Method for structuring hybridoma cell system for anti-human monoclonal antibody of hepatitis B virus and its application

A human monoclonal antibody and hybridoma cell line technology, applied in the field of bioengineering, can solve the problems of low antibody secretion and reduced antiviral effect, and achieve a large amount of secreted antibody, improve the effect of killing viruses, and have strong vitality. Effect

Inactive Publication Date: 2006-09-13
张永忠
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In the 03134618.9 and 03134617.0 patent applications, the construction of hybrid cell lines and the preparation of cross-linked bodies have few chances of obtaining antibody-positive hybridomas, low antibody secretion, and bec

Method used

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  • Method for structuring hybridoma cell system for anti-human monoclonal antibody of hepatitis B virus and its application
  • Method for structuring hybridoma cell system for anti-human monoclonal antibody of hepatitis B virus and its application
  • Method for structuring hybridoma cell system for anti-human monoclonal antibody of hepatitis B virus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] This example is a method for constructing a hybridoma cell line of anti-hepatitis B virus human monoclonal antibody (anti-HBs hMcAb).

[0061] 1. EBV transformation method to establish a transformed B lymphocyte line secreting anti-HBs hMcAb:

[0062] 1. Preparation of EBV:

[0063] ① B95-8 cells (lymphoma cell line, which can produce Epstein-Barr virus) were mixed with 2×10 5 The concentration of / ml is suspended in the RPMI1640 (complete culture medium) containing 20% ​​fetal calf serum;

[0064] ②Cultivate the cell solution in a 37°C incubator for 7 days;

[0065] ③ Centrifuge the cultured B95-8 cell solution at 3000rpm for 10 minutes;

[0066] ④Take 5ml of B95-8 supernatant for later use.

[0067] 2. Preparation of PBL (peripheral blood lymphocytes) of anti-HBs positive young women:

[0068] ① Take 5ml of peripheral blood from anti-HBs positive young women;

[0069] ② Centrifuge the 5ml peripheral blood density gradient to separate PBL;

[0070] ③ Collect PBL, ...

Embodiment 2

[0101] This embodiment is the hybridoma cell line (6-A) of anti-hepatitis B virus human monoclonal antibody 8 Systematic assay of cell lines).

[0102] 1. Proved by ELISA indirect method, polyacrylamide gel electrophoresis and enzyme-labeled anti-human IgM immunoblotting, 6-A 8 The cells secrete anti-HBs pentameric IgM hMcAb with a molecular weight of 96×10 4 (Standard human IgM is a pentamer with a molecular weight of 96×10 4 ), has strong specificity to HBsAg (hepatitis B surface antigen), and has no cross-reaction with other hepatitis B markers and some other viruses. See Figures 2, 3, 4, 5 and Tables 1 and 2.

[0103] Dilution

1

3

9

27

81

243

729

2187

6561

OD 3

3.37

3.20

2.33

0.83

0.33

0.20

0.17

0.12

0.11

P / N

63.6

60.4

43.9

15.6

6.2

3.7

2.8

2.4

2.2

[0104] Negative control: OD ...

Embodiment 3

[0124] This example is the production process of anti-HBs pentameric IgM hMcAb.

[0125] 1. Animal method produces anti-HBs pentamer IgM hMcAb, the procedure is as follows:

[0126] Cultivate enough 6-A8 cells in a rotating glass culture flask, inject 0.5ml of pristane into each nude mouse intraperitoneally, and inject 1×107 6-A8 cells into the nude mice after 1 week of pristine injection, and feed them continuously The nude mice were injected with cells for 2 weeks. After the abdominal cavity of the nude mice was extremely distended, the nude mice were sacrificed, and the 6-A8 cell solid tumors were removed by laparotomy, and stored in a -40°C low-temperature refrigerator for later use;

[0127] Then carry out the separation and purification of anti-HBs pentamer IgM hMcAb: take 1 gram of solid tumor, cut it into pieces, grind it, add 3ml of 0.01M PH7.4 PBS, ultrasonic homogenate for 15 minutes, centrifuge at 10000rpm for 25 minutes at low temperature, collect the supernatant,...

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PUM

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Abstract

The invention discloses an anti-hepatitis B monomer clone antibody hydridoma series construction method that adopts EBV transforming human B lymphocyte to gain transforming B lymphocyte cell series and taking cell melting with rat plasmocytoma cell series FO to construct excreting anti-HBshMcAb hydridoma series. The invention is easy to cultivate and has high melting. It could be used in cure lymphocyte B, HB advanced stage liver cancer.

Description

technical field [0001] The invention relates to a construction method of a hybridoma cell line of anti-hepatitis B virus human monoclonal antibody, a hepatitis B-directed interferon, a preparation method and application thereof, and belongs to the technical field of bioengineering. Background technique [0002] At present, the development of bioengineered antibody drugs is rapid. According to the report of Bioengineering Journal in December 2004, bioengineered antibody drugs accounted for 40% of the top 10 sales of bioengineered drugs in the world. The growth momentum of bioengineering antibody drugs is strong. Antibody drugs have become an extremely important member of the field of bioengineering pharmaceuticals. They are expected to become the largest class of biological products in 2005. It is estimated that their sales will exceed 20 billion US dollars in 2007, accounting for the entire biopharmaceutical industry. 1 / 3, it can be seen that the development prospect of bioe...

Claims

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Application Information

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IPC IPC(8): C12N5/28C07K16/00C07K19/00A61K38/17A61P35/00A61P1/16
Inventor 张永忠
Owner 张永忠
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