Antibody composition for detecting AML accompanied with CBF [beta] MYH11 and application

Abstract

The invention provides an antibody composition for detecting AML (acute myeloid leukemia) accompanied with CBF beta MYH11. The antibody composition comprises CD2, CD34, CD117, CD64, CD14, CD33, HLA-DR (human leukocyte antigen-DR), CD15, CD11b, CD13, CD38 and CD45 antibodies. During detection, the antibody composition is divided into two types of antibody combinations according to detection targets, and then samples are respectively added for flow cytometry detection; when the proportion of original myeloid cells / nucleated cells is between 6% and 78%, the proportion of mononuclear cells / nucleated cells is greater than 22%, and the proportion of CD2 / mononuclear cells is greater than 20%, determining that the sample is AML with CBF beta MYH11. The method provided by the invention is simple and low in cost, can be used for judging whether the AML patient has CBF beta MYH11 positive or not without further molecular biological PCR (Polymerase Chain Reaction) screening, and has a great practical value.

Description

Technical Field

[0001] This invention belongs to the field of acute myeloid leukemia detection technology, specifically relating to an antibody composition for detecting AML with CBFβMYH11 and its application. Background Technology

[0002] Acute myeloid leukemia (AML) is a malignant clonal disease characterized by the proliferation and expansion of myeloid cells and impaired differentiation, leading to ineffective hematopoiesis and life-threatening cytopenia and transfusion dependence. Most patients develop the disease after age 55, with approximately two-thirds developing it after age 65. AML with CBFβMYH11 is an independent subtype in the WHO 5th edition of the AML diagnosis, with a relatively good prognosis. In the traditional FAB diagnosis, it is considered AML-M4Eo, characterized by predominantly monocytic differentiation, with monocytic cells at all stages present.

[0003] Currently, the diagnostic method for this disease is flow cytometry combined with molecular biology techniques. Adding extensive molecular biology PCR screening to the flow cytometry-based diagnosis of AML can provide a definitive diagnosis. However, the need for PCR screening makes the testing process cumbersome and expensive. Chinese patent CN202210159447 discloses a combination reagent and system for detecting acute myeloid leukemia cells, but it does not specifically detect AML with CBFβMYH11. Similarly, Chinese patent CN201811555617 also discloses an antibody composition for immunophenotyping of myeloid tumors, which also fails to detect AML with CBFβMYH11.

[0004] Therefore, there is an urgent need to provide a method for detecting AML with CBFβMYH11 using only flow cytometry, in order to simplify the testing process and reduce the testing costs for patients. Summary of the Invention

[0005] In view of this, the present invention provides an antibody composition and its application for detecting AML with CBFβMYH11 through extensive screening and optimization, and evaluates the immunophenotypes of 108 AML patients. Combined with molecular biological diagnostic results, it is demonstrated that the antibody composition provided by the present invention can detect and diagnose AML with CBFβMYH11.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: An antibody composition for detecting AML with CBFβMYH11, the antibody composition comprising antibodies against CD2, CD34, CD117, CD64, CD14, CD33, HLA-DR, CD15, CD11b, CD13, CD38 and CD45.

[0007] In some specific embodiments, preferably, the antibodies in the antibody composition are all fluorescently labeled antibodies, specifically the following fluorescent labels: CD2-PE-Cy7, CD34-FITC, CD117-PE, CD64-FITC, CD14-APC-Cy7, CD33-APC, HLA-DR-PerCP, CD15-V450, CD11b-PE, CD13-PE-Cy7, CD38-BV421, and CD45-mFluor540.

[0008] A method for detecting AML with CBFβMYH11 using the above antibody composition includes the following steps: S1. Prepare antibody composition 1 and antibody composition 2 respectively, wherein: The antibody composition 1 comprises antibodies against CD34, CD117, CD33, HLA-DR, CD13, CD38, and CD45. The antibody composition 2 comprises antibodies against CD2, CD64, CD14, CD33, HLA-DR, CD15, CD11b, and CD45. S2. Prepare the samples to be tested. Add the samples to antibody composition 1 and antibody composition 2 respectively, mix and incubate, then add lysis buffer respectively, mix and incubate, centrifuge to remove supernatant, resuspend, and analyze by flow cytometry. Antibody composition 1 is used to evaluate the original cell population of the sample; Antibody composition 2 is used to evaluate sample mononuclear cells; S3. Analyze the above test results. If the ratio of primitive myeloid cells to nucleated cells is between 6% and 78%, the ratio of monocytes to nucleated cells is greater than 22%, and the ratio of CD2 to monocytes is greater than 20%, then the sample is determined to be AML with CBFβMYH11.

[0009] In some specific embodiments, preferably, the volume ratio of each antibody in antibody composition 1 in step S1 is CD34:CD117:CD33:HLA-DR:CD13:CD38:CD45 = 2:2:2:1:1:2:2; The volume ratio of each antibody in antibody composition 2 is CD2:CD64:CD14:CD33:HLA-DR:CD15:CD11b:CD45 = 1:2:1:2:2:1:2:1.

[0010] Furthermore, in step S2, the sample to be tested is bone marrow, with a cell concentration of 10. 6 cells / mL; The volume ratio of the sample to be tested to antibody composition 1 and antibody composition 2 is 25:6. The volume ratio of the lysis buffer to antibody composition 1 and antibody composition 2 is 125:6.

[0011] Furthermore, the incubation conditions after adding the sample in step S2 are: incubation in the dark for 15-20 minutes; the incubation conditions after adding the lysis buffer are: room temperature in the dark for 8-10 minutes.

[0012] A flow cytometry-based kit for detecting AML with CBFβMYH11, the kit comprising the above-described antibody composition.

[0013] The application of the above antibody composition or the above kit in the detection of AML with CBFβMYH11.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: The antibody combination provided by this invention can accurately distinguish whether AML patients are positive for CBFβMYH11, and it will not produce false positives for different types of CBFβMYH11-negative patients, demonstrating high accuracy. The entire detection process is simple and quick (based solely on flow cytometry results, if the ratio of primitive myeloid cells to nucleated cells is between 6% and 78%, the ratio of monocytes to nucleated cells is greater than 22%, and the ratio of CD2 to monocytes is greater than 20%, then the sample is determined to be AML with CBFβMYH11), and low in cost (no further molecular biological PCR screening is required), possessing great practical value. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the location of lymphocyte populations in Embodiment 1 of the present invention.

[0016] Figure 2 The figure shows the results of the proportion of primitive cells, the proportion of monocytes, and the expression of CD2 in Example 1 of the present invention.

[0017] Figure 3 This is a graph showing the detection results of the first type of CBFβMYH11 negative patients in Comparative Example 1 of this invention.

[0018] Figure 4 This is a graph showing the detection results of the second type of CBFβMYH11 negative patients in Comparative Example 1 of this invention. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products. The terminology used in this specification is for the purpose of describing specific embodiments only and is not intended to limit the present invention.

[0020] The technical concept of this invention is as follows: The inventors detected and analyzed the expression of leukocyte differentiation antigens in 108 newly diagnosed AML patients, and combined with the results of molecular biomolecular examinations, discovered that leukocyte differentiation antigens in AML patients with CBFβMYH11 exhibit a regular immunophenotype. Further screening of numerous antibody combinations for differentiating antigen detection yielded the optimal combination for detecting AML with CBFβMYH11, comprising Composition 1 and Composition 2. Composition 1 includes antibodies against CD34, CD117, CD33, HLA-DR, CD13, CD38, and CD45; Composition 2 includes antibodies against CD2, CD64, CD14, CD33, HLA-DR, CD15, CD11b, and CD45.

[0021] The test results of the above composition can be evaluated in the following ways: First, antibody composition 1 is used to evaluate the original cell population of the sample; to determine the ratio of primitive myeloid cells to nucleated cells. Then antibody composition 2 was used to evaluate the monocytes in the sample; the monocyte / nucleated cell ratio and the CD2 / monocyte ratio were determined; Based on the above results, if the ratio of primitive myeloid cells to nucleated cells is between 6% and 78%, the ratio of monocytes to nucleated cells is greater than 22%, and the ratio of CD2 to monocytes is greater than 20%, then the sample is determined to be AML with CBFβMYH11.

[0022] Key experimental material sources and physicochemical parameters: The sources of antibodies and their corresponding catalog numbers are shown in Table 1 below.

[0023]

[0024] The lysis buffer consists of methanol, diethylene glycol, formaldehyde, potassium chloride, magnesium sulfate, and sodium citrate dihydrate, and was purchased from Henan Kaipurui Biotechnology Co., Ltd.

[0025] Example 1 This embodiment provides an antibody composition for detecting AML with CBFβMYH11 and a detection method thereof. The antibody composition is used to detect CBFβMYH11-positive AML patient samples, as detailed below: 1. The combination of antibodies and fluorescein and their dosages are detailed in Table 2 below: Table 2. Details of specific combinations and dosages of antibodies labeled with various fluorescent dyes.

[0026] 2. Sample Preparation S1. Add the prepared panel 1 and panel 2 to flow cytometry tubes 1 and 2 respectively, then add 100µL of sample to flow cytometry tubes 1 and 2 respectively, mix well, and incubate in the dark for 15min. S2. Add 0.5 mL of lysis buffer to flow cytometry tubes 1 and 2 respectively, mix well, and lyse at room temperature in the dark for 8 min; S3. Centrifuge the pyrolysis mixture at 1500 rpm for 5 min, remove the supernatant, and obtain the pyrolysis precipitate; S4. Add 1 mL of PBS to the lysed precipitate and mix well. Then centrifuge at 1000 rpm for 5 min, and then resuspend in 150 μL of PBS to obtain the sample to be tested.

[0027] 3. Sample testing The prepared test samples were then tested using the instrument. The instrument conditions were adjusted according to the quality control samples to ensure the lymphocyte population was positioned as follows: Figure 1 At position P1, signals ranging from 20,000 to 50,000 were acquired, and the results were analyzed using BD Diva Software.

[0028] First, such as Figure 2 As shown in the CD45 / SSC scatter plot, CD45 is moderately positive, and primitive cells can be seen in a small area of ​​SSC. Figure 2 a Gate2). The combination of CD34 / CD117 / HLA-DR / CD13 / CD33 / CD38 antibodies can confirm whether this cell population is primitive myeloid cells and determine the proportion of nucleated cells they occupy. Generally, primitive myeloid cells are CD34 and CD117 positive. Figure 2 b).

[0029] Then, based on the antibody combination CD2 / CD64 / CD14 / CD33 / HLA-DR / CD15 / CD11b, monocytes were identified. Figure 2 cgate3), and calculate the proportion of nucleated cells it occupies. Monocytes are generally HLA-DR+CD64+CD14+CD33bri+CD15dim+CD11b+ cells, especially CD64+CD14+, which is a specific marker for identifying monocytes ( Figure 2 c).

[0030] Finally, the proportion of CD2-positive cells in monocytes was determined. Figure 2 d) This example meets the criteria of a primitive myeloid cell / nucleated cell ratio between 6% and 78%, a monocyte / nucleated cell ratio greater than 22%, and a CD2 / monocyte ratio greater than 20%. Comparative Example 1 This comparative example uses the same technical solution as Example 1 for detection, but the test sample is replaced with a sample from a CBFβMYH11 negative patient, to verify that the antibody composition provided by the present invention can accurately distinguish whether AML patients have CBFβMYH11.

[0031] 1. Select CBFβMYH11 negative patient samples and prepare the samples according to the same steps as in Example 1. Perform the test, and the test results are as follows: Figure 3 As shown.

[0032] The analysis was performed using the same method as in Example 1. In this case, the ratio of primitive myeloid cells to nucleated cells was between 6% and 78%, the ratio of monocytes to nucleated cells was less than 22%, and the ratio of CD2 to monocytes was less than 20%.

[0033] 2. Furthermore, to ensure the accuracy of the test, another CBFβMYH11-negative patient was selected, and a sample was prepared according to the same steps as in Example 1. The test results were as follows: Figure 4 As shown.

[0034] The analysis was performed using the same method as in Example 1. In this case, the ratio of primitive myeloid cells to nucleated cells was between 6% and 78%, the ratio of monocytes to nucleated cells was greater than 22%, but the ratio of CD2 to monocytes was less than 20%.

[0035] The above series of experiments demonstrate that the antibody combination provided by this invention can accurately distinguish whether AML patients are positive for CBFβMYH11. The detection process is simple and rapid, requiring only routine flow cytometry and eliminating the need for further molecular biological PCR screening. This application provides a novel method for detecting AML with CBFβMYH11, simplifying the detection process and reducing subsequent testing costs, making it highly valuable for application.

[0036] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0037] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody composition for detecting AML with CBFβMYH11, characterized in that, The antibody composition comprises antibodies against CD2, CD34, CD117, CD64, CD14, CD33, HLA-DR, CD15, CD11b, CD13, CD38, and CD45.

2. The antibody composition according to claim 1, characterized in that, The antibodies in the antibody composition are all fluorescently labeled antibodies, specifically as follows: CD2-PE-Cy7, CD34-FITC, CD117-PE, CD64-FITC, CD14-APC-Cy7, CD33-APC, HLA-DR-PerCP, CD15-V450, CD11b-PE, CD13-PE-Cy7, CD38-BV421, and CD45-mFluor540.

3. A method for detecting AML with CBFβMYH11 using the antibody composition according to claim 1 or 2, characterized in that, Includes the following steps: S1. Prepare antibody composition 1 and antibody composition 2 respectively, wherein: The antibody composition 1 comprises antibodies against CD34, CD117, CD33, HLA-DR, CD13, CD38, and CD45. The antibody composition 2 comprises antibodies against CD2, CD64, CD14, CD33, HLA-DR, CD15, CD11b, and CD45. S2. Prepare the samples to be tested. Add the samples to antibody composition 1 and antibody composition 2 respectively, mix and incubate, then add lysis buffer respectively, mix and incubate, centrifuge to remove supernatant, resuspend, and analyze by flow cytometry. Antibody composition 1 is used to evaluate the original cell population of the sample; Antibody composition 2 is used to evaluate sample mononuclear cells; S3. Analyze the above test results. If the ratio of primitive myeloid cells to nucleated cells is between 6% and 78%, the ratio of monocytes to nucleated cells is greater than 22%, and the ratio of CD2 to monocytes is greater than 20%, then the sample is determined to be AML with CBFβMYH11.

4. The method according to claim 3, characterized in that, In step S1, the volume ratio of each antibody in antibody composition 1 is CD34:CD117:CD33:HLA-DR:CD13:CD38:CD45 = 2:2:2:1:1:2:2; The volume ratio of each antibody in antibody composition 2 is CD2:CD64:CD14:CD33:HLA-DR:CD15:CD11b:CD45 = 1:2:1:2:2:1:2:

1.

5. The method according to claim 3, characterized in that, In step S2, the sample to be tested is bone marrow, with a cell concentration of 10. 6 cells / mL; The volume ratio of the sample to be tested to antibody composition 1 and antibody composition 2 is 25:

6. The volume ratio of the lysis buffer to antibody composition 1 and antibody composition 2 is 125:

6.

6. The method according to claim 5, characterized in that, The incubation conditions after adding the sample in step S2 are: incubation in the dark for 15-20 minutes; the incubation conditions after adding the lysis buffer are: room temperature in the dark for 8-10 minutes.

7. A kit for detecting AML with CBFβMYH11 based on flow cytometry, characterized in that, The kit comprises the antibody composition according to any one of claims 1 or 2.

8. The use of the antibody composition according to claim 1 or 2 or the kit according to claim 7 in the detection of AML with CBFβMYH11.

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