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Quantitative determination method for hepatitis b virus specificity cell toxicity T lymphocyte

A technology of hepatitis B virus and lymphocytes, which is applied in the field of immune detection, can solve the problems that need to be further improved, achieve reliable results, reduce non-specific binding, and reduce the effect of background staining

Inactive Publication Date: 2009-05-06
JIANGSU PROVINCE HOSPITAL
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  • Application Information

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Problems solved by technology

[0007] Therefore, the current methods for detecting specific CTLs need to be further improved in terms of specificity, sensitivity and stability.

Method used

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  • Quantitative determination method for hepatitis b virus specificity cell toxicity T lymphocyte
  • Quantitative determination method for hepatitis b virus specificity cell toxicity T lymphocyte
  • Quantitative determination method for hepatitis b virus specificity cell toxicity T lymphocyte

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Experimental program
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Embodiment 1

[0046] Embodiment 1: detection process of the present invention

[0047] 1. Marking of HLA-A2 and HLA-A24 gene subtypes: the purpose is to conduct preliminary screening of HLA-A2 and HLA-A24 gene subtypes in patients with hepatitis B, and the detection is carried out according to the instructions: the method is to add different Fluorescein thiocyanate (FITC)-labeled mouse anti-human immunoglobulin G1 (IgG1)- and phycoerythrin (PE)-labeled mouse anti-human IgG1 10 μl each, the reagents are all products of BD Company in the United States; in the detection tube Add 10 μl of mouse anti-human HLA-A2-FITC (the reagent is the product of BD Pharmingen, USA) and 10 μl of mouse anti-human HLA-A24-PE (the reagent is the product of Japan MBL Company) respectively, and incubate at 22—25°C in the dark 20-30min, with 3ml 0.01M pH 7.2 phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (bovine serum albumin, BSA), centrifuge at 500g for 5min, discard the supernatant, then sha...

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Abstract

The invention belongs to the field of immunodetection and discloses a method for the quantitative determination of specific cytotoxicityT lymphocyte of hepatitis B virus. The method adopts the technical proposal that the major histocompatibility compound of antigen peptide pentamer, a mouse anti-human CD3 monoclonal antibody and a mouse anti-human CD8 monoclonal antibody which are labeled by different fluoresceins are incubated with the anticoagulant peripheral blood of an HLA-A0201 or HLA-A2402 positive masculine hepatitis B virus carrier for a proper period; and after the operations of hematid schizolysis, centrifugal washing and cell fixation are completed, the CD3 gating technique is used for the quantitative determination of the specific cytotoxicityT lymphocyte of hepatitis B virus on a flow cytometry. The invention can increase the determination of the specificity, the sensitivity and the stability of the specific CTL.

Description

technical field [0001] The invention belongs to the field of immunoassay, and belongs to a method of detecting hepatitis B virus (HBV) specific cytotoxic T lymphocytes ( CTL) method. Background technique [0002] my country is a high-endemic area of ​​HBV infection, and chronic HBV infection is closely related to the occurrence of liver cirrhosis and primary liver cancer, but so far there is no drug to completely eliminate hepatitis B virus and cure chronic hepatitis B (CHB) and methods. The mechanism of chronic HBV persistent infection, domestic and foreign research recognized the most important factor is the lack of polyclonal, strong anti-HBV specific CTL immune response. The polyclonal specific CTL response in HBV-infected patients is the most important mechanism for clearing the virus, especially the virus in the liver cells. After HBV infection, different individuals have different immune response patterns, and their clinical outcomes are also different (Thio CL, Tho...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/576
Inventor 李军黄祖瑚韩亚萍刘源邢益平万玉峰孔练花周东辉卢山
Owner JIANGSU PROVINCE HOSPITAL
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