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Monoclonal antibody for resisting hepatitis B virus e antigen and application thereof

A technology of hepatitis B virus and monoclonal antibody, which is applied in the field of prevention and treatment, immunology, molecular virology, and diagnosis of hepatitis B virus, and can solve problems such as the limitation of HBeAg endpoint judgment

Active Publication Date: 2021-11-30
XIAMEN INNODX BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the reagents used to detect HBeAg at home and abroad are all antibodies against the 1-149aa epitope, and there is a certain cross-reactivity to HBcAg, which has a certain limiting effect on the judgment of the end point of HBeAg.

Method used

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  • Monoclonal antibody for resisting hepatitis B virus e antigen and application thereof
  • Monoclonal antibody for resisting hepatitis B virus e antigen and application thereof
  • Monoclonal antibody for resisting hepatitis B virus e antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Antigen Preparation

[0069] 1.1 Clone HBeAg -10-149, -10-152 and -10-183 amino acids

[0070] The -10-149 amino acid sequence of HBeAg is shown in SEQ ID NO:12, the -10-152 amino acid sequence is shown in SEQ ID NO:13, and the -10-183 amino acid sequence is shown in SEQ ID NO:14 Show. -10-149; -10-152; -10-183 antigens were prepared by using the Escherichia coli expression system.

[0071] 1.2 Preparation and purification -10-149; -10-152; -10-183 antigen

[0072] 1.2.1 Collect the bacterial liquid and ultrasonically crush it, centrifuge the crushed liquid at 12,000 rpm and 10°C for 10 min, and collect the supernatant.

[0073] 1.2.2 Precipitate the obtained supernatant with saturated thiamine, precipitate at 4 degrees for 1-2h, centrifuge at 13000rpm for 10min to collect the precipitate, and resuspend with 50mM TB8.8 buffer equal to the volume of the original supernatant, 1×PB7.4 Dialyze the sample. The sample was purified by medium-pressure DEAE-FF ...

Embodiment 2

[0074] Embodiment 2: Preparation of Anti-HBeAg mouse monoclonal antibody (hybridoma cell line 16D9, 9F10, 4C8, 14C12, 12D7, 8D1)

[0075] 2.1 Immunization of mice

[0076] 2.1.1 Preparation of the immunogen: the immunogen is -10-149, -10-152, -10-183 proteins expressed recombinantly in Escherichia coli. Take the completed antigen and dilute it to 0.4mg / mL, mix it with Freund's adjuvant in equal volume to form a water-in-oil emulsion (the method for judging whether the emulsification of the mixed solution is complete: a small drop of the mixed solution can be dropped on the liquid surface of clear water above, if the mixture does not condense, it can be considered to have been basically mixed). Complete Freund's adjuvant was used for the initial immunization, and incomplete Freund's adjuvant was used for subsequent booster immunization, and no adjuvant was added for the last booster immunization 72 hours before fusion.

[0077] 2.1.2 Basic immunization of mice: Use the ab...

Embodiment 3

[0092] Example 3: Evaluation of the binding activity of Anti-HBeAg mouse monoclonal antibody to different synthetic peptides at the N-terminus of HBeAg

[0093] 3.1 Polypeptide synthesis

[0094] Using the HBV sequence (GenBank ID: AAL15969.1) as a reference sequence, 3 polypeptides were synthesized (synthesized by Shanghai Sangon Biotechnology Co., Ltd.). These three polypeptides (S1-S3) together cover the 10 amino acids from minus 10 to minus 1 of HBeAg. The peptide information of S1-S3 is shown in the table below.

[0095] Table 1: Peptide information of S1-S3

[0096]

[0097]

[0098] 3.2 Reactivity analysis of Anti-HBeAg mouse monoclonal antibody and peptide S1-S3

[0099] 3.2.1 Preparation of reaction plate

[0100] The peptide was treated with 50mM CB buffer (NaHCO 3 / Na 2 CO 3 buffer, the final concentration is 50mM, the pH value is 9.6), and the final concentration is 5μg / mL; add 100μL of coating solution to each well of a 96-well microplate plate, and...

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Abstract

The invention relates to the fields of immunology and molecular virology, in particular to the fields of diagnosis, prevention and treatment of hepatitis B virus. Specifically, the invention relates to a monoclonal antibody of the hepatitis B virus e antigen, and also relates to a detection reagent (especially a chemiluminescence method) of the hepatitis B virus e antigen.

Description

technical field [0001] The invention relates to the fields of immunology and molecular virology, especially the fields of diagnosis, prevention and treatment of hepatitis B virus. Specifically, the present invention relates to the monoclonal antibody of hepatitis B virus, and also relates to the detection reagent of hepatitis B virus e antigen, especially the detection by chemiluminescence method. Background technique [0002] Hepatitis B virus (HBV for short) is a worldwide distribution and serious infectious disease. At present, hepatitis B virus infection has become a common and important public health problem worldwide. Clinically, abnormal liver function is the main feature and accompanied by diverse clinical manifestations. At present, there are 350 million to 400 million chronic HBV-infected people in the world, and 2 billion people have been infected with HBV, accounting for about 6% of the global population. A considerable part of chronic HBV carriers develop into...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N5/20C12N15/13G01N33/569
CPCC07K16/082G01N33/56983C07K2317/565C07K2317/56G01N2333/02
Inventor 陈自敏王邵娟刘嘉祺熊君辉袁权葛胜祥宋浏伟孙旭东
Owner XIAMEN INNODX BIOTECH CO LTD
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