Reagent kit for detecting hepatitis B virus e antigen and use

A hepatitis B virus and kit technology, applied in the field of virus immunology detection, can solve the problems of low sensitivity, high false positive rate, limited clinical application, etc.

Inactive Publication Date: 2011-05-18
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because monoclonal anti-HBeAg hybridoma cell lines are difficult to obtain, and because the anti-HBeAg monoclonal antibodies secreted by them are not paired, they cannot be used for ELISA detection. Therefore, when detecting HBeAg, coated monoclonal antibodies and enzyme-labeled polyclonal antibodies are often used , which will lead to technical defects such as high false positive rate and low sensitivity, so it is an inevitable trend to use double antibody sandwich-ELISA method to detect HBeAg
However, the double-antibody sandwich ELISA method requires two monoclonal antibodies that recognize different epitopes of HBeAg, and when using traditional methods to prepare anti-HBeAg monoclonal antibodies, only one dominant epitope of HBeAg (named as b epitope) can be obtained. Antibody, which has caused great limitations to the clinical application of sandwich ELISA detection of HBeAg

Method used

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  • Reagent kit for detecting hepatitis B virus e antigen and use
  • Reagent kit for detecting hepatitis B virus e antigen and use
  • Reagent kit for detecting hepatitis B virus e antigen and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1 The establishment of anti-HBeAg hybridoma cell line and the preparation of its monoclonal antibody

[0055] (1) Immunization of animals

[0056]BALB / C mice were immunized three times with recombinant HBeAg protein. One day before the first immunization, 0.5 mg of antibody ewb (product of Shanghai Kehua Co., Ltd.) recognizing the b epitope of HBeAg was intravenously injected. For the first immunization, Freund's complete adjuvant was mixed with equal amount of HBeAg protein, then emulsified, and injected intravenously, 0.1 mg per mouse, with an injection volume of 0.2-0.3 mL. For the second and third immunizations, use Freund's incomplete adjuvant and equal amounts of HBeAg protein to mix and then emulsify. The immunization method and dosage are the same as the first immunization. Before each immunization of mice, 0.1 mg of HBeAg and 0.5 mg of antibody ewb were mixed and emulsified with an equal amount of adjuvant. Each immunization interval is one month....

Embodiment 2

[0063] Embodiment 2 Preparation of anti-HBeAg monoclonal antibody S-29-3

[0064] The positive hybridoma cell line S-29-3 was mixed with 1×10 6 BABL / C female mice sensitized at the age of 8-10 weeks were intraperitoneally injected per mouse. After 7-10 days, the ascites of the mice was collected, and the titer of S-29-3 monoclonal antibody was detected by indirect ELISA method. The ascites was centrifuged at 4°C and 12000rpm for 15 minutes, and 1 part (volume) of ascites was mixed with 2 parts (volume) of 0.06 M pH4.8 acetate buffer, and n-octanoic acid 33 μL / mL was added dropwise with stirring at room temperature, and mixed for 30 minutes at room temperature. Stand at 4°C for more than 2 hours to fully precipitate, then centrifuge at 4°C and 12,000 rpm for 30 minutes, discard the precipitate, filter the supernatant through a sand core funnel, add 1 / 10 volume of 0.1M PBS with pH 7.4, and adjust the pH with 2M NaOH to 7.4. Add 0.277g / mL ammonium sulfate within 30min under ice...

Embodiment 3

[0065] Embodiment 3 Preparation of anti-HBeAg monoclonal antibody S-72-3

[0066] The positive hybridoma cell line S-72-3 was mixed with 1×10 6 BABL / C female mice sensitized in advance at the age of 8-10 weeks were intraperitoneally injected per mouse. After 7-10 days, the ascites of the mice was collected, and the remaining steps were the same as in Example 2 to prepare antibody S-72-3.

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Abstract

The present invention relates to a reagent kit for detecting hepatitis B virus e antigen and its use, and relates to the immunological virus detecting technology field. In order to overcome the technique defects of high false positive rate and low sensitivity in the HBeAg detection by using a coating monoclonal antibody and an enzyme labeled multi-resistance ELISA in the prior art, the invention provides the reagent kit for detecting the hepatitis B virus e antigen and its use, as well as the application in the HBeAg detection. The invention discloses the reagent kit for detectiong the hepatitis B virus e antigen, characterized by including monoclonal antibodies packed in containers to distinguish different epitopes of the hepatitis B virus e antigen, optionally one antibody labeled with horseradish peroxidase or fluorescein isothiocyanate or biotin. The reagent kit of the present invention is used clinically in detecting the hepatitis B virus, and has high specificity, sensitivity up to ng level, and excellent linear relation in the HBeAg concentration range of 3ng-95ng / ml.

Description

technical field [0001] The invention relates to the technical field of virus immunological detection, in particular to a kit for detecting hepatitis B virus e antigen and its application. Background technique [0002] In recent years, liver disease has increasingly become one of the major diseases threatening human health, among which viral hepatitis, especially hepatitis B, has the greatest impact on human beings. Hepatitis B virus (HBV) infection is a serious global public health problem and has become a current worldwide disease. Currently, there are 350 million HBV persistently infected people in the world, of which 75% are in Asia and the Western Pacific. my country is a high prevalence area of ​​hepatitis B, with 120 million people infected with HBV, and an average annual incidence of about 1.4 million, ranking third among legal infectious diseases. With the in-depth understanding of hepatitis B in our country and the gradual strengthening of self-care awareness, the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/576
Inventor 孙兵靖彩英颜凌晨黄超锋蔡兴锋
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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