Method for detecting secretory cells of peripheral blood hepatitis B surface antibody

A hepatitis B surface antibody and peripheral blood technology, applied in measuring devices, analytical materials, immunoassays, etc., can solve the problems of poor experimental repeatability, low activation efficiency, and long time consumption, so as to shorten the required time and reduce the amount of blood used , the effect of increasing sensitivity

Inactive Publication Date: 2017-06-06
NANJING DRUM TOWER HOSPITAL
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Problems solved by technology

[0005] (1) The number of anti-HBs secreting cells in peripheral blood is small, the detection rate is low, and the repeatability of the experiment is poor;
[0006] (2) The detection of antibody-secreting cells by the limited dilution method is cumbersome, time-consuming, and difficult, and requires a large amount of peripheral blood, making it difficult to popularize and apply;
[0007] (3) In addition to plasma cells directly secreting antibodies in peripheral blood, memory B cells need to be stimulated and activated to differentiate

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  • Method for detecting secretory cells of peripheral blood hepatitis B surface antibody

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Embodiment Construction

[0019] Technical thinking of the present invention is:

[0020] Peripheral blood human peripheral blood mononuclear cells (PBMC) were stimulated with R848, HBsAg and IL-2. After activation of B cells, the human peripheral blood anti-HBs secreting cells were captured by PVDF plate and counted and analyzed after color development.

[0021] The present invention is described in detail below:

[0022] 1. Stimulation and activation of B cells

[0023] (1) Sterile collection of human peripheral blood;

[0024] (2) Separation of lymphocyte separation liquid to obtain PBMC;

[0025] (3) PBMCs were resuspended in 1640 culture medium containing 10% fetal bovine serum, 1 μg / ml R848, 2 μg / ml HBsAg, 10 ng / ml IL-2, and the cell concentration was 2×10 6 / ml, put in 5% CO 2 , 37°C and saturated humidity incubator for 5 days.

[0026] 2. Detection of anti-HBs secreting cells in human peripheral blood by PVDF plate

[0027] (1) Coat PVDF plate with 10 μg / ml HBsAg, add 15 μg / ml anti-human ...

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Abstract

The invention relates to a method for detecting secretory cells of a peripheral blood hepatitis B surface antibody. According to the method disclosed by the invention, human Peripheral Blood Mononuclear Cells (PBMC) are stimulated and activated in vitro, and a pretreated Polyvinylidene Fluoride (PVDF) plate is used for detecting anti-HBs secretory cells in the PBMCs, to detect that a million of PBMCs contain one anti-HBs secretory cell. The method overcomes the detection difficulties in peripheral blood anti-HBs secretory cells, such as small quantity of the peripheral blood anti-HBs secretory cells, low detection rate, low experiment repetitiveness, complex operation, long time consumption and high difficulty, solves the problems that much peripheral blood needs to be adopted and in-vitro B cell activation methods such as CPG, CD40L and pokeweed mitogen are low in activation efficiency, and overcomes the defects of reduction of the detection rate of the anti-HBs secretory cells and the like. According to the method, R848, HBsAg and IL-2 are used for stimulating and activating peripheral blood B cells, and the HBsAg pre-coated PVDF plate is used for capturing the peripheral blood anti-HBs secretory cells, so that the blood amount for detection is reduced, and the detection sensitivity is improved.

Description

technical field [0001] The invention relates to a method for detecting special antibody-secreting cells in the technical field of biomedical engineering, in particular to a method for detecting antibody-secreting cells on the surface of hepatitis B in peripheral blood. Background technique [0002] Hepatitis B surface antibody (anti-HBs) is a sign of the body's immune protection against hepatitis B virus (HBV), and plays a very important role in the process of HBV clearance and prevention of reinfection. Anti-HBs in peripheral blood is secreted by plasma cells. After the body is stimulated by hepatitis B surface antigen (HBsAg), B cells mature and differentiate into plasma cells that secrete anti-HBs and memory B cells that do not secrete antibodies. The life span of plasma cells is short, while Memory B cells can survive for a long time without antigen stimulation, but their number is very small in peripheral blood. After the body is stimulated by HBsAg again, memory B cel...

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Application Information

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IPC IPC(8): G01N33/569
CPCG01N33/56966G01N2469/20
Inventor 吴超刘勇田辰陈雨欣黄睿
Owner NANJING DRUM TOWER HOSPITAL
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