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Reagent for permeating and immobilizing blood cells and an analysis method

An analysis method and technology of blood cells, applied in the field of cell and molecular analysis, can solve the problems of large changes in cell shape, destruction of cell surface antigenic determinants, and complicated steps.

Active Publication Date: 2021-03-16
GENERAL HOSPITAL OF NUCLEAR IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The previous permeabilization method for cells in slides or suspension was to treat the cells by diluting them with alcohol at low temperature (-20°C). Cell morphology changes greatly, destroying cell surface epitopes

Method used

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  • Reagent for permeating and immobilizing blood cells and an analysis method
  • Reagent for permeating and immobilizing blood cells and an analysis method
  • Reagent for permeating and immobilizing blood cells and an analysis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1, non-fixed cells and fixed cell surface antigen labeling

[0052] Table 1 provides 18 cases of whole blood cell membrane surface markers of healthy subjects treated with the reagent of the present invention. The results show that the non-specific binding of antibodies to fixed cells is slightly higher than that of non-fixed cells, but does not produce a large amount of non-specific Fluorescence (negative control) is not enough to interfere with the detection of cell surface marker antigens, and the cells treated with the reagents of the present invention can also maintain sufficient light scattering properties so that lymphocytes, monocytes and granulocytes can be distinguished, and the cell membrane antigens remain Complete and specific. However, when using 10% formaldehyde to fix cells in the prior art, it will increase the non-specific binding of cells (negative control), and change the binding of CD2 epitopes on lymphocytes (decrease in positive expression...

Embodiment 2

[0055] Example 2, Unfixed Cells and Fixed Infiltrated Cell Target Intraantigen Labeling

[0056] The experimental method was operated according to the above-mentioned steps, and 18 healthy subjects were labeled with whole blood cells. The results in the table are the average positive expression and signal-to-noise ratio (P / N) of some representative cell target antigens. The P / N is calculated by using the ratio of the special expression of the molecule in the target cell labeled with the monoclonal antibody to the mean fluorescence intensity (MFI) ratio of the corresponding molecule treated with the isotype control antibody. The internal protein is fixed and retained in place, and the fluorescently labeled macromolecular antibody can penetrate into the cell and bind to the corresponding protein antigen. Dimethyl sulfoxide helps to improve the detection of the intracellular antigen. Intracellular antigen labeling can increase the sensitivity of detection.

[0057] Table 2

[0...

Embodiment 3

[0060]Example 3, evaluating the effects of different concentrations of anionic surfactant BX, amino acid Glycine and different pH conditions on the expression of MPO and Lysozyme in neutrophil cells, the data show that Glycine can reduce the formaldehyde released by diazolidinyl urea The resulting fluorescence quenching of the labeled antibody conjugate improves the signal-to-noise ratio of the detection, and at the same time, the 1.2% BX concentration and the acidic condition of PH5.5 are more sensitive to cell staining.

[0061] table 3

[0062]

[0063]

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PUM

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Abstract

The invention discloses a reagent for permeating and immobilizing blood cells and an analysis method. The reagent comprises an immobilizing solution, a permeating solution and a cleaning solution, wherein the immobilizing solution comprises a transcellular membrane transport compound with the volume concentration of 1%-20%, a saccharide compound with the mass concentration of 0.1 mg / ml-10mg / ml, and one or more of a fatty aldehyde compound, an imine compound, a urea compound and an oxazolidine compound with the molar concentration of 0.3 mol / L-12. 3mol / L, wherein the permeation solution comprises a surfactant with the mass concentration of 0.01%-10% and protein or amino acid with the mass concentration of 0.2%-15%. The reagent can be used for effectively cracking red blood cells and permeabilizing leukocyte membranes without substantially damaging cell characteristics such as surface markers, forms and light scattering properties of the leukocyte membranes, and allows antibodies or other interested components to enter cells, so that cell contents are well preserved; protein, nucleic acid, enzyme, cytokines, virus particles and the like in cells are analyzed through a flow cytometer.

Description

technical field [0001] The invention relates to the technical field of cell molecule analysis, more specifically, it relates to reagents and analysis methods for infiltrating and fixing blood cells. Background technique [0002] The analysis of blood cell differentiation phenotype is used to assess the disease state and other health conditions of organisms is the latest development trend in the field of cell molecular analysis, and foreign countries have been able to provide commercial kits for detailed analysis of intracellular and cell surface targets. analyze. Staining of cell surface target antigens is readily accomplished using existing monoclonal antibody labeling techniques, such as by using specific cell surface markers to identify specific subpopulations of leukocytes, using flow cytometry, and determining the specificity Binder-labeled cells are analyzed to detect and differentiate multiple markers and physical characteristics of the cells, such as cell size and g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/30G01N1/34G01N15/14
CPCG01N1/30G01N1/34G01N15/14G01N2001/305
Inventor 俞秋兴张海方
Owner GENERAL HOSPITAL OF NUCLEAR IND
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