Indirect method for detecting lymphocyte subgroup with mono-clone antibody SPA hematid rosette method

A monoclonal antibody and lymphocyte technology, applied in the field of medical immunology, to avoid time-consuming and complicated operations, reduce cell damage, and simplify processing steps

Inactive Publication Date: 2011-07-20
甘肃省医学科学研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although the freeze-dried antibody sensitized erythrocyte rosette reagent adopted by the McAb-A-E indirect method itself has strong specificity, sensitivity, good stability, long validity period, convenient use, It only needs an ordinary optical microscope to carry out the test, and the test results can be stored for about two weeks. It has been widely used in clinical laboratories and related research units for more than ten years. However, the McAb-A-E indirect method as a clinical test method is due to Many inspection steps, relatively large sample volume, long cycle, easy to cause damage and loss of tested cells, no quantitative control indicators for individual important steps, difficulty in identifying non-specific rosettes and affecting counting results, etc., cannot adapt to clinical rapid, Accurate requirements make it difficult for many laboratories, especially clinical laboratories, to limit the application of this method

Method used

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  • Indirect method for detecting lymphocyte subgroup with mono-clone antibody SPA hematid rosette method
  • Indirect method for detecting lymphocyte subgroup with mono-clone antibody SPA hematid rosette method

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1 Add 1.0 mL of calcium- and magnesium-free Hank'S solution with a pH value of 7.0 to the lyophilized anti-CD3 monoclonal antibody (primary antibody) to dissolve to form a primary antibody storage solution; if the dissolved primary antibody storage solution cannot be used up, 0.1% NaN can be added 3 , sealed and stored at 4-8°C, it can be stored for 2-4 weeks.

[0030] Add 0.5mL calcium and magnesium-free Hank'S solution with a pH value of 7.0 to freeze-dried goat anti-mouse IgG (secondary antibody) sensitized red blood cell rosette reagent to dissolve to form a secondary antibody-sensitized red blood cell preservation solution; the dissolved cell suspension is as follows: Unusable, can add 0.1% NaN 3 , sealed and stored at 4-8°C for 2-4 weeks.

[0031] Draw 1.5 mL of fasting venous blood from the patient with ethylenediaminetetraacetic acid (EDTA) anticoagulant, add 4.5 mL of calcium- and magnesium-free Hank'S solution with a pH value of 7.0, mix well, and ca...

Embodiment 2

[0041] Example 2 Add 1.0 mL of calcium- and magnesium-free Hank'S solution with a pH value of 7.2 to the freeze-dried anti-CD4 monoclonal antibody to dissolve to form a primary antibody storage solution; if the dissolved primary antibody storage solution cannot be used up, add 0.1% NaN 3 , sealed and stored at 4-8°C, it can be stored for 2-4 weeks.

[0042] Add 0.5mL calcium- and magnesium-free Hank'S solution with a pH value of 7.2 to the freeze-dried rabbit anti-mouse IgG sensitized red blood cell rosette reagent to dissolve to form a secondary antibody-sensitized red blood cell preservation solution; if the dissolved cell suspension cannot be used up, 0.1% NaN can be added 3 , sealed and stored at 4-8°C for 2-4 weeks.

[0043] Draw 2 mL of fasting venous blood from the patient with ethylenediaminetetraacetic acid (EDTA) anticoagulant, add 4 mL of calcium- and magnesium-free Hank'S solution with a pH value of 7.2, mix well, and carefully add to 2.5 mL of lymphocyte separat...

Embodiment 3

[0052] Example 3 Add 1.0 mL of calcium- and magnesium-free Hank'S solution with a pH value of 7.4 to the freeze-dried anti-CD8 monoclonal antibody to dissolve to form a primary antibody storage solution; if the dissolved primary antibody storage solution cannot be used up, add 0.1% NaN 3 , sealed and stored at 4-8°C, it can be stored for 2-4 weeks.

[0053] Add 0.5mL calcium- and magnesium-free Hank'S solution with a pH value of 7.4 to the freeze-dried goat anti-mouse IgG sensitized red blood cell rosette reagent to dissolve to form a secondary antibody-sensitized red blood cell preservation solution; if the dissolved cell suspension cannot be used up, 0.1% NaN can be added 3 , sealed and stored at 4-8°C for 2-4 weeks.

[0054] Draw 2.5 mL of fasting venous blood from the patient with ethylenediaminetetraacetic acid (EDTA) anticoagulant, add 2.5 mL of calcium- and magnesium-free Hank'S solution with a pH value of 7.4, mix well, and carefully add to 2.5 mL of lymphocyte separ...

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Abstract

The invention relates to an indirect method for using a monoclonal antibody SPA red cell rosette to detect lymphocyte subsets. The method firstly prepares a primary antibody sensitized red blood cell preservation solution and a secondary antibody sensitized red blood cell preservation solution; secondly, the peripheral blood lymphocytes are separated, and then a secondary antibody sensitized red blood cell application solution and a primary antibody sensitized lymphocyte suspension are prepared by a calcium-free and magnesium-free Hanks balanced salt solution which contains 20 percent newborncalf serum; then the primary antibody sensitized lymphocyte suspension and the secondary antibody sensitized red blood cell application solution are mixed by the volume ratio of 1: 1 to form a mixed suspension; a centrifugation is carried out after the mixed suspension is statically settled under 18 - 28 DEG C, and then the mixed suspension is statically settled for 15 - 45 minutes at the temperature of 4 DEG C; finally, a pipette which is provided with a suction head is used for blowing and sucking the mixed suspension for 8 - 15 times, then cell smears are prepared and calculated by a high-power lens after natural drying and dyeing. Compared with the original McAb-A-E indirect method, the invention is characterized by simplified inspection process, short period, quantized control and satisfying the requirements of clinical inspection.

Description

technical field [0001] The invention relates to medical immunology, in particular to an indirect method for detecting lymphocyte subsets by a monoclonal antibody SPA erythrocyte rosette method. Background technique [0002] With the development of molecular immunology, T, B, and NK lymphocytes can be divided into several subgroups according to the different clusters of differentiation (CD) on the surface of lymphocyte membranes. , anti-CD8, anti-CD19, anti-CD20, anti-CD16, anti-CD56 and other monoclonal antibodies (McAb), to test the peripheral blood lymphocytes, according to the positive rate to determine the total T cells, T helper cells (Th), cells The distribution and changes of toxic T cell (Tc) subsets, B lymphocytes, NK cells, etc., so as to judge the immune function of the human body, and achieve the purpose of preventive health care and disease diagnosis, treatment, and prognosis. [0003] At present, the commonly used detection methods of lymphocyte subsets inclu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/531G01N1/30G01N15/10
Inventor 姚伯程
Owner 甘肃省医学科学研究院
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