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Method for detecting hemorrhagic Escherichia coli o157:h7 and its monoclonal antibody

A technology of Escherichia coli and O157 is applied in the field of detecting hemorrhagic Escherichia coli O157:H7, hybridoma cells, and two anti-hemorrhagic Escherichia coli O157:H7 monoclonal antibodies, which can solve the problems of complicated operation, long detection period, and detection products. Dependency on imports, etc.

Active Publication Date: 2015-10-14
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 hemorrhagic Escherichia coli O157:H7 mainly relies on the biochemical identification stipulated by 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.
Molecular biology and immunological detection are the fastest, accurate and stable rapid detection methods that have 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 uses hemorrhagic Escherichia coli O157:H7 as the detection target, and the claimed technology involves rapid detection products of hemorrhagic Escherichia coli O157:H7

Method used

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  • Method for detecting hemorrhagic Escherichia coli o157:h7 and its monoclonal antibody
  • Method for detecting hemorrhagic Escherichia coli o157:h7 and its monoclonal antibody
  • Method for detecting hemorrhagic Escherichia coli o157:h7 and its monoclonal antibody

Examples

Experimental program
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Embodiment 1

[0080] Example 1. Preparation of anti-hemorrhagic Escherichia coli O157:H7 monoclonal antibody GS2-D3 and 3A8F11B10E7

[0081] 1. Preparation of immunogen and positive standard

[0082] Hemorrhagic Escherichia coli O157:H7 (ATCC43895) was inoculated on a Sorbitol MacConkey (SMAC) plate, cultured at 37°C for 24 hours, picked a single colony in the improved E.C novobiocin enrichment broth, and cultured at 37°C, 150r / min shaking After 17 hours, count and add 0.3% formaldehyde solution to inactivate at room temperature for 1 day. Adjust the concentration of hemorrhagic Escherichia coli O157:H7 (ATCC43895) 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 hemorrhagic Escherichia coli O157:H7 bacteria solution was used as a positive control standard, and sterilized EC broth was used as a negative control standard.

[0083] 2. Preparation of monoclonal antibodies

[0084] 1) Experimental animals: Three 8-we...

Embodiment 2

[0109] Such as figure 1 As shown, lane 1 and lane 2 are GS2-D3 and 3A8F11B10E7, respectively. GS2-D3 has a heavy chain molecular weight of 48kDa and a light chain molecular weight of 26kDa; 3A8F11B10E7 has a heavy chain molecular weight of 48kDa and a light chain molecular weight of 28kDa. Example 2. Characterization of monoclonal antibodies GS2-D3 and 3A8F11B10E7

[0110] 1. Monoclonal Antibody Subclass Identification

[0111] 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.

[0112] 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.

[0113] 3. Add monoclonal antibody hybridoma cell supernatant, 8 microwells for each sample, 50 μl per well. Incubate for 1 hour at 37°C.

[0114] 4. After washing the plate 4 times, add specific binding rabbit anti-mous...

Embodiment 3

[0129] Example 3. The composition, preparation and application of the ELISA kit for detecting hemorrhagic Escherichia coli O157:H7

[0130] 1. The enzyme-linked immunosorbent assay kit consists of the following materials:

[0131] (1) Antibody pre-coated microtiter plate: Dilute with 0.02M acetic acid buffer (pH2.0) solution, and coat 96-well microtiter plate with anti-hemorrhagic Escherichia coli O157:H7 monoclonal antibody 3A8F11B10E7, 100 μl per well. Incubate overnight at 4°C, block and wash according to conventional ELISA methods.

[0132] (2) Hemorrhagic Escherichia coli O157:H7 positive control standard and negative control standard.

[0133] (3) Horseradish peroxidase-labeled monoclonal antibody GS2-D3 against hemorrhagic Escherichia coli O157:H7.

[0134] (4) Enzyme-labeled antibody diluent: 0.01M PBS, pH7.6.

[0135] (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 tim...

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Abstract

The invention belongs to the field of microbiological detection, and particularly relates to a method for detecting enterohemorrhagic E.coli O157:H7, and two monoclonal antibodies of the enterohemorrhagic E.coli O157:H7 generated in the preparation process of monoclonal antibodies at the same time for the method. The monoclonal antibodies can be combined with the enterohemorrhagic E.coli O157:H7 specifically, and are generated from mice hybridoma cell line GS2-D3, CGMCC No.6610 or 3A8F11B10E7 and CGMCC No.8458. The invention also provides an application of the monoclonal antibodies of the enterohemorrhagic E.coli O157:H7.

Description

technical field [0001] The invention belongs to the field of microorganism detection. Specifically, the present invention relates to a method for detecting hemorrhagic Escherichia coli O157:H7, two strains of anti-hemorrhagic Escherichia coli O157:H7 monoclonal antibodies used in the method and their use, and a hybridoma producing the monoclonal antibodies cell. Background technique [0002] Hemorrhagic Escherichia coli O157:H7 (Escherichia coli O157:H7) is the main serotype of intestinal Escherichia coli, which can be transmitted through beef and its products, milk and its products, chicken, vegetables, fruits, beverages, water, etc. Hemorrhagic Escherichia coli O157:H7 can cause enteritis. The typical clinical symptoms of infected patients are bloody stool, abdominal pain, low-grade fever or no fever. About 10% of the patients can develop hemolytic uremia and thrombotic thrombocytopenic purpura, and It can cause damage to the intestines, liver, spleen, kidneys, lymph, br...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/569C07K16/12C12N5/20
CPCC07K16/1232G01N33/56916G01N2333/265
Inventor 刘箐李浩林
Owner UNIV OF SHANGHAI FOR SCI & TECH