Antibody composition for solid tumor detection and application thereof

Abstract

The invention discloses an antibody composition for solid tumor detection and application thereof. The antibody composition comprises a CD45 antibody, a CD56 antibody, a CD9 antibody, a CD81 antibody, a CD36 antibody, a GD2 antibody, an HLA-DR antibody, a CD326 antibody, a CD7 antibody, a CD138 antibody, a CD19 antibody and a CD4 antibody. The antibody composition disclosed by the invention can be used for detecting solid tumor cells by flow cytometry, and can effectively define CD56 + tumor cells and CD56-tumor cells, so that the CD56 + tumor cells and the CD56-tumor cells cover the most complete solid tumor cell groups originated from ganglions, neuroendothelium, mesenchymal origin and the like, and the missed diagnosis probability is reduced.

Description

Technical Field

[0001] The invention relates to an antibody composition for solid tumor detection and application thereof, belonging to the technical field of disease monitoring. Background Art

[0002] Clinically, tumors can be divided into solid tumors and non-solid tumors. Solid tumors are tangible tumors that can be felt through clinical examinations such as X-rays, CT scans, B-ultrasound, or palpation. They are tumors that are not derived from the hematopoietic system. Non-solid tumors are tumors that cannot be seen or felt through X-rays, CT scans, B-ultrasound, or palpation, such as leukemia in blood diseases.

[0003] With the widespread application of flow cytometry in clinical practice, samples from patients with solid tumors are often encountered. Currently, only a single or a small number of antibody combinations are used to detect solid tumor samples, so it is difficult to meet the detection needs; secondly, patients with mid- and late-stage solid tumors often infiltrate the bone marrow or cerebrospinal fluid, such as neuroblastoma, retinoblastoma, small cell lung cancer, medulloblastoma, tumors of epithelial tissue origin, etc., and there is currently no antibody combination that can detect and identify solid tumors of different origins; thirdly, solid tumors usually have extramedullary masses, and diagnosis depends on tissue pathology, but the tissue pathology operation steps are cumbersome and the reporting cycle is long; fourthly, when receiving samples for inspection, the application form often lacks detailed medical history, clinical symptoms and other information, such as whether there is abdominal mass, visual impairment, lung shadows, etc. The lack of these key information will cause the tester to be unable to adjust or optimize the combination according to clinical information in the first time to meet the detection needs, which often leads to missed detection, making the test results inaccurate or false negatives. In order to reduce false negatives and improve the accuracy of detection, it is urgent for testers and clinicians to find better detection solutions to make up for the shortcomings of existing detection methods to meet clinical needs. Summary of the invention

[0004] In view of this, the main purpose of the present invention is to provide an antibody composition for solid tumor detection and its application. The technical problem to be solved is how to provide an antibody composition that can be used for flow cytometry detection of solid tumor cells and can effectively set a gate to circle CD56 + Tumor cells and CD56 - tumor cells, so that it covers the most complete solid tumor cells and reduces the probability of missed diagnosis.

[0005] The purpose of the present invention and the technical problem solved are achieved by adopting the following technical solutions. The present invention proposes an antibody composition for solid tumor detection, which comprises CD45 antibody, CD56 antibody, CD9 antibody, CD81 antibody, CD36 antibody, GD2 antibody, HLA-DR antibody, CD326 antibody, CD7 antibody, CD138 antibody, CD19 antibody and CD4 antibody.

[0006] The purpose of the present invention and the technical problems to be solved can be further achieved by adopting the following technical measures.

[0007] Preferably, the aforementioned antibody composition for solid tumor detection, wherein the antibody composition for solid tumor detection further comprises CK antibody and CD45.

[0008] Preferably, in the aforementioned antibody composition for solid tumor detection, the fluorescein label of the CD45 antibody is QB500; the fluorescein label of the CD56 antibody is PE-CY7; the fluorescein label of the CD9 antibody is FITC; the fluorescein label of the CD81 antibody is APC-CY7; the fluorescein label of the CD36 antibody is APC-CY7; the fluorescein label of the GD2 antibody is APC; the fluorescein label of the HLA-DR antibody is BV711; the fluorescein label of the CD326 antibody is PE; the fluorescein label of the CD7 antibody is APC-R700; the fluorescein label of the CD138 antibody is BV785; the fluorescein label of the CD19 antibody is BV421; the fluorescein label of the CD4 antibody is V605; and the fluorescein label of the CK antibody is FITC.

[0009] The purpose of the present invention and the technical problem to be solved are achieved by adopting the following technical solutions: The present invention proposes the use of the above antibody composition in the preparation of a product for detecting solid tumors.

[0010] The purpose of the present invention and the technical problems to be solved can be further achieved by adopting the following technical measures.

[0011] Preferably, the aforementioned antibody composition is used in the preparation of a product for detecting solid tumors, wherein the product is a kit.

[0012] The purpose of the present invention and the technical problem to be solved are achieved by adopting the following technical solutions: The present invention provides a kit for solid tumor detection, which comprises the above-mentioned antibody composition.

[0013] The purpose of the present invention and the technical problems to be solved can be further achieved by adopting the following technical measures.

[0014] Preferably, the aforementioned kit for solid tumor detection further comprises a red blood cell lysis solution and a membrane breaking agent.

[0015] The purpose of the present invention and the technical problem to be solved are achieved by adopting the following technical solutions: The present invention proposes the use of the above antibody composition in the preparation of flow cytometric samples for flow cytometry detection of solid tumors.

[0016] The purpose of the present invention and the technical problems to be solved can be further achieved by adopting the following technical measures.

[0017] Preferably, the aforementioned antibody composition is used in the preparation of a flow cytometric sample for flow cytometry detection of solid tumors, wherein the step of preparing the flow cytometric sample for flow cytometry detection of solid tumors comprises:

[0018] The CD45 antibody, CD56 antibody, CD9 antibody, CD81 antibody, CD36 antibody, GD2 antibody, HLA-DR antibody, CD326 antibody, CD7 antibody, CD138 antibody, CD19 antibody and CD4 antibody were added to the flow tube at a volume ratio of 2.5:2.5:5:2.5:2.5:2.5:2.5:5:2.5:2.5:2.5:2.5, and the sample to be tested was added so that the number of cells in the flow tube was 1×10 6 / tube, mix well and incubate in the dark; add lysis solution, mix well and incubate in the dark; centrifuge and remove supernatant, add buffer solution for washing and centrifuge again and remove supernatant; add membrane permeabilization agent a, mix well and incubate in the dark, centrifuge and remove supernatant; add the CK antibody and the membrane permeabilization agent b, mix well and incubate in the dark, add the buffer solution for washing, centrifuge and remove supernatant, and then add the buffer solution for resuspending to obtain a flow cytometer sample.

[0019] Preferably, the aforementioned antibody composition is used in the preparation of flow cytometry samples for flow cytometry detection of solid tumors, wherein the sample to be tested is bone marrow, peripheral blood, cerebrospinal fluid or tissue fluid; and the lysate is red blood cell lysate.

[0020] Compared with the prior art, the antibody composition for solid tumor detection and its application described in the present invention have the following beneficial effects:

[0021] The present invention not only provides an objective, highly sensitive and specific detection method for brain metastasis and bone marrow infiltration in patients with solid tumors of neural and endothelial origin, but also provides assistance for patient diagnosis, evaluation of efficacy after treatment and early prediction of recurrence. The antibody composition of the present invention can be used for flow cytometry detection of minimal residual disease in solid tumors of neural and endothelial origin, and can effectively set a gate to circle CD56 + Tumor cells and CD56 -of tumor cells, so that it covers the most complete tumor cell population and reduces the probability of missed diagnosis.

[0022] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The present invention shows the examination result of the bone marrow of a neuroblastoma patient after treatment;

[0024] Figure 2 The present invention shows the examination result of newly diagnosed neuroblastoma infiltrating the bone marrow;

[0025] Figure 3 The present invention shows the examination result of cerebrospinal fluid after the treatment of retinoblastoma;

[0026] Figure 4 The present invention shows the examination result of medullary thyroid carcinoma infiltrating the bone marrow;

[0027] Figure 5 The present invention shows the examination result of Ewing's sarcoma infiltrating the bone marrow;

[0028] Figure 6 This is a diagram showing the examination result of the present invention showing a tumor originating from a ganglion infiltrating the bone marrow;

[0029] Figure 7 This is a diagram showing the examination results of the present invention showing mesenchymal malignant tumor infiltrating the bone marrow. DETAILED DESCRIPTION

[0030] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the antibody composition for solid tumor detection proposed by the present invention and its application, its specific implementation, structure, characteristics and effects are described in detail below in combination with the preferred embodiments. In the following description, different "one embodiment" or "embodiment" does not necessarily refer to the same embodiment. In addition, specific features, structures or characteristics in one or more embodiments may be combined in any suitable form.

[0031] Unless otherwise specified, the materials, reagents, etc. involved below are all commercially available products well known to those skilled in the art; unless otherwise specified, the methods described are all methods known in the art. Unless otherwise defined, the technical terms or scientific terms used should have the usual meanings understood by ordinary technicians in the field to which the present invention belongs. If no specific experimental steps or conditions are specified below, the operations or conditions of the conventional experimental steps described in the literature in the field can be followed.

[0032] Some embodiments of the present invention provide an antibody composition for solid tumor detection, the antibody composition comprising CD45 antibody, CD56 antibody, CD9 antibody, CD81 antibody, CD36 antibody, GD2 antibody, HLA-DR antibody, CD326 antibody, CD7 antibody, CD138 antibody, CD19 antibody and CD4 antibody. These antibodies are mainly cell membrane antibodies, which are faster and more convenient to detect.

[0033] In some optional embodiments, the antibody composition for solid tumor detection may also include CK antibody and CD45. CK is a cytoplasmic antibody. If the above-mentioned membrane antibody is not detected abnormal, the cytoplasmic antibody is detected. If the above-mentioned membrane antibody can be confirmed, there is no need to detect the cytoplasmic antibody. CD45 is a membrane antibody used for gate setting.

[0034] Some embodiments of the present invention also provide a use of the above-mentioned antibody composition in preparing a product for detecting solid tumors.

[0035] In some optional embodiments, the product is a kit further comprising a red blood cell lysis solution and a membrane breaking agent.

[0036] Some embodiments of the present invention also provide a use of the above-mentioned antibody composition in preparing flow cytometry samples for detecting solid tumors by flow cytometry.

[0037] In some optional embodiments, the step of preparing a flow cytometer sample for flow cytometry detection of solid tumors comprises:

[0038] The CD45 antibody, CD56 antibody, CD9 antibody, CD81 antibody, CD36 antibody, GD2 antibody, HLA-DR antibody, CD326 antibody, CD7 antibody, CD138 antibody, CD19 antibody and CD4 antibody were added to the flow tube at a volume ratio of 2.5:2.5:5:2.5:2.5:2.5:2.5:5:2.5:2.5:2.5:2.5, and the sample to be tested was added so that the number of cells in the flow tube was 1×10 6 / tube, mix well and incubate in the dark; add lysis solution, mix well and incubate in the dark; centrifuge and remove supernatant, add buffer solution for washing and centrifuge again and remove supernatant; add membrane permeabilization agent a, mix well and incubate in the dark, centrifuge and remove supernatant; add the CK antibody and the membrane permeabilization agent b, mix well and incubate in the dark, add the buffer solution for washing, centrifuge and remove supernatant, and then add the buffer solution for resuspending to obtain a flow cytometer sample.

[0039] In some optional embodiments, the sample to be tested is bone marrow, peripheral blood, cerebrospinal fluid or tissue fluid; and the lysing solution is red blood cell lysing solution.

[0040] In some optional embodiments, the application specifically includes the following steps:

[0041] 1) Take a corresponding number of flow cytometry tubes, number them and indicate the specimen number;

[0042] 2) Add the corresponding antibodies to each tube. The amount of antibodies should be added according to the instructions for use of the antibodies (or the amount of reagents should be determined through antibody titration experiments). Note that the antibodies should be added to the bottom of the tube. The fluorescent labeling order of the antibodies is as follows:

[0043] A tube: CD45 / CD56 / CD9 / CD81 / CD36 / GD2 / HLA-DR / CD326 / CD7 / CD138 / CD19 / CD4;

[0044] B tube: CK / CD45;

[0045] Tube A mainly contains membrane antibodies, which are faster and more convenient to detect. Tube B contains cytoplasmic antibodies. If tube A does not detect abnormalities, tube B will be tested. If tube A can confirm the abnormality, tube B will not be tested.

[0046] 3) Add samples according to specimen count and add antibodies in the above order. Adding antibodies in order is to ensure that there is no mistake that affects the results.

[0047] 4) Shake, mix, and incubate at room temperature in the dark;

[0048] 5) Add red blood cell lysis buffer, shake and mix, lyse in the dark, centrifuge, and discard the supernatant;

[0049] 6) Add PBS buffer, shake gently to mix, centrifuge, and discard the supernatant; add PAS to tube A and resuspend, and wait for loading;

[0050] If tube A does not detect abnormality, step 6) may further include the following steps:

[0051] 7) Add PBS to tube B, mix well, centrifuge to remove supernatant, add membrane permeabilization solution A, and incubate at room temperature;

[0052] 8) Add PBS to tube B, centrifuge, remove the supernatant, add CK antibody, add membrane permeabilization agent b, mix well and protect from light;

[0053] 9) Add PBS to tube B, mix well, centrifuge, remove the supernatant, resuspend with PBS, and obtain on the machine;

[0054] 10) If there is no time to load the cells onto the machine, the processed cells can be fixed with formalin solution and preserved.

[0055] Table 1 Sources of the above mentioned antibodies against fluorescein

[0056]

[0057]

[0058] Use BD FACSDive software to analyze the data obtained in step 6) or step 9) above. First, use the area (A) and height (H) of forward scatter (FSC) to set the de-adhesion cell gate (usually represented by P1). Then display the cells in P1 and set the live cell gate P2 to obtain the best state of cells. Use FSC / side scatter (SSC) to set the live cell gate (usually represented by P3) to obtain single live cells. In the single live cell gate (P3 gate), CD45 / SSC is routinely used to set each blood cell gate first, and lymphocytes, monocytes, granulocytes, and whether there are obvious tumor cells or abnormal cells. In the single live cell gate (P3 gate), multiple marker combinations are used to set gates to observe non-hematopoietic cells, especially CD45. - CD56 + 、GD2 + CD56 + Cells: For samples in tube A, select CD56 / GD2, CD45 / CD56, CD45 / CD326, CD45 / CD138 or CD45 / GD2 to set the gate together. For samples in tube B, select CD45 / CK to set the gate to distinguish non-hematopoietic cells, etc., and judge benign or malignant based on the antigen phenotype.

[0059] The judgment is made in at least one of the following ways: by monitoring the phenotype of non-hematopoietic tumor cells, that is, tumor cells abnormally express CD56, CD326, CD9, CD81 and CK (Cytokeratin), do not express CD45, etc., and do not express various blood cell antigens; or live cells that do not express all detected antigens, these live cells need to be indicated as suspicious cells. CD45 is not expressed or weakly expressed in normal lymphocytes, monocytes, granulocytes, eosinophils, basophils, mast cells, dendritic cells and normal plasma cells, nucleated red blood cells, some acute B lymphocytic leukemia, a few acute myeloid leukemias, most plasma cell tumors and cells from non-hematopoietic tissues. Therefore, SSC / CD45 can be used to set a gate for preliminary screening of CD45. -Tumor cells. CD56 is usually expressed in normal NK cells, NK-T lymphocytes, a small number of normal plasma cells and 60%-70% of tumor plasma cells. GD2 is expressed in normal central nervous system, peripheral nerves, skin melanocytes and mesenchymal stem cells. It is highly expressed in tumors of neuroectodermal origin such as melanoma, glioma, neuroblastoma and small cell lung cancer. GD2 is considered a marker for certain cancer stem cells. Gating with CD56 / GD2 can be used as a reliable indicator for determining the origin of non-hematopoietic tissue. Hematopoietic cells usually do not express GD2, so CD45 / CD138 can be used for further gating analysis. CD138 is only expressed in plasma cells and myeloma cells, most of which are expressed in multiple myeloma patients. CD138 is also expressed in epithelial cells, mesenchymal cells, and tumor cells in the non-hematopoietic system, but CD45 is generally positive. CD326 is highly expressed in bone marrow, colon, lung, and most normal epithelial cells, and is expressed in gastrointestinal cancer. CD45 / CD326 can be used as an indicator of non-hematopoietic tissue origin. If CD56 is present, - GD2 - CD138 - CD326 - , further analysis is needed, CD7 / CD19 reverse gating can be used, CD19 is expressed in B lymphocytes and most plasma cells, CD7 is expressed in T cells, NK cells, thymocytes, hematopoietic stem cells and monocytes (weakly expressed), this reverse gating can remove the interference of T cells, B cells, some plasma cells and NK cells, and then HLA-DR / CD4 reverse gating can be used, HLA-DR is expressed in addition to B cells and activated T cells, as well as macrophages / monocytes and dendrites, CD4 is expressed in thymocytes, neutrophils, mature T lymphocytes, NK cells, macrophages, dendritic cells, this reverse gating can remove monocytes, CD4 + In order to eliminate the interference of helper T cells and monocytes / macrophages, CD45 / CD36 / CD81 / CD9 reverse gating was further used. CD81 is expressed in T lymphocytes, B lymphocytes, NK cells, monocytes, dendritic cells, thymocytes, endothelial cells and fibroblasts, CD36 is expressed in a variety of epithelial cells and endothelial cells, as well as red blood cells, platelets, macrophages / monocytes and some macrophage-derived dendritic cells, and CD9 is expressed in platelets, precursor B cells, activated lymphocytes, granulocytes, endothelial cells and epithelial cells. Through this step, the interference of some lymphocytes, some granulocytes, nucleated red blood cells and platelets can be removed. CD45 / CD138 gating was further used to analyze the expression of CD45 - CD138 -The position of FSC and SSC is used to determine whether the cells are of abnormal cell origin. If the FSC / SSC position of this group of cells coincides with that of lymphocytes, it is judged as negative. If the SSC and FSC are greater than those of lymphocytes, they are considered to be positive cells. CK can also be detected to determine whether they are of epithelial cell origin.

[0060] Some embodiments of the present invention also provide a kit for solid tumor detection, wherein the kit comprises the above-mentioned antibody composition.

[0061] In some optional embodiments, the kit further comprises a red blood cell lysis solution and a membrane breaking agent.

[0062] In some optional embodiments, the membrane-breaking agent includes membrane-breaking agent a and membrane-breaking agent b.

[0063] The present invention will be further described below in conjunction with specific embodiments, but this should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made to the present invention by technicians in this field based on the above-mentioned contents of the present invention still fall within the scope of protection of the present invention.

[0064] Example 1

[0065] This embodiment provides a kit for solid tumor detection. The kit comprises an antibody composition; the antibody composition comprises the antibodies shown in Table 2.

[0066] Furthermore, the kit also includes red blood cell lysis solution and membrane breaking agent as shown in Table 2.

[0067] The membrane-breaking agent includes a membrane-breaking agent a and a membrane-breaking agent b.

[0068] Table 2 Antibody composition for solid tumor detection and its labeling and dosage

[0069]

[0070] The use of the above antibody composition in preparing a flow cytometric sample for flow cytometry detection of solid tumors comprises the following steps:

[0071] 1) Take a corresponding number of flow cytometry tubes, number them and indicate the specimen number;

[0072] 2) Add the corresponding antibodies to each tube. The amount of antibodies should be added according to the instructions for use of the antibodies (or the amount of reagents should be determined through antibody titration experiments). Note that the antibodies should be added to the bottom of the tube. The fluorescent labeling order of the antibodies is as follows:

[0073] A tube: CD45 / CD56 / CD9 / CD81 / CD36 / GD2 / HLA-DR / CD326 / CD7 / CD138 / CD19 / CD4;

[0074] B tube: CK / CD45;

[0075] 3) Add samples according to specimen count and add antibodies in the above order;

[0076] 4) Shake and mix, and incubate at room temperature in the dark for 20 minutes;

[0077] 5) Add 1 ml of 10× red blood cell lysis buffer, shake and mix, lyse for 10 minutes in the dark; centrifuge at 1500 rpm for 5 minutes and discard the supernatant;

[0078] 6) Add 2 ml of PBS buffer, gently shake to mix, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant.

[0079] Add PAS to tube A and resuspend it, and wait for loading.

[0080] 7) Add 2 ml PBS to tube B, mix well, centrifuge to remove supernatant, add 100 μl membrane permeabilization solution A, and incubate at room temperature for 15 min;

[0081] 8) Add 2 ml PBS to tube B, centrifuge (5 min, 1500 rpm), remove the supernatant, add CK (keratin), add 50 μl membrane permeabilization agent B, mix well and protect from light for 15 min;

[0082] 9) Add 2 ml PBS to tube B, mix well, centrifuge (5 min, 1500 rpm), remove the supernatant, add 500 μl PBS, and wait for loading;

[0083] 10) The treated cells were fixed with 0.5% (v / v) formalin solution (300 μl / tube) and stored at 2-8°C for 5 days.

[0084] BD FACSDive software was used to analyze the data. First, the area (A) and height (H) of the forward angle light scatter (FSC) were used to set the gate for de-adhesion cells (usually represented by P1). Then, the cells in P1 were displayed, and the live cell gate P2 was set to obtain the best state of cells. The live cell gate (usually represented by P3) was set using FSC / side angle light scatter (SSC) to obtain single live cells. In the single live cell gate (P3 gate), CD45 / SSC was routinely used to set the gates for each blood cell, and lymphocytes, monocytes, granulocytes, and whether there were obvious tumor cells or abnormal cells. In the single live cell gate (P3 gate), a combination of multiple markers was used to set gates to observe non-hematopoietic cells, especially CD45. -CD56 + 、GD2 + CD56 + Cells: For samples in tube A, select CD56 / GD2, CD45 / CD56, CD45 / CD326, CD45 / CD138 or CD45 / GD2 to set the gate together. For samples in tube B, select CD45 / Cytokeratin to set the gate to distinguish non-hematopoietic cells, etc., and judge benign or malignant based on the antigen phenotype.

[0085] The judgment is made in the following way: by monitoring the phenotype of non-hematopoietic tumor cells, that is, tumor cells abnormally express CD56, CD326, CD9, CD81 and CK, do not express CD45, etc., and do not express various blood cell antigens; or live cells that do not express all detected antigens, these live cells need to be indicated as suspicious cells. CD45 is not expressed or weakly expressed in normal lymphocytes, monocytes, granulocytes, eosinophils, basophils, mast cells, dendritic cells and normal plasma cells, nucleated red blood cells, some acute B lymphocytic leukemias, a few acute myeloid leukemias, most plasma cell tumors and cells from non-hematopoietic tissues. Therefore, SSC / CD45 can be used to set a gate for preliminary screening of CD45. - Tumor cells. CD56 is usually expressed in normal NK cells, NK-T lymphocytes, a small number of normal plasma cells and 60%-70% of tumor plasma cells. GD2 is expressed in normal central nervous system, peripheral nerves, skin melanocytes and mesenchymal stem cells. It is highly expressed in tumors of neuroectodermal origin such as melanoma, glioma, neuroblastoma and small cell lung cancer. GD2 is considered a marker for certain cancer stem cells. Gating with CD56 / GD2 can be used as a reliable indicator for determining the origin of non-hematopoietic tissue. Hematopoietic cells usually do not express GD2, so CD45 / CD138 can be used for further gating analysis. CD138 is only expressed in plasma cells and myeloma cells, most of which are expressed in multiple myeloma patients. CD138 is also expressed in epithelial cells, mesenchymal cells, and tumor cells in the non-hematopoietic system, but CD45 is generally positive. CD326 is highly expressed in bone marrow, colon, lung, and most normal epithelial cells, and is expressed in gastrointestinal cancer. CD45 / CD326 can be used as an indicator of non-hematopoietic tissue origin. If CD56 is present, - GD2 - CD138 - CD326 -, further analysis is needed, CD7 / CD19 reverse gating can be used, CD19 is expressed in B lymphocytes and most plasma cells, CD7 is expressed in T cells, NK cells, thymocytes, hematopoietic stem cells and monocytes (weakly expressed), this reverse gating can remove the interference of T cells, B cells, some plasma cells and NK cells, and then HLA-DR / CD4 reverse gating can be used, HLA-DR is expressed in addition to B cells and activated T cells, as well as macrophages / monocytes and dendrites, CD4 is expressed in thymocytes, neutrophils, mature T lymphocytes, NK cells, macrophages, dendritic cells, this reverse gating can remove monocytes, CD4 + In order to eliminate the interference of helper T cells and monocytes / macrophages, CD45 / CD36 / CD81 / CD9 reverse gating was further used. CD81 is expressed in T lymphocytes, B lymphocytes, NK cells, monocytes, dendritic cells, thymocytes, endothelial cells and fibroblasts, CD36 is expressed in a variety of epithelial cells and endothelial cells, as well as red blood cells, platelets, macrophages / monocytes and some macrophage-derived dendritic cells, and CD9 is expressed in platelets, precursor B cells, activated lymphocytes, granulocytes, endothelial cells and epithelial cells. Through this step, the interference of some lymphocytes, some granulocytes, nucleated red blood cells and platelets can be removed. CD45 / CD138 gating was further used to analyze the expression of CD45 - CD138 - The position of FSC and SSC is used to determine whether the cells are of abnormal cell origin. If the FSC / SSC position of this group of cells coincides with that of lymphocytes, it is judged as negative. If the SSC and FSC are greater than those of lymphocytes, they are considered to be positive cells. CK can also be detected to determine whether they are of epithelial cell origin.

[0086] Example 2

[0087] The difference between this embodiment and embodiment 1 is that the kit of this embodiment includes an antibody composition, a red blood cell lysis solution, and a membrane permeabilizing agent; the antibody composition does not contain CK antibody, and the use of the antibody composition in preparing a flow cytometric sample for flow cytometry detection of solid tumors includes the following steps:

[0088] 1) Take a corresponding number of flow cytometry tubes, number them and indicate the specimen number;

[0089] 2) Add the corresponding antibodies to each tube. The amount of antibodies should be added according to the instructions for use of the antibodies (or the amount of reagents should be determined through antibody titration experiments). Note that the antibodies should be added to the bottom of the tube. The fluorescent labeling order of the antibodies is as follows:

[0090] A tube: CD45 / CD56 / CD9 / CD81 / CD36 / GD2 / HLA-DR / CD326 / CD7 / CD138 / CD19 / CD4;

[0091] 3) Add samples according to specimen count and add antibodies in the above order;

[0092] 4) Shake and mix, and incubate at room temperature in the dark for 20 minutes;

[0093] 5) Add 1 ml of 10× red blood cell lysis buffer, shake and mix, lyse for 10 minutes in the dark; centrifuge at 1500 rpm for 5 minutes and discard the supernatant;

[0094] 6) Add 2 ml of PBS buffer, gently shake to mix, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant.

[0095] Add PAS to tube A and resuspend it, and wait for loading.

[0096] The remaining components, steps and parameters are the same as those in Example 1.

[0097] Comparative Example 1

[0098] The difference between this comparative example and Example 1 is that the kit of this comparative example includes an antibody composition, a red blood cell lysate, and a membrane permeabilizing agent; the antibody composition includes CD7 antibody, CD9 antibody, CD56 antibody, CD81 antibody, GD2 antibody, CK antibody, CD326 antibody and CD45 antibody; the use of the antibody composition in preparing a flow cytometry sample for flow cytometry detection of solid tumors includes the following steps:

[0099] 1) Take a corresponding number of flow cytometry tubes, number them and indicate the specimen number;

[0100] 2) Add the corresponding antibodies to each tube. The amount of antibodies should be added according to the instructions for use of the antibodies (or the amount of reagents should be determined through antibody titration experiments). Note that the antibodies should be added to the bottom of the tube. The fluorescent labeling order of the antibodies is as follows:

[0101] Tube A: CD7 / CD9 / CD56 / CD81 / GD2 / CD45;

[0102] Tube B: CK / CD326 / CD56 / CD45;

[0103] 3) Add samples according to specimen count and add antibodies in the above order;

[0104] 4) Shake and mix, and incubate at room temperature in the dark for 20 minutes;

[0105] 5) Add 1 ml of 10× red blood cell lysis buffer, shake and mix, lyse for 10 minutes in the dark; centrifuge at 1500 rpm for 5 minutes and discard the supernatant;

[0106] 6) Add 2 ml of PBS buffer, gently shake to mix, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant.

[0107] Add PAS to tube A and resuspend it, and wait for loading.

[0108] 7) Add 2 ml PBS to tube B, mix well, centrifuge to remove supernatant, add 100 μl membrane permeabilization solution A, and incubate at room temperature for 15 min;

[0109] 8) Add 2 ml PBS to tube B, centrifuge (5 min, 1500 rpm), remove the supernatant, add CK (keratin), add 50 μl membrane permeabilization agent B, mix well and protect from light for 15 min;

[0110] 9) Add 2 ml PBS to tube B, mix well, centrifuge (5 min, 1500 rpm), remove the supernatant, add 500 μl PBS, and wait for loading;

[0111] 10) The treated cells were fixed with 0.5% (v / v) formalin solution (300 μl / tube) and stored at 2-8°C for 5 days;

[0112] The remaining components, steps and parameters are the same as those in Example 1.

[0113] Application example 1: Patient, male, 8 years old, clinical diagnosis: neuroblastoma patient after treatment, specimen type: bone marrow.

[0114] This application example uses the kit in Example 1 to detect the sample to be tested. The test results are as follows Figure 1 As shown. Gating strategy for tube A analysis: P1: De-adhesion cell gate P1; P2: Set the live cell gate to obtain the best state of cells; P3: Set the live cell gate to obtain a single live cell. The live cell marker combination gate in the P3 gate is used to observe plasma cells, lymphocytes, monocytes, granulocytes, and whether there are obvious tumor cells or abnormal cells; CD45 / SSC antibodies are used to set the gates of each group of cells in the P3 gate; multiple marker combinations are set in the P3 gate to observe cells of non-hematopoietic system origin; CD56 / GD2, CD7 / CD19, CD4 / HLA-DR, CD45 / CD36 / CD38, CD15 / CD138, CD45 / CD56 antibodies are used to set gates (P4, P5, P6, P7), and gates P10 and P11 are set in SSC / FSC. B tube gating strategy: P1: remove the adhesion cell gate P1; P2: set the live cell gate to obtain the best cell state; P3: set the live cell gate to obtain single live cells and use CD45 / CK to set the gate. In the figure, the above gates can be used to distinguish tubes A and B. Figure 1It can be seen that in CD56 / GD2, abnormal cells are 0.0%. Through the combined gating analysis of P4-P7, no abnormal cells are found in P11. Tube B: No abnormal cells are found. The conclusion is negative.

[0115] Application example 2: Patient, male, 11 years old, clinical diagnosis: neuroblastoma, specimen type: bone marrow.

[0116] This application example uses the kit in Example 2 to detect the sample to be tested. The test results are as follows Figure 2 As shown. Among them, tube A: P1: De-adhesion cell gate; P2: Set the live cell gate to obtain the best state of cells; P3: Set the live cell gate to obtain single live cells. The live cell marker combination in the P3 gate is used to set the gate to observe plasma cells, lymphocytes, monocytes, granulocytes, and whether there are obvious tumor cells or abnormal cells; CD45 / SSC antibodies are used to set the cell gates for each group in the P3 gate; CD56 / GD2 is used to set the gate; from Figure 3 CD56 + GD2 + The cell population expressed CD326 but not CD45, and its immunophenotype was consistent with neuroblastoma.

[0117] Application example 3: Patient, female, 5 years old, clinical diagnosis: retinoblastoma patient after treatment. Specimen type: cerebrospinal fluid.

[0118] This application example uses the kit in Example 2 to detect the sample to be tested. The test results are as follows Figure 3 As shown. In this application example, cerebrospinal fluid was detected after treatment of retinoblastoma, and the gate strategy was analyzed: P1: De-adhesion cell gate P1; P2: Setting the live cell gate to obtain the best state of cells; the number of cells in the cerebrospinal fluid sample was small, Figure 3 The CD45 / SSC graph showed cells in the lymphocyte and monocyte positions. No abnormal cells were found in CD45 / CD56 and GD2 / CD326. CD4, CD7, CD38, and HLA-DR antigens confirmed that the cells shown on the CD45 / SSC graph were lymphocytes and monocytes. Conclusion: No abnormal cells were detected.

[0119] Application example 4: Patient, male, 64 years old, clinical diagnosis: multiple myeloma, specimen type: bone marrow.

[0120] This application example uses the kit in Example 1 to detect the sample to be tested. The test results are as follows Figure 4 The patient was clinically suspected of multiple myeloma, so the test focused on the use of a combination of multiple myeloma-related antibodies, without specific non-hematopoietic tumor-related antibodies. Figure 4 CD45 was found on the CD45 / SSC plot dim / -Multiple myeloma cells can appear in this location, but the multiple myeloma cell-specific antibody CD138 is negative and CD56 is positive. The expression rate of this antibody on multiple myeloma cells is close to 60%. CD81 is negative and the cytoplasmic light chain is negative. Non-secretory multiple myeloma can also have a secondary phenotype. CD56 + CD138 - CD45 dim / - The cell group can also appear in non-hematopoietic tissues, so this group of cells needs to be tested with antibodies to further identify the cell source. After using a 12-color antibody combination to add CD326, CK, and GD2 antibodies, GD2 is negative, CD326 is positive, and CK is positive, which can be judged as an epithelial cell-derived tumor infiltrating the bone marrow. If a non-hematopoietic tissue-specific antibody of GD2 is added on the basis of the CD56 / CD81 / CD45 / CD138 detection antibody combination, it is not 100% possible to detect non-hematopoietic tumor cells. If only GD2 is tested in this case, it will be missed or the conclusion will be wrong. Therefore, the 12-color antibody combination scheme of the present invention has a wide coverage, avoiding the risk of missed detection in this case. The bone marrow biopsy of this patient confirmed a neuroendocrine tumor and medullary thyroid carcinoma.

[0121] Application example 5: Patient, female, 7 years old, clinical diagnosis: acute leukemia, specimen type: bone marrow.

[0122] This application example uses the kit in comparative example 1 to test the sample to be tested. The test results are as follows Figure 5 As shown. A total of 8 antibodies, CD7 / CD9 / CD56 / CD81 / GD2 / CK / CD326 / CD45, were used for detection. Figure 5 The results showed that a group of cells were found in the CD45 negative area, which did not express CD56, GD2, CD9, CD326, and CK, and only expressed CD81. They were considered to be cells of non-hematopoietic tissue origin. If only the CD45 / GD2 / CD9 / CD56 detection scheme was used, this case would be missed. The patient's bone marrow histopathology confirmed that it was Ewing's sarcoma, and the bone marrow morphology showed clustered cell groups.

[0123] Application example 6: Patient, male, 64 years old, clinical diagnosis: anemia, multiple myeloma to be excluded. Specimen type: bone marrow.

[0124] This application example uses the kit in Example 1 to detect the sample to be tested. The test results are as follows Figure 6 The patient was clinically suspected of multiple myeloma, so the test focused on the use of a combination of multiple myeloma-related antibodies, without specific non-hematopoietic tumor-related antibodies. Figure 6 CD45 was found on the CD45 / SSC dot plot - CD56 dimThe cell population is CD81 positive and cytoplasmic light chain negative. Non-secretory multiple myeloma cells may have this phenotype. CD56 dim CD138 - CD45 - CD81 + The cell group can also appear in non-hematopoietic tissues, and this phenotype cannot determine the cell origin. After further adding CD326, CK, and GD2 antibodies, GD2 is positive, CD326 is partially positive, and CK is positive, which can be judged as a tumor of epithelial cell origin infiltrating the bone marrow. This example is detected using the kit in Example 1, and the result can determine that the non-hematopoietic tissue is of neural origin. If CK is further added, it can be determined that the tumor cell is of neuroepithelial cell origin. The present invention comprehensively covers non-hematopoietic tissue-related specific antibodies, which can not only detect tumor cells, but also determine their origin.

[0125] Application example 7: Patient, male, 18 years old, clinical diagnosis: lymphoma, bone destruction, bone marrow necrosis. Specimen type: bone marrow.

[0126] This application example uses the kit in Example 1 to detect the sample to be tested. The test results are as follows Figure 7 As shown. Figure 7 It can be seen that 25.44% of the patient's CD45 / SSC chart showed abnormal cells, which expressed CK, GD2, and CD81. A few expressed CD56 and CD326. The abnormal cells were metastatic cancer cells and were finally diagnosed as mesenchymal malignant tumors.

[0127] In the specification of the present invention, a lot of specific details are described. However, it is understood that embodiments of the present invention can be practiced without these specific details. In some embodiments, well-known methods, structures and techniques are not shown in detail so as not to obscure the understanding of this specification.

[0128] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0129] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

[0130] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. An antibody composition for solid tumor detection, characterized in that: The antibody composition includes CD45 antibody, CD56 antibody, CD9 antibody, CD81 antibody, CD36 antibody, GD2 antibody, HLA-DR antibody, CD326 antibody, CD7 antibody, CD138 antibody, CD19 antibody and CD4 antibody.

2. The antibody composition for solid tumor detection according to claim 1, characterized in that: The antibody composition for solid tumor detection also includes CK antibody and CD45.

3. The antibody composition for solid tumor detection according to claim 2, characterized in that: The fluorescein marker of the CD45 antibody is QB500; the fluorescein marker of the CD56 antibody is PE-CY7; the fluorescein marker of the CD9 antibody is FITC; the fluorescein marker of the CD81 antibody is APC-CY7; the fluorescein marker of the CD36 antibody is APC-CY7; the fluorescein marker of the GD2 antibody is APC; the fluorescein marker of the HLA-DR antibody is BV711; the fluorescein marker of the CD326 antibody is PE; the fluorescein marker of the CD7 antibody is APC-R700; the fluorescein marker of the CD138 antibody is BV785; the fluorescein marker of the CD19 antibody is BV421; the fluorescein marker of the CD4 antibody is V605; and the fluorescein marker of the CK antibody is FITC.

4. Use of the antibody composition according to any one of claims 1 to 3 in the preparation of a product for detecting solid tumors.

5. Use of the antibody composition according to claim 4 in preparing a product for detecting solid tumors, characterized in that: The product is a test kit.

6. A kit for solid tumor detection, characterized in that: The kit comprises the antibody composition according to any one of claims 1 to 3.

7. The kit for solid tumor detection according to claim 6, characterized in that: The kit also contains red blood cell lysis solution and membrane breaking agent.

8. Use of the antibody composition according to any one of claims 1 to 3 in preparing flow cytometric samples for detecting solid tumors by flow cytometry.

9. Use of the antibody composition according to claim 8 in preparing flow cytometric samples for flow cytometry detection of solid tumors, characterized in that: The steps of preparing a flow cytometer sample for flow cytometry detection of solid tumors include: The CD45 antibody, CD56 antibody, CD9 antibody, CD81 antibody, CD36 antibody, GD2 antibody, HLA-DR antibody, CD326 antibody, CD7 antibody, CD138 antibody, CD19 antibody and CD4 antibody were added to the flow tube at a volume ratio of 2.5:2.5:5:2.5:2.5:2.5:2.5:5:2.5:2.5:2.5:2.5, and the sample to be tested was added so that the number of cells in the flow tube was 1×10 6 / tube, mix well and incubate in the dark; add lysis solution, mix well and incubate in the dark; centrifuge and remove supernatant, add buffer solution for washing and centrifuge again and remove supernatant; add membrane permeabilization agent a, mix well and incubate in the dark, centrifuge and remove supernatant; add the CK antibody and the membrane permeabilization agent b, mix well and incubate in the dark, add the buffer solution for washing, centrifuge and remove supernatant, and then add the buffer solution for resuspending to obtain a flow cytometer sample.

10. Use of the antibody composition according to claim 9 in preparing flow cytometry samples for flow cytometry detection of solid tumors, characterized in that: The sample to be tested is bone marrow, peripheral blood, cerebrospinal fluid or tissue fluid; and the lysing solution is red blood cell lysing solution.

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