Antibody compositions for detecting naive monocytes and uses thereof

Abstract

The application provides an antibody composition for detecting naive monocytes and application thereof, and the antibody composition for detecting naive monocytes comprises a first group of antibodies and a second group of antibodies, the first group of antibodies comprises CD15 antibodies, CD117 antibodies, CD34 antibodies, CD33 antibodies, CD14 antibodies, HLA-DR antibodies, CD13 antibodies, CD38 antibodies, CD64 antibodies, CD19 antibodies, CD56 antibodies, CD7 antibodies and CD45 antibodies; and the second group of antibodies comprises HLA-DR antibodies, CD64 antibodies, CD33 antibodies, CD14 antibodies, CD65 antibodies and CD45 antibodies. The application uses flow cytometry for detection, and the antibody composition of the first group of antibodies and the second group of antibodies is given, so that the detection sensitivity and specificity are improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antibody composition for detecting immature monocytes and its application. Background Technology

[0002] Acute myelomonocytic leukemia, acute monocytic leukemia, and chronic myelomonocytic leukemia all involve abnormalities in monocytes. In the diagnosis of these diseases and the monitoring of MRD in acute leukemia, attention should be paid to the ratio and immunophenotype of myeloid blasts, myeloid immature cells, and mature monocytes. The ratio and phenotype of myeloid blasts are relatively simple and well-established. However, immature monocytes are often difficult to identify due to their low proportion and nonspecific phenotype. Summary of the Invention

[0003] In view of this, the present invention provides an antibody composition for detecting immature monocytes and its application, which can accurately determine the proportion and immunophenotype of immature monocytes.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an antibody composition for detecting immature monocytes, comprising a first group of antibodies and a second group of antibodies, wherein the first group of antibodies comprises CD15 antibody, CD117 antibody, CD34 antibody, CD33 antibody, CD14 antibody, HLA-DR antibody, CD13 antibody, CD38 antibody, CD64 antibody, CD19 antibody, CD56 antibody, CD7 antibody, and CD45 antibody; and the second group of antibodies comprises HLA-DR antibody, CD64 antibody, CD33 antibody, CD14 antibody, CD65 antibody, and CD45 antibody.

[0005] Preferably, the fluorescent labeling order of the CD15 antibody, CD117 antibody, CD34 antibody, CD33 antibody, CD14 antibody, HLA-DR antibody, CD13 antibody, CD38 antibody, CD64 antibody, CD19 antibody, CD56 antibody, CD7 antibody, and CD45 antibody in the first group of antibodies is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC-Cy7, APC-iFluor700, BrilliantViolet 605, Brilliant Violet 421, Brilliant Violet 650, Brilliant Violet 750, ECD, APC, and QB500. In the second group of antibodies, the fluorescent labeling order of HLA-DR antibody, CD64 antibody, CD33 antibody, CD14 antibody, CD65 antibody, and CD45 antibody was FITC, PE, PE-Cy7, APC-Cy7, Brilliant Violet 421, and PerCP, respectively.

[0006] Preferably, the volume ratio of CD15 antibody, CD117 antibody, CD34 antibody, CD33 antibody, CD14 antibody, HLA-DR antibody, CD13 antibody, CD38 antibody, CD64 antibody, CD19 antibody, CD56 antibody, CD7 antibody, and CD45 antibody in the first group of antibodies is 7:7:2:2:2:4:2:2:2:2:4:2:2; In the second group of antibodies, the volume ratio of HLA-DR antibody, CD64 antibody, CD33 antibody, CD14 antibody, CD65 antibody, and CD45 antibody was 5:1:1:1:2:5.

[0007] In a second aspect, the present invention provides the use of the antibody composition described herein in the preparation of flow cytometry samples for detecting immature mononuclear cells in bone marrow or peripheral blood.

[0008] Thirdly, the present invention provides the application of the antibody composition described herein in the preparation of a detection product for detecting immature mononuclear cells in bone marrow or peripheral blood.

[0009] Fourthly, the present invention provides a reagent kit comprising a first container and a second container, wherein the first container contains the first group of antibodies and the second container contains the second group of antibodies.

[0010] Preferably, it also includes other containers for holding red blood cell lysis buffer and PBS buffer, respectively.

[0011] Preferably, it also includes flow cytometry tubes for use with the flow cytometer.

[0012] Fifthly, the present invention provides an application of the kit described herein in detecting immature mononuclear cells in bone marrow or peripheral blood.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention addresses the challenges of low abundance and nonspecific phenotype of immature monocytes by employing flow cytometry for detection and provides antibody compositions of first and second groups of antibodies, improving detection sensitivity and specificity. By simultaneously covering myeloid lineage markers, immature / differentiation markers, and monocyte-related markers, complementary immunophenotypic coordinates are formed, clearly distinguishing myeloid progenitors, immature monocytes, and mature monocytes, reducing missed detections and misdiagnoses caused by the low proportion and nonspecific phenotype of immature monocytes. Attached Figure Description

[0014] Figure 1 This is a diagram illustrating the immature mononuclear cells in a normal bone marrow sample provided in Example 2 of the present invention; Figure 2 This is a diagram illustrating the immature mononuclear cells in an abnormal bone marrow sample provided in Example 2 of the present invention. Detailed Implementation

[0015] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0016] Acute myelomonocytic leukemia, acute monocytic leukemia, and chronic myelomonocytic leukemia all involve abnormalities in monocytes. In the diagnosis of these diseases and the monitoring of MRD in acute leukemia, attention should be paid to the ratio and immunophenotype of myeloid blasts, myeloid immature cells, and mature monocytes. The ratio and phenotype of myeloid blasts are relatively simple and well-established. However, immature monocytes are often difficult to identify due to their low proportion and nonspecific phenotype.

[0017] To address the aforementioned technical problems, the present invention provides an antibody composition for detecting immature monocytes, comprising a first group of antibodies and a second group of antibodies. The first group of antibodies includes CD15 antibody, CD117 antibody, CD34 antibody, CD33 antibody, CD14 antibody, HLA-DR antibody, CD13 antibody, CD38 antibody, CD64 antibody, CD19 antibody, CD56 antibody, CD7 antibody, and CD45 antibody. The second group of antibodies includes HLA-DR antibody, CD64 antibody, CD33 antibody, CD14 antibody, CD65 antibody, and CD45 antibody.

[0018] Furthermore, in the first group of antibodies, the fluorescent labeling order of CD15 antibody, CD117 antibody, CD34 antibody, CD33 antibody, CD14 antibody, HLA-DR antibody, CD13 antibody, CD38 antibody, CD64 antibody, CD19 antibody, CD56 antibody, CD7 antibody, and CD45 antibody is FITC, PE, PerCP-Cy5.5, PE-Cy7, APC-Cy7, APC-iFluor700, BrilliantViolet 605, Brilliant Violet 421, Brilliant Violet 650, Brilliant Violet 750, ECD, APC, and QB500; In the second group of antibodies, the fluorescent labeling order of HLA-DR antibody, CD64 antibody, CD33 antibody, CD14 antibody, CD65 antibody, and CD45 antibody was FITC, PE, PE-Cy7, APC-Cy7, Brilliant Violet 421, and PerCP, respectively.

[0019] Furthermore, in the first group of antibodies, the volume ratio of CD15 antibody, CD117 antibody, CD34 antibody, CD33 antibody, CD14 antibody, HLA-DR antibody, CD13 antibody, CD38 antibody, CD64 antibody, CD19 antibody, CD56 antibody, CD7 antibody, and CD45 antibody is 7:7:2:2:2:4:2:2:2:2:2:4:2:2; and in the second group of antibodies, the volume ratio of HLA-DR antibody, CD64 antibody, CD33 antibody, CD14 antibody, CD65 antibody, and CD45 antibody is 5:1:1:1:2:5.

[0020] In a second aspect, the present invention provides the use of the antibody composition described herein in the preparation of flow cytometry samples for detecting immature mononuclear cells in bone marrow or peripheral blood.

[0021] Thirdly, the present invention provides the application of the antibody composition described above in the preparation of a detection product for detecting immature mononuclear cells in bone marrow or peripheral blood.

[0022] Fourthly, the present invention provides a reagent kit comprising a first container and a second container, wherein the first container contains the first group of antibodies and the second container contains the second group of antibodies.

[0023] Furthermore, it also includes other containers for holding red blood cell lysis buffer and PBS buffer, respectively.

[0024] Fifthly, the present invention also provides an application of the kit described herein in detecting immature mononuclear cells in bone marrow or peripheral blood.

[0025] This invention addresses the challenges of low abundance and nonspecific phenotype of immature monocytes by employing flow cytometry for detection and provides antibody compositions of first and second groups of antibodies, improving detection sensitivity and specificity. By simultaneously covering myeloid lineage markers, immature / differentiation markers, and monocyte-related markers, complementary immunophenotypic coordinates are formed, clearly distinguishing myeloid progenitors, immature monocytes, and mature monocytes, reducing missed detections and misdiagnoses caused by the low proportion and nonspecific phenotype of immature monocytes.

[0026] In some embodiments, the step of preparing flow cytometry samples for detecting immature mononuclear cells in bone marrow or peripheral blood includes: adding CD15 antibody, CD117 antibody, CD34 antibody, CD33 antibody, CD14 antibody, HLA-DR antibody, CD13 antibody, CD38 antibody, CD64 antibody, CD19 antibody, CD56 antibody, CD7 antibody, and CD45 antibody from tube 1 to flow cytometry tube 1 in a volume ratio of 7:7:2:2:2:4:2:2:2:2:2:4:2:2; adding HLA-DR antibody, CD64 antibody, CD33 antibody, CD14 antibody, CD65 antibody, and CD45 antibody from tube 2 to flow cytometry tube 2 in a volume ratio of 5:1:1:1:2:5; and adding the sample to be tested to each tube, so that the cell count in the flow cytometry tube is 2 × 10⁻⁶. 6 Each tube contains one antibody, which is mixed and incubated in the dark. Red blood cell lysis buffer is added, mixed, and incubated in the dark. The mixture is centrifuged, the supernatant is discarded, and the cells are washed with PBS buffer, centrifuged again, and the supernatant is discarded. Finally, the cells are resuspended in the aforementioned buffer to obtain the flow cytometry sample. The sample to be tested is bone marrow or peripheral blood. In this invention, patient samples are prepared into cell suspensions containing the above antibody composition, and then analyzed using a flow cytometer to determine the presence of abnormal myeloid blast cells and abnormal monocytes, including abnormal immature monocytes and abnormal mature monocytes.

[0027] Example 1 Reagent Information This embodiment provides a fluorescently labeled antibody composition, which includes a first group of antibodies and a second group of antibodies. The first group of antibodies includes CD15 antibody, CD117 antibody, CD34 antibody, CD33 antibody, CD14 antibody, HLA-DR antibody, CD13 antibody, CD38 antibody, CD64 antibody, CD19 antibody, CD56 antibody, CD7 antibody, and CD45 antibody. The second group of antibodies includes HLA-DR antibody, CD64 antibody, CD33 antibody, CD14 antibody, CD65 antibody, and CD45 antibody. In the first group of antibodies, the fluorescent labeling order of CD15 antibody, CD117 antibody, CD34 antibody, CD33 antibody, CD14 antibody, HLA-DR antibody, CD13 antibody, CD38 antibody, CD64 antibody, CD19 antibody, CD56 antibody, CD7 antibody, and CD45 antibody was FITC, PE, PerCP-Cy5.5, PE-Cy7, APC-Cy7, APC-iFluor700, Brilliant Violet 605, Brilliant Violet 421, Brilliant Violet 650, Brilliant Violet 750, ECD, APC, and QB500. In the second group of antibodies, the fluorescent labeling order of HLA-DR antibody, CD64 antibody, CD33 antibody, CD14 antibody, CD65 antibody, and CD45 antibody was FITC, PE, PE-Cy7, APC-Cy7, Brilliant Violet 421, and PerCP, respectively.

[0028] These fluorescently labeled antibodies can be purchased directly through public channels; see Table 1 for specific sources.

[0029] Table 1

[0030] Example 2 Sample Detection The test samples were tested using the kits listed in Table 1, including the following steps: Preparing the test samples: Adjusting the concentration of the bone marrow sample to 3 × 10⁻⁶. 7 -4×10 7 Cells / mL. Following the dosages specified in Table 1, add the following antibodies sequentially to flow cytometry tube 1: FITC-labeled CD15 antibody, PE-labeled CD117 antibody, PerCP-Cy5.5-labeled CD34 antibody, PE-Cy7-labeled CD33 antibody, APC-Cy7-labeled CD14 antibody, APC-iFluor700-labeled HLA-DR antibody, Brilliant Violet 605-labeled CD13 antibody, Brilliant Violet 421-labeled CD38 antibody, Brilliant Violet 650-labeled CD64 antibody, Brilliant Violet 750-labeled CD19 antibody, APC-labeled CD7 antibody, ECD-labeled CD56 antibody, and QB500-labeled CD45 antibody. FITC-labeled HLA-DR antibody, PE-labeled CD64 antibody, PE-Cy7-labeled CD33 antibody, APC-Cy7-labeled CD14 antibody, Brilliant Violet 421-labeled CD65 antibody, and PerCP-labeled CD45 antibody were sequentially added to flow cytometry tube 2, followed by the sample to be tested, to bring the cell count in the flow cytometry tube to 2 × 10⁻⁶ cells / mL. 6Mix the samples thoroughly and incubate in the dark for 20 minutes. Add 2-3 mL of 1×Lysing solution for FACS 10* erythrocyte lysis buffer, mix gently, and incubate at room temperature in the dark for 10 minutes. Then, centrifuge the flow cytometry tube at 400 rcf / min for 5 minutes and discard the supernatant. Add 2 mL of PBS buffer to the flow cytometry tube, mix well, and centrifuge at 400 rcf / min for 5 minutes. Discard the supernatant and resuspend the sample in 150-300 μL of PBS buffer. Place the flow cytometry tube on a flow cytometer for detection and obtain 500,000-1,000,000 events. Export the data in FCS format and analyze the data using flow cytometry software to determine the proportion of immature monocytes in the bone marrow.

[0031] The erythrocyte lysis buffer was Lysing solution for FACS 10* erythrocyte lysis buffer (manufactured by Hangzhou Lianke Co., Ltd., catalog number: LSB03). The PBS buffer had a phosphate concentration of 0.02 mol / L and a pH of 7.2-7.6.

[0032] During the gating process, debris and adhesions are first removed using FSC-A / FSC-H and FSC-A / SSC-A. Then, the CD45 / SSC dot map is used to gating each cell region, respectively circling the regions of lymphocytes, mature monocytes, granulocytes, primitive regions, and nucleated erythrocytes. First, CD117+ cells are identified using a CD45 / CD117 dot plot. Then, CD34 / CD117, CD34 / CD38, CD34 / HLA-DR, and CD13 / CD33 dot plots are used to examine the differentiation of early myeloid cells and the presence of cross-lineage expression of CD19, CD7, and CD56. These characteristics are used to determine whether abnormal early myeloid cells exist. Next, CD33 / HLA-DR and CD64 / HLA-DR dot plots are used to identify CD33-positive, CD64-positive, and HLA-DR-positive monocytes. Based on CD14 positivity / positivity, immature monocytes and mature monocytes are classified, and the ratio of immature to mature monocytes is calculated. By analyzing 40 AML residual specimens from normal bone marrow samples, the mean percentage of immature monocytes was 0.64%, with a standard deviation (SD) of 0.4%. Based on the mean + 1.96 × SD, the upper limit of the reference range for immature monocytes in bone marrow was 1.4%. The immunophenotype of normal immature monocytes is characterized by a low expression of CD117, positive expression of CD13, CD15, and CD38, strong HLA-DR positivity, and expression or weak expression of CD65. Mature monocytes are CD117 negative, HLA-DR positive, CD13 and CD38 positive, CD15 weakly positive, and CD65 negative. Abnormal immature monocytes may be characterized by an increased proportion or an abnormal immunophenotype.

[0033] The normal bone marrow samples processed in Example 2 were analyzed, and the results are as follows: Figure 1 As shown, in this patient's bone marrow, CD34+CD117+ myeloid blasts accounted for approximately 0.6% of the nucleated cells, with a normal phenotype; 1.4% immature monocytes (dark green in the image) were also observed, with a normal phenotype. The results of the bone marrow sample containing abnormal immature monocytes are as follows. Figure 2 As shown, two groups of immature monocytes were observed in the patient's bone marrow. One group was dark green, accounting for 0.91%, expressing CD117, CD13, CD33, and CD64, and partially expressing HLA-DR, CD15, and CD65, but not CD14. The other group was red, accounting for 1.08%, expressing CD13, CD33, CD15, HLA-DR, and CD64, and partially expressing CD65, but not CD14. The dark green immature monocytes were abnormal immature monocytes.

[0034] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0035] 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 immature monocytes, characterized in that, include: The first group of antibodies includes CD15 antibody, CD117 antibody, CD34 antibody, CD33 antibody, CD14 antibody, HLA-DR antibody, CD13 antibody, CD38 antibody, CD64 antibody, CD19 antibody, CD56 antibody, CD7 antibody, and CD45 antibody. The second group of antibodies includes HLA-DR antibody, CD64 antibody, CD33 antibody, CD14 antibody, CD65 antibody, and CD45 antibody; In the first group of antibodies, the fluorescent labeling order of CD15 antibody, CD117 antibody, CD34 antibody, CD33 antibody, CD14 antibody, HLA-DR antibody, CD13 antibody, CD38 antibody, CD64 antibody, CD19 antibody, CD56 antibody, CD7 antibody, and CD45 antibody was FITC, PE, PerCP-Cy5.5, PE-Cy7, APC-Cy7, APC-iFluor700, Brilliant Violet 605, Brilliant Violet 421, Brilliant Violet 650, Brilliant Violet 750, ECD, APC, and QB500. In the second group of antibodies, the fluorescent labeling order of HLA-DR antibody, CD64 antibody, CD33 antibody, CD14 antibody, CD65 antibody, and CD45 antibody was FITC, PE, PE-Cy7, APC-Cy7, Brilliant Violet 421, and PerCP, respectively.

2. The antibody composition according to claim 1, characterized in that, In the first group of antibodies, the volume ratio of CD15 antibody, CD117 antibody, CD34 antibody, CD33 antibody, CD14 antibody, HLA-DR antibody, CD13 antibody, CD38 antibody, CD64 antibody, CD19 antibody, CD56 antibody, CD7 antibody, and CD45 antibody is 7:7:2:2:2:4:2:2:2:2:4:2:2; In the second group of antibodies, the volume ratio of HLA-DR antibody, CD64 antibody, CD33 antibody, CD14 antibody, CD65 antibody, and CD45 antibody was 5:1:1:1:2:

5.

3. The use of the antibody composition according to any one of claims 1-2 in the preparation of flow cytometry samples for detecting immature mononuclear cells in bone marrow or peripheral blood.

4. The use of the antibody composition according to any one of claims 1-2 in the preparation of a detection product for detecting immature mononuclear cells in bone marrow or peripheral blood.

5. A reagent kit, characterized in that, It includes a first container and a second container, the first container containing a first group of antibodies as described in any one of claims 1-2, and the second container containing a second group of antibodies as described in any one of claims 1-2.

6. The reagent kit according to claim 5, characterized in that, It also includes other containers for holding red blood cell lysis buffer and PBS buffer, respectively.

7. The reagent kit according to claim 5, characterized in that, It also includes flow cytometry tubes used with flow cytometers.

8. The use of the kit according to any one of claims 5-7 in detecting immature mononuclear cells in bone marrow or peripheral blood.

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