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B cell malignant tumor-associated antigen expression quantity detection kit and detection method

A detection kit and a technology for malignant tumors, applied in the biological field, can solve problems such as the limitation of the half-life of nuclides, and achieve the effects of less sample volume, no environmental pollution, and simple operation

Inactive Publication Date: 2017-01-04
北京海思特医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the disadvantage is that it is limited by the half-life of the nuclide and needs to be specially prevented from radioactive contamination
Moreover, radioimmunoassay kits for CD20 and CD22 detection have not yet been developed in China

Method used

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  • B cell malignant tumor-associated antigen expression quantity detection kit and detection method
  • B cell malignant tumor-associated antigen expression quantity detection kit and detection method
  • B cell malignant tumor-associated antigen expression quantity detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] An embodiment of a method for detecting the expression level of a B-cell malignancy-associated antigen of the present invention, such as figure 1 shown, including the following steps:

[0042] S1. Collect test specimens: extract peripheral blood or bone marrow from B-NHL patients as test specimens;

[0043] S2. Adding antibodies and fluorescent staining: Add different fluorescently labeled anti-CD45 antibodies, anti-CD19 antibodies and anti-CD20 antibodies to the test specimen and mix well;

[0044] S3. Flow cytometry detection: After incubating the test specimen and lysing red blood cells, use flow cytometry to detect, and set a gate to obtain CD19 + CD20 + target cell fluorescence intensity;

[0045] S4. Determination of the standard curve: use flow cytometry to measure the fluorescence of QuantiB RITE PEBeads under the same conditions as the test specimen, and obtain the standard curve of the average fluorescence intensity of the Beads and the expression level of ...

Embodiment approach

[0050] As a preferred embodiment, the specific operation process is:

[0051] In step S1, the peripheral blood of the B-NHL patient is taken as a test sample, the white blood cell count is performed on the test sample, and the cell concentration is adjusted to 3×10 7 / mL;

[0052] In step S2, take a dedicated flow tube, add 10 μL of CD45-Percp, 5 μL of CD19APC, and 10 μL of CD20PE into the flow tube; add 70 μL of the test sample into the flow tube, and vortex at low speed to mix;

[0053] In step S3, incubate the mixed test specimen at room temperature in the dark for 20 minutes; add 1 mL of erythrocyte lysate solution to the flow tube, vortex at low speed to mix, and lyse the red blood cells at room temperature in the dark for 10 minutes; After the end, discard the supernatant, then add 1mL PBS to the tube, vortex at low speed to mix, and centrifuge at 1500r / min for 5min; Within 24 hours, use the flow cytometer to test on the machine; during the test, adjust the voltage of ...

Embodiment 2

[0058] The kit and detection process used in this example are basically the same as in Example 1, the difference is that the anti-CD20 antibody added to the flow tube reagent is replaced with an anti-CD22 antibody, that is, the fluorescently labeled antibody uses CD45 Percp / CD19 APC / CD22 PE ; When setting the gate, the setting for CD20 will also be changed for CD22.

[0059] In step S5, BD FACSDIVA software is used to analyze the flow tube of the test specimen, such as Figure 5 Shown:

[0060] Set a gate in the CD45Percp-SSC map to obtain CD45-positive lymphocytes with small SSC, and set a gate to CD45 in the CD45Percp-CD19 APC map + CD19 + B lymphocytes, CD19 was obtained in the CD19 APC-CD22 PE map + CD22 + For the target cells, read the mean value of the fluorescence intensity of the target cells, take the logarithm and substitute it into the standard curve of the Beads fluorescence intensity and antigen expression level to obtain the logarithmic value of the CD22 anti...

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Abstract

The invention provides a B cell malignant tumor-associated antigen expression quantity detection kit and a detection method. Reagents of the kit include a Percp marked CD45 antibody, an APC marked CD19 antibody and a PE marked CD20 or CD22 antibody. Based on flow cytometry, the antigen expression quantity of peripheral blood and normal or abnormal B lymphocyte CD20 and CD22 in bone marrow is detected by a PE micro-sphere introduction method. Compared with existing other detection methods, the method has the advantages that quantitative detection can be realized, the antigen expression quantity of cell surfaces can be directly detected, characteristics of B lymphocyte non-Hodgkin lymphoma can be visually reflected, fewer specimens are used, operation is simple and rapid, and environmental pollution is avoided. Quantitative detection results can be used for instructing targeted therapy of B-NHL by clinical CD20 and CD22 monoclonal antibody-based drugs and judging treatment prognosis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for detecting the expression level of B-cell malignant tumor-associated antigens. The invention also relates to a method for detecting the expression of B cell malignant tumor-associated antigens, which uses flow cytometry to detect the expression of CD20 and CD22 antigens in B-NHL tumor cells. Background technique [0002] Malignant lymphoma is a tumor that occurs in lymph nodes and (or) extranodal lymphoid tissue, and is a group of highly curable tumors. Lymphoma is currently divided into two categories internationally, namely, Hodgkin Lymphoma (Hodgkin Lymphoma, HL) and Non-Hodgkin Lymphoma (Non-Hodgkin Lymphoma, NHL). According to the different origins of lymphocytes, NHL can be divided into B cell, T cell and natural killer (Natural Killer, NK) cell lymphoma, and about 70% to 85% of NHL is derived from B lymphocytes. The 2008 WHO pathological classification divides NHL i...

Claims

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Application Information

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IPC IPC(8): G01N33/50G01N33/574
CPCG01N33/5052G01N33/57407
Inventor 肖思颖朱丽丹王绪华刘丽媛秦晓明王显凤吴纯斌梁超陈忠黄士昂
Owner 北京海思特医学检验实验室有限公司
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