Amplification method of polyclonal anti-HBV immune cells

A nuclear cell, hepatitis B technology, applied in the field of immune cells, can solve the problem of low amplification efficiency of antigen-specific T cells, and achieve the effect of increasing efficiency, improving amplification efficiency, and improving detection efficiency

Inactive Publication Date: 2021-02-02
THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In order to solve the problem of low expansion efficiency of antigen-specific T cells, the present invention provides a combination of antigen-specific peptide library combined with cytokine interleukin-7 (IL-7) and Anti-CD28 antibody for cell expansion in vitro Methods

Method used

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  • Amplification method of polyclonal anti-HBV immune cells
  • Amplification method of polyclonal anti-HBV immune cells
  • Amplification method of polyclonal anti-HBV immune cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The selection of embodiment 1 material

[0043] Collect 3 blood samples from patients with acute hepatitis B convalescence in the 302nd Hospital of the People's Liberation Army, and select the hepatitis B serological marker test result (HBV serological marker test) as: HBsAg - , HBsAb + , HBeAb + , HBcAb + samples for subsequent experiments.

Embodiment 2

[0044] Example 2 Peripheral Blood Mononuclear Cell Separation

[0045] 1. Take 8ml of blood (the blood in Example 1) from a vein, add it into a test tube containing heparin solution (10-50u / m1 blood sample), mix well, and make the blood anticoagulant. The anticoagulant blood was diluted 1-fold with pH7.2 Hanks solution.

[0046] 2. Take 4ml of lymphocyte stratification solution and place it in a graduated centrifuge tube, then tilt the centrifuge tube at an angle of 45 degrees, and slowly add the diluted whole blood to the separation solution along the tube wall with a capillary dropper. Care should be taken to maintain the interface between the two clear.

[0047] 3. Centrifuge at 2000r / min with a horizontal centrifuge for 20min at 18°C ​​to 20°C.

[0048] 4. Use a capillary pipette to gently insert into the turbid zone, gently suck out the layer of cells along the tube wall, and transfer to another centrifuge tube. That is to say, all mononuclear cells should be absorbed,...

Embodiment 4

[0053] Example 4 HBV peptide library stimulated cells and ELISPOT detection

[0054] 4.1 Solution preparation

[0055] PBS: Prepared with analytical grade reagents, Millipore-grade pure water, and autoclaved.

[0056] PBST: Add 0.05% Tween-20 to PBS, pay attention to aseptic operation. Stored at 20-25°C, it can be stored for one month.

[0057] 70% ethanol: analyze pure ethanol 70mL, add Millipore grade pure water to 100mL.

[0058] 30% ethanol: 30mL of analytical pure ethanol, add Millipore grade pure water to 100mL.

[0059] Coating antibody (mO-11-31-87): According to the instructions of U-Cytech, add double distilled water to dissolve. When used, dilute 50 times with PBS, 50 μL per well.

[0060] Blocking solution: Dilute the blocking storage solution in the kit 10 times with PBS, 200 μL per well.

[0061] Antibody Diluent: Dilute the diluted stock solution in the kit 10 times with PBS.

[0062] Detection antibody (mB-12-34-38): According to the instructions of U-Cy...

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Abstract

The invention relates to the technical field of immune cells, particularly to an amplification method of polyclonal anti-HBV (hepatitis B virus) immune cells. The amplification method comprises the following steps: separating peripheral blood mononuclear cells of convalescent patients suffering from acute hepatitis B; synergistically stimulating by using HBV antigenic peptide library to unite IL-7 and Anti-CD28 antibody, so as to effectively amplify antigen-specific cells. The efficient amplification method of antigen-specific T cells established by the invention has important significance for studying the immunologic mechanism of HBV removal and effectively judging clinical prognosis.

Description

technical field [0001] The invention relates to the technical field of immune cells, and mainly relates to a method for amplifying polyclonal anti-hepatitis B virus immune cells. Background technique [0002] Chronic hepatitis B (CHB) is an infectious disease with high incidence in my country, and the most common "trilogy" of disease progression is from CHB to liver cirrhosis and finally to liver cancer. In my country, more than 80% of patients with liver cancer are positive for hepatitis B virology (HBV), and studies have shown that HBsAg-positive hepatitis B patients are more than 100 times more likely to develop liver cancer than negative patients. It can be seen that effective removal of HBV is important for blocking disease progression and preventing disease. The occurrence of liver cancer is very important. Acute hepatitis B and 1-2% chronic hepatitis B patients can effectively clear the virus through their own anti-HBV immune response, complete HBeAg seroconversion an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783A61K38/20A61K39/395A61K39/29A61P31/20C12Q1/02
Inventor 王福生徐若男张纪元福军亮金磊
Owner THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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