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Method for detecting weak antigen expression level based on geometric mean fluorescence index

A technology of geometric mean fluorescence and expression level, applied in the field of flow cytometry, can solve the problem of inability to accurately detect weak antigen expression, and achieve the effect of improving authenticity and objectivity, avoiding experimental errors, and improving accuracy and precision.

Inactive Publication Date: 2015-05-20
JIANGSU PROVINCE HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the above-mentioned problem that flow cytometry cannot accurately detect the expression of weak antigens in the prior art, the present invention provides a method for detecting the expression level of weak antigens by using flow cytometry

Method used

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  • Method for detecting weak antigen expression level based on geometric mean fluorescence index
  • Method for detecting weak antigen expression level based on geometric mean fluorescence index
  • Method for detecting weak antigen expression level based on geometric mean fluorescence index

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Detection of ZAP-70 expression level in chronic lymphocytic leukemia tumor cells

[0028] 1. Preparation of mononuclear cells: freshly submitted chronic lymphocytic leukemia peripheral blood samples 2mL, lymphocyte separation medium to separate mononuclear cells, adjust the concentration of mononuclear cell suspension to ±10 6 cells / 100 μL.

[0029] The above-mentioned target cells can be prepared by selecting different existing technologies according to different samples in actual situations, and the samples can be bone marrow, peripheral blood or lymph nodes.

[0030] 2. Immunofluorescence staining of cells:

[0031] 2.1 Add 100 μL of the prepared single cell suspension to each tube, a total of two tubes. Add 20 μL each of CD3-CD16-CD56-FITC / CD5-APC / CD19-PerCP mouse anti-human monoclonal antibody (from BD Pharmingen, USA) to both tubes, vortex and mix well, and react in the dark at room temperature for 30 minutes.

[0032] 2.2 Wash once with PBS, 1000 rp...

Embodiment 2

[0052] Example 2: Detection of IgG expression level of bone marrow hematopoietic cell autoantibody

[0053] 1. Preparation of mononuclear cells: 2ml of freshly drawn bone marrow samples from patients with suspected immune-related cytopenias, and the concentration of the mononuclear cell suspension is ±10 6 cells / 100 μL.

[0054] 2. Immunofluorescence staining of cells.

[0055] 2.1 Add 100 μL of the prepared single cell suspension, 3 tubes in total. Add 20 μL each of CD15-FITC / IgG-PE / CD19-PerCP / and CD235a-FITC / IgG-PE / CD19-PerCP mouse anti-human monoclonal antibody (from BD Pharmingen, USA) to the tube to be tested, and add CD15- 20ul each of FITC / IgG1-PE / CD19-PerCP and CD235a-FITC / IgG1-PE / CD19-PerCP were mixed by vortexing and reacted at room temperature for 30 minutes in the dark.

[0056] 2.2 Wash once with PBS, 1000 rpm, centrifuge to remove supernatant.

[0057] 3. Add 500uLPBS, and test and analyze on the machine.

[0058] 3.1 Set the resolution of the fluorescence ...

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Abstract

The invention discloses a method for detecting a weak antigen expression level based on a geometric mean fluorescence index. The method comprises the following steps: at first, carrying out immunofluorescence dyeing on a target cell, then detecting the dyed cell on a flow cytometer, and carrying out data calculation and analysis, wherein when setting the parameters of the flow cytometer, reducing the resolution of a fluorescence channel to be detected; after setting the geometric mean fluorescence strength of isotype control of an antigen to be detected of the target cell as a fixed value, obtaining the geometric mean fluorescence strength of antigens to be detected of a positive cell and the target cell, calculating the geometric mean fluorescence index of the antigen, if the geometric mean fluorescence index of the antigen to be detected of the target cell is higher than a defined value, the antigen to be detected is expressed. The method disclosed by the invention is used for effectively improving the detection accuracy and precision of the target cell, reducing the experimental errors and subjective judgment errors in the detection of weak antigen expression to the uttermost and effectively promoting the practical value of the flow cytometer in a lot of fields.

Description

technical field [0001] The invention relates to the technical field of flow cytometry, and more specifically, relates to a method for detecting the expression level of weak antigens based on a geometric mean fluorescence index using a flow cytometer. Background technique [0002] Flow cytometry (Flow Cytometry, FCM) is a detection method for quantitative analysis and sorting of single cells or other biological particles at the functional level. It uses laser as the excitation light source, can analyze tens of thousands of cells at high speed, and can measure multiple parameters from one cell at the same time. It has the advantages of fast speed, high precision, and good accuracy. It has become the most advanced cell quantitative analysis technology , is widely used in clinical medicine and various biological research fields. At present, most flow cytometers believe that they can detect weak antigens with as low as 100 fluorescent molecules on a single cell in terms of theor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N15/14
Inventor 吴雨洁何广胜陈梓灵李建勇徐卫王慧沈安俐
Owner JIANGSU PROVINCE HOSPITAL