An MRD detection product

Abstract

The present invention relates to the field of medicine, and in particular to a MRD detection product, wherein the MRD detection product includes an MRD detection reagent, and the MRD detection reagent includes anti-CD38 antibodies, anti-CD45 antibodies, anti-CD229 antibodies, anti-CD269 antibodies, anti-CD138 antibodies, anti-CD81 antibodies, anti-CD117 antibodies, anti-CD20 antibodies, anti-CD200 antibodies, anti-CD56 antibodies, anti-CD19 antibodies, anti-CD27 antibodies, anti-Kappa light chain antibodies or anti-Lambda light chain antibodies. The present invention adopts a 2-tube 8-color scheme, which can effectively set a gate to circle out CD38-plasma cells and CD38+ plasma cells, covering the most complete plasma cell population, especially CD229, CD269 indicators, which are rarely used in the detection of residual multiple myeloma, and can greatly reduce the false positive rate and the probability of missed diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to an MRD detection product. Background Art

[0002] The incidence of multiple myeloma (MM) has increased significantly, and it has become one of the important diseases affecting human health. It is the second most common blood tumor in terms of incidence, accounting for 10% of blood system malignancies. In the past decade, MM treatment regimens have been continuously optimized, and the efficacy has increased significantly, while also increasing the complete remission rate and survival rate. In addition to traditional chemotherapy, targeted drugs and cell immunotherapy have become new development directions for current treatment methods. Chimeric Antigen Receptions T cells (CAR-T) achieve specific recognition of tumor-associated antigens through genetically modified T cells, allowing effector T cells to fully exert their anti-tumor effects. It is the focus of current cell immunotherapy and the most cutting-edge science in the treatment of malignant tumors in the current medical field. It has achieved significant results in the treatment of blood system diseases such as multiple myeloma, and significantly improved the remission rate of refractory relapsed diseases that are ineffective with conventional treatment, improving patient prognosis. However, due to many reasons, the proportion of long-term remission achieved by this method is less than 50%. Pay attention to monitoring after treatment, early detection of minimal residual disease (MRD), and early bridging transplantation or secondary CAR-T treatment are top priorities. Flow cytometry (FCM) is a detection method that can achieve quantitative analysis of single cells. It has the advantages of rapidity, high precision, and multi-parameters. It is one of the most advanced cell quantitative analysis methods. FCM has been widely used in clinical and scientific research fields, especially in clinical in vitro diagnosis. It has been widely and deeply used and is the gold standard for clinical diagnosis of malignant diseases such as leukemia and lymphoma. The use of FCM to detect bone marrow MRD in MM patients is an important prognostic variable. The use of specific antibody compositions in combination with multicolor flow cytometry can effectively evaluate prognosis, detect recurrence, and guide the selection of treatment options. It is an important clinical evaluation indicator for MM.

[0003] However, the correct selection of detection antibodies is the key to achieving the above clinical evaluation. For example, the most conventional analysis scheme is to use the membrane pan-plasma cell marker CD38 supplemented with SSC or CD45 (CD38 / SSC or CD38 / CD138 dual parameters) to set the gate to select plasma cells. However, for patients after CD38 monoclonal antibody or CD138 monoclonal antibody treatment, or patients after CD38-CAR-T or CD138-Car-T cell infusion treatment, the use of CD38 or CD138 for gating is no longer applicable. The reason is that after treatment, some normal or abnormal plasma cells undergo clonal evolution, and their surface CD38 antigen and CD138 antigen have lost or weakened expression, that is, CD38 and CD138 antigens will show negative expression. Therefore, there is no new and effective scheme in clinical practice to set the gate to select the target plasma cells. To solve this problem, researchers have conducted research explorations, including Tembhare et al.'s study using CD38 combined with CD45 / CD138 / SSC to gate plasma cells to detect MRD after monoclonal antibody and CAR-T cell infusion therapy, but it still has the following defects: 1. The expression rate of CD138 in MM is 99.5%, which affects the gating efficiency of plasma cells in EDTA anticoagulated blood samples. 2. The expression rate of CD38 in MM is >80%, which is expressed on monocytes. In some MMs, the expression of CD38 is weakened, which will affect the gating efficiency of plasma cells. In addition, the CD38 antigen is strongly expressed in the precursor stage of B cells during the maturation of normal B cells, while CD38 is strongly expressed on plasma cells in MM, which affects the sensitivity of CD38 gating to select plasma cells. 3. It is not suitable for patients treated with CD38-CAR-T cells or CD38-CD138-CAR-T dual-target cell infusion therapy and patients after CD38 monoclonal antibody and CD138 monoclonal antibody treatment.

[0004] More importantly, when receiving samples for post-treatment residual monitoring, the test application form lacks a detailed treatment history, such as whether traditional chemoradiotherapy or biological therapy is used, and which specific biological therapy is used. Therefore, due to the lack of these key information, the tester cannot adjust or optimize the test plan according to the specific treatment plan in the first time, which often leads to false negative test results. In order to reduce the occurrence of false negatives and improve the accuracy of detection, as a tester, it is urgent to find a better detection plan to make up for the shortcomings of existing detection methods and meet clinical needs. Although there are literature reports that antigens such as CD56 are expressed on plasma cells and can be used to detect plasma cells, the current detection plan generally does not include many indicators in the post-treatment residual detection plan for multiple myeloma patients. Different testing institutions have different detection plans. There is no unified detection plan at present. The commonly used ones are mostly a combination of 4 to 8 antibodies. Some only detect plasma cell membrane antigens, and some only detect plasma cell cytoplasmic light chains. These detection plans cannot solve the problem of false negatives. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide an MRD detection product to solve the problems in the prior art.

[0006] To achieve the above objectives and other related objectives, the present invention provides uses of anti-CD38 antibodies, anti-CD45 antibodies, anti-CD229 antibodies, anti-CD269 antibodies and anti-CD138 antibodies in the preparation of multiple myeloma minimal residual disease (MRD) detection products.

[0007] The present invention also provides an MRD detection product, which includes an MRD detection reagent, and the MRD detection reagent includes anti-CD38 antibody, anti-CD45 antibody, anti-CD229 antibody, anti-CD269 antibody and anti-CD138 antibody.

[0008] The present invention also provides a method for screening MRD therapeutic drugs, the screening method comprising using the MRD detection product to detect an in vitro sample, the in vitro sample being from a subject after being acted upon by an alternative drug.

[0009] As described above, the MRD detection product of the present invention has the following beneficial effects: the present invention adopts a 2-tube 8-color scheme, which can effectively set gates to circle out CD38- plasma cells and CD38+ plasma cells, covering plasma cell membrane antigens and cytoplasmic light chains, that is, covering the most complete plasma cell population, especially the CD229 and CD269 indicators. These two indicators are currently rarely used in the detection of residual multiple myeloma, and can greatly reduce the false positive rate and the probability of missed diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Shown is a graph of normal plasma cell analysis results;

[0011] The gating strategy for tube A is as follows: P1: remove the adhesion cell gate P1; P2: set the live cell gate to obtain a single live cell. Use CD45 / SSC antibodies to set the blood cell gates in the P2 gate; set a multi-marker combination gate in the P2 gate to observe plasma cells, lymphocytes, monocytes, granulocytes, and whether there are obvious tumor cells or abnormal cells; use CD45 / CD38, CD38 / CD138, and SSC / CD38 antibodies to set the plasma cell gate (P8, P9, and P11);

[0012] The gating strategy for tube B is as follows: P1: remove adhesion cell gate P1; P2: set the live cell gate to obtain single live cells; use CD45 / SSC antibodies to set the gates for each blood cell in gate P2; set a multi-marker combination gate in gate P2 to observe plasma cells, lymphocytes, monocytes, granulocytes, and whether there are obvious tumor cells or abnormal cells; use CD45 / CD38, CD38 / CD229, and SSC / CD38 antibodies to set gates for plasma cells (P8, P9, and P10), and use CD45 / CD19 antibodies to set gates for B cells (CD19+Bcells) as an internal control to judge the staining of cytoplasmic antibodies.

[0013] In the figure, tube A and tube B can be distinguished by the above gating strategy.

[0014] Figure 2 Shown is the MRD test results for multiple myeloma patients after traditional chemotherapy.

[0015] Figure 3 Shown is the MRD detection results of multiple myeloma patients after CD269 monoclonal antibody treatment.

[0016] Figure 4 Shown is the MRD detection results of multiple myeloma patients after CD38 monoclonal antibody treatment.

[0017] Figure 5 The figure shows the test results of MM-MRD samples detected by traditional methods.

[0018] Figure 6 Shown is a graph showing the detection results of MM-MRD samples using the method of the present invention. DETAILED DESCRIPTION

[0019] The present invention provides uses of anti-CD38 antibodies, anti-CD45 antibodies, anti-CD229 antibodies, anti-CD269 antibodies and anti-CD138 antibodies in preparing a multiple myeloma minimal residual disease (MRD) detection product.

[0020] The expression intensity of CD38 in normal plasma cells is significantly higher than that in other hematopoietic cells. In multiple myeloma, CD38 is strongly expressed or partially weakly expressed.

[0021] CD45 is generally positively expressed. There are a small number of CD45- / dim plasma cells in normal bone marrow, and tumor plasma cells appear CD45- / dim with a small number of CD45+ subpopulations.

[0022] CD229 is expressed on normal plasma cells and T cells, weakly expressed on B cells and monocytes, and the average fluorescence intensity of its expression on abnormal plasma cells is significantly higher than that on normal plasma cells. It can be used as a reliable indicator for plasma cell gating and can be used for residual evaluation after CD38 and CD138 biological therapy.

[0023] CD269 is a B cell maturation antigen (BCMA), which is only expressed on the surface of germinal center B cells, malignant and normal plasma cells. It is relatively highly expressed on the surface of myeloma cells, with an expression rate of 70%-80% in bone tumor patients. It is a target for immunotherapy of multiple myeloma.

[0024] CD138 is only expressed in plasma cells and myeloma cells. It is a more specific plasma cell marker than CD38 and is mostly expressed in multiple myeloma patients. CD138 is expressed in epithelial cells, mesenchymal cells and tumor cells in the non-hematopoietic system.

[0025] In certain embodiments of the present invention, the MRD detection product also includes any one or more of the following: anti-CD81 antibody, anti-CD117 antibody, anti-CD20 antibody, anti-CD200 antibody, anti-CD56 antibody, anti-CD19 antibody, anti-CD27 antibody, anti-Kappa light chain antibody or anti-Lambda light chain antibody.

[0026] CD117, CD20 and CD200 are not expressed in normal plasma cells, but are partially expressed in neoplastic plasma cells.

[0027] CD56 is usually not expressed in normal plasma cells but is often expressed in neoplastic plasma cells.

[0028] CD19 is expressed in most plasma cells, with a lower expression intensity than B cells. Some subpopulations of cells are CD19 negative, and neoplastic plasma cells generally do not express CD19.

[0029] CD27 is expressed in normal plasma cells, but not in multiple myeloma patients, and is an important indicator for evading the body's immune system.

[0030] Kappa and Lambda are basically not expressed in normal and abnormal plasma cells. Intracellular Kappa and Lambda antigens need to be detected when distinguishing plasma cell clonality. Malignant plasma cells are mostly monoclonal, while normal plasma cells are polyclonal. If the expression pattern is different from the normal one, it is suspected to be malignant. After excluding other factors, MRD positivity is diagnosed.

[0031] The MRD detection product is an MRD diagnostic product. The MRD detection product is a product detected by flow cytometry.

[0032] The present invention also provides an MRD detection product, which includes an MRD detection reagent, and the MRD detection reagent includes anti-CD38 antibody, anti-CD45 antibody, anti-CD229 antibody, anti-CD269 antibody and anti-CD138 antibody.

[0033] In one embodiment, the MRD detection reagent further includes any one or more of anti-CD81 antibody, anti-CD117 antibody, anti-CD20 antibody, anti-CD200 antibody, anti-CD56 antibody, anti-CD19 antibody, anti-CD27 antibody, anti-Kappa light chain antibody or anti-Lambda light chain antibody.

[0034] In one embodiment, the MRD detection reagent includes a first MRD detection reagent and a second MRD detection reagent, wherein the first MRD detection reagent includes anti-CD38 antibody, anti-CD45 antibody, and anti-CD229 antibody, and the second MRD detection reagent includes anti-CD38 antibody, anti-CD45 antibody, and anti-CD138 antibody.

[0035] The first MRD detection reagent also includes any one or more of anti-CD269 antibody, anti-CD81 antibody, anti-CD117 antibody, anti-CD20 antibody, anti-CD200 antibody, anti-CD56 antibody, anti-CD19 antibody, anti-CD27 antibody, anti-Kappa light chain antibody or anti-Lambda light chain antibody, and the second MRD detection reagent includes the remaining antibodies among anti-CD269 antibody, anti-CD81 antibody, anti-CD117 antibody, anti-CD20 antibody, anti-CD200 antibody, anti-CD56 antibody, anti-CD19 antibody, anti-CD27 antibody, anti-Kappa light chain antibody or anti-Lambda light chain antibody except the antibodies contained in the first MRD detection reagent.

[0036] In one embodiment, the first MRD detection reagent also includes five antibodies selected from anti-CD269 antibody, anti-CD81 antibody, anti-CD117 antibody, anti-CD20 antibody, anti-CD200 antibody, anti-CD56 antibody, anti-CD19 antibody, anti-CD27 antibody, anti-Kappa light chain antibody or anti-Lambda light chain antibody, and the second MRD detection reagent includes the remaining five antibodies selected from anti-CD269 antibody, anti-CD81 antibody, anti-CD117 antibody, anti-CD20 antibody, anti-CD200 antibody, anti-CD56 antibody, anti-CD19 antibody, anti-CD27 antibody, anti-Kappa light chain antibody or anti-Lambda light chain antibody except the five antibodies contained in the first MRD detection reagent.

[0037] In one embodiment, the first MRD detection reagent includes anti-CD38 antibody, anti-CD45 antibody, anti-CD229 antibody, anti-CD269 antibody, anti-CD81 antibody, anti-CD117 antibody, anti-CD20 antibody and anti-CD200 antibody.

[0038] In one embodiment, the second MRD detection reagent includes anti-CD38 antibody, anti-CD45 antibody, anti-CD138 antibody, anti-CD56 antibody, anti-CD19 antibody, anti-CD27 antibody, anti-Kappa light chain antibody and anti-Lambda light chain antibody.

[0039] In one embodiment, the MRD detection product further includes any one or more of the following: red blood cell lysate, buffer or membrane permeabilizing agent. The buffer is, for example, PBS, physiological saline and other buffers commonly used in flow cytometry.

[0040] The red blood cell lysis solution or membrane-breaking agent can be purchased commercially.

[0041] The present invention also provides a method for screening MRD therapeutic drugs, the screening method comprising using the MRD detection product to detect an in vitro sample, the in vitro sample being from a subject after being acted upon by an alternative drug.

[0042] Specifically, the MRD therapeutic drug screening method is to detect samples from subjects after being acted upon by the MRD therapeutic drug to observe the effectiveness of the MRD therapeutic drug.

[0043] The screening method is to design the scheme and judge the results in the following manner: by monitoring the phenotype of abnormal plasma cells, that is, tumor cells abnormally acquire markers that are not normally expressed or lose normally expressed markers.

[0044] The ex vivo sample is a bone marrow or peripheral blood sample of a multiple myeloma patient after treatment. The treatment method is selected from traditional chemotherapy, CD269 monoclonal antibody therapy, CD38 monoclonal antibody therapy, CD38 monoclonal antibody therapy, CD138 Car-T therapy or CD38 Car-T therapy. There are a small number of normal plasma cells remaining in the sample to be tested, and the normal plasma cells remaining during the detection process can be used as internal quality control for the detection.

[0045] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0046] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0047] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0048] Example 1 Detection of residual disease cells in multiple myeloma by flow cytometry

[0049] Experimental principle:

[0050] The antibody labeled with fluorescein is incubated with the single-cell suspension at room temperature to allow the fluorescent antibody to bind to the corresponding antigen on the cell surface; the single-cell suspension forms a single-cell liquid flow under the wrapping of the sheath fluid and flows through the detection window; the laser generated by the instrument will excite the fluorescein labeled on the antibody to emit light of a specific wavelength; the instrument detects light of different wavelengths separately and converts them into electrical signals; the electrical signals can be analyzed after being processed by the computer system.

[0051] Experimental instruments and reagents:

[0052] BD CantoII flow cytometer, CD45 percp-cy5.5, CD117-BV510, CD81-FITC, CD27-BV510, CD200-APC, CD269-PE, CD38-PE-CY7, CD138-BV421, CD56-PE, CD38-FITC, CD229-BV421, CD20-APC-CY7. The first to seventh reagent manufacturers are Biolegend, and the catalog numbers are 304028, 313219, 349504, 302835, 329208, and 357504, respectively. , 356608; Antibodies 8 to 13 are BD products, with catalog numbers 562935, 8308891, 9217298, 744901, and 560734, respectively; CD19-PE-CY7 is Beckman product, catalog number: IM3628; Kappa-APC is Dako product, catalog number: C0222; cLambda-APC-CY7 is Cytogons product, catalog number: CYT-LAC750; Red blood cell lysis buffer and membrane permeabilization agent are BD products, with catalog numbers 70-LSB0 and 641776, respectively. Specimen:

[0053] 1) Peripheral blood of healthy people (including normal plasma cells);

[0054] 2) Peripheral blood specimens of multiple myeloma patients after traditional chemotherapy;

[0055] 3) Peripheral blood specimens of multiple myeloma patients after CD269 monoclonal antibody treatment;

[0056] 4) Peripheral blood specimens of multiple myeloma patients after CD38 monoclonal antibody treatment.

[0057] Experimental steps:

[0058] 1. Take the corresponding number of flow cytometry tubes, number them and indicate the specimen number.

[0059] 2. Add the corresponding antibodies to each tube. The amount of antibodies should be added according to the instructions for use of the antibodies, and be careful to add the antibodies to the bottom of the tube. The fluorescent labeling order of the antibodies is as follows:

[0060] A tube: CD81 / CD269 / CD38 / CD45 / CD229 / CD117 / CD20 / CD200

[0061] B tube: CD38 / CD56 / CD19 / CD45 / CD138 / CD27 / cLambda / cKappa

[0062] 3. Add samples according to specimen count and add antibodies in the above order.

[0063] 4. Vortex, mix well, and incubate at room temperature in the dark for 20 minutes.

[0064] 5. Add 1-3 ml of 10× lysis buffer, shake and mix, and lyse for 10 minutes in the dark. Centrifuge at 1500 rpm for 5 minutes and discard the supernatant.

[0065] 6. Add 1-3 ml PBS buffer, shake gently to mix, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant. Resuspend Tube 1 with PAS and put it on the machine.

[0066] 7. Add 2 ml PBS to tube B, mix well, centrifuge to remove the supernatant, add membrane permeabilization solution a, add 100 μL PBMC to each tube, and incubate at room temperature for 15 minutes.

[0067] 8. Add 2 ml PBS to tube B, centrifuge (5 min, 1500 rpm), remove the supernatant, add Kappa, Lambda, add 100 μL of membrane permeabilization agent b, mix well and protect from light for 15 min.

[0068] 9. Add 2 ml of PBS to tube B, mix well, centrifuge (5 min, 1500 rpm), remove the supernatant, add 500 μL of PBS, and wait for loading.

[0069] 10. The treated cells were fixed with 0.5% formalin solution (300 μL / tube) and stored at 2-8°C for 5 days.

[0070] 11. BD FACSDive Software was used to analyze the data. First, the area (A) and height (H) of forward scatter (FSC) were used to set the gate for de-adhesion cells (denoted by P1). Then, the cells in P1 were displayed, and the live cell gate (denoted by P2) was set using FSC / side scatter (SSC) to obtain single live cells. In the single live cell gate (P2 gate), CD45 / SSC was first used to set the gates for each blood cell, and lymphocytes, monocytes, granulocytes, and whether there were obvious tumor cells or abnormal cells were observed. Multiple marker combinations are gated within the single live cell gate (P2 gate) to observe plasma cells, especially monoclonal plasma cells: CD38 / CD138, CD45 / CD38, and SSC / CD38 are used as gates for samples in tube A; CD38 / CD229, CD45 / CD38 / CD138, SSC / CD229, SSC / CD38 combined with cKappa and cLambda antigens are used as gates for samples in tube B to distinguish plasma cells, and the benign and malignant nature of the cells can be determined based on their phenotypes.

[0071] Experimental results:

[0072] The results are as follows Figure 1As shown, all plasma cells expressed CD19, CD27st, CD38, CD45dim, CD81, CD138, and CD229, but did not express CD20, CD56, CD117, and CD200. cLambda and cKappa were expressed in polyclonal ways, and this specimen was MRD negative.

[0073] The results are as follows Figure 2 As shown in the figure, this specimen is a specimen after traditional chemotherapy. All plasma cells express CD20dim, CD27, CD38, CD45dim, CD56, CD138, and cLambda, and some cells express CD200 and CD269dim, but do not express CD19, CD81, and cKappa.

[0074] The results are as follows Figure 3 As shown, all plasma cells expressed CD27, CD38, CD45dim, CD138, CD229, and cKappa, some cells expressed CD20dim and CD81, a few expressed CD19, and no CD56 or cLambda was expressed. In this specimen, CD269 was negative after treatment.

[0075] The results are as follows Figure 4 As shown, all plasma cells expressed CD229 and cKappa, some CD27 and CD138 were weakly expressed, and CD19, CD38, CD45, CD56, CD117, CD81, CD20, CD200, CD269, and cLambda were all negative. In this specimen, CD45 / CD229 and cKappa played a key role in capturing monoclonal plasma cells.

[0076] The solution of the present invention has the following advantages:

[0077] (1) Using a combination of multiple markers to set gates can avoid missed diagnosis caused by weakened or lost expression of CD38 and / or CD138 after treatment. The scheme of the present invention found that cases with negative CD38, CD138, CD56, and CD19 all expressed CD229 and cKappa or cLambda. The six markers can cover 100% of the cases and cover the most complete plasma cell population;

[0078] (2) In addition to preventing missed diagnoses, the combination of multiple markers can also help discover special clones and weak clones. Although most tumors are single clones, a small number of tumors can have 2-3 clones at the same time. Generally speaking, small clones are easily overlooked. These small clones are of great significance for future recurrence, phenotypic changes, and even the evolution of the main clone at a certain moment, whether to diagnose secondary tumors, and revealing the laws of tumor occurrence and development and targeted treatment;

[0079] (3) The combined gating is different from the traditional CD38 gating and will not be interfered by basophils, plasmacytoid dendritic cells, mast cells, granulocytes, etc., and avoids the effect of sodium citrate on CD138;

[0080] (4) This combined gate will not be affected by biological targeted therapy. In the combined gate of the present invention, CD229 is expressed on plasma cells, T and B cells, and cytoplasmic Kappa and Lambda are expressed intracellularly. Targeted therapy for such antigens has predictable toxic side effects, or targeting cytoplasmic antigens has great technical difficulty, which is a difficult marker for future targeted biological therapies such as CAR-T targeting new targets. This combined gate is almost not affected by the selection or change of treatment options, and is urgently needed for the current targeted treatment of multiple myeloma, and is suitable for the MRD detection and analysis scheme for widespread application and promotion.

[0081] Example 2 Two methods for detecting residual disease in multiple myeloma samples after treatment

[0082] 1) Detection of MM-MRD using traditional methods

[0083] The antibodies used are anti-CD38 antibody, anti-CD45 antibody, anti-CD56 antibody, anti-CD138 antibody, anti-CD27 antibody, anti-CD19 antibody, anti-Kappa light chain antibody, and anti-Lambda light chain antibody. The other steps are the same as those in Example 1.

[0084] like Figure 5 As shown, plasma cells fully expressed CD38, CD138, CD27, and CD19dim, but did not express CD56, ckappa / cLambda=0.7, which was polyclonal expression. No light chain-restricted expression was observed in plasma cells, and the result was MRD negative.

[0085] 2) Detection of MM-MRD using the method of the present invention

[0086] The experimental steps are the same as in Example 1.

[0087] The results are as follows Figure 6 As shown, 0.008% of abnormal plasma cells (shown in dark green) expressed CD200, CD27, CD38, CD138, CD117, CD229, and cKappa, partially expressed CD56, and did not express CD19, cLambda, and CD269.

[0088] In summary, the traditional method detected a false negative, while the method of the present invention successfully captured abnormal cells and the test result was MRD positive.

[0089] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. Use of anti-CD38 antibody, anti-CD45 antibody, anti-CD229 antibody, anti-CD269 antibody and anti-CD138 antibody in the preparation of an MRD detection product, wherein the MRD detection product comprises a first MRD detection reagent and a second MRD detection reagent, wherein the first MRD detection reagent comprises anti-CD38 antibody, anti-CD45 antibody, anti-CD229 antibody, anti-CD269 antibody, anti-CD81 antibody, anti-CD117 antibody, anti-CD20 antibody and anti-CD200 antibody; the second MRD detection reagent comprises anti-CD38 antibody, anti-CD45 antibody, anti-CD138 antibody, anti-CD56 antibody, anti-CD19 antibody, anti-CD27 antibody, anti-Kappa light chain antibody and anti-Lambda light chain antibody, and the MRD detection product is a product detected by flow cytometry; the anti-CD38 antibody is CD38-PE-CY7 or CD38-FITC, and the anti-CD45 antibody is CD45 percp-cy5.5, the anti-CD229 antibody is CD229-BV421, the anti-CD269 antibody is CD269-PE, the anti-CD81 antibody is CD81-FITC, the anti-CD117 antibody is CD117-BV510, the anti-CD20 antibody is CD20-APC-CY7, the anti-CD200 antibody is CD200-APC, the anti-CD138 antibody is CD138-BV421, the anti-CD56 antibody is CD56-PE, the anti-CD19 antibody is CD19-PE-CY7, the anti-CD27 antibody is CD27-BV510, the anti-Kappa light chain antibody is Kappa-APC, and the anti-Lambda light chain antibody is cLambda-APC-CY7.

2. An MRD detection product, characterized in that: The MRD detection product includes an MRD detection reagent, and the MRD detection product includes a first MRD detection reagent and a second MRD detection reagent. The first MRD detection reagent includes anti-CD38 antibody, anti-CD45 antibody, anti-CD229 antibody, anti-CD269 antibody, anti-CD81 antibody, anti-CD117 antibody, anti-CD20 antibody and anti-CD200 antibody; the second MRD detection reagent includes anti-CD38 antibody, anti-CD45 antibody, anti-CD138 antibody, anti-CD56 antibody, anti-CD19 antibody, anti-CD27 antibody, anti-Kappa light chain antibody and anti-Lambda light chain antibody. The MRD detection product is a product detected by flow cytometry; the anti-CD38 antibody is CD38-PE-CY7 or CD38-FITC, the anti-CD45 antibody is CD45 percp-cy5.5, the anti-CD229 antibody is CD229-BV421, and the anti-CD269 antibody is CD269-PE , the anti-CD81 antibody is CD81-FITC, the anti-CD117 antibody is CD117-BV510, the anti-CD20 antibody is CD20-APC-CY7, the anti-CD200 antibody is CD200-APC, the anti-CD138 antibody is CD138-BV421, the anti-CD56 antibody is CD56-PE, the anti-CD19 antibody is CD19-PE-CY7, the anti-CD27 antibody is CD27-BV510, the anti-Kappa light chain antibody is Kappa-APC, and the anti-Lambda light chain antibody is cLambda-APC-CY7.

3. The MRD detection product according to claim 2, characterized in that: The MRD detection product also includes any one or more of the following: red blood cell lysate, buffer or membrane permeabilizing agent.

4. A method for screening MRD therapeutic drugs, characterized in that: The screening method comprises detecting an in vitro sample using the MRD detection product described in any one of claims 2 or 3, wherein the in vitro sample is from a subject after being acted upon by a candidate drug.

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