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Hybridoma cell line of Escherichia-coli O157:H7 resistant monoclonal antibody

A monoclonal antibody, O157 technology, applied in anti-bacterial immunoglobulin, biochemical equipment and methods, instruments, etc., can solve the problems of high cost, high cost, not easy to popularize in grassroots units, etc., to improve detection sensitivity and detection. high efficiency, strict prevention of missed detection and false detection, and good sensitivity

Inactive Publication Date: 2014-07-02
中华人民共和国吉林出入境检验检疫局
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of PCR method for detection requires more expensive instruments, which is expensive and not easy to promote at grassroots units
Monoclonal antibodies have the advantages of good specificity, stability and reproducibility, but reliable EHEC O157: H7 monoclonal antibody hybridomas are not easy to prepare. At present, my country has been relying on imports

Method used

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  • Hybridoma cell line of Escherichia-coli O157:H7 resistant monoclonal antibody
  • Hybridoma cell line of Escherichia-coli O157:H7 resistant monoclonal antibody
  • Hybridoma cell line of Escherichia-coli O157:H7 resistant monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Escherichia coli O157: Preparation of H7 Antigen Bacteria Liquid and BALB / c Mouse Immunization

[0022] The standard Escherichia coli O157:H7 (ATCC11229) stored at -80°C in this study was taken, cultured in broth, cultured on LB by streaking method, enriched by conventional methods, centrifuged at 6000r / min for 10min, and the bacterial precipitate was collected. Colony counting (2.0 × 10 8 CFU / mL), adding formaldehyde with a final concentration of 0.3% overnight at 4°C to inactivate the bacteria. The next day, the eluted bacterial liquid was subjected to ultrasonic waves under the conditions of 20KHz and 150W ice bath to break the bacterial cells, each time for 10 seconds, with an interval of 10 seconds, and a total time of 20 minutes. BCA protein assay kit was used to measure the bacterial protein content, and the result was 2.636 mg / mL.

[0023] Eight-week-old female BALB / c mice were used for immunization. The above-mentioned inactivated bacteria soluti...

Embodiment 2

[0024] The mensuration of embodiment 2 immune mouse serum antibody titer

[0025] After 3 immunizations, blood was collected from the tail of the mice, and the serum titer was detected by conventional indirect ELISA method. Wherein, the coating antigen is the Escherichia coli O157:H7 bacterium liquid prepared in Example 1, the dilution is 1:50, the mouse serum concentration is the gradient concentration starting from 200× dilution, and the enzyme-labeled secondary antibody is HRP (horseradish peroxidase) labeled rabbit anti-mouse IgG (1:15000 dilution). The assay results showed that the antibody titer was 1:25600.

Embodiment 3

[0026] Example 3 Escherichia coli O157: Preparation of H7 monoclonal antibody

[0027] (1) Establishment of hybridoma cell lines

[0028] ①Preparation of feeder cells: Normal BABL / C mice were killed by dislocation of the neck, and 8 mL of HAT culture solution was injected into the peritoneal cavity, and the culture solution was drawn out after gently shaking the mouse for a few times, and the cell concentration was adjusted to 10 5 cells / mL, added to a 96-well cell culture plate, 100 μL per well, and cultured overnight in a cell culture incubator for later use. These are feeder cells.

[0029] ②Cultivation of myeloma cells and preparation of cell suspension: Myeloma cells (SP2 / 0) were resuscitated one week before fusion, passaged every other day, and fusion was carried out one day after passage. Take about 4 bottles (25cm 2 ) SP2 / 0 cells, blow down the cells in each bottle with RPMI-1640 culture medium, centrifuge at 1200r / min for 10min, repeat 2-3 times, and count with a ...

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Abstract

The invention discloses a hybridoma cell line of Escherichia-coli O157:H7 resistant monoclonal antibody. By adoption of the hybridoma cell line, the problem that the monoclonal antibody is not easily obtained; by passage for many years and times, the monoclonal antibody can be stably secreted, and the secreted monoclonal antibody is applied to a liquid-phase chip and a colloidal gold method to detect Escherichia-coli O157:H7 in foods, and has the advantages of good sensitivity, specificity, stability and repeatability and the like. Pathogens also can be detected under the condition of low concentration, so that the detection sensitivity and the detection rate are further increased and the leaking detection and the false detection are prevented strictly.

Description

technical field [0001] The invention belongs to the field of biotechnology, specifically anti-escherichia coli O157:H7 monoclonal antibody and its application. Background technique [0002] Enterohemorrhagic Escherichia coli (Enterohaemorrhagic Escherichia coli , EHEC) mainly includes several serotypes such as O157:H7, O26:H11 and O111, among which O157:H7 is a typical strain. O157:H7 infection can cause serious gastrointestinal complications such as hemorrhagic colitis (HC), appendicitis, esophageal stricture, and colonic perforation, and can also cause hemolytic uremic syndrome and thrombotic platelets in children and the elderly Severe complications such as reduced purpura epilepsy can cause death in severe cases. Enteric infectious disease caused by O157: H7 has become a serious global public health problem, resulting in multiple outbreaks in many countries. O157: H7 can cause hemorrhagic enteritis, about 10% of patients develop hemolytic uremic syndrome and thromboti...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/12G01N33/577G01N33/569
Inventor 孟日增宋战昀芦春梅刘韬赵庆松王玮
Owner 中华人民共和国吉林出入境检验检疫局
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