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Single clone antibody of antimutagen hepatitis B virus surface antigen and preparation method thereof

A hepatitis B virus, monoclonal antibody technology, applied in antiviral immunoglobulin, cells modified by introducing foreign genetic material, fusion cells, etc. and other problems to achieve the effect of ensuring the quality of blood source

Inactive Publication Date: 2006-11-15
徐钧
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, these diagnostic reagents have a fatal flaw, that is, they cannot detect the mutated hepatitis B virus, especially the hepatitis B virus with a mutated surface antigen HBsAg, which cannot be diagnosed with conventional reagents, which will cause missed diagnosis.
So that hepatitis B virus carriers or hepatitis B patients can not be diagnosed, so they can not get treatment
What's more serious is that when screening blood donors, if routine kits are used, blood from hepatitis B patients may be transfused to recipients as healthy blood, resulting in serious cross-infection

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment one: the monoclonal antibody of anti-hepatitis B virus surface antigen (HBsAg) is prepared by hybridoma technology

[0021] 1. Materials

[0022] BALB / C pure line mice, female, 8 weeks old. SP2 / 0 myeloma cell line, RPMI1640 medium, newborn calf serum, hypoxanthine-methotrexate-thymine (HAT), hypoxanthine-thymine (HT), polyethylene glycol (PEG4000), (all of the above Courtesy of Huamei Bioengineering Corporation). Horseradish peroxidase (HRP)-labeled goat anti-mouse immunoglobulin (Wuhan Boster Biological Products Company).

[0023] 2. Method

[0024] 1. Immunogen preparation 10 μg (5 μl, 2 μg / μl) antigen was dropped on a 2 mm×2 mm nitrocellulose membrane (NC), and dried in the air.

[0025] 2. Immunization was implanted in the spleen, the mouse was anesthetized with ketamine, the spleen area was plucked after the limbs were fixed, alcohol was disinfected, a 5 mm incision was made below the costal edge of the left midaxillary line, the lower edge of the sp...

Embodiment 2

[0035] Example 2: Anti-HBsAg monoclonal antibody detects variant surface antigen

[0036] 1. Construction and expression of recombinant plasmids expressing variant surface antigens

[0037]A pair of primers were designed for two tests of the HBV S gene, and 21 pairs of site-specific mutation primers were designed according to the mutation sites of the S gene reported in the literature, and the HBV S gene was site-directedly mutated by PCR. The mutated S gene was cloned into the eukaryotic expression vector PCR3.1, and it was used in the experiment after being confirmed by sequencing. A total of 21 recombinant plasmids expressing variant HBsAg were constructed.

[0038] 21 recombinant plasmids expressing variant HBsAg were transfected into COS-7 cells, and the supernatant was collected.

[0039] 2. Detection of reactivity of anti-HBsAg monoclonal antibody E1C with variant surface antigen

[0040] Coat the purified anti-HBs monoclonal antibody E1C with an appropriate dilution...

Embodiment 1

[0045] The identification result of embodiment 1 and embodiment 2 is as follows:

[0046] 1. The establishment and cloning of hybridoma cell lines and the ELISA titer of immunized mouse blood reached 1:10 4 As above, the splenocytes were fused with SP2 / 0 cells and cultured in HAT medium. Cell clonal growth was seen in some wells about 1 week later. After 2 weeks, the cells grew to more than 1 / 3 of the bottom area of ​​the well. The success rate of fusion was 75%, and the specificity The positive rate of antibody was 28.3%. One best hybridoma cell line E1C was retained after three times of cloning.

[0047] 2. Identification of anti-mutant HBsAg monoclonal antibody

[0048] 2.1 Titer The monoclonal antibody titers in E1C ascites type monoclonal antibody and cell culture supernatant were detected by indirect ELISA method were 1∶6.4×10 4 , 1:160.

[0049] E 1C

[0050] Note: Positive standard: OD value of detection hole / OD value of control group > 2.1

[0051]...

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PUM

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Abstract

A monoclonal antibody against mutant hepatitis B virus surface antigen secreted by hybridoma cell line CGMCC HBSP2. The anti-mutated hepatitis B surface antigen monoclonal antibody prepared according to the present invention can simultaneously detect hepatitis B viruses including 14 kinds of mutant strains and wild strains, and can replace the HBsAb in the conventional hepatitis B surface antigen detection kit. Into the hepatitis B surface antigen diagnostic kit. The use of the antibody can avoid the missed diagnosis caused by conventional reagents, so that the carriers of the mutated and non-mutated hepatitis B virus or hepatitis B patients can be diagnosed and treated in time. Especially when screening blood donors, it can detect mutant virus carriers that cannot be detected by conventional kits, avoid transfusing the blood of hepatitis B patients as healthy blood to recipients, and ensure the quality of blood sources.

Description

technical field [0001] The invention belongs to hepatitis B virus antibody, in particular to a monoclonal antibody against wild and mutated multifunctional hepatitis B virus surface antigens. Background technique [0002] Hepatitis B is a global health problem. There are about 350 million chronic carriers of HBV in the world, and 2 million patients die from hepatitis B every year, making it the ninth leading cause of death in the world. my country's HBV chronic carriers account for 1 / 3 of the world's total, and it is the country with the highest HBV virus carrier rate in the world. The estimated population HBsAg positive rate was 10.3%. There are more than 100 million asymptomatic HBsAg carriers, and more than 30 million people with current symptoms, the incidence rate is about 30.41%. Hepatitis B not only seriously threatens the health of our people, but also brings serious economic burden to the society. [0003] The mutation rate of HBV is relatively high, and some st...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/08C12N5/16C07K16/10G01N33/577
Inventor 徐钧解军聂继盛
Owner 徐钧
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