Detection kit for evaluating NK cell culture purity using mass spectrometry technology and its application

Abstract

The present invention belongs to the field of mass spectrometry flow cytometry technology, specifically a detection kit and application for evaluating the purity of NK cell culture using mass spectrometry flow cytometry technology. The present invention discloses a kit for detecting the immune status of human NK cells. The kit can be used to monitor changes in the immune status of the human body and evaluate the purity of its NK cell culture. Based on a specific or unified culture method, the difference in the purity of NK cell culture in samples is evaluated, thereby reducing the overactivation of NK cells. For samples with low NK cell culture purity, the types of additives or culture medium components are controlled to be added, and the NK cells are specifically purified and activated, thereby reducing the difference between samples with high-purity NK cell culture and samples with low-purity NK cell culture. 37 antibodies can be used to quickly and accurately perform in-depth analysis of immune cells in peripheral blood and evaluate the purity of NK cell culture by collecting peripheral blood intravenously on a mass spectrometry flow cytometer platform.

Description

Technical Field

[0001] The present invention relates to the technical field of mass spectrometry flow cytometry, and in particular to a detection kit for evaluating the purity of NK cell culture by mass spectrometry flow cytometry and its application. Background Art

[0002] Mass spectrometry flow cytometry is a flow technology that uses the principle of mass spectrometry to perform multi-parameter detection on single cells. It inherits the high-speed analysis characteristics of traditional flow cytometers and has the high-resolution capability of mass spectrometry detection. It is a new development direction of flow cytometry technology.

[0003] Mass cytometry technology can achieve accurate immunophenotyping of cell populations, comprehensive analysis of intracellular signal transduction networks, analysis of functional connections between cell subpopulations, and high-throughput multi-parameter detection of large numbers of samples. Most of the existing mass cytometry technologies are aimed at the culture medium components in the NK cell culture process, or the addition of additives in vitro, or the optimization of separation methods, while ignoring the differences between sample donors. The introduction of either culture medium components or additives will over-activate NK cells, which will cause heterogeneity in downstream NK cell research. Summary of the invention

[0004] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract of the specification and the title of the invention of this application to avoid blurring the purpose of this section, the abstract of the specification and the title of the invention, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0005] In view of the problems existing in the existing NK cell culture, the present invention is proposed.

[0006] To solve the above technical problems, according to one aspect of the present invention, the present invention provides the following technical solutions: a detection kit for evaluating the purity of NK cell culture by mass spectrometry flow cytometry, the present invention discloses a kit for detecting the immune status of human NK cells, the kit can be used to monitor the changes in the immune status of the human body and evaluate the purity of its NK cell culture, the kit comprises 37 labeled antibodies;

[0007] The 37 antibodies are monoclonal antibodies CD45, CD196 / CCR6, CD123 / IL-3R, CD11c, CD16, CD45RO, CD27, CD183 / CXCR3, CD185 / CXCR5, CD28, TCRgd, CD294 / CRTH2, CD197 / CCR7, CD14, CD3, CD20, HLA-DR, CD127 / IL-7Ra, CD69, CD137, CD62L, Ki67, NKG2A, NKG2D, NKp30, CD19, CD4, CD8, CD45RA, CD161, CD194 / CCR4, CD25 / IL-2R, CD38, CD56 / NCAM, CD66b, CD11b and NKp44.

[0008] A method of using the mass spectrometry flow cytometry detection kit to evaluate the purity of NK cell culture comprises the following steps:

[0009] S1. Blood collection;

[0010] S2.FcR-blocking;

[0011] S3. Staining, the staining is divided into two parts, the first part is for membrane protein staining, and the second part is for cytoplasmic protein staining;

[0012] S4. Get on the machine;

[0013] S5. Data analysis and interpretation.

[0014] As a preferred solution for the application of the detection kit for evaluating the purity of NK cell culture by mass spectrometry flow cytometry technology described in the present invention, the method comprises the following steps:

[0015] 1) Collect 5 ml of blood in an anticoagulant tube to prevent coagulation and platelet activation. Add 5 ml of blood and an equal volume of D-Hanks solution to an empty 15 ml centrifuge tube, mix and shake well. Take another empty 50 ml centrifuge tube, add 10 ml of Ficoll to it, and then use a dropper to draw the diluted blood and slowly drip it onto the Ficoll along the tube wall to form layers. Perform density gradient centrifugation at room temperature with a brake speed of 1,400 g x 20 min.

[0016] 2) Add 50ul blockmix to the sample, resuspend the cells, and place at room temperature for 10 minutes. There is no need to remove the blocking solution after the incubation.

[0017] 3) Membrane protein staining: Add 50μl antibody cocktail, 37 antibody combinations, and 50ul blocking solution to each tube of sample after blocking. The volume is now 100ul. Gently pipette to mix the cells. Place at room temperature for 15 minutes. Gently vortex to mix the cells. Continue to place at room temperature for 15 minutes. Add 2ml Cell Staining Buffer to each tube of sample. Centrifuge at room temperature for 300gX5min and discard the supernatant.

[0018] Cytoplasmic protein staining: Prepare FixI solution, dilute 5XFixI stock solution to 1X with PBS, each sample requires 1ml1XFixI solution, add 1ml1XFixI solution to each tube, mix well, let stand at room temperature for 10-30min, add 2mlPerm-Sbuffer to each tube, centrifuge at 800gX5min, discard the supernatant, add 50μl antibody cocktail to each tube of cells, gently vortex the cells, let stand at room temperature for 30min, add 2ml Cell Staining Buffer to each tube, centrifuge at 800gX5min at room temperature, discard the supernatant, repeat the above steps, and discard the supernatant;

[0019] 4) Centrifuge at room temperature for 800g for 5 min, discard the supernatant, add 2ml Cell Staining Buffer to each sample to resuspend the cells, centrifuge at room temperature for 800g for 5 min, discard the supernatant, add 1ml Cell Acquisition Solution to each sample, resuspend the cells with CAS solution, take 10μl for cell counting, centrifuge the rest of the suspension at room temperature for 800g for 5 min, discard the supernatant, place the cell pellet on ice until resuspending before loading. According to the cell counting results, resuspend the cells with CAS solution to 1.0X10 6 / mlHelios suspension, add 10% EQBeads to the cell suspension, start quality control - collect data on the machine WB Injector;

[0020] 5) The data collected directly by the instrument is the most original data and needs to be processed before data analysis.

[0021] As a preferred scheme for the application of the detection kit for evaluating the purity of NK cell culture using the mass spectrometry flow technology described in the present invention, wherein: after the centrifugation is completed, the cells in the centrifuge tube are divided into four layers from top to bottom, the first layer, the plasma layer, the second layer, the mononuclear cell layer, the third layer, the transparent separation liquid layer, the fourth layer: the red blood cell layer, and the mononuclear cell layer is aspirated with a dropper for washing.

[0022] As a preferred solution for the application of the mass spectrometry flow cytometry detection kit for evaluating the purity of NK cell culture, the 10% EQBeads are prepared from 1 part of EQbeads and 9 parts of CAS, and the cell suspension is filtered using a flow tube with a 35μm filter.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. Based on a specific or unified culture method, evaluate the difference in NK cell culture purity in the sample, thereby reducing NK cell overactivation. For samples with low NK cell culture purity, control the addition of additives or culture medium components to specifically purify and activate NK cells, thereby reducing the difference between samples with high NK cell purity and samples with low NK cell purity.

[0025] 2. By using 37 kinds of antibodies, the immune cells in the peripheral blood can be quickly and accurately analyzed in depth and the purity of NK cell culture can be evaluated by collecting peripheral blood intravenously on the mass spectrometry flow cytometer platform, making the kit highly specific, sensitive and accurate in evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail below in combination with the accompanying drawings and detailed embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0027] Figure 1 It is an application flow chart of the detection kit for evaluating the purity of NK cell culture by mass spectrometry technology of the present invention;

[0028] Figure 2 This is a data quality control result diagram of the detection kit for evaluating the purity of NK cell culture using the mass spectrometry technology of the present invention;

[0029] Figure 3 This is a data quality control result diagram of the detection kit for evaluating the purity of NK cell culture using the mass spectrometry technology of the present invention;

[0030] Figure 4 This is a cell dimensionality reduction analysis result diagram of a detection kit for evaluating the purity of NK cell culture using mass spectrometry technology of the present invention;

[0031] Figure 5 This is a thermal diagram of the analysis results of the detection kit for evaluating the purity of NK cell culture using the mass spectrometry technology of the present invention;

[0032] Figure 6This is a diagram showing the results of differential cell subpopulation analysis of a detection kit for evaluating the purity of NK cell culture using mass spectrometry technology of the present invention. DETAILED DESCRIPTION

[0033] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0034] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0035] Secondly, the present invention is described in detail with reference to schematic diagrams. When describing the embodiments of the present invention in detail, for the sake of convenience, the cross-sectional diagrams showing the device structure will not be partially enlarged according to the general scale, and the schematic diagrams are only examples, which should not limit the scope of protection of the present invention. In addition, in actual production, the three-dimensional dimensions of length, width and depth should be included.

[0036] Example

[0037] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0038] A detection kit for evaluating the purity of NK cell culture by mass spectrometry technology. The present invention discloses a kit for detecting the immune status of human NK cells. The kit can be used to monitor changes in the immune status of the human body and evaluate the purity of its NK cell culture. The kit contains 37 labeled antibodies.

[0039] The 37 antibodies were monoclonal antibodies CD45, CD196 / CCR6, CD123 / IL-3R, CD11c, CD16, CD45RO, CD27, CD183 / CXCR3, CD185 / CXCR5, CD28, TCRgd, CD294 / CRTH2, CD197 / CCR7, CD14, CD3, CD20, HLA-DR, CD127 / IL-7Ra, CD69, CD137, CD62L, Ki67, NKG2A, NKG2D, NKp30, CD19, CD4, CD8, CD45RA, CD161, CD194 / CCR4, CD25 / IL-2R, CD38, CD56 / NCAM, CD66b, CD11b, and NKp44.

[0040] An application of a detection kit for evaluating the purity of NK cell culture using mass spectrometry technology as claimed in claim 1, comprising the following steps;

[0041] S1. Blood collection;

[0042] S2.FcR-blocking;

[0043] S3. Staining, the staining is divided into two parts, the first part is for membrane protein staining, and the second part is for cytoplasmic protein staining;

[0044] S4. Get on the machine;

[0045] S5. Data analysis and interpretation.

[0046] In this example, the application of mass spectrometry technology with a detection kit for evaluating the purity of NK cell culture includes the following steps:

[0047] 1) Collect 5 ml of blood in an anticoagulant tube to prevent coagulation and platelet activation. Add 5 ml of blood and an equal volume of D-Hanks solution to an empty 15 ml centrifuge tube, mix and shake well. Take another empty 50 ml centrifuge tube, add 10 ml of Ficoll to it, and then use a dropper to draw the diluted blood and slowly drip it onto the Ficoll along the tube wall to form layers. Perform density gradient centrifugation at room temperature with a brake speed of 1,400 g x 20 min.

[0048] 2) Add 50ul blockmix to the sample, resuspend the cells, and place at room temperature for 10 minutes. There is no need to remove the blocking solution after the incubation.

[0049] 3) Membrane protein staining: Add 50μl antibody cocktail, 37 antibody combinations, and 50μl blocking solution to each tube of sample after blocking, then the volume is 100μl, gently pipette to mix the cells, let it stand at room temperature for 15 minutes, gently vortex to mix the cells, let it stand at room temperature for another 15 minutes, add 2ml Cell Staining Buffer to each tube of sample, centrifuge at room temperature for 300gX5min, and discard the supernatant;

[0050] Cytoplasmic protein staining: Prepare FixI solution, dilute 5XFixI stock solution to 1X with PBS, each sample requires 1ml1XFixI solution, add 1ml1XFixI solution to each tube, mix well, let stand at room temperature for 10-30min, add 2mlPerm-Sbuffer to each tube, centrifuge at 800gX5min, discard the supernatant, add 50μl antibody cocktail to each tube of cells, gently vortex the cells, let stand at room temperature for 30min, add 2ml Cell Staining Buffer to each tube, centrifuge at 800gX5min at room temperature, discard the supernatant, repeat the above steps, and discard the supernatant;

[0051] 4) Centrifuge at room temperature for 800g for 5 min, discard the supernatant, add 2ml Cell Staining Buffer to each sample to resuspend the cells, centrifuge at room temperature for 800g for 5 min, discard the supernatant, add 1ml Cell Acquisition Solution to each sample, resuspend the cells with CAS solution, take 10ul for cell counting, centrifuge the rest of the suspension at room temperature for 800g for 5 min, discard the supernatant, place the cell pellet on ice until resuspending before loading. According to the cell counting results, resuspend the cells with CAS solution to 1.0X10 6 / mlHelios suspension, add 10% EQBeads to the cell suspension, start quality control - collect data on the machine WBInjector;

[0052] 5) The data collected directly by the instrument is the most original data and needs to be processed before data analysis.

[0053] In this example, after centrifugation, the cells in the centrifuge tube are divided into four layers from top to bottom, the first layer, the plasma layer, the second layer, the mononuclear cell layer, the third layer, the transparent separation liquid layer, and the fourth layer: the red blood cell layer, and the mononuclear cell layer is aspirated with a dropper, washed, and passed.

[0054] In this example, 10% EQBeads was prepared from 1 part EQBeads and 9 parts CAS, and the cell suspension was filtered using a flow tube with a 35 um filter.

[0055] Example 1

[0056] According to the kit instructions, dilute the antibody according to the number of cells;

[0057] Example 2

[0058] Data quality control: Mass spectrometry detection is mainly for single cells. If there are a lot of cell fragments / cell polymers / dead cells during the analysis, it is necessary to circle out the live single cells and perform corresponding analysis on the live single cells. The results are as follows: Figure 2 and Figure 3 shown.

[0059] Example 3

[0060] Data analysis

[0061] Cell dimensionality reduction analysis, based on the purpose of analysis, imports the data into the bioinformatics analysis website through the client for corresponding analysis.

[0062] Dimensionality reduction analysis; 46 subgroups were classified, and the results were as follows Figure 4 As shown;

[0063] The results of heat map analysis are as follows Figure 5 As shown;

[0064] T cells (1, 3-6, 29-40, 43, and 44)

[0065] B cells (27 and 28)

[0066] NK cells (2, 7-12, 20, and 23)

[0067] NKT cells (15, 34, and 17)

[0068] γδ T cells (14, 16, 45, and 46)

[0069] Monocytes (19 and 21-22)

[0070] Granulocytes (18)

[0071] DC cells (24-26)

[0072] Others (13, 41, 42)

[0073] Differential cell subpopulation analysis, the results are as follows Figure 6 shown.

[0074] Although the present invention has been described above with reference to the embodiments, various modifications may be made thereto and parts thereof may be replaced by equivalents without departing from the scope of the present invention. In particular, as long as there is no structural conflict, the various features in the embodiments disclosed in the present invention may be used in combination with each other in any manner, and the fact that these combinations are not exhaustively described in this specification is only for the sake of omitting space and saving resources. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. Use of an antibody composition in the preparation of a detection kit for evaluating the purity of NK cell culture based on mass spectrometry flow cytometry, characterized in that; The antibody composition comprises 37 labeled antibodies; The 37 antibodies are monoclonal antibodies CD45, CD196 / CCR6, CD123 / IL-3R, CD11c, CD16, CD45RO, CD27, CD183 / CXCR3, CD185 / CXCR5, CD28, TCRgd, CD294 / CRTH2, CD197 / CCR7, CD14, CD3, CD20, HLA-DR, CD127 / IL-7Ra, CD69, CD137, CD62L, Ki67, NKG2A, NKG2D, NKp30, CD19, CD4, CD8, CD45RA, CD161, CD194 / CCR4, CD25 / IL-2R, CD38, CD56 / NCAM, CD66b, CD11b and NKp44.

2. The use of the antibody composition according to claim 1 in the preparation of a detection kit for evaluating the purity of NK cell culture based on mass spectrometry flow cytometry, characterized in that: It includes the following steps: S1. Blood collection; S2.FcR-blocking; S3. Staining, the staining is divided into two parts, the first part is for membrane protein staining, and the second part is for cytoplasmic protein staining; S4. Get on the machine; S5. Data analysis and interpretation.

3. Use of the antibody composition according to claim 2 in the preparation of a detection kit for evaluating the purity of NK cell culture based on mass spectrometry flow cytometry, characterized in that: It includes the following step; Collect 5 ml of blood in an anticoagulant tube to prevent coagulation and platelet activation. Add 5 ml of blood and an equal volume of D-Hanks solution to an empty 15 ml centrifuge tube, mix and shake well. Take another empty 50 ml centrifuge tube, add 10 ml of Ficoll to it, and then use a dropper to draw the diluted blood and slowly drip it onto the Ficoll along the tube wall to form layers. Perform density gradient centrifugation at room temperature with a brake speed of 1,400 g x 20 min. Add 50 μl of block mix to the sample, resuspend the cells, and place at room temperature for 10 minutes. It is not necessary to remove the blocking solution after the incubation. Membrane protein staining: add 50μl lantibody cocktail, 37 antibody combinations, and 50μl blocking solution to each tube of sample after blocking, the volume is now 100μl, gently pipette to mix the cells, let it stand at room temperature for 15 minutes, gently vortex to mix the cells, let it stand at room temperature for another 15 minutes, add 2ml Cell Staining Buffer to each tube of sample, centrifuge at room temperature for 300gX5min, and discard the supernatant; Cytoplasmic protein staining: Prepare FixI solution, dilute 5XFixI stock solution to 1X with PBS, each sample requires 1ml1XFixI solution, add 1ml1XFixI solution to each tube, mix well, let stand at room temperature for 10-30min, add 2mlPerm-Sbuffer to each tube, centrifuge at 800gX5min, discard the supernatant, add 50μl antibody cocktail to each tube of cells, gently vortex the cells, let stand at room temperature for 30min, add 2ml Cell Staining Buffer to each tube, centrifuge at 800gX5min at room temperature, discard the supernatant, repeat the above steps, and discard the supernatant; Centrifuge at room temperature for 800gX5min, discard the supernatant, add 2ml Cell Staining Buffer to each sample to resuspend the cells, centrifuge at room temperature for 800gX5min, discard the supernatant, add 1ml Cell Acquisition Solution to each sample, resuspend the cells with CAS solution, take 10ul for cell counting, centrifuge the rest of the suspension at room temperature for 800gX5min, discard the supernatant, place the cell pellet on ice until resuspending before loading on the machine, according to the cell counting results, resuspend the cells with CAS solution to 1.0X10 6 / mlHelios suspension, add 10%EQ Beads to the cell suspension, start quality control - collect data on the machine WBInjector; The data collected directly by the instrument is the most original data and needs to be processed before data analysis.

4. Use of the antibody composition according to claim 3 in the preparation of a detection kit for evaluating the purity of NK cell culture based on mass spectrometry flow cytometry, characterized in that: After centrifugation, the cells in the centrifuge tube are divided into four layers from top to bottom: the first layer is the plasma layer, the second layer is the mononuclear cell layer, the third layer is the transparent separation liquid layer, and the fourth layer is the red blood cell layer. The mononuclear cell layer is aspirated with a dropper and washed.

5. Use of the antibody composition according to claim 3 in the preparation of a detection kit for evaluating the purity of NK cell culture based on mass spectrometry flow cytometry, characterized in that: The 10% EQBeads were prepared from 1 part EQbeads and 9 parts CAS, and the cell suspension was filtered using a flow tube with a 35 um filter.

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