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Double-antibody sandwich method for detecting enterobacter sakazakii in food

A technology of Enterobacter sakazakii and double antibody sandwich

Inactive Publication Date: 2014-04-09
中华人民共和国淮安出入境检验检疫局 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the immunogenicity of LPS is weak, and it is difficult to elicit a sufficient immune response in mice

Method used

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  • Double-antibody sandwich method for detecting enterobacter sakazakii in food

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0037] The present invention is further illustrated by the following examples.

[0038] 1. Instrument:

[0039] TGL-40B desktop low-speed centrifuge, Shanghai Anting Scientific Instrument Factory

[0040] KFLOW pure water machine, KFLOW company

[0041] ZD-9556 horizontal shaker, Taicang Science and Education Equipment Factory

[0042] 96-well 8×12 detachable ELISA plate, Xiamen Yijiamei Laboratory Equipment Co., Ltd.

[0043] MuLtiska Mks Microplate Reader, Thermo Labsystems

[0044] Adjustable pipette, Thermo Labsystems

[0045] Vortex mixer, Shanghai Huxi Instrument Analysis Factory

[0046] 2. Reagents:

[0047] Tetramethylbenzidine (TMB), Shanghai Jingchun Industrial Co., Ltd.

[0048] Other reagents are analytical reagents

[0049] 3. Steps

[0050] 1. Preparation of monoclonal antibodies

[0051] (1) Experimental animals: 5 8-week-old BALB / c mice were selected for immunization;

[0052] (2) Antigen configuration: Dilute the immunogen with normal saline to prep...

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Abstract

The invention relates to a double-antibody sandwich method for detecting enterobacter sakazakii in food. The method is characterized in that the enterobacter sakazakii (ATCC29544) and lipopolysaccharide (LPS) extracted by coupling the enterobacter sakazakii (ATCC29544) with BSA (bovine serum albumin) are respectively used as immunogen to simultaneously immunize BALB / c mice, and 11 enterobacter sakazakii monoclonal antibodies are obtained through immunization, fusion and screening to respectively label HRP (horse radish peroxidase) and are paired through the enterobacter sakazakii. A No.7 monoclonal antibody CGMCC No. 7207 is used as a coating antibody, a No.8 monoclonal antibody CGMCCNO.7208 is used as an enzyme labeled antibody, a sandwich ELISA method of the enterobacter sakazakii is established by adopting the ATCC29544 as a standard product, and LOD is 53000CFU / Ml. By adopting the monoclonal antibody with high physicochemical property, good uniformity, good specificity and capability of being in mass preparation, the established sandwich method is high in sensitivity and low in cost, no cross reaction with salmonella enteritidis, E.coli, E.coliO157: H7, salmonella typhimurium, staphylococcus aureus and listeria monocytogenes exists, and thus a rapid and high efficient analysis means is provided for detecting the enterobacter sakazakii in the food.

Description

technical field [0001] The invention relates to a double-antibody sandwich method for detecting Enterobacter sakazakii in food, belonging to the field of immune analysis. Background technique [0002] Enterobacter sakazakii (Enterobacter sakazakii) is a pathogen in infant formula, and its contamination and control attracted attention at the 35th CCFH Congress. Biologically, Enterobacter sakazakii is a kind of perinatal flagellar, motile, sporeless Gram-negative bacteria that parasitizes in the intestinal tract of humans and animals, and can cause human and animal pathogenicity under certain conditions. The main antigens are O antigen, H antigen and Vi antigen. Sporadic and outbreak cases of meningitis, sepsis and necrotizing colitis caused by it in infants and premature infants have appeared one after another in the world. Several research reports have shown that infant formula milk powder is the main source of infection currently found to cause meningitis, sepsis, and n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/02G01N33/577
Inventor 冯民王小晋匡华孔德昭胥传来徐丽广马伟刘丽强
Owner 中华人民共和国淮安出入境检验检疫局
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