Antibody composition for detecting peripheral blood circulating tumor plasma cells based on full-spectrum flow cytometry and application of antibody composition

Abstract

The invention provides an antibody composition for detecting peripheral blood circulating tumor plasma cells based on full-spectrum flow cytometry and application of the antibody composition, and belongs to the technical field of multiple myeloma detection. The antibody composition includes, but is not limited to, CD38, CD56, CD138, CD19, CD117, ckappa, clambda, CD45, and BCMA. The antibody composition comprises, but is not limited to, CD38, CD56, CD138, CD19, CD117, ckappa, clambda, CD45, and BCMA According to the method, the tumor plasma cells in peripheral blood can be accurately marked by selecting a high-specificity antibody combination, the tumor plasma cells and normal cells can be effectively distinguished, multicolor staining and spectrum deconvolution are carried out through full spectrum flow cytometry, dye overlapping is avoided, the detection capacity and the resolution ratio of multiple markers are improved, and sensitive detection of the tumor plasma cells is ensured.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and particularly relates to the technical field of detection of plasma cell diseases such as multiple myeloma. Specifically, it relates to an antibody composition for detecting circulating tumor plasma cells (CTPCs) in peripheral blood based on full-spectrum flow cytometry and its application. Background Art

[0002] Multiple myeloma (MM) is a common plasma cell malignancy characterized by the accumulation of abnormal plasma cells inside and outside the bone marrow, which in turn affects normal hematopoiesis and leads to a series of clinical complications. In recent years, with the development of liquid biopsy technology, "circulating tumor plasma cells (CTPCs)" in peripheral blood, as an important biomarker, are being widely studied. The detection of CTPCs provides important clinical information for the early diagnosis, disease progression monitoring, and treatment response evaluation of multiple myeloma.

[0003] Traditional CTPCs detection techniques mainly rely on single fluorescence labeling and flow cytometry analysis. Although this method can reveal the presence and quantitative changes of CTPCs to a certain extent, there are many deficiencies. First, traditional techniques usually use a limited number of fluorescence channels and cannot detect multiple biomarkers simultaneously, which limits the in-depth analysis of CTPCs subsets and functions. Second, traditional flow cytometry is relatively cumbersome in data processing and analysis, requiring manual setting of gating and thresholds, which is easily affected by subjective factors, resulting in limited consistency and accuracy of the results. In addition, traditional techniques lack efficient cell enrichment means, making the detection sensitivity and specificity to be improved.

[0004] Therefore, developing an antibody composition based on full-spectrum flow cytometry and its application, which can effectively solve the deficiencies of traditional detection methods in sensitivity, specificity, and data analysis, has important clinical application value. Summary of the Invention

[0005] The object of the present invention is to provide an antibody composition for detecting circulating tumor plasma cells in peripheral blood based on full-spectrum flow cytometry and its application, aiming to solve the deficiencies of traditional detection methods in sensitivity, specificity, and data analysis. By optimizing the antibody combination, applying full-spectrum flow cytometry, sample processing procedures, data analysis algorithms, and introducing absolute counting technology, the efficient identification and accurate counting of extremely low-content CTPCs are achieved. It not only improves the detection sensitivity and specificity but also realizes multi-dimensional cell analysis and automated data processing, providing strong support for the early diagnosis, treatment monitoring, and prognosis evaluation of multiple myeloma and other plasma cell tumors.

[0006] The present invention provides the following technical solutions to solve the above technical problems:

[0007] Specifically, the antibody composition of the present invention for detecting peripheral blood circulating tumor plasma cells based on full-spectrum flow cytometry includes, but is not limited to, CD38, CD56, CD138, CD19, CD117, ckappa, clambda, CD45, BCMA.

[0008] In a preferred embodiment, the antibodies in the antibody composition are all fluorescently labeled antibodies, and the fluorescent labels include, but are not limited to, cFlour TM R720, PE-Cy5, BV-421, cFlour TM BYG710, cFlour TM BYG610, AlexarFlour488, PE, PERCP-CY5.5, cFlour TM V547.

[0009] In full-spectrum flow cytometry, the selection of fluorophores has a great impact on the detection results. By specifically labeling the antibodies with fluorophores, when the antibody composition of the present invention is applied to detect and subtype peripheral blood circulating tumor plasma cells by full-spectrum flow cytometry, all fluorophores in each channel can achieve the best staining effect, thereby achieving an excellent population separation effect and making the detection results more accurate.

[0010] The second aspect of the present invention is to provide the application of the antibody composition in the preparation of a product for detecting peripheral blood circulating tumor plasma cells based on full-spectrum flow cytometry.

[0011] In a preferred embodiment, the product is a kit.

[0012] The third aspect of the present invention is to provide a kit for detecting peripheral blood circulating tumor plasma cells based on full-spectrum flow cytometry, and the kit contains the antibody composition.

[0013] In a preferred embodiment, the kit further includes a red blood cell lysate, a permeabilizing agent, and a fluorescence stabilizer.

[0014] In a preferred embodiment, the ratio of the antibodies in the antibody composition is 1:1:1:1:1:1:1:1:1.

[0015] The fourth aspect of the present invention is to provide a method for using a kit, which at least includes the following steps:

[0016] (1) Take 2 flow cytometry tubes and label them as sample tube and control tube;

[0017] (2) First add Brilliant StainBuffer to the sample tube, and then add the antibody conjugated with a fluorophore, and mix well;

[0018] (3) Add the sample to be tested into the sample tube and the control tube respectively, and incubate at room temperature;

[0019] (4) Add red blood cell lysate to the sample tube and the control tube respectively for lysis;

[0020] (5) Centrifuge and wash;

[0021] (6) Mix again and detect on the machine.

[0022] In a preferred embodiment, the incubation time is 10 - 20 minutes, and the lysis time is 10 - 15 minutes.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects:

[0024] By selecting a highly specific antibody combination (CD38, CD56, CD138, CD19, CD117, ckappa, clambda, CD45, BCMA), the present invention can accurately label tumor plasma cells in peripheral blood, effectively distinguish them from normal cells, perform multi-color staining and spectral deconvolution through full-spectrum flow cytometry, avoid dye overlap, improve the detection ability and resolution of multiplex labeling, and ensure the sensitive detection of tumor plasma cells. All detections of the present invention are convenient, fast, time-saving, labor-saving, and simple to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings described herein are used to provide a further understanding of the present invention, and constitute a part of this application. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0026] Figures 1 to 6 Show the detection results of using the kit in Example 2 to detect samples of multiple myeloma patients by full-spectrum flow cytometry;

[0027] Figure 7a Show the detection results of using the BV421-labeled CD138 antibody in Example 2 to detect samples of multiple myeloma patients; Figure 7b Show the detection results of using the APC-labeled CD138 antibody in Comparative Example 1 to detect samples of multiple myeloma patients. DETAILED DESCRIPTION OF THE INVENTION

[0028] The following content describes the technical solution of the present invention clearly and completely in combination with embodiments, 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 to the following embodiments by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

[0029] The ordinal numbers used herein, such as "first", "second", etc., are only for descriptive purposes to distinguish similar objects, and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features.

[0030] The sources of the fluorescein-labeled antibodies used in the following examples are shown in Table 1.

[0031] Table 1 Sources of Fluorescein-Labeled Antibodies in Examples

[0032]

[0033]

[0034] In the PBS buffer solution used in the examples, the concentration of phosphate is 0.02 mol / L, and pH = 7.2 - 7.6.

[0035] Example 1

[0036] This example provides a kit for detecting peripheral blood circulating tumor plasma cells by using full-spectrum flow cytometry. The kit contains an antibody composition as shown in Table 2.

[0037] Table 2 Antibody Composition, Labeling and Dosage for Detecting Peripheral Blood Circulating Tumor Plasma Cells by Full-Spectrum Flow Cytometry

[0038]

[0039] In this example, each antibody in Table 2 is a fluorescein-labeled antibody. The fluorescein labeling of each antibody is as shown in Table 2, and the fluorescein corresponds to the antibody one by one.

[0040] Further, the kit also contains red blood cell lysate, membrane-breaking agent and fluorescence stabilizer. The red blood cell lysate is one of BD lysing Buffer 10×Concentrate (produced by BD, catalog number: 555899) and Kaprui hemolysin (produced by Henan Kaprui Biotechnology Co., Ltd.). The membrane-breaking 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 StainBuffer.

[0041] Example 2

[0042] In this example, the kit in Example 1 was used to detect the sample to be tested, including the following steps:

[0043] Prepare the sample to be tested: Collect 5-10 ml of peripheral blood from the patient's elbow vein according to aseptic operation, and separate mononuclear cells by density gradient centrifugation.

[0044] Adjust the concentration of the sample to 5×10 6 ~5×10 7 cells / mL.

[0045] Place the antibody to be used on an ice box for standby, and mix it well before use;

[0046] Number the sample tubes as XXX1 and XXX2 according to the patient's name and experimental Panel;

[0047] Add envelope staining antibodies to the sample tubes according to the Panel, and the specific dosage of each antibody shall be subject to the actual recommended dosage;

[0048] Add the well-mixed sample according to the counting result;

[0049] Mix gently at low speed for 3 seconds, continue to mix on a horizontal shaker, and incubate at room temperature in the dark for 30 minutes;

[0050] Add 100 μl of Reagent A to the sample tube, mix gently at low speed, and incubate at room temperature in the dark for 5 minutes;

[0051] Add an appropriate amount of red blood cell lysate to the sample tube, mix well, and incubate at room temperature in the dark for 15 minutes;

[0052] Centrifuge at 800 g for 5 minutes at room temperature, aspirate and discard the supernatant;

[0053] Add an appropriate amount of PBS, centrifuge at 800 g for 5 minutes, aspirate and discard the supernatant;

[0054] Add 50 μl of Reagent B to the sample tube; add cytoplasmic staining antibody. The antibody needs to be mixed well before use (the specific dosage of the antibody shall be subject to the actual recommended amount), mix well at low speed, and keep it away from light at room temperature for 20 minutes;

[0055] Add an appropriate amount of PBS and mix well with a pipette, centrifuge at 800 g for 5 min, and discard the supernatant;

[0056] Resuspend with 0.3 ml of PBS, mix well at low speed for 3 seconds, and wait in the dark for loading onto the machine.

[0057] Place the flow cytometry tube on the Cytek NL-CLC flow cytometer for detection. Adjust the instrument conditions according to each quality control product, and obtain 1 million signals. After exporting the data in fcs format, analyze it using Infinicyt software. The analysis results are as Figures 1 to 6 shown. Figures 1 to 6 In the MM group (shown in orange in the figure) is the abnormal cell population, which is positively expressed for CD38, CD138, and CD45, and the phenotype is an abnormal plasma cell population, which can be diagnosed as multiple myeloma.

[0058] Example 3

[0059] In this example, a method for detecting abnormal plasma cells in multiple myeloma patients using a traditional flow cytometer (Wei Shuangyu. Analysis of the diagnostic value of flow cytometry immunophenotyping in multiple myeloma [J]. Electronic Journal of Clinical Medical Literature, 2020.) was used to verify the detection results in Example 2. The test sample was a peripheral blood sample. Through the detection of 500 samples, it was shown that compared with the method using a traditional flow cytometer, the diagnostic accuracy rate of the method in Example 2 was 100%, that is, abnormal cells could be detected in all samples. Comparing the proportion of abnormal plasma cells in the nucleated cells of multiple myeloma patients in the two groups of tests, there was no significant difference (P > 0.05). The antigen types and expression intensities were the same, and there was no significant difference in the positive antigen expression rate.

[0060] Comparative Example 1

[0061] In this Comparative Example 1, the CD138 antibody labeled with BV421 in Example 2 was replaced with an APC-labeled CD138 antibody, and the remaining steps were the same as those in Example 2 for detecting the test sample (blood sample). The detection results are shown in Figure 7. In Figure 7

[0062] (a) The figure shows the cell population results of the CD138 antibody in Example 2,

[0063] (b) The figure shows the cell population results of the CD138 antibody in this comparative example.

[0064] It can be seen from Figure 7 that the detection result of the CD138 antibody in Example 2 was positive, while the detection result of the CD138 antibody in this comparative example became negative, affecting the judgment of the detection results.

[0065] As described above, it is only the preferred embodiment of the present invention and does not impose any formal restrictions on the present invention; any ordinary technician in the industry can smoothly implement the present invention as shown in the accompanying drawings of the specification and as described above; however, any minor changes, modifications, and equivalent variations made by those skilled in the art within the scope of the technical solution of the present invention by using the technical content disclosed above are equivalent embodiments of the present invention; at the same time, any changes, modifications, and equivalent variations made to the above embodiments based on the essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. An antibody composition for detecting circulating tumor plasma cells in peripheral blood based on full-spectrum flow cytometry, characterized in that: The antibody composition includes, but is not limited to, CD38, CD56, CD138, CD19, CD117, ckappa, clambda, CD45, BCMA.

2. The antibody composition according to claim 1, wherein: Each antibody is a fluorescein-labeled antibody.

3. The antibody composition according to claim 1, wherein: The antibodies in the antibody composition are all fluorescently labeled antibodies, and the fluorescent labels include but are not limited to cFlour TM R720, PE-Cy5, BV-421, cFlour TM BYG710, cFlour TM BYG610, Alexar Flour488, PE, PERCP-CY5.5, cFlour TM V547 4. Use of the antibody composition according to any one of claims 1 to 3 in the preparation of a product for detecting circulating tumor plasma cells in peripheral blood based on full-spectrum flow cytometry.

5. The application according to claim 4, wherein: The product is a kit.

6. A kit for detecting peripheral blood circulating tumor plasma cells based on full-spectrum flow cytometry, characterized in that: The kit contains the antibody composition according to any one of claims 1 to 3.

7. The kit according to claim 6, wherein: The kit includes a red blood cell lysate, a permeabilizing agent, and a fluorescence stabilizer.

8. The kit according to claim 6, characterized in that: The concentration range of the antibodies in the antibody composition is 0.1 - 10 μg / mL.

9. The kit according to claim 6, characterized in that: The ratio of the antibodies in the antibody composition is 1:1:1:1:1:1:1:1:

1.

10. The method of using the kit according to claim 6, characterized in that: It includes at least the following steps: (1) Take 2 flow cytometry tubes, labeled as the sample tube and the control tube; (2) First add Brilliant Stain Buffer to the sample tube, then add the fluorescein-conjugated antibody, and mix well; (3) Add the test sample to the sample tube and the control tube respectively, and incubate at room temperature for 10 - 20 minutes; (4) Add the red blood cell lysate to the sample tube and the control tube respectively for lysis, and the lysis time is 10 - 15 minutes; (5) Centrifuge and wash; (6) Mix again and detect on the machine.

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