Antibody combination for CD123 + dendritic cell typing and application thereof

Abstract

The invention discloses an antibody combination for typing CD123 + dendritic cells and application of the antibody combination. An antibody in the antibody combination consists of a CD45 antibody, a CD3 antibody, a CD20 antibody, a CD16 antibody, an HLA-DR antibody and a CD123 antibody. When the kit formed by the antibody combination provided by the invention is used for typing the CD123 + dendritic cells in the peripheral blood single cells, the quantity and the stability of the collected cells are remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an antibody combination for CD123+ dendritic cell typing and its application. Background Technology

[0002] Dendritic cells (DCs) are the core sentinels and commanders of the immune system, named for the tree-like protrusions on their surface. They were first discovered in 1973 by Ralph Steinman, who was awarded the Nobel Prize in Physiology or Medicine in 2011 for this discovery. As the most specialized antigen-presenting cells (APCs), DCs bridge the gap between innate and adaptive immunity, acting as initiators and regulators of the immune response.

[0003] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease, mainly divided into cutaneous lupus erythematosus (CLE) and systemic lupus erythematosus (SLE). CLE primarily affects the skin, while SLE can involve multiple systems and organs. CLE has a long course; although limited to the skin, studies have shown that 10%–40% of patients experience overlap and transition from CLE to SLE. Accurate diagnosis of CLE is a prerequisite for treatment. However, CLE has numerous subtypes and complex and diverse clinical manifestations, sometimes mimicking inflammatory diseases or even tumors such as mycosis fungoides, making diagnosis difficult. To address this diagnostic challenge, recent studies have revealed specific presence and distribution patterns of CD123+ plasmacytoid dendritic cells (pDCs) in CLE histopathology, which can serve as a useful diagnostic tool and clue for different CLE subtypes and their differential diagnosis.

[0004] CN114391102B discloses a kit for analyzing the phenotype and function of CD303+ dendritic cell subsets. CD303 (also known as BDCA-2) is another biomarker for pDCs, but its specificity is not as good as CD123. CD303 is expressed on pDCs, but may also be expressed at low levels on other immune cells. Therefore, using CD303+ plasmacytoid dendritic cells as a detection indicator may have the risk of poor specificity.

[0005] Currently, researchers are continuously developing anti-BDCA-2 antibodies or antibody-drug conjugates for the treatment of lupus erythematosus, and related non-clinical and clinical trials are ongoing. Cell typing and receptor occupancy data after drug administration are key indicators of efficacy. Because the CD303 surface antigen binds to drugs, the published CD303+ dendritic cell subset phenotype methods are not applicable to the efficacy analysis of these drugs. Furthermore, CD123+ plasmacytoid dendritic cells constitute a very low proportion of peripheral blood mononuclear cells, making flow cytometry segmentation using whole blood samples difficult. Additionally, whole blood samples are unstable and cannot be transported for extended periods, posing inconveniences for clinical trial transport and testing. Therefore, developing an accurate, stable, and highly specific kit for CD123+ dendritic cell typing in monkey peripheral blood mononuclear cells is crucial, laying the foundation for non-clinical and clinical efficacy evaluation of related drugs. Summary of the Invention

[0006] This invention provides a kit for analyzing CD123+ dendritic cell typing. The technical problem to be solved is to overcome the problems of insufficient target cells collected by existing CD123+ plasma cell-like dendritic cell typing methods, which leads to difficulties in grouping and instability of the test samples. The kit aims to determine the optimal experimental conditions to obtain accurate results.

[0007] Based on the flow cytometry platform, the inventors first extracted PBMCs from whole blood. Suitable samples were taken and the operation was performed according to Implementation Case 1. If the extracted PBMCs were not to be tested immediately, they could be frozen for later testing. The flow cytometry analysis logic used FVS780 dye to circle live cells, CD45⁺CD3⁻CD20⁻CD16⁻ to exclude non-target cells, HLA-DR⁺ to screen antigen-presenting cells (APCs), and CD123⁺ to specifically label pDC cells.

[0008] A first aspect of the present invention provides an antibody combination for CD123+ dendritic cell typing, wherein the antibodies in the antibody combination are composed of CD45 antibody, CD3 antibody, CD20 antibody, CD16 antibody, HLA-DR antibody and CD123 antibody.

[0009] In this document, the antibody combination contains no other antibodies besides CD45 antibody, CD3 antibody, CD20 antibody, CD16 antibody, HLA-DR antibody and CD123 antibody.

[0010] In some embodiments, the antibodies in the antibody combination are fluorescently labeled antibodies, wherein the fluorescently labeled antibodies are selected from FITC, PE, APC, PE-Cy5, PE-Cy7, APC-Cy7, PerCP-Cy5.5, Alexa Fluor 488, Alexa Fluor 647, Cy5, Cy7, BV421, BV605, BV786, and BV711; different antibodies use different fluorescent labels, enabling the separation of cells bound by the antibodies by flow cytometry in a single sorting step.

[0011] In some embodiments, the fluorescein is selected from APC, FITC, PerCP-Cy5.5, BV711, BV786, PE-Cy7, and PE.

[0012] In some implementations, the antibody combination satisfies one or more of the following:

[0013] 1) The CD45 antibody is an APC-labeled antibody;

[0014] 2) The CD3 antibody is a FITC-labeled antibody;

[0015] 3) The CD20 antibody is a PerCP-Cy5.5 labeled antibody;

[0016] 4) The CD16 antibody is a BV711-labeled antibody;

[0017] 5) The HLA-DR antibody is a BV786-labeled antibody; and,

[0018] 6) The CD123 antibody is a PE-Cy7 labeled antibody.

[0019] In some implementations, the antibody combination satisfies one or more of the following:

[0020] 1) The CD45 antibody mentioned is BD Biosciences' 561290;

[0021] 2) The CD3 antibody mentioned is BD Biosciences' 556611;

[0022] 3) The CD20 antibody mentioned is BD Biosciences 560736;

[0023] 4) The CD16 antibody mentioned is BD Biosciences' 563127;

[0024] 5) The HLA-DR antibody is BD Biosciences' 564041; and,

[0025] 6) The CD123 antibody mentioned is BD Biosciences 560826.

[0026] In some embodiments, the mass ratio of CD45 antibody:CD3 antibody:CD20 antibody:CD16 antibody:HLA-DR antibody:CD123 antibody in the antibody combination is (1-2):(90-110):(40-60):(90-110):(90-110):(20-30).

[0027] In some implementations, the mass ratio of CD45 antibody:CD3 antibody:CD20 antibody:CD16 antibody:HLA-DR antibody:CD123 antibody is 1.57:100:50:100:100:25.

[0028] In some implementations, the concentrations of the antibodies in the antibody combination are as follows: CD45 antibody: 1.575 μg / mL; CD3 antibody: 100.000 μg / mL; CD16 antibody: 100.000 μg / mL; CD20 antibody: 50.000 μg / mL; CD123 antibody: 25.000 μg / mL; and HLA-DR antibody: 100.000 μg / mL.

[0029] Another aspect of the present invention provides a staining mixture comprising the antibody combination provided by the present invention and FVS780 working solution.

[0030] In some embodiments, the volume ratio of the antibody combination to the FVS780 working solution is (20-25):(70-90), for example, 23:77.

[0031] Another aspect of the present invention provides a kit for CD123+ dendritic cell typing, the kit comprising the antibody combination provided by the present invention or the staining mixture provided by the present invention.

[0032] Another aspect of the present invention provides a method for CD123+ dendritic cell typing, the method comprising labeling the surface of cells to be sorted using the antibody combination provided by the present invention or the staining mixture provided by the present invention, and then sorting the identified cells.

[0033] In some implementations, the cells to be sorted are derived from PBMC samples.

[0034] In some implementations, the method includes the following steps:

[0035] 1) Add Fc receptor blocking agent to the cells to be sorted;

[0036] 2) Use the antibody combination or the staining mixture provided by the present invention to stain and label the surface of the cells to be sorted obtained in step 1);

[0037] 3) Wash and centrifuge the cells obtained in step 2); and,

[0038] 4) The cells obtained in step 3) are sorted using a flow cytometer.

[0039] In some implementations, the method first obtains single cells during machine sorting, then obtains live single cells, then identifies antigen-presenting cells, and finally obtains CD123+ dendritic cells.

[0040] In some implementations, the sorting is performed using flow cytometry, with the following gate logic applied to the flow cytometry data during sorting:

[0041] a'. Using All Events as the parent group, delineate the white blood cell population in the flow cytometry plot;

[0042] b'. Using the above cells as the parent population, delineate the single-cell population;

[0043] c'. Delineate the viable cell population using single cells as the parent population;

[0044] d. Using live cells as the parent population, delineate cells that are CD45+ and CD3-;

[0045] e'. Using CD45+CD3- cells as the parent population, delineate cells that are both CD16- and CD20-; and,

[0046] f'. Using CD16-CD20- cells as the parent population, the final delineation of CD123+ and HLA-DR+ cells is the CD123+ dendritic cell population.

[0047] In this article, "CD45+" means CD45 positive and "CD3-" means CD3 negative.

[0048] In some implementations, the method uses the following gate logic:

[0049] a'. Using All Events as the parent group, delineate the white blood cell population in the FSC-A-SSC-A diagram;

[0050] b'. Using the white blood cell population as the parent population, delineate the single cell population in the FSC-A-FSC-H diagram;

[0051] c'. Using the single-cell population as the parent population, delineate the viable cell population in the FVS780-A-SSC-A diagram;

[0052] d'. Using the live cell population as the parent population, delineate CD45+ and CD3- cells in the CD45 APC-A–CD3 FITC-A diagram;

[0053] e'. Using the CD45+CD3- cell population as the parent population, delineate CD16- and CD20- cells in the CD16 BV711-A–CD20 PerCP-Cy5.5-A diagram; and,

[0054] f'. Using the CD16-CD20- cell population as the parent population, the cells that are CD123+ and HLA-DR+ are finally delineated in the CD123 PE-Cy7-A–HLA-DR BV786-A diagram, which are the CD123+ dendritic cell population.

[0055] In some implementations, the method uses the following gate logic:

[0056] a'. Using ALL Events as the parent group, delineate the location of white blood cells in the FSC-A - SSC-A scatter plot using the "WBC" gate;

[0057] b'. Using WBC as the parent group, delineate the location of individual cells in the FSC-A - FSC-H scatter plot using the "Singlets" gate;

[0058] c'. Using Singlets as the parent group, circle the location of live cells in the FVS780–SSC-A scatter plot with the "Live" gate;

[0059] d'. Using Live as the parent population, in the CD45 APC-A–CD3 FITC-A scatter plot, cells that are CD45 positive and CD3 negative are delineated by the "CD45+CD3-" gate;

[0060] e'. Using CD45+CD3- as the parent population, in the CD16 BV711-A–CD20 PerCP-Cy5.5 scatter plot, cells that are both CD16- and CD20- are delineated by the "CD16-CD20-" gate;

[0061] f'. Using CD16-CD20- as the parent population, in the CD123 PE-Cy7–HLA-DR BV786-A scatter plot, cells that are both CD123-positive and HLA-DR-positive are identified by the "CD123+HLA-DR+" gate, which is the CD123+ dendritic (pDC) cell population.

[0062] First, non-target cells are excluded. CD45+ can identify all white blood cells (such as lymphocytes, monocytes, granulocytes, etc.), while the combination of CD3-, CD20-, and CD16- is used to exclude T cells, B cells, NK cells, neutrophils, etc., retaining other white blood cell populations, such as monocytes and dendritic cell subsets. Next, antigen-presenting cells (APCs) are screened. HLA-DR+ can identify and enrich APC populations such as dendritic cells and monocytes. Finally, pDCs are specifically labeled. pDCs highly express CD123, and excessive activation of pDCs is associated with autoimmune diseases such as lupus erythematosus.

[0063] In some implementations, the method satisfies one or more of the following:

[0064] 1) Step 1) is preceded by a step of staining the sample using an FVS780;

[0065] 2) Step 1) After adding the Fc receptor blocking agent, an incubation step is also included;

[0066] 3) Step 2, after adding the above antibody combination or the above staining mixture, also includes an incubation step; and,

[0067] 4) Step 4) Before sorting on the machine, there is also a step of fixing using PFA.

[0068] In some implementations, the cells to be sorted in step 1) of the method are derived from PBMC samples and obtained through the following steps:

[0069] a-1. Collect whole blood and PBS (Ca2+-free) 2+ Mg 2+ (Ions) are diluted and mixed in a certain proportion;

[0070] b-2. Add an appropriate amount of sample density separation solution to a 15 mL high-performance centrifuge tube and centrifuge;

[0071] c-3. Slowly add the diluted whole blood from step a-1 to a high-performance centrifuge tube (containing sample density separation solution) and centrifuge;

[0072] d-4. Transfer the supernatant (including the cloud layer) from each tube to a newly labeled 15 mL centrifuge tube, and gently invert it 3-5 times to obtain the PBMC sample;

[0073] e-5. Add PBS to the newly labeled 15 mL centrifuge tube to bring the volume to 10 mL, and centrifuge at either speed (both rising and falling); and,

[0074] f-6. Discard the supernatant, add 1 mL of cryopreservation solution, resuspend and mix well, then count. If needed for testing, store in a cryopreservation box at -80℃.

[0075] In some implementations, the centrifugation in step b-2. is performed at a speed of 1000-2000 rpm, a temperature of 10-30°C, and for 1-5 minutes.

[0076] In some implementations, the centrifugation speed in step c-3 is 1000-3000 rpm, the temperature is 20°C-25°C, the acceleration / deceleration rate is 6:1, and the centrifugation time is 30-60 min.

[0077] In some implementations, the centrifugation in step e-5. is performed at 9 rpm for both upward and downward acceleration, at 1000-3000 rpm, at a temperature of 10-30°C, for 5-20 minutes.

[0078] In some implementations, step 1) is preceded by a step of staining the sample using the FVS780 via the following steps:

[0079] a) Live / dead staining: Take a PBMC sample, add 100µL of FVS780 working solution (BD), mix well by pipetting, and incubate at 2~8°C in the dark for 10 min;

[0080] b) Centrifugation: Centrifugation conditions: temperature 4℃, speed 500×g, time 5 min, discard supernatant; and...

[0081] c) Centrifugation after washing: Add 600 µL of 1×PBS (pre-cooled) to wash, mix well, and centrifuge under the following conditions: temperature 4℃, speed 500×g, time 5 min. Discard the supernatant.

[0082] In some implementation schemes, step 1) of adding the cells to be sorted to the Fc receptor blocking agent involves the following steps:

[0083] Add 50 µL of Fc receptor blocking agent to each well, mix well by pipetting, and incubate at room temperature in the dark for 10 min.

[0084] In some implementation schemes, the specific steps of step 2) are as follows:

[0085] Add 100 µL of staining mixture (CD45 antibody, CD3 antibody, CD20 antibody, CD16 antibody, HLA-DR antibody, CD123 antibody) to each well, mix well by pipetting, and incubate at 2-8°C in the dark for 30 min ± 5 min.

[0086] In some implementations, the CD45 antibody fluorescein is APC, the CD3 antibody fluorescein is FITC, the CD20 antibody fluorescein is PerCP-Cy5.5, the CD16 antibody fluorescein is BV711, the HLA-DR antibody fluorescein is BV786, and the CD123 antibody fluorescein is PE-Cy7.

[0087] In some implementation schemes, the specific steps of step 3) are as follows:

[0088] After washing, centrifuge: Add 600 µL of 1×PBS (pre-cooled) to wash, mix well, and centrifuge under the following conditions: temperature 4℃, speed 500×g, time 5 min. Discard the supernatant. Repeat twice.

[0089] In some implementation schemes, 300 μL of 1×PBS (pre-cooled) is added to the sample before sorting and resuspended, then the sample is shaken to mix.

[0090] In some implementations, flow cytometry is used for sorting, with the flow cytometry parameters set to distinguish positive cell populations: FITC voltage set to 400-500, PE voltage set to 400-500, PerCP-Cy5.5 voltage set to 550-650, PE-Cy7 voltage set to 550-650, APC voltage set to 300-400, BV711 voltage set to 400-500, BV786 voltage set to 500-600, and FVS780 voltage set to 700-800.

[0091] In some implementations, the cells to be sorted are derived from PBMC samples obtained from whole blood selected from human and non-human primates, such as cynomolgus monkeys, rhesus monkeys, or macaques.

[0092] In some implementations, the settings of the flow sorter in step 4) of the method satisfy one or more of the following:

[0093] 1) The forward scattered light voltage value is set to 270-275;

[0094] 2) The side-scattered light voltage value is set to 455-460;

[0095] 3) The FITC voltage value is set to 475-485;

[0096] 4) The voltage value of PE is set to 485-490;

[0097] 5) The voltage value of PerCP-Cy5.5 is set to 593-570;

[0098] 6) The voltage value of PE-Cy7 is set to 588-592;

[0099] 7) Set the voltage value of APC to 355-360;

[0100] 8) The voltage value of BV711 is set to 485-490;

[0101] 9) The voltage value of BV786 is set to 533-540; and,

[0102] 10) The voltage value of FVS780 is set to 753-759.

[0103] In some implementations, the voltage values ​​are set as follows: FITC 479.3, PE 488.2, PerCP-Cy5.5 696.3, PE-Cy7 590.3, APC 357.7, BV711 487.0, BV786 536.8, and FVS780 755.5.

[0104] This invention has developed a kit for typing CD123+ dendritic cells in monkey peripheral blood mononuclear cells by optimizing experimental conditions, and determined the antibody combination, providing a reference for the establishment of related methods.

[0105] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0106] The reagents and raw materials used in this invention are all commercially available.

[0107] The positive and progressive effects of this invention are as follows: a kit for typing CD123+ dendritic cells in peripheral blood mononuclear cells has been developed, which significantly improves the number and stability of pDC cells collected. At the same time, by counting the extracted PBMCs and then loading them for detection, the proportion of pDC cells in different animals can be compared. On the one hand, this lays the foundation for the efficacy evaluation of non-clinical and clinically relevant drugs, and on the other hand, it can establish background data for disease diagnosis. Attached Figure Description

[0108] Figure 1 Flow cytometry for FITC Mouse Anti-NHP CD45 single-stain tube.

[0109] Figure 2 Flow cytometry for a single-staining tube of BV605 Mouse Anti-Human CD3.

[0110] Figure 3 Flow cytometry for a single-staining tube of BV786 Mouse Anti-Human CD20.

[0111] Figure 4 Flow cytometry plot of BV510 Mouse Anti-Human CD16 single-stain tube.

[0112] Figure 5Flow cytometry for AF647 Mouse Anti-Human HLA-DR single-stain tube.

[0113] Figure 6 The flow cytometry plot for BV421 Mouse Anti-Human CD123 single-stain tube is shown.

[0114] Figure 7 This is voltage gain data for different channels.

[0115] Figure 8 Compensation data for different channels.

[0116] Figure 9 Flow cytometry plot of CD123+ dendritic cells in a fresh whole blood sample.

[0117] Figure 10 Flow cytometry plot for CD123+ dendritic cell typing in PBMC samples. Detailed Implementation

[0118] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0119] Antibody information:

[0120] FITC Mouse Anti-Human CD3 (BD, 556611);

[0121] APC Mouse Anti-NHP CD45 (BD, 561290);

[0122] BV711 Mouse Anti-Human CD16 (BD, 563127);

[0123] PerCP-Cy5.5 Mouse Anti-Human CD20 (BD, 560736);

[0124] PE-Cy7 Mouse Anti-Human CD123 (BD, 560826);

[0125] BV786 Mouse Anti-Human HLA-DR (BD, 564041);

[0126] PBMC Extraction

[0127] (1) Invert and mix the sample, collect 10 mL of fresh monkey whole blood (Guangxi Xiongsen Primate Experimental Animal Breeding and Development Co., Ltd.), and dilute the whole blood with PBS at a ratio of 1:1.

[0128] (2) Add 4 mL of sample density separation solution (Haoyang Huake, catalog number LTS10770125) to a 15 mL high-efficiency centrifuge tube and centrifuge at 1500 rpm and 20℃ for 1 min.

[0129] (3) Take the diluted whole blood from step (1) and slowly add it to the high-efficiency centrifuge tube (containing sample density separation solution) from step (2). The volume of the diluted whole blood added should be consistent with the volume of the sample density separation solution from step (2).

[0130] (4) Centrifugation conditions: 2200 rpm, 20℃~25℃, 6 increments and 1 decrement, centrifuge for 30 min.

[0131] (5) Aspirate the supernatant (including the cloud layer) from each tube into a marked 15mL centrifuge tube, and gently invert it 3 to 5 times to obtain the PBMC sample.

[0132] Example 1: Different antibody titrations, channel voltages, compensation, and gating confirmation

[0133] 1. Experimental Procedure

[0134] 1.1. Target cell staining treatment:

[0135] a) Live / dead staining: Take a PBMC sample, add 100µL of FVS780 working solution (BD, 1:900, v / v), mix well by pipetting, and incubate at 2~8°C in the dark for 10 min;

[0136] b) Centrifugation: Centrifugation conditions: temperature 4℃, speed 500×g, time 5 min, discard supernatant.

[0137] c) Centrifugation after washing: Add 600 µL of 1×PBS (pre-cooled) to wash, mix well, and centrifuge under the following conditions: temperature 4℃, speed 500×g, time 5 min. Discard the supernatant.

[0138] d) Blocking: Add 50µL of Fc receptor blocking agent solution (BD PHARMINGEN, catalog number 564220) to each well, mix well by pipetting, and incubate at 2-8°C in the dark for 10min±3min.

[0139] e). Staining: Add 100 µL of total volume (total volume includes antibody volume + PBS dilution volume) of CD45 antibody staining solution, CD3 antibody staining solution, CD20 antibody staining solution, CD16 antibody staining solution, HLA-DR antibody staining solution and CD123 antibody staining solution to each well according to the concentrations in Table 1-6. Mix well by pipetting and incubate at 2-8°C in the dark for 30 min ± 5 min.

[0140] f) Centrifugation after washing: Add 600 µL of 1×PBS (pre-cooled) to wash, mix well, and centrifuge under the following conditions: temperature 4℃, speed 500×g, time 5 min. Discard the supernatant. Repeat twice.

[0141] g). Instrumental testing: Add 300 μL of 1×PBS (pre-cooled) to resuspend and vortex to mix.

[0142] 1.2 Flow cytometry analysis: The flow cytometry parameters were set to distinguish positive cell populations.

[0143] 2. Experimental Results

[0144] 2.1 Antibody titration results

[0145] According to the results in Tables 1 to 6, the optimal concentrations for CD45 antibody, CD3 antibody, CD16 antibody, CD20 antibody, CD123 antibody, and HLA-DR antibody are 1.575 μg / mL, 100.000 μg / mL, 100.000 μg / mL, 60.000 μg / mL, 30.000 μg / mL, and 100.000 μg / mL, respectively.

[0146] Table 1. Titration results of APC Mouse Anti-NHP CD45 antibody

[0147]

[0148] Table 2. Results of FITC Mouse Anti-Human CD3ε Antibody Titration

[0149]

[0150] Table 3. Titration results of BV711 Mouse Anti-Human CD16 antibody

[0151]

[0152] Table 4. Results of PerCP-Cy™ 5.5 Mouse Anti-Human CD20 Antibody Titration

[0153]

[0154] Table 5. Titration results of PE-Cy™7 Mouse Anti-Human CD123 antibody

[0155]

[0156] Table 6. Titration results of BV786 Mouse Anti-Human HLA-DR antibody

[0157]

[0158] 2.2 Confirmation of different channel voltages, compensation, and gate settings

[0159] Use the optimal antibody concentration described above for single-stain tube detection. Figures 1 to 6 The flow cytometry results for single-staining tubes with different antibodies are shown in the figure. The target cell population is entirely within the gating zone. Optimal voltage compensation is as follows: Figure 7 and Figure 8 As shown.

[0160] Example 2: Comparison of techniques for CD123+ dendritic cell typing in whole monkey blood and extracted peripheral blood mononuclear cells.

[0161] 1. Experimental Procedure

[0162] 1.1 PBMC Extraction

[0163] (1) Invert and mix the sample, collect 10 mL of fresh monkey whole blood (Guangxi Xiongsen Primate Experimental Animal Breeding and Development Co., Ltd.), and dilute the whole blood with PBS at a ratio of 1:1.

[0164] (2) Add 4 mL of sample density separation solution (Haoyang Huake, catalog number LTS10770125) to a 15 mL high-efficiency centrifuge tube and centrifuge at 1500 rpm and 20℃ for 1 min.

[0165] (3) Take the diluted whole blood from step (1) and slowly add it to the high-efficiency centrifuge tube (containing sample density separation solution) from step (2). The volume of the diluted whole blood added should be consistent with the volume of the sample density separation solution from step (2).

[0166] (4) Centrifugation conditions: 2200 rpm, 20℃~25℃, 6 increments and 1 decrement, centrifuge for 30 min.

[0167] (5) Aspirate the supernatant (including the cloud layer) from each tube into a marked 15 mL centrifuge tube, and gently invert it 3 to 5 times to obtain the PBMC sample.

[0168] 1.2 Flow cytometry analysis

[0169] (1) Staining: Take 100 μL of counted PBMC samples or whole blood samples of monkeys after erythropoiesis, add 100 µL of FVS780 working solution, mix by pipetting, and incubate at 2~8°C in the dark for 10 min.

[0170] (2) After washing, centrifuge: Add 1000 µL of 1×PBS (pre-cooled) to wash, mix well, and centrifuge under the following conditions: temperature 4℃, speed 500×g, time 5 min. Discard the supernatant.

[0171] (3) Add 50 µL of Fc receptor blocking agent (BD PHARMINGEN, catalog number 564220) to each well, mix well by pipetting, and incubate at room temperature in the dark for 10 min.

[0172] (4) Staining: Add 100 µL of staining mixture to each well (the staining mixture is prepared according to the ratio of 5 µL FITC Mouse Anti-Human CD3 (BD, 556611) + 5 µL APC Mouse Anti-NHP CD45 (BD, 561290) + 5 µL BV711 Mouse Anti-Human CD16 (BD, 563127) + 1.5 µL PerCP-Cy™5.5 Mouse Anti-Human CD20 (BD, 560736) + 1.5 µL PE-Cy™7 Mouse Anti-Human CD123 (BD, 560826) + 5 µL BV786 Mouse Anti-Human HLA-DR (BD, 564041) + 77 µL Fixable Viability Stain 780 working solution, and use immediately after preparation). After mixing by pipetting, incubate at 2-8°C in the dark for 30 min ± 5 min.

[0173] (5) After washing, centrifuge: Add 1000 µL of 1×PBS (pre-cooled) to wash, mix well, and centrifuge under the following conditions: temperature 4℃, speed 500×g, time 5 min. Discard the supernatant.

[0174] (6) Fixation: Add 1000 μL of 2% PFA to each flow cytometer tube and incubate at 2-8℃ for 20 min. Centrifugation conditions: 4℃, speed 500×g, time 5 min. Remove the centrifuged sample, discard the supernatant, add 1000 μL of 1×PBS (pre-cooled), and mix well. Centrifugation conditions: 4℃, speed 500×g, time 5 min.

[0175] (7) Detection on the instrument: Add 300 μL of 1×PBS (pre-cooled) to resuspend and shake to mix.

[0176] 2. Experimental Results

[0177] according to Figure 9 and Figure 10 The flow cytometry results indicate that this method clearly distinguishes cell populations. However, whole blood samples showed a smaller number of CD123+ dendritic cells, and these small cell populations may be misclassified as false negatives due to errors or operational procedures, hindering genotyping. PBMC samples, on the other hand, showed a significant number of CD123+ dendritic cells. Furthermore, because cell counting was performed during PBMC sample loading, the number of CD123+ dendritic cells obtained from PBMC samples with the same cell count is more meaningful than that obtained from whole blood samples with an unknown cell count. This allows for the later detection of CD123+ dendritic cell counts based on a large number of normal individuals' PBMC samples to obtain background data, laying the foundation for subsequent drug evaluation or diagnosis of abnormal individuals.

[0178] Example 3: Comparison of stability of CD123+ dendritic cell typing samples from whole monkey blood and extracted peripheral blood mononuclear cells.

[0179] Fresh monkey whole blood samples and fresh peripheral blood mononuclear cell samples extracted from fresh monkey whole blood were tested using the detection steps in Example 2 as 0h data. Each sample was tested in triplicate. The remaining monkey whole blood samples were divided into 3 aliquots and used to detect the data at 6h, 24h and 48h after being placed in the same 2-8℃ refrigerator. The remaining fresh peripheral blood mononuclear cell samples extracted from fresh monkey whole blood were divided into 4 aliquots and used to detect the data at 6h, 24h, 48h and 72h after being frozen in liquid nitrogen. The stability results are shown in Tables 7 and 8.

[0180] Table 7: Results of CD123+ dendritic cell stability in whole blood of monkeys

[0181]

[0182] Table 8: Results of CD123+ dendritic cell stability in monkey PBMCs

[0183]

[0184] The results showed that CD123+ dendritic cells in monkey whole blood were stable for 6 hours at 2-8℃, and their stability did not pass the test after 24 hours, with a CV% greater than 25%. CD123+ dendritic cells in monkey PBMCs were stable for 72 hours under liquid nitrogen cryopreservation conditions, which greatly improved their stability.

[0185] The specific embodiments described above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several improvements or substitutions without departing from the principle of the present invention, and these improvements or substitutions should also be considered within the scope of protection of the present invention.

Claims

1. An antibody combination for use in CD123+ dendritic cell typed, characterized in that, The antibodies in the antibody combination consist of CD45 antibody, CD3 antibody, CD20 antibody, CD16 antibody, HLA-DR antibody and CD123 antibody.

2. The antibody combination of claim 1, wherein The antibodies in the antibody combination are fluorescein-labeled antibodies, the fluorescein in the fluorescein-labeled antibodies is selected from FITC, PE, APC, PE-Cy5, PE-Cy7, APC-Cy7, PerCP-Cy5.5, Alexa Fluor 488, Alexa Fluor 647, Cy5, Cy7, BV421, BV605, BV786 and BV711; different antibodies are labeled with different fluoresceins, so that cells bound by the antibodies can be separated by flow cytometry in a single sorting step; Preferably, the antibody combination meets one or more of the following: 1) the CD45 antibody is an APC-labeled antibody; 2) the CD3 antibody is a FITC-labeled antibody; 3) the CD20 antibody is a PerCP-Cy5.5-labeled antibody; 4) the CD16 antibody is a BV711-labeled antibody; 5) the HLA-DR antibody is a BV786-labeled antibody; and, 6) the CD123 antibody is a PE-Cy7-labeled antibody.

3. The antibody combination of claim 1 or 2, wherein The antibody combination meets one or more of the following: 1) the CD45 antibody is 561290 of BD Biosciences; 2) the CD3 antibody is 556611 of BD Biosciences; 3) the CD20 antibody is 560736 of BD Biosciences; 4) the CD16 antibody is 563127 of BD Biosciences; 5) the HLA-DR antibody is 564041 of BD Biosciences; and, 6) the CD123 antibody is 560826 of BD Biosciences.

4. The antibody combination according to any one of claims 1 to 3, wherein The mass ratio of CD45 antibody:CD3 antibody:CD20 antibody:CD16 antibody:HLA-DR antibody:CD123 antibody in the antibody combination is (1-2):(90-110):(40-60):(90-110):(90-110):(20-30); Preferably, the mass ratio of CD45 antibody:CD3 antibody:CD20 antibody:CD16 antibody:HLA-DR antibody:CD123 antibody is 1.57:100:50:100:100:25; More preferably, the concentration of each antibody in the antibody combination is CD45 antibody: 1.575 μg / mL; CD3 antibody: 100.000 μg / mL; CD16 antibody: 100.000 μg / mL; CD20 antibody: 50.000 μg / mL; CD123 antibody: 25.000 μg / mL and HLA-DR antibody: 100.000 μg / mL, respectively.

5. A dyeing mixture, characterized by The staining mixture comprises the antibody combination according to any one of claims 1-4, and FVS780 working solution; The volume ratio of the antibody combination to FVS780 working solution is (20-25):(70-90), for example 23:

77.

6. A kit for CD123+ dendritic cell typed, characterized in that, The kit comprises the antibody combination according to any one of claims 1-4 or the staining mixture according to claim 5.

7. A method of CD 123+ dendritic cell typing, characterized by, The method comprises using the antibody combination according to any one of claims 1-4 or the staining mixture according to claim 5 to label the surface of the cells to be sorted, and then sorting the identified cells; the cells to be sorted are preferably derived from a PBMC sample; Preferably, the method comprises the following steps: 1) adding the cells to be sorted to an Fc receptor blocking agent; 2) using the antibody combination according to any one of claims 1-4 or the staining mixture according to claim 5 to label the surface of the cells to be sorted obtained in step 1); 3) washing and centrifuging the cells obtained in step 2); and 4) sorting the cells obtained in step 3) on a machine, preferably using a flow cytometer.

8. The method of claim 7, wherein, The method first acquires single cells, then acquires live single cells, then identifies antigen presenting cells, and finally acquires CD123+ dendritic cells when sorting on a machine; Preferably, the sorting is performed using a flow cytometer, and the following circle gate logic is used for the flow cytogram when sorting: a’. Circle the white blood cell group in the flow cytogram with All Events as the parent group; b’. Circle the single cell group with the above cells as the parent group; c’. Circle the live cell group with single cells as the parent group; d. Circle the CD45+ and CD3- cells with live cells as the parent group; e’. Circle the CD16- and CD20- cells with CD45+ and CD3- cells as the parent group; and f’. Finally, circle the CD123+ and HLA-DR+ cells with CD16- and CD20- cells as the parent group, which are the CD123+ dendritic cell group; More preferably, the method uses the following circle gate logic: a’. Circle the white blood cell group in the FSC-A-SSC-A graph with All Events as the parent group; b’. Circle the single cell group in the FSC-A-FSC-H graph with the white blood cell group as the parent group; c’. Circle the live cell group in the FVS780-A-SSC-A graph with the single cell group as the parent group; d’. Circle the CD45+ and CD3- cells in the CD45 APC-A-CD3 FITC-A graph with the live cell group as the parent group; e’. Circle the CD16- and CD20- cells in the CD16 BV711-A-CD20 PerCP-Cy5.5-A graph with the CD45+ and CD3- cell group as the parent group; and f’. Finally, circle the CD123+ and HLA-DR+ cells in the CD123 PE-Cy7-A-HLA-DR BV786-A graph with the CD16- and CD20- cell group as the parent group, which are the CD123+ dendritic cell group.

9. The method of claim 7 or 8, wherein, The method meets one or more of the following: 1) Before step 1), the method further comprises a step of staining the sample with FVS780; 2) After step 1), the method further comprises a step of incubation after adding the Fc receptor blocking agent; 3) After step 2), the method further comprises a step of incubation after adding the antibody combination or the staining cocktail; and 4) Before step 4), the method further comprises a step of fixation using PFA.

10. The method according to any one of claims 7 to 9, characterized in that, The settings of the flow cytometer in step 4) of the method satisfy one or more of the following: 1) The forward scatter voltage is set to 270-275; 2) The side scatter voltage is set to 455-460; 3) The FITC voltage is set to 475-485; 4) The PE voltage is set to 485-490; 5) The PerCP-Cy5.5 voltage is set to 593-570; 6) The PE-Cy7 voltage is set to 588-592; 7) The APC voltage is set to 355-360; 8) The BV711 voltage is set to 485-490; 9) The BV786 voltage is set to 533-540; and 10) The FVS780 voltage is set to 753-759.

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