Antibody composition and application thereof for detecting multiple myeloma based on full spectrum flow cytometry

Abstract

The present invention provides an antibody composition for detecting multiple myeloma based on full-spectrum flow cytometry and its application, belonging to the technical field of multiple myeloma detection. The antibody composition includes CD3 antibody, CD20 antibody, HLA-DR antibody, CD8 antibody, CD11b antibody, CD10 antibody, CD71 antibody, CD28 antibody, CD14 antibody, CD13 antibody, Igκ antibody, CD15 antibody, Igλ antibody, CD117 antibody, CD56 antibody, CD45 antibody, CD19 antibody, CD34 antibody, CD138 antibody, CD4 antibody, CD38 antibody, CD22 antibody and CD27 antibody. The kit containing the antibody composition can be applied to a full-spectrum flow cytometer to accurately perform preliminary screening on patients with multiple myeloma.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease detection, in particular to the technical field of multiple myeloma detection, and specifically to an antibody composition for detecting multiple myeloma based on full-spectrum flow cytometry and an application thereof. Background Art

[0002] Multiple Myeloma (MM) is a serious hematological malignancy, in which malignant cells originate from abnormal plasma cells in the bone marrow. The main symptoms of this disease include hypercalcemia, bone disease, renal insufficiency and anemia. Disease progression and subsequent relapse are characterized by subclonal evolution and drug resistance, and almost all patients eventually develop into relapsed and refractory types. In recent years, the application of a variety of high-sensitivity detection technologies to perform laboratory tests on patients with multiple myeloma has provided a basis for the early diagnosis of multiple myeloma, which is a research hotspot in the discipline of clinical testing.

[0003] Chinese patent application publication CN111912983A discloses an antibody composition, a kit and an application for detecting minimal residual lesions in multiple myeloma. The antibody composition is composed of antibody CD319 or antibody CD269, and antibodies CD138, CD38, CD184, CD27, CD19, CD56, ckappa and clamda. The kit containing the antibody composition has been verified to be useful for detecting minimal residual lesions after conventional chemotherapy drugs. However, this scheme is only applicable to the detection of minimal residual lesions after treatment of confirmed patients, and cannot be used for the preliminary diagnosis of the disease. Summary of the invention

[0004] In order to solve the above problems, the first aspect of the present invention is to provide an antibody composition for detecting multiple myeloma based on full-spectrum flow cytometry. The antibody composition of the present invention can be used for preliminary diagnosis when a patient is suspected of having multiple myeloma, which is beneficial for clinical classification of the disease.

[0005] Specifically, the technical solution of the present invention to achieve the above object is as follows:

[0006] An antibody composition for detecting multiple myeloma based on full-spectrum flow cytometry, the antibody composition comprising CD3 antibody, CD20 antibody, HLA-DR antibody, CD8 antibody, CD11b antibody, CD10 antibody, CD71 antibody, CD28 antibody, CD14 antibody, CD13 antibody, Igκ antibody, CD15 antibody, Igλ antibody, CD117 antibody, CD56 antibody, CD45 antibody, CD19 antibody, CD34 antibody, CD138 antibody, CD4 antibody, CD38 antibody, CD22 antibody and CD27 antibody.

[0007] In a preferred embodiment, each antibody is a fluorescein-labeled antibody.

[0008] In a further preferred embodiment, the fluorescein label of the CD3 antibody is cFluor V420, the fluorescein label of the CD20 antibody is cFluor V450, the fluorescein label of the HLA-DR antibody is BV510, the fluorescein label of the CD8 antibody is cFluor V547, the fluorescein label of the CD11b antibody is BV570, the fluorescein label of the CD10 antibody is BV605, the fluorescein label of the CD71 antibody is BV650, the fluorescein label of the CD28 antibody is BV711, the fluorescein label of the CD14 antibody is BV750, the fluorescein label of the CD13 antibody is BV785, the fluorescein label of the Igκ antibody is AF488, the fluorescein label of the CD15 antibody is cFluor B548, the fluorescein label of the Igλ antibody is PE, and the fluorescein label of the CD117 antibody is cFluor BYG610, the fluorescein label of the CD56 antibody is PE-Cy5, the fluorescein label of the CD45 antibody is PerCP-Cy5.5, the fluorescein label of the CD19 antibody is cFluor BYG710, the fluorescein label of the CD34 antibody is PE-Cy7, the fluorescein label of the CD138 antibody is BV421, the fluorescein label of the CD4 antibody is cFluor R668, the fluorescein label of the CD38 antibody is cFluorR720, the fluorescein label of the CD22 antibody is APC-Fire750, and the fluorescein label of the CD27 antibody is APC-Fire810.

[0009] The second aspect of the present invention is to provide the use of the antibody composition in preparing a product for detecting multiple myeloma based on full-spectrum flow cytometry.

[0010] In a preferred embodiment, the product is a kit.

[0011] The third aspect of the present invention is to provide a kit for detecting multiple myeloma based on full-spectrum flow cytometry, wherein the kit comprises the antibody composition.

[0012] In a preferred embodiment, the kit comprises hemolysin, a membrane permeabilizing agent and a fluorescence stabilizer.

[0013] In a preferred embodiment, the volume ratio of the HLA-DR antibody, the CD8 antibody, the CD11b antibody, the CD10 antibody, the CD71 antibody, the Igκ antibody, the Igλ antibody, the CD117 antibody, the CD34 antibody, the CD22 antibody, the CD27 antibody, the CD45 antibody, the CD3 antibody, the CD28 antibody, the CD14 antibody, the CD13 antibody, the CD19 antibody, the CD38 antibody, the CD20 antibody, the CD138 antibody, the CD4 antibody, the CD56 antibody, and the CD15 antibody is 5:5:5:5:5:5:5:5:5:5:5:5:3:2.5:2.5:2.5:2.5:2.5:2.5:2.5:2:2:2:1.25:1.

[0014] The fourth aspect of the present invention is to provide the use of the antibody composition in preparing flow cytometry samples for detecting multiple myeloma based on full-spectrum flow cytometry.

[0015] In a preferred embodiment, the step of preparing a flow cytometer sample for detecting multiple myeloma based on full-spectrum flow cytometry comprises:

[0016] The CD3 antibody, the CD20 antibody, the HL-ADR antibody, the CD8 antibody, the CD11b antibody, the CD10 antibody, the CD71 antibody, the CD28 antibody, the CD14 antibody, the CD13 antibody, the CD15 antibody, the CD117 antibody, the CD56 antibody, the CD45 antibody, the CD19 antibody, the CD34 antibody, the CD138 antibody, the CD4 antibody, the CD38 antibody, the CD22 antibody, and the CD27 antibody were added to a flow tube at a volume ratio of 2.5:2:5:5:5:5:5:2.5:2.5:2.5:1:5:1.25:3:2.5:5:2:2:2.5:5:5, and the sample to be tested was added so that the number of cells in the flow tube was 2×10 5 / tube, mix well and incubate in the dark; add red blood cell lysis solution, mix well and incubate in the dark; centrifuge, remove supernatant, add buffer solution for washing and centrifuge again, remove supernatant; add membrane permeabilization agent, mix well and incubate in the dark, centrifuge, and remove supernatant; add the Igκ antibody and the Igλ antibody, mix well and incubate in the dark, add the buffer solution for washing, centrifuge, remove supernatant, then add the buffer solution for resuspending, add fluorescence stabilizer, and obtain flow cytometry sample; wherein the sample to be tested is bone marrow, peripheral blood or body cavity effusion (such as pleural effusion).

[0017] In the present invention, the patient sample is prepared into a cell suspension containing the above-mentioned antibody composition, and the data obtained are analyzed by full-spectrum flow cytometry detection to determine whether there is abnormal expression in the cells. Igκ, Igλ, CD117, CD56, CD45, CD19, CD138, CD27, and CD38 antibodies are used to determine whether plasma cells are abnormal. Usually, the phenotype of plasma cells is CD38+ / CD56+ / CD19- / CD138+. After finding this group of cells, Igκ / Igλ is analyzed for judgment. Igκ / Igλ<0.5 or Igκ / Igλ>3 are abnormal plasma cells. Then, the remaining antibodies are used to determine whether there are abnormalities in other cell lines besides plasma cell abnormalities. T lymphocyte lineage-related antigens are: CD3, CD8, CD4; B lymphocyte lineage-related antigens are: CD20, CD19, CD22; myeloid lineage-related antigens are: CD11b, CD14, CD13, CD15, CD117, CD56; stem cell lineage antigens are: HLA-DR, CD10, CD117, CD34, CD38; and erythrocyte lineage antigens are: CD71. If there are abnormalities in the above series of antigens, other test kits need to be selected according to different series for further diagnosis.

[0018] In a preferred embodiment, each antibody is a fluorescein-labeled antibody.

[0019] In a further preferred embodiment, the fluorescein label of the CD3 antibody is cFluor V420, the fluorescein label of the CD20 antibody is cFluor V450, the fluorescein label of the HLA-DR antibody is BV510, the fluorescein label of the CD8 antibody is cFluor V547, the fluorescein label of the CD11b antibody is BV570, the fluorescein label of the CD10 antibody is BV605, the fluorescein label of the CD71 antibody is BV650, the fluorescein label of the CD28 antibody is BV711, the fluorescein label of the CD14 antibody is BV750, the fluorescein label of the CD13 antibody is BV785, the fluorescein label of the Igκ antibody is AF488, the fluorescein label of the CD15 antibody is cFluor B548, the fluorescein label of the Igλ antibody is PE, and the fluorescein label of the CD117 antibody is cFluor BYG610, the fluorescein label of the CD56 antibody is PE-Cy5, the fluorescein label of the CD45 antibody is PerCP-Cy5.5, the fluorescein label of the CD19 antibody is cFluor BYG710, the fluorescein label of the CD34 antibody is PE-Cy7, the fluorescein label of the CD138 antibody is BV421, the fluorescein label of the CD4 antibody is cFluor R668, the fluorescein label of the CD38 antibody is cFluorR720, the fluorescein label of the CD22 antibody is APC-Fire750, and the fluorescein label of the CD27 antibody is APC-Fire810.

[0020] Compared with the prior art, the present invention has the following beneficial effects: the antibody composition in the present invention is dedicated to the full-spectrum flow cytometer, and can be used for the full-spectrum flow cytometer to perform the initial screening of multiple myeloma patients. Normal and abnormal plasma cells can be distinguished through one tube, which can save a lot of precious samples. And because one tube is rich in enough detection indicators, the detection rate can be effectively improved. At the same time, the repeated application of the skeleton antibody is reduced, which greatly saves costs. Through the data statistics of 362 multiple myeloma patient tests, combined with clinical data and comparison with traditional flow cytometers, the kit can be used to accurately find malignant tumor cells. Compared with the test results of traditional flow cytometers, it was found that the diagnostic accuracy was 100%, that is, all samples could detect abnormal cells. Compared with the proportion of abnormal plasma cells in the nucleated cells of multiple myeloma patients, there was no statistically significant difference (P>0.05). The types and expression intensities of each antigen were consistent, and there was no statistically significant difference in the positive antigen expression rate (P>0.05). At the same time, the invention of the kit can make up for the current lack of full-spectrum flow cytometer technology for diagnosing multiple myeloma. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figures 1 to 6 The test results of the kit in Example 2 using full-spectrum flow cytometry to detect samples from multiple myeloma patients;

[0022] Figure 7 This is a comparison chart of the test results of the kit in Comparative Example 1 and the kit in Example 2 using full-spectrum flow cytometry to detect samples from multiple myeloma patients. DETAILED DESCRIPTION

[0023] The following content is combined with the embodiments to clearly and completely describe the technical solution of the present invention so that those skilled in the art can fully understand the present invention. Obviously, the described embodiments are only some preferred embodiments of the present invention, rather than all embodiments. Any equivalent transformation or substitution made by ordinary technicians in this field to the following implementation methods without creative work belongs to the protection scope of the present invention.

[0024] The sources of the fluorescein-labeled antibodies used in the following examples are shown in Table 1.

[0025] Table 1 Sources of fluorescein-labeled antibodies in the examples

[0026] Antibody Name Fluorescein Procurement Sources Part Number 1 CD3 cFluor V420 Cytek Biosciences R7-10053 2 CD20 cFluor V450 Cytek Biosciences R7-20015 3 HLADR BV510 BioLegend 307646 4 CD8 cFluor V547 Cytek Biosciences R7-10063 5 CD11b BV570 BioLegend 301325 6 CD10 BV605 BioLegend 312222 7 CD71 BV650 BioLegend 334116 8 CD28 BV711 BioLegend 302948 9 CD14 BV750 BioLegend 367136 10 CD13 BV785 BioLegend 301726 11 Igκ AF488 BioLegend 316512 12 CD15 cFluor B548 Cytek Biosciences R7-10025 13 Igλ PE BioLegend 316608 14 CD117 cFluor BYG610 Cytek Biosciences R7-10023 15 CD56 PE-Cy5 BioLegend 362516 16 CD45 PerCP-Cy5.5 Cytek Biosciences R7-10006 17 CD19 cFluor BYG710 Cytek Biosciences R7-20009 18 CD34 PE-Cy7 BioLegend 343516 19 CD138 BV-421 BD Biosciences 562935 20 CD4 cFluor R668 Cytek Biosciences R7-10049 21 CD38 cFluor R720 Cytek Biosciences R7-20061 22 CD22 APC-Fire750 BioLegend 363522 23 CD27 APC-Fire810 BioLegend 393214

[0027] The phosphate concentration of the PBS buffer in the example is 0.02 mol / L, and the pH is 7.2 to 7.6.

[0028] Example 1

[0029] This embodiment provides a kit for detecting multiple myeloma using full-spectrum flow cytometry. The kit comprises an antibody composition as shown in Table 2.

[0030] Table 2 Antibody composition and its label and dosage for detecting multiple myeloma based on full spectrum flow cytometry

[0031]

[0032]

[0033] Further, each antibody in Table 2 is a fluorescein-labeled antibody. The fluorescein labeling of each antibody is shown in Table 2, and the fluorescein corresponds to the antibody one by one.

[0034] Furthermore, the kit also includes a red blood cell lysis solution, a membrane breaking agent and a fluorescence stabilizer.

[0035] Specifically, the red blood cell lysis buffer is one of BD lysing Buffer 10×Concentrate (produced by BD, catalog number: 555899) and Keprey hemolysin (produced by Henan Keprey Biotechnology Co., Ltd.). The membrane permeabilization agent is one of BD Cytofix / Cytoperm Fixation and Permeabilization Solution (catalog number: 554722), Beckman PerFix-nc, and BD IntraSure. The fluorescence stabilizer is BD Brilliant Stain Buffer.

[0036] Example 2

[0037] This embodiment uses the kit in Example 1 to detect the sample to be tested, including the following steps:

[0038] Prepare the sample to be tested: adjust the concentration of the bone marrow sample to 5×10 6 ~5×10 7 cells / mL.

[0039] According to the dosage in Table 2, CD3 antibody labeled with cFluor V420, CD20 antibody labeled with cFluor V450, HL-ADR antibody labeled with BV510, CD8 antibody labeled with cFluor V547, CD11b antibody labeled with BV570, CD10 antibody labeled with BV605, CD71 antibody labeled with BV650, CD28 antibody labeled with BV711, CD14 antibody labeled with BV750, CD13 antibody labeled with BV785, CD15 antibody labeled with cFluor B548, CD117 antibody labeled with cFluor BYG610, CD56 antibody labeled with PE-Cy5, CD45 antibody labeled with PerCP-Cy5.5, CD19 antibody labeled with cFluor BYG710, CD34 antibody labeled with PE-Cy7, CD138 antibody labeled with BV421, cFluor CD4 antibody labeled with R668, CD38 antibody labeled with cFluor R720, CD22 antibody labeled with APC-Fire750, and CD27 antibody labeled with APC-Fire810 were added to the flow tube in sequence, and the sample to be tested was added to make the number of cells in the flow tube 2×10 5 , mix well, incubate in dark for 15 min, add 1 mL 1× BD Lysing Buffer 10× Concentrate red blood cell lysis buffer, mix gently, and incubate in dark for 15 min at room temperature.

[0040] Add 1 mL of 1×BD Lysing Buffer 10×Concentrate red blood cell lysis buffer to the flow tube, mix well and incubate at room temperature in the dark for 8 minutes. Then place the flow tube in a low-speed centrifuge at 300 rcf / min for 5 minutes and remove the supernatant.

[0041] Add 1mL PBS buffer to the flow tube, mix well, then centrifuge at 150rcf / min for 5 minutes, remove the supernatant; then add 0.5mL BD Cytofix / Cytoperm Fixation and Permeabilization Solution, mix gently, and incubate at room temperature in the dark for 10 minutes. Centrifuge at 300rcf / min for 5 minutes on a low-speed centrifuge, remove the supernatant; add AF488-labeled Igκ antibody and PE-labeled Igλ antibody in the amounts in Table 2, mix gently, and incubate in the dark for 30 minutes. Then add 1mL PBS, mix well, centrifuge at 150rcf / min for 5 minutes, remove the supernatant; add 150μL PBS buffer to resuspend; then add 50μL BD Brilliant Stain Buffer fluorescent stabilizer.

[0042] The flow tube was placed on the Cytek NL-CLC flow cytometer for detection. The instrument conditions were adjusted according to the quality control products, and 50,000 signals were obtained. After the data were exported into FCS format, Beckman Kaluza software was used for analysis. The analysis results were as follows: Figures 1 to 6 shown. Figures 1 to 6 The middle MM group (shown in red) is an abnormal cell group, which positively expresses CD38, CD138, CD56, and Igκ. Its phenotype is an abnormal plasma cell group, which can be diagnosed as multiple myeloma.

[0043] It should be noted that, in addition to bone marrow samples, peripheral blood samples or body cavity effusion samples (e.g., pleural effusion) can also be used as the test sample according to actual conditions. The concentration of the test sample is first adjusted to 5×10 6 ~5×10 7 cells / mL and then used for detection.

[0044] Example 3

[0045] This example uses a method for detecting abnormal plasma cells in patients with multiple myeloma using a traditional flow cytometer (Wei Shuangyu. Analysis of the diagnostic value of flow cytometric immunophenotyping for multiple myeloma [J]. Electronic Journal of Clinical Medical Literature, 2020.) to verify the test results in Example 2. The sample to be tested is a bone marrow sample. The detection of 362 samples showed that the diagnostic accuracy of the method in Example 2 was 100% compared with the method using a traditional flow cytometer, that is, all samples could detect abnormal cells. The proportion of abnormal plasma cells in nucleated cells of multiple myeloma patients in the two groups of experiments was compared, and the difference was not statistically significant (P>0.05). The types and expression intensities of each antigen were consistent, and there was no statistically significant difference in the expression rate of positive antigens (P>0.05).

[0046] Comparative Example 1

[0047] In this comparative example, the CD138 antibody labeled with BV421 in Example 2 was replaced with the CD138 antibody labeled with APC, and the remaining methods were the same as those in Example 2 to detect the sample to be tested (bone marrow sample). The test results are shown in FIG. Figure 7 shown. Figure 7 Figure (a) shows the clustering result of CD138 antibody in Example 2, and Figure (b) shows the clustering result of CD138 antibody in this comparative example. Figure 7 It can be seen that the detection result of the CD138 antibody in Example 2 is positive, while the detection result of the CD138 antibody in this comparative example becomes negative, which affects the judgment of the detection result.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. For any person skilled in the art, the present invention may have various changes and modifications. Any simple equivalent changes and modifications made according to the scope of protection of the present invention and the contents of the specification should be included in the scope of protection of the present invention.

Claims

1. An antibody composition for detecting multiple myeloma based on full-spectrum flow cytometry, characterized in that: The antibody composition comprises CD3 antibody, CD20 antibody, HLA-DR antibody, CD8 antibody, CD11b antibody, CD10 antibody, CD71 antibody, CD28 antibody, CD14 antibody, CD13 antibody, Igκ antibody, CD15 antibody, Igλ antibody, CD117 antibody, CD56 antibody, CD45 antibody, CD19 antibody, CD34 antibody, CD138 antibody, CD4 antibody, CD38 antibody, CD22 antibody and CD27 antibody; each antibody is a fluorescein-labeled antibody; the fluorescein label of the CD3 antibody is cFluor V420, the fluorescein label of the CD20 antibody is cFluor V450, the fluorescein label of the HLA-DR antibody is BV510, the fluorescein label of the CD8 antibody is cFluor V547, the fluorescein label of the CD11b antibody is BV570, the fluorescein label of the CD10 antibody is BV605, the fluorescein label of the CD71 antibody is BV650, the fluorescein label of the CD28 antibody is BV711, the fluorescein label of the CD14 antibody is BV750, the fluorescein label of the CD13 antibody is BV785, the fluorescein label of the Igκ antibody is AF488, the fluorescein label of the CD15 antibody is cFluor B548, the fluorescein label of the Igλ antibody is PE, the fluorescein label of the CD117 antibody is cFluor BYG610, the fluorescein label of the CD56 antibody is PE-Cy5, the fluorescein label of the CD45 antibody is PerCP-Cy5.5, the fluorescein label of the CD19 antibody is cFluor BYG710, the fluorescein label of the CD34 antibody is PE-Cy7, the fluorescein label of the CD138 antibody is BV421, the fluorescein label of the CD4 antibody is cFluor R668, the fluorescein label of the CD38 antibody is cFluor R720, the fluorescein label of the CD22 antibody is APC-Fire750, and the fluorescein label of the CD27 antibody is APC-Fire810; the volume ratio of the HLA-DR antibody, the CD8 antibody, the CD11b antibody, the CD10 antibody, the CD71 antibody, the Igκ antibody, the Igλ antibody, the CD117 antibody, the CD34 antibody, the CD22 antibody, the CD27 antibody, the CD45 antibody, the CD3 antibody, the CD28 antibody, the CD14 antibody, the CD13 antibody, the CD19 antibody, the CD38 antibody, the CD20 antibody, the CD138 antibody, the CD4 antibody, the CD56 antibody, and the CD15 antibody is 5:5:5:5:5:5:5:5:5:5:5:5:3:2.5:2.5:2.5:2.5:2.5:2:2:2:1.25:1。.

2. Use of the antibody composition according to claim 1 in the preparation of a product for detecting multiple myeloma based on full-spectrum flow cytometry.

3. The use according to claim 2, characterized in that: The product is a test kit.

4. A kit for detecting multiple myeloma based on full-spectrum flow cytometry, characterized in that: The kit comprises the antibody composition of claim 1.

5. The kit according to claim 4, characterized in that The kit comprises hemolysin, a membrane-breaking agent and a fluorescence stabilizer.

6. Use of the antibody composition according to claim 1 in preparing flow cytometry samples for detecting multiple myeloma based on full-spectrum flow cytometry.

7. The use according to claim 6, characterized in that: The steps of preparing a flow cytometer sample for detecting multiple myeloma based on full-spectrum flow cytometry include: The CD3 antibody, the CD20 antibody, the HLA-DR antibody, the CD8 antibody, the CD11b antibody, the CD10 antibody, the CD71 antibody, the CD28 antibody, the CD14 antibody, the CD13 antibody, the CD15 antibody, the CD117 antibody, the CD56 antibody, the CD45 antibody, the CD19 antibody, the CD34 antibody, the CD138 antibody, the CD4 antibody, the CD38 antibody, the CD22 antibody, and the CD27 antibody were added to a flow tube at a volume ratio of 2.5:2:5:5:5:5:5:2.5:2.5:2.5:1:5:1.25:3:2.5:5:2:2:2.5:5:5, and the sample to be tested was added so that the number of cells in the flow tube was 2×10 5 / tube, mix well and incubate in the dark; add red blood cell lysis solution, mix well and incubate in the dark; centrifuge and remove the supernatant, add buffer solution for washing and centrifuge again and remove the supernatant; add membrane permeabilization agent, mix well and incubate in the dark, centrifuge and remove the supernatant; add the Igκ antibody and the Igλ antibody, mix well and incubate in the dark, add the buffer solution for washing, centrifuge and remove the supernatant, then add the buffer solution for resuspending, add a fluorescence stabilizer to obtain a flow cytometer sample; wherein the sample to be tested is bone marrow, peripheral blood or body cavity effusion.

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