CN113252904A - WAS protein detection method - Google Patents
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Abstract
The invention relates to a WAS protein detection method, wherein the detection of a sample to be detected comprises the following steps: taking 400-; centrifuging, removing the fixing agent in the first product, washing with 1 × phosphate buffer solution, mixing with erythrocyte lysate, placing in a water bath kettle at 36.5-37.5 deg.C for 4-6min to lyse erythrocyte, adding perforating agent, and breaking membrane at room temperature for 14-16min to obtain a second product; mixing the second product with mouse anti-human WAS protein IgG2 alpha antibody, and incubating at room temperature for 30 min; and eluting unbound free antibody, mixing with rat anti-mouse IgG2 alpha antibody, incubating at room temperature for 30min, washing, detecting by a flow type computer to obtain detection data, and analyzing the detection data. The invention can directly detect the WAS protein level in peripheral blood, skips the step of separating PBMC from the peripheral blood, and saves the using amount of a sample to be detected; the method has simple and quick steps and is more suitable for wide application of WAS protein detection.
Description
Technical Field
The application relates to the technical field of WAS protein detection, in particular to a WAS protein detection method.
Background
WAS protein (Wiskott-Aldrich Syndrome protein) is specifically expressed in a hematopoietic system, is a key regulator for cytoskeleton and immune synapse formation, and is essential in the processes of lymphocyte and bone marrow cell migration, cell signaling, cytotoxicity, phagocytosis and the like.
WAS protein is specifically expressed in immune cells other than erythrocytes; subcellular localization of WAS proteins WAS primarily within the cytoplasm and, to a lesser extent, on the nucleus and cell membrane.
Lymphocytes are an important class of immune cell lines produced by lymphoid organs. According to the phenotype and functional characteristics, the cells can be divided into different categories, such as T cells, B cells, NK cells and the like. The lymphocytes cooperate and restrict each other in the immune response process to complete the recognition, response and elimination of antigen substances together, thereby maintaining the stable environment in the organism. Different types of lymphocytes can be distinguished by using different cell surface antigens and carrying out fluorescence labeling.
The expression of WAS protein in blood sample has important relation with the body's immune system performance. By quantitative analysis of WAS protein in blood samples, the immune function of an organism can be evaluated more accurately, and the occurrence, development and treatment effect evaluation of certain diseases can be better researched. Meanwhile, the expression conditions of WAS proteins of different lymphocytes are known, which is helpful for more detailed analysis.
At present, the methods commonly used for the quantitative analysis of WAS proteins in blood samples are mainly: flow cytometry and Western immunoblotting (Western blot). Traditional flow cytometry for the detection of WAS proteins requires first isolation of mononuclear lymphocytes (PBMCs) from peripheral blood, followed by cell fixation, membrane rupture and perforation, incubation with antibodies and fluorescent labeling of the PBMCs. Then detecting by flow cytometry to obtain detection data, and analyzing the detection data. The western blotting method is one of techniques for analyzing proteins, and is a method in which a protein sample is separated by polyacrylamide electrophoresis according to molecular weight, transferred to a hybridization membrane (blot), and then subjected to specific detection of a target protein by a primary antibody/secondary antibody complex. The operation steps comprise cell lysis, protein quantification and denaturation, gel preparation, electrophoresis, membrane transfer, antibody incubation and exposure and development.
Traditional flow cytometry for the detection of WAS proteins requires first isolating mononuclear lymphocytes (PBMCs) from peripheral blood, which is a large requirement for the detection of samples, at least 2-5ml, and a high requirement for gradient density centrifugation of PBMCs, which takes about 2 hours. The western blotting method also needs to separate PBMC, further extracts proteins from the lysed PBMC, has complicated operation steps, is time-consuming and labor-consuming, generally needs 1 day, and is not suitable for wide application.
Therefore, it is a major problem to be solved to provide a method for detecting WAS protein that does not require PBMC isolation from peripheral blood.
Disclosure of Invention
The application provides a WAS protein detection method, which aims to solve the problems that the process is complex and the time is long due to the fact that PBMC is required to be separated from peripheral blood to detect the WAS protein.
The technical scheme adopted by the application is as follows:
the invention provides a WAS protein detection method, and the detection of a sample to be detected comprises the following steps:
taking 400-;
centrifuging, removing the fixing agent in the first product, washing with 1 × phosphate buffer solution, mixing with erythrocyte lysate, placing in a water bath kettle at 36.5-37.5 deg.C for 4-6min to lyse erythrocyte, adding perforating agent, and breaking membrane at room temperature for 14-16min to obtain a second product;
mixing the second product with a mouse anti-human WAS protein IgG2 alpha antibody, and incubating for 30min at room temperature, wherein the mouse anti-human WAS protein IgG2 alpha antibody is a primary antibody;
and eluting unbound free antibody, mixing with a rat anti-mouse IgG2 alpha antibody, incubating at room temperature for 30min, washing, detecting by a flow type computer to obtain detection data, and analyzing the detection data, wherein the rat anti-mouse IgG2 alpha antibody is a secondary antibody.
Further, washing with 1 x phosphate buffer solution, mixing with erythrocyte lysate, placing in water bath at 36.5-37.5 deg.C for 4-6min to lyse erythrocytes, comprising:
washing with 1 × phosphate buffer solution, centrifuging to remove supernatant, adding 500ul erythrocyte lysate, mixing, placing in water bath at 36.5-37.5 deg.C for 4-6min to lyse erythrocyte, processing until the liquid turns transparent, centrifuging, and removing supernatant.
And further, detecting the reference substance according to the detection step of the sample to be detected to obtain the detection data of the reference substance, and analyzing and comparing the detection data of the reference substance with the detection data of the sample to be detected.
Further, the second product WAS mixed with mouse anti-human WAS protein IgG2 α antibody and incubated for 30min at room temperature, including:
taking 2-4ul of mouse anti-human WAS protein IgG2 alpha antibody, taking 40-60ul of second product, and mixing the mouse anti-human WAS protein IgG2 alpha antibody and the second product in a vortex manner to obtain a third product;
taking 2-4ul of mouse anti-human nonspecific IgG2 alpha isotype antibody, taking 40-60ul of second product, and mixing the mouse anti-human nonspecific IgG2 alpha isotype antibody with the second product in a vortex manner to obtain a fourth product;
and standing the rest second product at room temperature for 30min to obtain a fifth product.
Further, the second product WAS mixed with mouse anti-human WAS protein IgG2 α antibody and incubated at room temperature for 30min, which further included:
adding 1 x phosphate buffer solution into the third product, the fourth product and the fifth product respectively for washing;
the washed product was centrifuged and the supernatant discarded.
Further, the mixture was mixed with rat anti-mouse IgG2 α antibody and incubated at room temperature for 30min, including:
adding 1.0-2.0ul rat anti-mouse IgG2 alpha antibody into the third product, the fourth product and the fifth product after discarding the supernatant respectively, vortexing and mixing uniformly, and incubating for 30min at normal temperature and in the dark.
Further, the method further comprises the following steps after mixing with the rat anti-mouse IgG2 alpha antibody and incubating for 20-40min at room temperature:
respectively adding 1 Xphosphate buffer solution into the third product, the fourth product and the fifth product after incubation for 30min at normal temperature in a dark place for washing;
centrifuging the washed product, and removing the supernatant;
150-250ul of 1 XPhosphate buffer was added to the product after the supernatant was discarded.
Further, 500ul of fixing agent is mixed with 50ul of peripheral blood of the sample to be detected, and the mixture is fixed for more than 30min at the temperature of 3-5 ℃, so that a first product is obtained, and the method comprises the following steps:
mixing 1-3ul of each of a plurality of different fluorescence labeled antibodies and 0.1-0.3ul of Fixable visual Dye with 40-60ul of peripheral blood of a sample to be detected, uniformly mixing by vortex, and incubating the antibodies for 30min at 3-5 ℃;
respectively adding 0.5-1.5ml of 1 XPBS, uniformly mixing by vortex, centrifuging, removing supernatant, and eluting unbound free antibody;
respectively adding 400-600ul of fixing agent, uniformly mixing by vortex, and fixing at 3-5 ℃ for more than 30min to obtain a first product.
Further, the plurality of different fluorescently labeled antibodies comprises:
mouse anti-human WAS protein IgG2 alpha antibody, mouse anti-human non-specific IgG2 alpha isotype antibody, PE-labeled rat anti-mouse IgG2 alpha antibody, FITC-labeled mouse anti-human CD45 IgG1 antibody, BV 421-labeled mouse anti-human CD3 IgG1 antibody, PE-CY 7-labeled mouse anti-human CD4 IgG1 antibody, BV 510-labeled mouse anti-human CD8 IgG1 antibody, PerCP/CY 5.5-labeled mouse anti-human CD19 IgG1 antibody, and APC-labeled mouse anti-human CD56 IgG1 antibody.
Further, the multiple different fluorescently-labeled antibodies are antibodies homologous to the sample to be tested or antibodies non-homologous to the sample to be tested.
Further, the Fixable visual Dye can label dead cells to distinguish dead from live cells;
when the cell surface marker is CD45+, all leukocytes can be immunophenotyped; when the cell surface marker is CD45+ CD3+ CD4+, the CD4+ T cells can be subjected to immune typing; when the cell surface marker is CD45+ CD3+ CD8+, the CD8+ T cells can be subjected to immune typing; when the cell surface marker is CD45+ CD19+, B cells can be subjected to immune typing; when the cell surface marker is CD45+ CD56+, NK cells can be subjected to immune typing.
Further, when the detection data is positive, the sample is a sample expressing the WAS protein;
and when the detection data is negative, the WAS protein is not expressed.
The technical scheme of the application has the following beneficial effects:
the invention relates to a WAS protein detection method, wherein the detection of a sample to be detected comprises the following steps: taking 400-; centrifuging, removing the fixing agent in the first product, washing with 1 × phosphate buffer solution, mixing with erythrocyte lysate, placing in a water bath kettle at 36.5-37.5 deg.C for 4-6min to lyse erythrocyte, adding perforating agent, and breaking membrane at room temperature for 14-16min to obtain a second product; mixing the second product with mouse anti-human WAS protein IgG2 alpha antibody, and incubating at room temperature for 30 min; and eluting unbound free antibody, mixing with rat anti-mouse IgG2 alpha antibody, incubating at room temperature for 30min, washing, detecting by a flow type computer to obtain detection data, and analyzing the detection data.
The invention can directly take peripheral blood to detect the WAS protein level, skips the step of separating PBMC from the peripheral blood, saves the dosage of a sample to be detected and only needs 50 ul; the method has simple and quick steps, only needs 2 hours in the whole process, and is more suitable for wide application of WAS protein detection.
Drawings
In order to more clearly explain the technical solution of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious to those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of a method of detecting WAS protein according to the present invention;
FIG. 2 shows the results of a normal sample (from top to bottom, a negative control tube, an isotype control tube, and a detection tube, respectively) of a WAS protein detection method according to a first embodiment of the present invention;
FIG. 3 shows the results of a sample to be tested (from top to bottom, a negative control tube, a isotype control tube, and a detection tube, respectively) in a WAS protein detection method according to an embodiment of the present invention;
fig. 4 is a superposition of the peak images of the WAS protein of the normal sample (left side) and the sample to be tested (right side) in the method for detecting the WAS protein according to the first embodiment of the present invention, wherein the dotted line is the negative control tube, the dotted line is the isotype control tube, and the solid line is the detection tube;
FIG. 5 is a diagram of the WAS protein detection method according to the present invention, which can distinguish different types of lymphocytes by fluorescence labeling based on different lymphocyte surface antigens;
FIG. 6 is a diagram showing the WAS protein expression in different lymphocytes according to the WAS protein detection method of the first embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, like numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following examples do not represent all embodiments consistent with the present application. But merely as exemplifications of systems and methods consistent with certain aspects of the application, as recited in the claims.
The experimental materials of the present application include: the sample to be detected and the anticoagulated peripheral blood sample of the normal sample. The fixed punch Kit BD Cytofix/Cytoperm Soln Kit (BD Biosciences, cat 554714). The antibody comprises: mouse anti-human WAS protein IgG2 a antibody (BD Biosciences, cat 557773), mouse anti-human nonspecific IgG2 a isotype antibody (Biolegend, cat 400202), PE-labeled rat anti-mouse IgG2 a antibody (Biolegend, cat407108), Fixable viatility Dye-labeled dead cells (Dye bioscience, cat 65-0865-18), FITC-labeled mouse anti-human CD45 IgG1 antibody (BD Biosciences, cat 555482), BV 421-labeled mouse anti-human CD3 IgG1 antibody (Biolegend, cat 300434), PE-CY 7-labeled mouse CD4 IgG1 antibody (Biolegend, cat 300512), BV 510-labeled mouse anti-human IgG 84 IgG 42 antibody (Biolegend, cat 4732), cp/345.5-labeled mouse anti-human CD19 IgG 4 antibody (Biolegend, cat 19 antibody (Biolegend, mouse anti-human CD 4624) and BV 510-labeled mouse anti-human CD 4624 IgG1 antibody (Biolegend, mouse bioscience, cat 4624). 1 XPBS (phosphate buffered saline). Erythrocyte lysate (TIANGEN, cat RT 122-02).
The application provides a WAS protein detection method, which comprises the following steps:
s01: mixing 40-60ul of whole blood of a sample to be detected and a normal sample with 600ul of a fixing agent, and fixing at 3-5 ℃ for more than 30min to obtain first products;
the method specifically comprises the following steps:
preparing 2 flow tubes, marking as Test (sample to be detected) and HC (normal sample), respectively adding 40-60ul of whole blood of the sample to be detected and the normal sample, respectively adding 1-3ul of various fluorescence-marked antibodies and 0.1-0.3ul of Fixable visual Dye, uniformly mixing by vortex, and incubating the antibodies for 30min at 3-5 ℃;
respectively adding 0.5-1.5ml of 1 XPBS, uniformly mixing by vortex, centrifuging, removing supernatant, and eluting unbound free antibody;
respectively adding 400-600ul of fixing agent, uniformly mixing by vortex, and fixing at 3-5 ℃ for more than 30min to obtain first products.
Wherein the plurality of different fluorescently labeled antibodies comprises:
mouse anti-human WAS protein IgG2 alpha antibody, mouse anti-human non-specific IgG2 alpha isotype antibody, PE-labeled rat anti-mouse IgG2 alpha antibody, FITC-labeled mouse anti-human CD45 IgG1 antibody, BV 421-labeled mouse anti-human CD3 IgG1 antibody, PE-CY 7-labeled mouse anti-human CD4 IgG1 antibody, BV 510-labeled mouse anti-human CD8 IgG1 antibody, PerCP/CY 5.5-labeled mouse anti-human CD19 IgG1 antibody, and APC-labeled mouse anti-human CD56 IgG1 antibody.
Wherein, the multiple different fluorescence labeled antibodies are antibodies homologous with the sample to be detected or antibodies non-homologous with the sample to be detected.
In particular, the Fixable visual Dye can label dead cells to distinguish dead from live cells; when the cell surface marker is CD45+, all leukocytes can be immunophenotyped; when the cell surface marker is CD45+ CD3+ CD4+, the CD4+ T cells can be subjected to immune typing; when the cell surface marker is CD45+ CD3+ CD8+, the CD8+ T cells can be subjected to immune typing; when the cell surface marker is CD45+ CD19+, B cells can be subjected to immune typing; when the cell surface marker is CD45+ CD56+, NK cells can be subjected to immune typing.
S02: centrifuging, discarding the fixing agent in the first product, washing with 1 x phosphate buffer solution, mixing with erythrocyte lysate, placing in a water bath kettle at 36.5-37.5 ℃ for 4-6min to lyse erythrocytes, processing until the liquid turns transparent, adding a perforating agent to rupture the membrane and perforate for 14-16min at room temperature, centrifuging, discarding the supernatant, and obtaining a second product, wherein the second product has the volume of 100 plus 200 ul;
s03: mixing the second product with a mouse anti-human WAS protein IgG2 alpha antibody, and incubating for 30min at room temperature, wherein the mouse anti-human WAS protein IgG2 alpha antibody is a primary antibody;
mixing the second product with mouse anti-human WAS protein IgG2 alpha antibody, and incubating at room temperature for 30min, which specifically comprises:
preparing 4 flow tubes, marking as Test (+) and HC (+) as detection tubes, using Test (-) and HC (-) as homotype control tubes, and using original Test and HC as negative control tubes;
adding 2-4ul of mouse anti-human WAS protein IgG2 alpha antibody into the bottom of a detection tube, taking 40-60ul of a second product from a negative control tube into a corresponding detection tube, and performing vortex mixing to obtain a third product;
adding 2-4ul of mouse anti-human nonspecific IgG2 alpha isotype antibody to the bottom of a isotype control tube, taking 40-60ul of a second product from a negative control tube to the bottom of the corresponding isotype control tube, and performing vortex mixing to obtain a fourth product;
standing the rest second product in the negative control tube at room temperature for 30min to obtain a fifth product;
respectively adding 1 x phosphate buffer solution into a detection tube, a homotype control tube and a negative control tube for washing;
the washed product was centrifuged and the supernatant discarded.
S04: and eluting unbound free antibody, mixing with a rat anti-mouse IgG2 alpha antibody, incubating at room temperature for 30min, washing, detecting by a flow type computer to obtain detection data, and analyzing the detection data, wherein the rat anti-mouse IgG2 alpha antibody is a secondary antibody.
Mixing with rat anti-mouse IgG2 alpha antibody, and incubating at room temperature for 30min, including:
respectively adding 1.0-2.0ul of rat anti-mouse IgG2 alpha antibody into a detection tube, a homotypic control tube and a negative control tube after supernatant liquid of S03 is removed, vortexing and mixing uniformly, and incubating for 30min at normal temperature in a dark place;
respectively adding 1 Xphosphate buffer solution into a detection tube, a isotype control tube and a negative control tube which are incubated for 30min at normal temperature in a dark place for washing;
centrifuging the washed product, and removing the supernatant;
150-250ul of 1 XPhosphate buffer was added to the product after the supernatant was discarded.
S05: and analyzing and comparing the detection data of the products in the S04 final detection tube, the isotype control tube and the negative control tube.
Wherein, when the detection data is negative, the WAS protein is not expressed in the sample.
In conclusion, the method provided by the invention realizes the WAS protein detection and quantitative analysis of the sample to be detected by adopting less samples to be detected, and can perform fine analysis of different lymphocytes.
The WAS protein detection method can directly detect the WAS protein level in peripheral blood, skips the step of separating PBMC from the peripheral blood, saves the using amount of a sample to be detected, has short time consumption, and is more suitable for wide application of WAS protein detection. Meanwhile, the invention can also detect the expression condition of WAS protein of different lymphocytes by combining a cellular immune grouping method, thereby being beneficial to more precise analysis.
Example one
1) Preparing 2 flow tubes, marking as Test (sample to be detected) and HC (normal sample), respectively adding the sample to be detected, 50ul of whole blood of the normal sample, 2ul of each of a plurality of different fluorescence-labeled antibodies and 0.2ul of Fixable visual Dye, uniformly mixing by vortex, and fixing at4 ℃ for more than 30 min;
2) then respectively adding 0.5-1.5ml of 1 XPBS, uniformly mixing by vortex, centrifuging, removing supernatant, respectively adding 500ul of fixing agent, uniformly mixing by vortex, and fixing for more than 30min at 3-5 ℃;
3) centrifuging (3500rpm, 3min, unlimited temperature), discarding the supernatant fixative, and keeping the tube bottom cells;
4) adding 1ml of 1 XPBS (phosphate buffer), mixing uniformly by vortex, centrifuging (3500rpm, 3min, unlimited temperature) and then discarding the supernatant;
5) respectively adding 5ml of erythrocyte lysate, blowing and uniformly mixing by using a gun head, and carrying out cracking treatment in a water bath kettle at 37 ℃ for 4-6min until the liquid in the flow tube is transparent;
6) centrifuging (3500rpm, 3min, unlimited temperature), discarding supernatant, adding 1ml of 1 × perforating agent, mixing by vortex, and perforating at room temperature in dark place for 15 min; if more erythrocytes still appear after centrifugation, the step 5 can be repeated to crack the erythrocytes as much as possible and then the holes are punched;
7) preparing 4 flow tubes, marking as Test (+) and HC (+) as detection tubes, using Test (-) and HC (-) as homotype control tubes, and using original Test and HC as negative control tubes;
8) incubating the primary antibody: centrifuging the negative control tube (3500rpm, 3min, unlimited temperature), and discarding the supernatant, wherein the liquid volumes of the Test (sample to be detected) and HC (normal sample) flow tubes are about 100-; firstly, 3ul of mouse anti-human WAS protein IgG2 alpha antibody is sucked and added to the bottom of a detection tube, then 50ul of cell sap is sucked from a negative control tube and is added to the bottom of the corresponding detection tube, and the cell sap is swirled and mixed evenly; adding 3ul of mouse anti-human non-specific IgG2 alpha isotype antibody into the isotype control tube, sucking 50ul of cell fluid from the negative control tube to the bottom of the corresponding isotype control tube, and uniformly mixing by vortex; negative control tubes were left without anything; incubating for 30min at normal temperature in dark place;
9) cleaning: adding 1ml of 1 XPBS, centrifuging (3500rpm, 3min, unlimited temperature), and discarding the supernatant;
10) repeating the step 9;
11) incubation of secondary antibody: sucking 1.5ul of rat anti-mouse IgG2 alpha antibody, respectively adding into the bottom of the detection tube, the isotype control tube and the negative control tube, vortexing, mixing, and incubating for 30min at normal temperature in a dark place;
12) cleaning: adding 1ml of 1 XPBS, centrifuging (3500rpm, 3min, unlimited temperature), and discarding the supernatant;
13) repeating the step 12;
14) finally, 200ul of 1 XPBS is added, mixed evenly by vortex, and detected on a flow cytometer.
15) Quantitative analysis result of WAS protein: analyzing original data by Flowjo software;
FIG. 2 shows WAS protein detection results of normal samples, from top to bottom, which are respectively a negative control tube, a isotype control tube and a detection tube;
FIG. 3 shows WAS protein detection results of a sample to be detected, which are a negative control tube, a isotype control tube and a detection tube from top to bottom;
FIG. 4 is a peak diagram showing WAS protein (WASP) detection results of a normal sample and a test sample. And (3) superposing WAS protein peak images of a normal sample (left side) and a sample to be detected (right side), wherein a dotted line is a negative control tube, a dotted line is a homotype control tube, and a solid line is a detection tube. The WASP peak image of the normal sample detection tube is completely separated from the negative control tube and the isotype control tube, which indicates that the detection result of the WASP of the normal sample detection tube is positive, and the percentage of the WASP + is 87.6%, which indicates that the WASP in the normal sample is expressed. The WASP peak image of the detection tube of the sample to be detected is almost overlapped with a negative control tube and a homotype control tube, the percentage of the WASP + is 1.29 percent, and the detection result of the WASP of the detection tube of the sample to be detected is negative, which indicates that the expression of the WASP in the sample to be detected is reduced or not expressed.
FIG. 5 is a schematic representation of the labeling of different classes of lymphocytes with surface antigens. Different types of lymphocytes can be distinguished by carrying out fluorescent labeling according to different lymphocyte surface antigens.
Figure 6 shows WAS protein expression in different lymphocytes.
Example two
1) Preparing 2 flow tubes, marking as Test (sample to be detected) and HC (normal sample), respectively adding the sample to be detected, 40ul of normal sample whole blood, 1ul of each of a plurality of different fluorescence-labeled antibodies and 0.1ul of Fixable visual Dye, uniformly mixing by vortex, and fixing at4 ℃ for more than 30 min;
2) then respectively adding 0.5ml of 1 XPBS, uniformly mixing by vortex, centrifuging, removing supernatant, respectively adding 400ul of fixing agent, uniformly mixing by vortex, and fixing for more than 30min at3 ℃;
3) centrifuging (3500rpm, 3min, unlimited temperature), discarding the supernatant fixative, and keeping the tube bottom cells;
4) adding 1ml of 1 XPBS (phosphate buffer), mixing uniformly by vortex, centrifuging (3500rpm, 3min, unlimited temperature) and then discarding the supernatant;
5) respectively adding 5ml of erythrocyte lysate, blowing and uniformly mixing by using a gun head, and carrying out cracking treatment in a water bath kettle at 37 ℃ for 4-6min until the liquid in the flow tube is transparent;
6) centrifuging (3500rpm, 3min, unlimited temperature), discarding supernatant, adding 1ml of 1 × perforating agent, mixing by vortex, and perforating at room temperature in dark place for 15 min; if more erythrocytes still appear after centrifugation, the step 5 can be repeated to crack the erythrocytes as much as possible and then the holes are punched;
7) preparing 4 flow tubes, marking as Test (+) and HC (+) as detection tubes, using Test (-) and HC (-) as homotype control tubes, and using original Test and HC as negative control tubes;
8) incubating the primary antibody: centrifuging the negative control tube (3500rpm, 3min, unlimited temperature), and discarding the supernatant, wherein the liquid volumes of the Test (sample to be detected) and HC (normal sample) flow tubes are about 100-; firstly sucking 2ul of mouse anti-human WAS protein IgG2 alpha antibody to add to the bottom of a detection tube, sucking 40ul of cell sap from a negative control tube to the bottom of the corresponding detection tube, and uniformly mixing by vortex; adding 2ul of mouse anti-human non-specific IgG2 alpha isotype antibody into the isotype control tube, sucking 40ul of cell fluid from the negative control tube to the bottom of the corresponding isotype control tube, and uniformly mixing by vortex; negative control tubes were left without anything; incubating for 30min at normal temperature in dark place;
9) cleaning: adding 1ml of 1 XPBS, centrifuging (3500rpm, 3min, unlimited temperature), and discarding the supernatant;
10) repeating the step 9;
11) incubation of secondary antibody: sucking 0.5ul rat anti-mouse IgG2 alpha antibody, respectively adding into the bottom of the detection tube, the isotype control tube and the negative control tube, vortexing, mixing, and incubating for 30min at normal temperature in a dark place;
12) cleaning: adding 1ml of 1 XPBS, centrifuging (3500rpm, 3min, unlimited temperature), and discarding the supernatant;
13) repeating the step 12;
14) finally, 150ul of 1 XPBS is added, mixed evenly by vortex, and detected on a flow cytometer.
EXAMPLE III
1) Preparing 2 flow tubes, marking as Test (sample to be detected) and HC (normal sample), respectively adding the sample to be detected, 60ul of normal sample whole blood, 3ul of various fluorescence-labeled antibodies and 0.3ul of Fixable visual Dye, uniformly mixing by vortex, and fixing at4 ℃ for more than 30 min;
2) then respectively adding 1.5ml of 1 XPBS, uniformly mixing by vortex, centrifuging, removing supernatant, respectively adding 400-600ul of fixing agent, uniformly mixing by vortex, and fixing for more than 30min at the temperature of 3-5 ℃;
3) centrifuging (3500rpm, 3min, unlimited temperature), discarding the supernatant fixative, and keeping the tube bottom cells;
4) adding 1ml of 1 XPBS (phosphate buffer), mixing uniformly by vortex, centrifuging (3500rpm, 3min, unlimited temperature) and then discarding the supernatant;
5) respectively adding 5ml of erythrocyte lysate, blowing and uniformly mixing by using a gun head, and carrying out cracking treatment in a water bath kettle at 37 ℃ for 4-6min until the liquid in the flow tube is transparent;
6) centrifuging (3500rpm, 3min, unlimited temperature), discarding supernatant, adding 1ml of 1 × perforating agent, mixing by vortex, and perforating at room temperature in dark place for 15 min; if more erythrocytes still appear after centrifugation, the step 5 can be repeated to crack the erythrocytes as much as possible and then the holes are punched;
7) preparing 4 flow tubes, marking as Test (+) and HC (+) as detection tubes, using Test (-) and HC (-) as homotype control tubes, and using original Test and HC as negative control tubes;
8) incubating the primary antibody: centrifuging the negative control tube (3500rpm, 3min, unlimited temperature), and discarding the supernatant; firstly, sucking 4ul of mouse anti-human WAS protein IgG2 alpha antibody, adding the antibody to the bottom of a detection tube, sucking 60ul of cell sap from a negative control tube to the bottom of the corresponding detection tube, and uniformly mixing by vortex; adding 4ul of mouse anti-human nonspecific IgG2 alpha isotype antibody into the isotype control tube, sucking 60ul of cell fluid from the negative control tube to the bottom of the corresponding isotype control tube, and uniformly mixing by vortex; negative control tubes were left without anything; incubating for 30min at normal temperature in dark place;
9) cleaning: adding 1ml of 1 XPBS, centrifuging (3500rpm, 3min, unlimited temperature), and discarding the supernatant;
10) repeating the step 9;
11) incubation of secondary antibody: 2.5ul of rat anti-mouse IgG2 alpha antibody is respectively added into the bottom of the detection tube, the homotype control tube and the negative control tube, vortexed and mixed uniformly, and incubated for 30min at normal temperature in a dark place;
12) cleaning: adding 1ml of 1 XPBS, centrifuging (3500rpm, 3min, unlimited temperature), and discarding the supernatant;
13) repeating the step 12;
14) finally, 250ul of 1 XPBS is added, mixed evenly by vortex, and detected on a flow cytometer.
Comparative example 1
1) Preparing 2 flow tubes, marking as Test (sample to be detected) and HC (normal sample), respectively adding 50ul of whole blood of the sample to be detected and the normal sample and 500ul of fixing agent, uniformly mixing by vortex, and fixing at4 ℃ for more than 30 min;
2) centrifuging (3500rpm, 3min, unlimited temperature), discarding the supernatant fixative, and keeping the tube bottom cells;
3) adding 1ml of 1 XPBS (phosphate buffer), mixing uniformly by vortex, centrifuging (3500rpm, 3min, unlimited temperature) and then discarding the supernatant;
4) respectively adding 1ml of erythrocyte lysate, uniformly mixing by vortex, adding 4ml of erythrocyte lysate, and treating with 37 ℃ water bath for 4-6min until the liquid in the flow tube is transparent;
5) centrifuging (3500rpm, 3min, unlimited temperature), discarding supernatant, adding 1ml of 1 × perforating agent, mixing by vortex, and perforating at room temperature in dark place for 15 min; if more erythrocytes still appear after centrifugation, repeating the step 4 to crack the erythrocytes as much as possible and then perforating;
6) preparing 2 flow tubes, marking as Test (+) and HC (+) as detection tubes, using Test (-) and HC (-) as homotype control tubes, and using original Test and HC as negative control tubes;
7) incubating the primary antibody: centrifuging the negative control tube (3500rpm, 3min, unlimited temperature), and discarding the supernatant, wherein the liquid volumes of the Test (sample to be detected) and HC (normal sample) flow tubes are about 100-; firstly, 3ul of mouse anti-human WAS protein IgG2 alpha antibody is sucked and added to the bottom of a detection tube, then 50ul of cell sap is sucked from a negative control tube and is added to the bottom of the corresponding detection tube, and the cell sap is swirled and mixed evenly; adding 3ul of mouse anti-human non-specific IgG2 alpha isotype antibody into the isotype control tube, sucking 50ul of cell fluid from the negative control tube to the bottom of the corresponding isotype control tube, and uniformly mixing by vortex; negative control tubes were left without anything; incubating for 30min at normal temperature in dark place;
8) cleaning: adding 1ml of 1 XPBS, centrifuging (3500rpm, 3min, unlimited temperature), and discarding the supernatant;
9) repeating the step 8;
10) incubation of secondary antibody: sucking 1.5ul of rat anti-mouse IgG2 alpha antibody, respectively adding into the bottom of the detection tube, the isotype control tube and the negative control tube, vortexing, mixing, and incubating for 30min at normal temperature in a dark place;
11) cleaning: adding 1ml of 1 XPBS, centrifuging (3500rpm, 3min, unlimited temperature), and discarding the supernatant;
12) repeating the step 11;
13) finally, 200ul of 1 XPBS is added, mixed evenly by vortex, and detected on a flow cytometer.
14) Quantitative analysis result of WAS protein: analyzing original data by Flowjo software;
FIG. 2 shows WAS protein detection results of normal samples, which are negative control tube, isotype control tube and detection tube from top to bottom. The left panel is an FSC-SSC scattergram of all cells after red blood lysis in peripheral blood, the lymphocyte populations (Lymphocytes) account for 22.1%, 22.3% and 20.9%, respectively, the middle panel is a scattergram showing the lymphocyte populations by setting gate (Set gate), and the right panel is the expression of WAS protein (WASP) in the lymphocyte populations.
FIG. 3 shows WAS protein detection results of a sample to be detected, which are a negative control tube, a isotype control tube and a detection tube from top to bottom. The left panel is a FSC-SSC scattergram of all cells after red blood lysis in peripheral blood, the lymphocyte population (Lymphocytes) accounts for 32.9%, 31.2% and 32.5%, respectively, the middle panel is a scattergram showing the lymphocyte population by setting gate (Set gate), and the right panel is the expression of WAS protein (WASP) in the lymphocyte population.
FIG. 4 is a peak diagram showing WAS protein (WASP) detection results of a normal sample and a test sample. And (3) superposing WASP peak images of a normal sample (left side) and a sample to be detected (right side), wherein a dotted line is a negative control tube, a dotted line is a homotype control tube, and a solid line is a detection tube. The WASP peak image of the normal sample detection tube is completely separated from the negative control tube and the isotype tube, which indicates that the detection result of the WASP of the normal sample detection tube is positive, and the percentage of the WASP + is 87.6%, which indicates that the WASP in the normal sample is expressed. The WASP peak image of the detection tube of the sample to be detected is almost overlapped with a negative control tube and a homotype control tube, the percentage of the WASP + is 1.29 percent, and the detection result of the WASP of the detection tube of the sample to be detected is negative, which indicates that the expression of the WASP in the sample to be detected is reduced or not expressed.
Comparative example No. two
1) Preparing 2 flow tubes, marking as Test (sample to be detected) and HC (normal sample), respectively adding 50ul of whole blood of the sample to be detected and the normal sample, respectively adding 0.2ul of Fixable visual Dye, FITC-labeled mouse anti-human CD45 IgG1 antibody, BV 421-labeled mouse anti-human CD3 IgG1 antibody, PE-CY 7-labeled mouse anti-human CD4 IgG1 antibody, BV 510-labeled mouse anti-human CD8 IgG1 antibody, PerCP/CY 5.5-labeled mouse anti-human CD19 IgG1 antibody and APC-labeled mouse anti-human CD56 IgG1 antibody, respectively 2ul, mixing by vortex, and fixing at4 ℃ for 30 min;
2) adding 1ml of 1 XPBS (phosphate buffer), mixing by vortex, centrifuging (3500rpm, 3min, 4 ℃), and discarding the supernatant;
3) repeating the step 2;
4) respectively adding 500ul of fixing agent, mixing uniformly by vortex, and fixing at4 deg.C for more than 30 min;
5) the operations of lysing erythrocytes, breaking membranes, perforating, incubating primary antibody and incubating secondary antibody after cell fixation are the same as in example 1; detecting detection data by a streaming machine, and analyzing the detection data;
6) quantitative analysis result of WAS protein: raw data were analyzed using Flowjo software.
FIG. 5 is a schematic representation of the labeling of different classes of lymphocytes with surface antigens. Different types of lymphocytes can be distinguished by carrying out fluorescent labeling according to different lymphocyte surface antigens.
FIG. 6 shows WAS protein (WASP) expression in different lymphocytes. It can be seen that the percentage of WASP + in the different lymphocyte subpopulations CD45+, CD4+, CD8+, CD19+ and CD56+ in the normal sample (left) was 95.8%, 98.6%, 99.2%, 98.8% and 98.1%, respectively, suggesting that there was WASP expression in the different lymphocyte subpopulations in the normal sample. The percentages of WASP + in the different lymphocyte subsets CD45+, CD4+, CD8+, CD19+ and CD56+ in the test sample (right side) were 46.0%, 3.74%, 82.3%, 8.67% and 33.0%, respectively, indicating that the expression of WASP was different in the different lymphocyte subsets in the test sample.
As can be seen by comparing comparative example one with comparative example two: the first comparative example can be used for detecting and quantitatively analyzing WAS protein of a sample to be detected, and the second comparative example can be used for finely analyzing different lymphocytes. The combination of comparative example one and comparative example two, as shown in example one, example two and example three, allows for a fine analysis of different lymphocytes of the WAS protein.
In addition, it should be noted that all assays of the present application can be performed without isotype control, increasing isotype control effects: the influence of non-specific fluorescence signals is eliminated, so that the test result is more reliable.
The method is easy to operate, reliable in result, and capable of quickly detecting the WAS protein (WASP) expression condition in the sample to be detected and further analyzing different WASP expression conditions in different lymphocyte subpopulations.
The embodiments provided in the present application are only a few examples of the general concept of the present application, and do not limit the scope of the present application. Any other embodiments extended according to the scheme of the present application without inventive efforts will be within the scope of protection of the present application for a person skilled in the art.
Claims (12)
1. The method for detecting the WAS protein is characterized in that the detection of a sample to be detected comprises the following steps:
taking 400-;
centrifuging, removing the fixing agent in the first product, washing with 1 × phosphate buffer solution, mixing with erythrocyte lysate, placing in a water bath kettle at 36.5-37.5 deg.C for 4-6min to lyse erythrocyte, adding perforating agent, and breaking membrane at room temperature for 14-16min to obtain a second product;
mixing the second product with a mouse anti-human WAS protein IgG2 alpha antibody, and incubating for 30min at room temperature, wherein the mouse anti-human WAS protein IgG2 alpha antibody is a primary antibody;
and eluting unbound free antibody, mixing with a rat anti-mouse IgG2 alpha antibody, incubating at room temperature for 30min, washing, detecting by a flow type computer to obtain detection data, and analyzing the detection data, wherein the rat anti-mouse IgG2 alpha antibody is a secondary antibody.
2. The method for detecting WAS protein according to claim 1, wherein the washing with 1 XPhosphate buffer solution is mixed with erythrocyte lysate, and the mixture is placed in a water bath kettle at 36.5-37.5 ℃ for 4-6min to lyse erythrocytes, comprising:
washing with 1 × phosphate buffer solution, centrifuging to remove supernatant, adding 500ul erythrocyte lysate, mixing, placing in water bath at 36.5-37.5 deg.C for 4-6min to lyse erythrocyte, processing until the liquid turns transparent, centrifuging, and removing supernatant.
3. The method for detecting WAS protein according to claim 1 or 2, further comprising detecting a control according to the step of detecting the sample to be detected, obtaining detection data of the control, and comparing the detection data of the control with the detection data of the sample to be detected.
4. The method for detecting WAS protein according to claim 3, wherein the second product is mixed with mouse anti-human WAS protein IgG2 α antibody and incubated at room temperature for 30min, comprising:
taking 2-4ul of mouse anti-human WAS protein IgG2 alpha antibody, taking 40-60ul of second product, and mixing the mouse anti-human WAS protein IgG2 alpha antibody and the second product in a vortex manner to obtain a third product;
taking 2-4ul of mouse anti-human nonspecific IgG2 alpha isotype antibody, taking 40-60ul of second product, and mixing the mouse anti-human nonspecific IgG2 alpha isotype antibody with the second product in a vortex manner to obtain a fourth product;
and standing the rest second product at room temperature for 30min to obtain a fifth product.
5. The method for detecting WAS protein according to claim 4, wherein the second product is mixed with mouse anti-human WAS protein IgG2 α antibody and incubated at room temperature for 30min, and further comprising:
adding 1 x phosphate buffer solution into the third product, the fourth product and the fifth product respectively for washing;
the washed product was centrifuged and the supernatant discarded.
6. The method for detecting WAS protein according to claim 5, wherein the mixing with rat anti-mouse IgG2 α antibody and incubating for 30min at room temperature comprises:
adding 1.0-2.0ul rat anti-mouse IgG2 alpha antibody into the third product, the fourth product and the fifth product after discarding the supernatant respectively, vortexing and mixing uniformly, and incubating for 30min at normal temperature and in the dark.
7. The method for detecting WAS protein according to claim 6, wherein the method further comprises mixing with rat anti-mouse IgG2 α antibody, and after incubating for 20-40min at room temperature:
respectively adding 1 Xphosphate buffer solution into the third product, the fourth product and the fifth product after incubation for 30min at normal temperature in a dark place for washing;
centrifuging the washed product, and removing the supernatant;
150-250ul of 1 XPhosphate buffer was added to the product after the supernatant was discarded.
8. The method for detecting WAS protein according to claim 1 or 2, wherein 500ul of fixative is mixed with 50ul of peripheral blood of a sample to be detected, and the mixture is fixed at 3-5 ℃ for more than 30min to obtain a first product, comprising:
mixing 1-3ul of each of a plurality of different fluorescence labeled antibodies and 0.1-0.3ul of Fixable visual Dye with 40-60ul of peripheral blood of a sample to be detected, uniformly mixing by vortex, and incubating the antibodies for 30min at 3-5 ℃;
respectively adding 0.5-1.5ml of 1 XPBS, uniformly mixing by vortex, centrifuging, removing supernatant, and eluting unbound free antibody;
respectively adding 400-600ul of fixing agent, uniformly mixing by vortex, and fixing at 3-5 ℃ for more than 30min to obtain a first product.
9. The method for detecting WAS protein according to claim 8, wherein the plurality of different fluorescently labeled antibodies comprises:
mouse anti-human WAS protein IgG2 alpha antibody, mouse anti-human non-specific IgG2 alpha isotype antibody, PE-labeled rat anti-mouse IgG2 alpha antibody, FITC-labeled mouse anti-human CD45 IgG1 antibody, BV 421-labeled mouse anti-human CD3 IgG1 antibody, PE-CY 7-labeled mouse anti-human CD4 IgG1 antibody, BV 510-labeled mouse anti-human CD8 IgG1 antibody, PerCP/CY 5.5-labeled mouse anti-human CD19 IgG1 antibody, and APC-labeled mouse anti-human CD56 IgG1 antibody.
10. The method for detecting WAS protein according to claim 8, wherein the plurality of different fluorescently labeled antibodies are antibodies that are homologous to the sample to be tested or antibodies that are non-homologous to the sample to be tested.
11. The method for detecting WAS protein according to claim 8, wherein the fitable visual ability Dye can label dead cells to distinguish dead from live cells;
when the cell surface marker is CD45+, all leukocytes can be immunophenotyped; when the cell surface marker is CD45+ CD3+ CD4+, the CD4+ T cells can be subjected to immune typing; when the cell surface marker is CD45+ CD3+ CD8+, the CD8+ T cells can be subjected to immune typing; when the cell surface marker is CD45+ CD19+, B cells can be subjected to immune typing; when the cell surface marker is CD45+ CD56+, NK cells can be subjected to immune typing.
12. The method for detecting WAS protein according to claim 11, wherein when the detection data is positive, the sample is a sample expressing WAS protein;
and when the detection data is negative, the WAS protein is not expressed.
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