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Preparation and screening of monoclonal antibody against hepatitis B surface antigen

A hepatitis B surface antigen, monoclonal antibody technology, applied in biochemical equipment and methods, chemical instruments and methods, antiviral immunoglobulin, etc., to achieve the effect of improving accuracy, reducing false negative test results, and broad binding ability

Inactive Publication Date: 2018-10-09
ABON BIOPHARM HANGZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many mutant HBsAgs that have not been recognized, therefore, there is a great demand for monoclonal antibodies that can bind both wild-type and mutant HBsAg

Method used

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  • Preparation and screening of monoclonal antibody against hepatitis B surface antigen
  • Preparation and screening of monoclonal antibody against hepatitis B surface antigen
  • Preparation and screening of monoclonal antibody against hepatitis B surface antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Preparation of hybridoma cell lines (preservation numbers are CCTCC No: C2016198 and CCTCC No: C2016210)

[0049] 1.1 Immunization of animals

[0050] Animal preparation: The immunized animals used to prepare hybridoma cells are not particularly limited, and mice, rats, hamsters, rabbits, and BALB / c mice can be used as immunization objects. In this example, 6-8 week old female BALB / c mice (purchased from Shanghai Animal Center) were selected for immunization according to the pre-established immunization scheme.

[0051] 1.2 Antigen preparation

[0052] The two serotypes of HBsAg antigens (purchased from MP Biomedicals Asia Pacific Pte Ltd) are Adr and Ay respectively, Adr is a recombinant protein expressed in Escherichia coli, and Ay is purified from biological samples of HBV-infected patients)

[0053] 1.3 Immunization scheme

[0054] The immunogenicity of a specific antigen is affected by many factors, but the immunogenicity can be enhanced by the use of...

Embodiment 2

[0076] Example 2. Mass production of monoclonal antibodies

[0077] There are mainly two methods for mass production of monoclonal antibodies using hybridoma cells: in vivo and in vitro culture. The cost of culturing hybridoma cells in vitro is relatively high, and experimental animals are not used. Currently, in vivo methods are commonly used to prepare monoclonal antibodies. And the hybridoma cells were injected into the peritoneal cavity of pre-sensitized BALB / c mice.

[0078] The stages of in vivo preparation of monoclonal antibody ascites are as follows:

[0079] (1) Liquid paraffin presensitized mice: the volume of liquid paraffin injected is usually 0.5ml.

[0080] (2) Injection of hybridoma cells: the interval between injection of liquid paraffin and hybridoma cells is optimal at 7-20 days, and the number of injected hybridoma cells is 0.5×10 6 -2×10 6 optimal. Usually, mouse ascites can be formed 7-10 days after injection of hybridoma cells.

[0081] Collection...

Embodiment 3

[0082] Example 3. Purification, Identification and Application of Monoclonal Antibodies in Test Kits

[0083] 3.1 Purification of monoclonal antibodies

[0084] The method of purifying monoclonal antibody from ascites is basically the same as the method of purifying immunoglobulin. A salt precipitation method, molecular sieve, etc. may be used. If the antibody is mouse IgG, affinity purification methods such as Protein A and Protein G can also be used.

[0085] Antibodies 15G6E1 and 5C7 in the present invention were purified using Protein A.

[0086] After mixing equal volumes of ascitic fluid and loading buffer, load the sample on the Protein A column that has been equilibrated with loading buffer. After loading the sample, use 0.01M PBS solution to elute the impurity protein, and stop the elution when the OD280 of the effluent is <0.03. Then use the eluent to elute the target antibody, and collect the effluent. When collecting, add hydrochloric acid solution dropwise to...

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Abstract

The present invention relates to a monoclonal antibody capable of binding to hepatitis B surface antigen, a hybridoma cell strain capable of producing the antibody and having the preservation number of CCTCC No:C2016198, a preparation method of the antibody, and applications of the antibody in hepatitis B surface antigen detection kits. According to the present invention, the antibody capable of binding to hepatitis B surface antigen is provided, can bind to wild type hepatitis B surface antigen or mutant hepatitis B surface antigen, has wide binding ability, can effectively reduce false negative test results, and further can improve the accuracy of hepatitis B virus detection.

Description

technical field [0001] The invention relates to the preparation and screening of monoclonal antibodies, in particular to monoclonal antibodies capable of binding to the surface antigen of hepatitis B virus. Background technique [0002] Hepatitis B virus (HBV) is a double-stranded DNA virus belonging to the family Hepadnaviridae, which is a spherical particle with a diameter of about 42 nm. It has two parts, the shell and the nucleocapsid, the shell is 7-8nm thick, has S protein, M protein and L protein, and the surface of the S protein has hepatitis B surface antigen (HBsAg) related to virus infection. [0003] Hepatitis B virus is the most common pathogen that causes viral hepatitis. It can be transmitted through blood, mother to child, close contact and sexual contact. After human infection with hepatitis B virus, it is easy to cause chronic hepatitis B, and then lead to liver cirrhosis and / or liver cancer . At present, there are about 400 million chronic hepatitis B vi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/08G01N33/576G01N33/577G01N33/569C12R1/91
CPCC07K16/082G01N33/56983G01N33/5764G01N33/577G01N2333/02
Inventor 杜付彬戈东平陆启明
Owner ABON BIOPHARM HANGZHOU
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