CN116679059A - Quantitative method for single cell surface antigen expression quantity - Google Patents

Quantitative method for single cell surface antigen expression quantity Download PDF

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CN116679059A
CN116679059A CN202310662465.2A CN202310662465A CN116679059A CN 116679059 A CN116679059 A CN 116679059A CN 202310662465 A CN202310662465 A CN 202310662465A CN 116679059 A CN116679059 A CN 116679059A
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target
cell
cells
buffer
antigen
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梅恒
胡豫
雒文静
李成功
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Tongji Medical College of Huazhong University of Science and Technology
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Tongji Medical College of Huazhong University of Science and Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention belongs to the technical field of biological detection, and particularly discloses a quantitative method for the expression quantity of single cell surface antigens, which comprises the steps of S0, drawing a standard curve and constructing a standard equation; s1, target cell treatment: pretreating target cells, incubating the target cells with fluorescein-conjugated antibodies of the same class as the fluorescent quantitative kit, wherein the target cells can be detected by the corresponding fluorescein-conjugated antibodies; s2, obtaining the number of target antigen molecules on the surface of the cell: and detecting the average fluorescence intensity MFI of the corresponding fluorescence channel of the target antigen molecule on the surface of the target cell by using a fluorescence quantification kit through a flow meter on-machine, and according to a formula II: absolute number of antigen molecules of interest m=10 (b+a×log) 10 MFI) calculation of the individual orderThe absolute number m of antigen molecules of interest on the surface of the target cells. The invention makes the detection of the expression number of the target antigen molecules on the surface of the single cell simpler and more feasible, and provides a feasible scheme for closely monitoring the expression of the target antigen on the surface of the tumor cell, the expression of the CAR on the surface of the immune cell and the expression of the target antigen on the surface of the rest target cell in vitro and in vivo.

Description

Quantitative method for single cell surface antigen expression quantity
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a quantitative method for the expression quantity of single cell surface antigens.
Background
In targeted immunotherapy of malignant tumors, the expression number of antigen molecules of interest on the surface of tumor cells (such as CD19, CD7, BCMA, CD38, CD20, CD22, CD30, GD2 and the like) is related to the therapeutic effect, and quantitative analysis and monitoring of the change of the expression level of the antigen have certain guiding significance for clinical management. Chimeric antigen receptor T cells (Chimeric antigen receptor-T cells, CAR-T) are an important component of targeted immunotherapy, significant achievement is achieved in treatment of hematological tumors, and research shows that the expression density of CAR on the surface of T cells is related to differentiation and exhaustion of CAR-T cells, and meanwhile, the differentiation and exhaustion of CAR-T cells are related to clinical outcome of patients, so that monitoring the expression quantity and change of CAR on the surface of CAR-T cells in clinical therapy has a certain indication effect on prognosis of patients.
At present, no standardized operation flow is available for evaluating the expression number of CAR-T cell surface CAR, and flow cytometry and immunohistochemistry are often adopted for evaluating the relative expression level of tumor cell surface antigen of a patient, and no method for evaluating the absolute number of target antigen expression is available. Thus, we set up this methodology to absolute quantify the number of single cell surface antigen of interest expression using a uniform standard.
Disclosure of Invention
The invention provides a quantitative method for the expression quantity of single cell surface antigens, which aims to solve the problem that the prior art cannot detect the expression quantity of target antigens on the surface of single cells accurately.
In order to solve the problems, the invention adopts the following technical scheme:
description of the first aspect of the inventionThe quantitative analysis method for the expression quantity of the single cell surface antigen provides a universal analysis method, can be used for carrying out analysis based on different kits and instruments, and comprises the following steps
A quantitative analysis method for the expression quantity of single cell surface antigen comprises the following steps
S0, drawing a standard curve and constructing a standard equation:
s01, resuspending fluorescent quantitative microsphere dry powder by using Buffer, detecting the average fluorescence intensity MFI on a flow meter by using a fluorescent quantitative kit, collecting quantitative microspheres with total number of n,
s02, according to the average fluorescence intensity MFI of the corresponding fluorescence channel of each fluorescent quantitative microbead and the number of the represented quantitative microbeads, using Log 10 (absolute number of antigen molecules of interest) is Y-axis, log 10 And drawing a standard curve for the X-axis plot point by using the MFI to obtain a standard equation I: log (Log) 10 Absolute number of antigen molecules of interest = a Log 10 Mfi+b; on the contrary, if the X, Y axis information is intentionally replaced, the method should be considered as the same as the method, because the Cartesian coordinate system is a virtual coordinate, the whole replacement of the transverse and longitudinal axis parameters does not generate a new scheme, and the equivalent scheme is considered to be adopted when the method has no specific physical meaning;
s1, target cell treatment: pretreating target cells, and incubating the target cells with a fluorescein-conjugated antibody of the same class as a fluorescence quantification kit (the same class indicates that the kit is the same as fluorescein related to the conjugated antibody), wherein the target cells can be detected by the fluorescein-conjugated antibody (the fluorescein-conjugated antibody is considered to be the corresponding fluorescein-conjugated antibody of the target cells at the moment);
s2, obtaining the number of target antigen molecules on the surface of the cell: and detecting the average fluorescence intensity MFI of the corresponding fluorescence channel of the target antigen molecule on the surface of the target cell by using a fluorescence quantification kit through a flow meter on-machine, and according to a formula II: m=10 (b+a×log) 10 MFI), m is the absolute number of antigen molecules, and the absolute number m of target antigen molecules on the surface of a single target cell is calculated.
In some embodiments, the surface of the target cell expresses an antigen molecule of interest, and the antigen molecule of interest is detectable by a corresponding fluorescein-conjugated antibody; the target cell may be at least one of an immune cell and a tumor cell, but it is not limited to either of the above, and may be other cells that can be detected by the fluorescein-conjugated antibody.
In some embodiments, the target cell may be at least any one of a chimeric antigen receptor T cell, a human external Zhou Xiezhong tumor cell, and a tumor cell line, but it is also not limited to any one of the three, and may be other cells that can be detected by the fluorescein-conjugated antibody.
In some embodiments, the step of treating the target cell
When the target cell is a chimeric antigen receptor T cell and the target antigen molecule is a CAR, the step of preprocessing the target cell is as follows: incubating chimeric antigen receptor T cells with biotin-conjugated anti-mouse FMC63scFv, washing with Buffer, removing supernatant, incubating with anti-CD 3-APC and fluorescein-labeled streptavidin, washing with Buffer, and re-suspending;
when the target cell is a human external Zhou Xiezhong tumor cell, the target antigen molecule is any one of CD19, CD7, BCMA and oTAA (the rest antigen molecules, othertumor associated antigen and oTAA), and the steps of treating the target cell are as follows: human peripheral blood and erythrocyte lysate are incubated at room temperature, and then supernatant is removed by centrifugation, after Buffer washing, the human peripheral blood and erythrocyte lysate are incubated with corresponding fluorescein-coupled antibodies (such as anti-CD 19-fluorescein: fluorescein-coupled CD19 antibody, CD 7-fluorescein, BCMA-fluorescein, oTAA-fluorescein, according to target antigen molecule adaptability), and anti-CD 45-FITC, and Buffer is added for re-suspension after Buffer washing;
when the target cell is a tumor cell and the target antigen molecule is any one of CD19, BCMA and oTAA, the steps of treating the target cell are as follows: tumor cells were washed with PBS and centrifuged to remove supernatant, incubated with the corresponding fluorescein-conjugated antibodies (e.g., anti-CD 19-fluorescein (i.e., fluorescein-conjugated CD19 antibody), BCMA-fluorescein, oTAA-fluorescein, selected adaptively according to the antigen molecule of interest), buffer washed and resuspended in Buffer.
In any of the foregoing examples, CD45-FITC and CD3-APC are not limited to both, and CD45 and CD 3-conjugated fluorescein are adaptively adjusted on the basis of conforming to the color matching rule of flow cytometry, and are equally within the scope of the invention under the conditions of close range and similar effect to the present invention,
in some embodiments, the fluorescent quantitative kit is at least one of PE, APC, PE-cy7, BV421, BV605, and BV510 fluorescent quantitative kits of BD Biosciences, but the fluorescent quantitative kit is not limited to any one of the above, and may be other kits having a detection function.
In some embodiments, the Buffer is formulated using PBS+0.02% sodium azide+0.5% bovine serum albumin, or a reagent having a similar function to the formulation used to form the Buffer is used.
In some modes, when the target cell is a chimeric antigen receptor (chimeric antigen receptor, CAR) T cell and the cell is co-incubated with biotin-conjugated anti-mouse FMC63scFv, anti-CD 3-APC and fluorescein-labeled streptavidin, the chimeric antigen receptor T cell is obtained according to the cd3+ car+ loop gate strategy, respectively, and MFI of the antigen molecule of interest of the population of cells is detected as the target cell is a Zhou Xiezhong tumor cell outside human, and the target cell is incubated with any one of anti-CD 19-fluorescein (representing fluorescein-conjugated antibody), CD 7-fluorescein, BCMA-fluorescein, ostaa-fluorescein (the specific type of fluorescein varies accordingly depending on the kit, keeping the fluorescein involved in both consistent) and anti-CD 45-FITC, a population of tumor cells is obtained according to the cd45dimcd19+ or CD45dimcd7+ or CD45 dimcdma+ loop gate strategy, respectively, and MFI of the antigen molecule of interest of the population of cells is detected; when the target cells are tumor cell lines and the cells are incubated with any one of anti-CD 19-fluorescein, BCMA-fluorescein, oTAA-fluorescein, a tumor cell population is obtained according to CD19+ or BCMA+ or oTAA+ loop gate strategy, respectively, and the MFI of the antigen molecules of interest of the cell population is detected.
Description of the second aspect of the inventionThe method for quantifying the expression quantity of the single cell surface antigen based on PE fluorescence quantification comprises the following steps of
S1, target cell treatment: pretreating target cells, incubating the target cells with PE-conjugated antibodies of target antigen molecules, wherein the PE-conjugated antibodies can detect the target cells,
s2, detecting the average fluorescence intensity MFI of the PE channel of the target cell surface target antigen molecule by using a PE fluorescence kit through a flow meter on-machine, and according to a formula I: absolute number of antigen molecules of interest m=10 (-0.3175+1.0007×log) 10 MFI), the absolute number m of antigen molecules of interest on the surface of a single target cell is calculated.
In some embodiments, the step of treating the target cell
When the target cells are chimeric antigen receptor T cells and the target antigen molecules are anti-CD 19-CAR, the steps of preprocessing the target cells are as follows: chimeric antigen receptor T cells incubated with biotin-conjugated anti-mouse FMC63scFv, buffer washed and supernatant removed, incubated with anti-CD 3-APC and PE-labeled streptavidin, buffer washed and Buffer resuspended;
when the target cell is a human external Zhou Xiezhong tumor cell and the target antigen molecule is any one of CD19, CD7, BCMA and oTAA, the steps of treating the target cell are as follows: after incubating human peripheral blood and erythrocyte lysate, centrifuging to remove supernatant, washing with Buffer, incubating with any corresponding PE coupling antibody of anti-CD 19-PE, CD7-PE, BCMA-PE and oTAA-PE and anti-CD 45-FITC, washing with Buffer, and re-suspending with Buffer;
when the target cell is a tumor cell line and the target antigen molecule is any one of CD19, BCMA and oTAA, the steps of treating the target cell are as follows: tumor cell lines were washed with PBS and centrifuged to remove supernatant, incubated with PE-conjugated antibodies directed against any of CD19-PE, BCMA-PE, oTAA-PE, and Buffer washed followed by Buffer resuspension.
In some embodiments, the step of treating the target cell is specifically:
when the target cell is a chimeric antigen receptor T cell: chimeric antigen receptor T cells incubated with biotin-conjugated anti-mouse FMC63scFv at 4deg.C for 40 min, buffer washed and supernatant removed, incubated with anti-CD 3-APC and PE-labeled streptavidin for 30 min at room temperature, buffer washed and resuspended in 200 μl Buffer;
when the target cell is a human external Zhou Xiezhong tumor cell: 100 μl of human peripheral blood is incubated with 1ml of erythrocyte lysate for 10 min at room temperature, the supernatant is removed by centrifugation, after Buffer washing, the supernatant is incubated with any one of anti-CD 19-PE, CD7-PE, BCMA-PE and oTAA-PE and anti-CD 45-FITC for 30 min at room temperature, and 200 μl of Buffer is added for resuspension after Buffer washing;
when the target cell is a tumor cell line: tumor cell lines were washed with PBS and centrifuged to remove supernatant, and incubated with any one of anti-CD 19-PE, BCMA-PE, oTAA-PE for 30 minutes at room temperature, and 200. Mu.l Buffer was added after Buffer washing to resuspend.
In some ways, it can be appreciated in particular with reference to fig. 4-6 that when the target cells are chimeric antigen receptor (chimeric antigen receptor, CAR) T cells and the cells are incubated with biotin-conjugated anti-mouse FMC63scFv, anti-CD 3-APC and fluorescein-labeled streptavidin, chimeric antigen receptor T cells are obtained according to the cd3+ car+ loop gate strategy, accordingly, and MFI of the antigen molecule of interest of the cell population is detected, wherein the corresponding then indicates an adaptive selection depending on the conjugated antibody used, etc.; when the target cells are human external Zhou Xiezhong tumor cells and the target cells are incubated with any one of anti-CD 19-fluorescein, CD 7-fluorescein, BCMA-fluorescein, oTAA-fluorescein and anti-CD 45-FITC, obtaining a tumor cell population according to the CD45dimCD19+, CD45dimCD7+, CD45dimBCMA+ or CD45 dimTAA+ loop gate strategy, respectively, and detecting the MFI of the antigen molecules of interest of the cell population; when the target cells are tumor cell lines and the cells are incubated with any one of anti-CD 19-fluorescein, BCMA-fluorescein, oTAA-fluorescein, a tumor cell population is obtained according to CD19+, BCMA+ or oTAA+ loop gate strategy, respectively, and the MFI of the antigen molecule of interest of the cell population is detected. The main purpose of the loop gate strategy is to define target cells and avoid the interference of other non-target cells, for example, when the target cells are Zhou Xiezhong tumor cells outside human, the target tumor cells need to be locked by the loop gate strategy, so that the interference of other cells in the peripheral blood of human is avoided.
In some embodiments, the PE fluorescence quantification kit is a BD Biosciences-Cat #34495 kit or a kit having properties similar thereto, and the standard is provided with a number of antigen molecules of different grades and corresponding fluorescence intensities on top of the beads, and the flow cytometer is a BD FACSVia or an instrument similar thereto, such that the standard curve obtained therefrom is capable of generating formula I.
The beneficial effects of the invention are as follows:
the method has the advantages that the detection of the expression number of the target antigen molecules on the surface of the single cell is simpler and easier, a feasibility scheme is provided for closely monitoring the expression of the target antigen on the surface of the tumor cell, the expression of the CAR on the surface of the immune cell and the expression of the target antigen on the surface of the rest target cell in vitro and in vivo, and the method has higher accuracy.
Drawings
FIG. 1 is a standard graph;
FIG. 2 is a graph showing the fit of standard curve on days 0, 7, and 14 during the precision test;
FIG. 3 is a graph showing fitting of the standard product for 0 month, 1 month, 2 months and 3 months in long-term freezing stability detection;
FIG. 4 is a schematic representation of CAR-T cell surface CAR expression level loop gate strategy;
FIG. 5 shows the external Zhou Xiezhong tumor cell surface antigen molecule of interest expression level loop gate strategy;
FIG. 6 shows the tumor cell line surface tumor antigen molecule expression level loop gate strategy.
Detailed Description
The following description is made in connection with specific examples of investigation:
1. material instrument
The main reagent comprises: PE fluorescence quantitative kit (BD Biosciences, cat # 34495)
anti-mouse FMC63 scFv(BioSwan,Cat#500019)
anti-human CD3 APC(Biolegend,Cat#317318)
PE Streptavidin(Biolegend,Cat#405204)
anti-human CD19 PE(Biolegend,Cat#303308)
anti-human CD7 PE(Biolegnd,Cat#343106)
anti-human CD45 FITC(Biolegend,Cat#304006)
anti-human BCMAPE(Biolegend,Cat#357504)
The main instrument is as follows: flow cytometer (BD FACSVia)
2. Method of
A. PBS+0.02% sodium azide+0.5% bovine serum albumin is used for preparing a Buffer required by an experiment;
B. re-suspending fluorescent quantitative microsphere dry powder by using 500 μl Buffer, performing on-machine detection on BD FACSVia flow meter, and collecting 10000 quantitative microspheres in total;
C. drawing a standard curve according to the average fluorescence intensity (MFI) of each quantitative microbead PE channel and the quantitative microbead number represented by the microbeads;
D. chimeric Antigen Receptor (CAR) -T cells were incubated with biotin-conjugated anti-mouse FMC63scFv for 40 min at 4 ℃, buffer washed and supernatant removed, incubated with anti-CD 3-APC and PE strepitavidine for 30 min at room temperature, buffer washed and resuspended in 200 μl Buffer, and the average fluorescence intensity of CAR was detected and calculated on-stream (Mean Fluorescence Intensity, MFI); blank groups are not subjected to incubation and dyeing treatment of all antibodies, and the rest steps are the same; the control group was not incubated with anti-mouse FMC63scFv, the remaining steps were the same;
e.100 mu l of human peripheral blood is incubated with 1ml of erythrocyte lysate for 10 minutes at room temperature, the supernatant is removed by centrifugation, after Buffer washing, the mixture is incubated with anti-CD 45-FITC and anti-CD 19-PE, CD7-PE or BCMA-PE for 30 minutes at room temperature, 200 mu l of Buffer is added after Buffer washing, and the MFI of target molecules is detected and calculated on-line in a flow meter;
F. washing tumor cell lines with different treatment conditions, centrifuging to remove supernatant, incubating with anti-CD 19-PE or BCMA-PE at room temperature for 30 min, washing with Buffer, adding 200 μl Buffer for resuspension, detecting on a flow meter, and calculating MFI of target molecules; blank groups were not stained for all antibody incubations, the remaining steps were the same.
3. Results
3.1 Standard Curve experiment results
3.2 degree of Standard Fit
3.3 precision detection of Standard Curve
And (5) detecting the standard products on a flow meter on the 0 th day, the 7 th day and the 14 th day of the time sequence.
3.4 detection of Long-term freezing stability of Standard substance (-75+ -5 ℃ C.)
And (3) diluting the standard substance, split charging the diluted standard substance into four parts, placing the four parts in a refrigerator at the temperature of minus 75+/-5 ℃, respectively carrying out flow detection and analysis on the fluorescence intensity of the standard substance for 0 month, 1 month, 2 months and 3 months, and calculating the fitting degree of a standard curve.
3.5 detection of CAR-T cell surface CAR molecule expression level
The number of single cell surface CARs in the blank and control groups was below the lower limit of the assay, so the results were valid.
3.6 detection of target antigen molecule expression amount on external Zhou Xiezhong tumor cell surface
Obtaining a tumor cell population according to the CD45dimCD19+, CD45dimCD7+ or CD45dimBCMA+ loop gate strategy, and calculating the antigen molecule PE-MFI of interest of the cell population of interest
3.7 detection of the expression level of surface antigen molecules of tumor cell lines
5.7.1 detection of the number of molecules expressed on the surface of Nalm6 cells under treatment with different drug concentrations
The number of target antigen molecules on the surface of single cells in the blank group is lower than the lower limit of the detection method, so that the result is effective.
5.7.2 detection of the number of BCMA molecules expressed on the cell surface of U266B1 under treatment with different drug concentrations
The number of target antigen molecules on the surface of single cells in the blank group is lower than the lower limit of the detection method, so that the result is effective.
4. Conclusion(s)
During the course of the establishment of the methodology for quantifying the amount of expression of a single cell surface antigen by flow cytometry,
a: the degree of fit, precision and stability of the PE fluorescence quantification kit (BD Biosciences) kit standard were first evaluated,
b: then detecting the expression quantity of the target molecules on the surfaces of the CAR-T cells, the Zhou Xiezhong tumor cells outside the human body and the tumor cell line,
the result shows that the sample to be tested logs 10 The PE-MFI measurement value is in the standard curve detection range, and the detection method can sensitively reflect the change of the expression level of the antigen molecule.
The method makes the detection of the expression number of the target antigen molecules on the surface of the single cell simpler and easier, and provides feasibility for closely monitoring the expression condition of the antigen molecules on the surface of the tumor cell in vitro and in vivo.
It will be apparent to those skilled in the art that various modifications to the above embodiments may be made without departing from the general spirit and concepts of the invention. Which fall within the scope of the present invention. The protection scheme of the invention is subject to the appended claims.

Claims (10)

1. A method for quantitatively analyzing the expression level of a single cell surface antigen, comprising the steps of
S0, drawing a standard curve and constructing a standard equation:
s01, resuspending fluorescent quantitative microsphere dry powder by using Buffer, detecting the average fluorescence intensity MFI on a flow meter by using a fluorescent quantitative kit, collecting quantitative microspheres with total number of n,
s02, according to the average fluorescence intensity MFI of the corresponding fluorescence channel of each fluorescent quantitative microbead and the number of the represented quantitative microbeads, using Log 10 The absolute number of the target antigen molecules is Y-axis and Log 10 And drawing a standard curve for the X-axis plot point by using the MFI to obtain a standard equation I: log (Log) 10 Absolute number of antigen molecules of interest = a Log 10 MFI+b；
S1, target cell treatment: pretreating target cells, incubating the target cells with a fluorescein-conjugated antibody of the same class as a fluorescence quantification kit, wherein the target cells can be detected by the fluorescein-conjugated antibody;
s2, obtaining the number of target antigen molecules on the surface of the cell: and detecting the average fluorescence intensity MFI of the corresponding fluorescence channel of the target antigen molecule on the surface of the target cell by using a fluorescence quantification kit through a flow meter on-machine, and according to a formula II: absolute number of antigen molecules of interest m=10 (b+a×log) 10 MFI) the absolute number m of antigen molecules of interest on the surface of a single target cell is calculated.
2. The method for quantitative analysis of the expression amount of single cell surface antigen according to claim 1, wherein the surface of the target cell expresses an antigen molecule of interest, and the antigen molecule of interest is detected by a corresponding fluorescein-conjugated antibody; wherein the target cell is at least any one of an immune cell and a tumor cell.
3. The method according to claim 2, wherein the target cell is at least any one of a chimeric antigen receptor T cell, a human external Zhou Xiezhong tumor cell, and a tumor cell line.
4. The method for quantitative analysis of the expression level of a single cell surface antigen according to claim 3, wherein the step of treating the target cells
When the target cell is a chimeric antigen receptor T cell and the target antigen molecule is a CAR, the step of preprocessing the target cell is as follows: incubating chimeric antigen receptor T cells with biotin-conjugated anti-mouseFMC63scFv, washing with Buffer, removing supernatant, incubating with anti-CD 3-APC and fluorescein-labeled streptavidin, washing with Buffer, and re-suspending;
when the target cell is a human external Zhou Xiezhong tumor cell and the target antigen molecule is any one of CD19, CD7, BCMA and oTAA, the steps of treating the target cell are as follows: after incubating human peripheral blood and erythrocyte lysate at room temperature, centrifuging to remove supernatant, washing with Buffer, incubating with corresponding fluorescein-conjugated antibody and anti-CD 45-FITC, washing with Buffer, and re-suspending with Buffer;
when the target cell is a tumor cell and the target antigen molecule is any one of CD19, BCMA and oTAA, the steps of treating the target cell are as follows: tumor cells were washed with PBS and centrifuged to remove supernatant, incubated with the corresponding fluorescein-conjugated antibodies, washed with Buffer and resuspended with Buffer.
5. The method for quantitative analysis of the expression level of single cell surface antigens according to any of claims 1 to 3, wherein said fluorescent quantitative kit is at least one of the fluorescent quantitative kits PE, APC, PE-cy7, BV421, BV605 and BV510 of BDbiosciences.
6. The method for quantitative analysis of the expression level of a single cell surface antigen according to claim 4,
when the target cells are chimeric antigen receptor T cells and the cells are incubated with biotin-conjugated anti-mouse FMC63scFv, anti-CD 3-APC and fluorescein-labeled streptavidin, obtaining chimeric antigen receptor T cells according to a CD3+CAR+ loop gate strategy correspondingly, and detecting MFI of antigen molecules of interest of the target cell population;
when the target cells are human external Zhou Xiezhong tumor cells and the target cells are incubated with any one of anti-CD 19-fluorescein, CD 7-fluorescein, BCMA-fluorescein, oTAA-fluorescein and anti-CD 45-FITC, obtaining a tumor cell population according to the CD45dimCD19+, CD45dimCD7+, CD45dimBCMA+ or CD45 dimTAA+ loop gate strategy, respectively, and detecting the MFI of the antigen molecules of interest of the cell population;
when the target cells are tumor cell lines and the cells are incubated with any one of anti-CD 19-fluorescein, BCMA-fluorescein, oTAA-fluorescein, a tumor cell population is obtained according to CD19+, BCMA+ or oTAA+ loop gate strategy, respectively, and the MFI of the antigen molecule of interest of the cell population is detected.
7. The method for quantifying the expression quantity of the single cell surface antigen based on PE fluorescence quantification is characterized by comprising the following steps of
S1, target cell treatment: pretreating target cells, incubating the target cells with PE-conjugated antibodies of target antigen molecules, wherein the PE-conjugated antibodies can detect the target cells,
s2, detecting the average fluorescence intensity MFI of the PE channel of the target cell surface target antigen molecule by using a PE fluorescence kit through a flow meter on-machine, and according to a formula I: absolute number of antigen molecules of interest m=10 (-0.3175+1.0007×log) 10 MFI), the absolute number m of antigen molecules of interest on the surface of a single target cell is calculated.
8. The method for quantifying the amount of expression of a single cell surface antigen based on PE fluorescence quantification according to claim 7, wherein the step of treating the target cells
When the target cells are chimeric antigen receptor T cells and the target antigen molecules are anti-CD 19-CAR, the steps of preprocessing the target cells are as follows: chimeric antigen receptor T cells incubated with biotin-conjugated anti-mouseFMC63scFv, buffer washed and then supernatant removed, incubated with anti-CD 3-APC and PE-labeled streptavidin, buffer washed and then resuspended in Buffer;
when the target cell is a human external Zhou Xiezhong tumor cell and the target antigen molecule is any one of CD19, CD7, BCMA and oTAA, the steps of treating the target cell are as follows: after incubating human peripheral blood and erythrocyte lysate, centrifuging to remove supernatant, washing with Buffer, incubating with any corresponding PE coupling antibody of anti-CD 19-PE, CD7-PE, BCMA-PE and oTAA-PE and anti-CD 45-FITC, washing with Buffer, and re-suspending with Buffer;
when the target cell is a tumor cell line and the target antigen molecule is any one of CD19, BCMA and oTAA, the steps of treating the target cell are as follows: tumor cell lines were washed with PBS and centrifuged to remove supernatant, incubated with PE-conjugated antibodies directed against any of CD19-PE, BCMA-PE, oTAA-PE, and Buffer washed followed by Buffer resuspension.
9. The method for quantifying the amount of expression of a single cell surface antigen based on PE fluorescence quantification according to claim 7, wherein the step of treating the target cell is specifically:
when the target cell is a chimeric antigen receptor T cell: chimeric antigen receptor T cells incubated with biotin-conjugated anti-mouseFMC63scFv at 4deg.C for 40 min, buffer washed and supernatant removed, incubated with anti-CD 3-APC and PE-labeled streptavidin for 30 min at room temperature, buffer washed and resuspended in 200. Mu.l Buffer;
when the target cell is a human external Zhou Xiezhong tumor cell: 100 μl of human peripheral blood is incubated with 1ml of erythrocyte lysate for 10 min at room temperature, the supernatant is removed by centrifugation, after Buffer washing, the supernatant is incubated with any one of anti-CD 19-PE, CD7-PE, BCMA-PE and oTAA-PE and anti-CD 45-FITC for 30 min at room temperature, and 200 μl Buffer is added for resuspension after Buffer washing;
when the target cell is a tumor cell line: tumor cell lines were washed with PBS and centrifuged to remove supernatant, and incubated with any one of anti-CD 19-PE, BCMA-PE, oTAA-PE for 30 minutes at room temperature, and Buffer washed and resuspended in 200. Mu.l Buffer.
10. The method for quantifying the amount of single cell surface antigen expression based on PE fluorescence quantification according to claim 7, wherein when the target cell is a chimeric antigen receptor T cell and the cell is co-incubated with biotin-conjugated anti-mouseFMC63scFv, anti-CD 3-APC and fluorescein-labeled streptavidin, the chimeric antigen receptor T cell is obtained according to the cd3+ car+ loop gate strategy accordingly, and the MFI of the antigen molecule of interest of the population of target cells is detected; when the target cells are human external Zhou Xiezhong tumor cells and the cells are incubated with any one of anti-CD 19-PE, CD7-PE, BCMA-PE, oTAA-PE and anti-CD 45-FITC, obtaining a tumor cell population according to the round door strategy of CD45dimCD19+, CD45dimCD7+, CD45dimBCMA+ or CD45 dimTAA+, and detecting PE-MFI of the antigen molecules of interest of the cell population; when the target cells are tumor cell lines and the cells are incubated with any one of anti-CD 19-PE, BCMA-PE and oTAA-PE, a tumor cell population is obtained according to CD19+, BCMA+ or oTAA+ loop gate strategy correspondingly, and PE-MFI of antigen molecules of interest of the cell population is detected.
The method for quantifying the amount of single cell surface antigen expression based on PE fluorescence quantification according to any one of claims 7 to 10, wherein the PE fluorescence quantification kit is a BDBiosciences-cat#34495 kit and the flow cytometer is BDFACSVia; the Buffer solution was prepared using PBS+0.02% sodium azide+0.5% bovine serum albumin.
CN202310662465.2A 2023-06-06 2023-06-06 Quantitative method for single cell surface antigen expression quantity Pending CN116679059A (en)

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