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Anti-helicobacter pylori protein monoclonal antibody and its cell line, preparation method and application

A Helicobacter pylori and monoclonal antibody technology, applied in the field of biomedical engineering, can solve the problems of large differences in polyclonal antibodies, stability and practical application effects are yet to be tested, and difficult to stabilize applications, etc., to achieve high-sensitivity effects

Active Publication Date: 2021-06-04
FUZHOU MAIXIN BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to improve the sensitivity of detection, you can choose to prepare polyclonal antibodies to obtain high-affinity antibodies, but the differences between batches of polyclonal antibodies are large, and it is difficult to use them stably. In practice, the cocktail method of combining multiple monoclonal antibodies is often used. At the technical level of antibodies, the above goals can also be achieved by preparing bispecific antibodies. Fan Guirong and others used VacA and HpaA recombinant proteins to immunize separately, and obtained a stable cell line that can recognize the two proteins by the method of secondary fusion. This attempt expanded the antibody recognition ability, but its stability and practical application effect have yet to be tested (Fan Guirong, Preparation of bispecific monoclonal antibody against Helicobacter pylori VacA and HpaA, Chongqing Medical University, master's degree thesis, 2009)

Method used

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  • Anti-helicobacter pylori protein monoclonal antibody and its cell line, preparation method and application
  • Anti-helicobacter pylori protein monoclonal antibody and its cell line, preparation method and application
  • Anti-helicobacter pylori protein monoclonal antibody and its cell line, preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0025] Embodiment 1 Helicobacter pylori culture and thalline protein preparation

[0026] Put the obtained Helicobacter pylori (ATCC 700392, Strain Designations: 26695) into a 37°C constant temperature water bath, shake it to thaw quickly, and use an inoculation loop to inoculate an appropriate amount of strain on the prepared Columbia medium (according to the ratio of 1000ml: 41g respectively Add pure water and Columbia culture medium (Beijing Suo Laibao Technology Co., Ltd., add 8% defibrated sheep blood), according to the conditions of the primary culture, place the plate in a microaerophilic tank and cultivate it for 2 days. A milky white viscous bacterial lawn grows, wash the bacterial lawn with PBS buffer pH7.4, centrifuge at 5000rpm for 5 minutes, discard the supernatant, then add 500μl of PBS, ultrasonically disrupt the bacterial body, and centrifuge at 14000rpm for 15 minutes after ultrasonication. The supernatant was transferred to a new centrifuge tube, the pellet w...

Embodiment 2

[0027] The establishment of embodiment 2 hybridoma cell lines

[0028] 1. Immunity

[0029] The supernatant and precipitated thalline protein obtained in Example 1 were respectively emulsified with complete Freund's adjuvant (Sigma company), and after mixing according to the ratio of 1:1, immunized 4-6 week old female Balb / c mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), and each mouse was injected subcutaneously at 6 points in the abdomen, with a dose of 60 μg / only. Immunization was boosted every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma Company) at a dose of 30 μg per mouse. 7 days after the third booster immunization, indirect ELISA (wavelength 450nm) was used to detect the polyantibody titer of the anti-immunogen in the mouse serum. 50μg / only.

[0030] 2. Cell Fusion

[0031] Aseptically prepare the mouse splenocyte suspension that reaches the immune standard, mix it with mouse myeloma cell s...

Embodiment 3

[0036] Example 3 Preparation of Monoclonal Antibody by Ascites Induction Method

[0037] 1. Ascites preparation

[0038] The cells in the logarithmic growth phase were washed with serum-free medium and suspended, counted about 5×10 5 , 1ml. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The removed ascites was centrifuged at 4000rpm for 10min at 4°C. Carefully suck out the ascites in the middle and collect in a centrifuge tube, and store at 4°C or -20°C.

[0039] 2. Purification of monoclonal antibodies

[0040] Antibody was purified from ascitic fluid by HiTrap rProtein A FF (GE Company) affinity chromatography according to the manual. The purity was identified by SDS-PAGE gel, and the concentration was determined by Bradford method. Purified antibodies were stored at -20°C.

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Abstract

The invention provides a Helicobacter pylori antibody and its secreting cell line, preparation method and application. In the present invention, the ultrasonic supernatant of the cultured Helicobacter pylori protein is used as an immunogen to immunize mice, and the antibody 15A5 that can specifically bind to the Helicobacter pylori protein is obtained after screening, and the antibody is IgG2a subtype. This antibody detects infected H. pylori in tissue or serum.

Description

technical field [0001] The invention relates to the field of biomedical engineering, in particular to an anti-helicobacter pylori protein monoclonal antibody and its cell line, preparation method and application. Background technique [0002] Helicobacter pylori is a Gram-negative bacillus, helical, microaerophilic, and Marshall et al. isolated Helicobacter pylori (Hp for short) from gastric mucosal tissue for the first time in 1983, and reported that it was associated with type B gastritis and digestive disorders. Sexual ulcers are closely related. Since 1994, the World Health Organization has classified Helicobacter pylori infection as the first type of cancer risk. More than half of the world's population has been infected by Helicobacter pylori. Helicobacter pylori parasitizes in the gastric mucosal tissue. The infection first causes chronic gastritis, and leads to gastric ulcer and gastric atrophy, and may develop into gastric cancer. Helicobacter pylori infection and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/12C12N15/13C12N5/20G01N33/577G01N33/569
CPCC07K16/121C07K2317/33C07K2317/56C07K2317/92G01N33/56911G01N33/577G01N2333/20G01N2469/10
Inventor 杨清海陈惠玲高惠然李婷王小亚
Owner FUZHOU MAIXIN BIOTECH CO LTD
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