerated food, blood and brain tissue infections, etc.

Active Publication Date: 2016-04-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Infection with Listeria can cause symptoms such as muscle pain, nausea, and diarrhea. In severe cases, it can cause blood and brain tissue infections. Moreover, since the bacteria can still grow and reproduce at a refrigeration temperature of 4°C, contaminated refrigerated food is harmful to human health. big threat

Method used

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  • Double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) method for detecting listeria in food on basis of monoclonal antibodies
  • Double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) method for detecting listeria in food on basis of monoclonal antibodies
  • Double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) method for detecting listeria in food on basis of monoclonal antibodies

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Embodiment Construction

[0037] specific implementation plan

[0038] The present invention is further illustrated by the following examples.

[0039] 1. Instrument:

[0040] TGL-40B desktop low-speed centrifuge, Shanghai Anting Scientific Instrument Factory

[0041] KFLOW pure water machine, KFLOW company

[0042] ZD-9556 horizontal shaker, Taicang Science and Education Equipment Factory

[0043] 96-well 8×12 detachable ELISA plate, Xiamen Yijiamei Laboratory Equipment Co., Ltd.

[0044] MuLtiskaMks microplate reader, ThermoLabsystems

[0045] Adjustable pipette, ThermoLabsystems

[0046] Vortex mixer, Shanghai Huxi Instrument Analysis Factory

[0047] 2. Reagents:

[0048] Tetramethylbenzidine (TMB), Shanghai Jingchun Industrial Co., Ltd.

[0049] Other reagents are analytical reagents

[0050] 3. Steps

[0051] 1. Preparation of monoclonal antibodies

[0052] (1) Experimental animals: 5 8-week-old BALB / c mice were selected for immunization;

[0053] (2) Antigen configuration: Dilute Li...

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Abstract

The invention discloses a double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) method for detecting listeria in food on the basis of monoclonal antibodies, belonging to the technical field of immunoassay. The method comprises the following steps: immunizing 8-week BALB mouse with prokaryotically expressed listeria monocytogenes P60 protein, and obtaining 15 strains of listeria specific monoclonal antibodies by immunization, fusion and multiple screening; respectively marking horse radish peroxidase HRP, and carrying out pairwise coupling by taking the listeria monocytogenes CMCC54003 as targets. The sandwich ELISA method which is established by screening different paired cross reactions and taking 2G7 as a coating antibody and 2H8-HRP as an enzyme labelled antibody has cross reactions to listeria including the listeria monocytogenes, and has no cross reactions to tested staphylococcus aureus, escherichia coli, escherichia coli O157, salmonella, enterobacter sakazakii, campylobacter jejuni and campylobacter coli. According to the method disclosed by the invention, the monoclonal antibodies having listeria specificity are prepared and the double-antibody sandwich ELISA method is established, and the method provides a technical means for specific rapid detection of the listeria in food.

Description

technical field [0001] The invention relates to a double-antibody sandwich ELISA method for detecting Listeria in food based on a monoclonal antibody, and belongs to the technical field of immune analysis. Background technique [0002] Listeria monocytogenes is a facultative anaerobic Gram-positive bacterium belonging to the genus Listeria. It is primarily food-borne and is one of the deadliest foodborne pathogens worldwide. Listeria is widely distributed in the environment, and meat, eggs, poultry, dairy products, vegetables, etc. have all been proven to be the source of Listeria infection. Infection with Listeria can cause symptoms such as muscle pain, nausea, and diarrhea. In severe cases, it can cause blood and brain tissue infections. Moreover, since the bacteria can still grow and reproduce at a refrigeration temperature of 4°C, contaminated refrigerated food is harmful to human health. Big threat. Therefore, many countries have taken measures to control Listeria in...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569G01N33/543G01N33/535C07K16/12
CPCC07K16/1296G01N33/535G01N33/543G01N33/56911G01N33/577
Inventor 匡华王文彬胥传来徐丽广马伟刘丽强吴晓玲宋珊珊胡拥明
Owner JIANGNAN UNIV
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