A method and a kit for detecting the biological activity of human immunoglobulin antibody-dependent cell-mediated phagocytosis

Abstract

The application provides a detection method and a detection kit for biological activity of human immunoglobulin antibody-dependent cell-mediated phagocytosis, and belongs to the technical field of biological detection. The application uses hPBMC-induced hMDMs as effector cells, uses Sp2 / 0-Ag14 cells expressing alpha-Gal antigen as target cells, and realizes accurate detection of the biological activity of immunoglobulin products ADCP under a suitable effector-target ratio and a concentration gradient of a test product. The detection method is intuitive, has high precision and accuracy, fills the blank of the current ADCP detection of immunoglobulin products, and has important significance for improving the quality control and clinical application of immunoglobulin products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to a method for detecting biological activity of human immunoglobulin antibody-dependent cell-mediated phagocytosis and a detection kit. BACKGROUND

[0002] Human immunoglobulin products are high-concentration human immunoglobulin preparations prepared from a large number of mixed raw plasma of healthy people through steps such as separation and purification, virus inactivation / removal, etc. The mainstream human immunoglobulin products on the market are divided into two types: intravenous human immunoglobulin (Human Immunoglobulin (pH4) for Intravenous Injection, hereinafter referred to as IVIG) and subcutaneous injection of human immunoglobulin (Subcutaneous injection of human immunoglobulin, hereinafter referred to as SCIG). The main component of both is immunoglobulin G (Immunoglobulin G, IgG) secreted and expressed by B lymphocytes, including IgG1, IgG2, IgG3 and IgG4 four subtypes, containing a broad spectrum of resistance. It is clinically used for the treatment of various primary and secondary immunoglobulin deficiency diseases, infectious diseases, inflammatory diseases and autoimmune diseases. Studies have shown that human immunoglobulin products contain two main functional regions: Fab segment for binding antigen and Fc segment for initiating human body effect. The Fc segment binds to immune cell surface Fc gamma receptors (Fc gamma RI, Fc gamma RIIa, Fc gamma RIIb, Fc gamma RIIc, Fc gamma RIIIa and Fc gamma RIIIb), FcRn receptors and complements, and then exerts corresponding immune responses, such as antibody-dependent cell-mediated cytotoxicity (Antibody dependent cell-mediated cytotoxicity, ADCC), antibody-dependent cell-mediated phagocytosis (Antibody dependent cellular phagocytosis, ADCP), complement-dependent cytotoxicity (Complement dependent cytotoxicity, CDC), etc.

[0003] At present, the quality standard of the biological activity of IgG Fc segment in the current "Chinese Pharmacopoeia" and "European Pharmacopoeia" is only one CDC detection, which can only reflect the activation ability of Fc segment and complement. In addition, the Chinese invention patent with publication number CN115537448A discloses a method for detecting antibody-dependent cell-mediated cytotoxicity of intravenous human immunoglobulin, which fills the blank of ADCC detection of IVIG at present. However, the research on the detection method of the biological activity of ADCC of human immunoglobulin product is still limited, and no relevant patents or quality standards have been found. The Chinese invention patent with publication number CN113186167A discloses a method for determining the biological activity of ADCP of anti-CD20 monoclonal antibody drug. The method constructs stable expression CD32a-FcεRIγ fusion protein receptor and effector cell Jurkat / NFAT / CD32a-FcεRIγ expressing luciferase driven by NFAT response element, uses Raji cell as target cell, and evaluates ADCP activity by detecting the expression level of reporter gene (such as luciferase activity or fluorescence intensity). Although the method is simple to operate and has a short test period, the transfection efficiency of effector cells may affect the experimental conditions, and the expression of reporter gene may be affected by other factors, and there is interference of background signal. In addition, the method depends on engineered cells, and the level of activated reporter gene in the signal pathway is detected to characterize the ADCP biological activity of the antibody, which cannot fully characterize the ADCP biological activity of the antibody in human body.

[0004] In addition, flow cytometry detection method is also a commonly used method for detecting ADCP. This method can directly use cells from human body as effector cells to detect the biological activity of ADCP. Compared with the reporter gene method, the effector cells from human body can more accurately simulate the in vivo immune response process, so as to reflect the actual biological effect of antibody drug; and the phagocytosis of effector cells on target cells can be directly observed, and the detection result is more intuitive. However, there is no report on the use of flow cytometry detection method for ADCP detection of human immunoglobulin products. Human immunoglobulin products contain a wide range of antibodies against pathogens and heterologous species proteins, and the detection of various parameters will affect the precision and accuracy of the detection. How to obtain an ADCP detection method with good precision and accuracy needs further research. SUMMARY

[0005] The purpose of the present application is to provide a detection method and kit for antibody-dependent cell-mediated phagocytosis biological activity of human immunoglobulin.

[0006] The present application provides a detection method for the ADCP activity of human immunoglobulin, which comprises the following steps:

[0007] (1) preparing fluorescently labeled Sp2 / 0-Ag14 cells expressing α-Gal antigen as target cells;

[0008] (2) diluting human immunoglobulin to an initial working concentration, and then performing gradient dilution;

[0009] (3) adding the human immunoglobulin after dilution in step (2) to the target cells for incubation;

[0010] (4) adding effector cells to the system after incubation in step (3) according to an effector-to-target ratio to obtain a final reaction system, and incubating;

[0011] (5) collecting cells after incubation in step (4), adding flow detection antibodies for incubation, and staining the effector cells;

[0012] (6) collecting cells after staining in step (5), detecting using a flow cytometer, and determining the ADCP activity of the human immunoglobulin using the detection data.

[0013] Further, in step (1), the fluorescent labeling is PKH26 fluorescent labeling.

[0014] And / or, in step (3), the temperature for incubation is 25-37°C, and the time for incubation is 30-60 min.

[0015] And / or, in step (4), the effector cells are macrophages.

[0016] Further, in step (4), the macrophages are macrophages induced from monocytes in human primary peripheral blood mononuclear cells.

[0017] Further, in step (4), the effector-to-target ratio of the effector cells and the target cells is 3:1-5:1.

[0018] Further, in step (4), the effector-to-target ratio of the effector cells and the target cells is 5:1.

[0019] Further, in step (4), in the final reaction system, the initial concentration of the human immunoglobulin is 20 mg / ml, and there are 8 concentration points in a 4-fold dilution gradient.

[0020] And / or, in step (4), in the final reaction system, the number of target cells is 200 cells / μl.

[0021] And / or, in step (4), in the final reaction system, RPMI 1640 culture medium containing 10% fetal bovine serum is used as an experimental buffer for dilution.

[0022] Further, in step (4), the temperature of the incubation is 25-37℃, and the time of the incubation is 1-5h.

[0023] Further, in step (5), the flow detection antibody is a CD11b antibody.

[0024] Further, in step (5), the incubation is light-avoiding incubation, the temperature of the incubation is 0-4℃, and the time of the incubation is 15-30min.

[0025] Further, in step (6), when the detection is performed, the target cells are detected through a PE fluorescence channel, and the effector cells are detected through an APC fluorescence channel.

[0026] The present application also provides a kit for determining the ADCP activity of human immunoglobulin, which comprises human immunoglobulin, fluorescently labeled target cells, effector cells, a flow detection antibody, and an experimental buffer.

[0027] Further,

[0028] The fluorescently labeled target cells are PKH26 fluorescently labeled target cells.

[0029] Further, the effector cells are macrophages.

[0030] Further, the flow detection antibody is a CD11b antibody.

[0031] Further, the experimental buffer is RPMI 1640 culture medium containing 10% fetal bovine serum.

[0032] Preferably, the macrophages are macrophages induced from monocytes in human primary peripheral blood mononuclear cells.

[0033] In the present application, monocytes are sorted from hPBMCs and induced into macrophages (human Monocytes-Derived Macrophages, hMDMs) as effector cells, Sp2 / 0-Ag14 cells expressing α-Gal antigen are used as target cells, and the effector cells and the target cells are labeled with two different fluorescences respectively, and the effector cells will present double-positive fluorescence signals after phagocytizing the target cells; the proportion of the number of cells presenting double-positive fluorescence signals to the total number of target cells can represent the ADCP activity of the antibody.

[0034] In the present application, the effector-target ratio refers to the number ratio of effector cells (Effector Cells) to target cells (Target Cells).

[0035] The present application has the following beneficial effects:

[0036] The present invention provides an ADCP detection method for human immunoglobulin products. The method adopts Sp2 / 0-Ag14 cells as target cells, utilizes monocytes sorted from human PBMCs, and uses human monocyte-derived macrophages (hMDMs) as effector cells. The method accurately detects the ADCP biological activity of human immunoglobulin products under an appropriate effector-target ratio (5:1) and a test sample concentration gradient (the starting concentration of SCIG is set to 20 mg / ml, with a 4-fold dilution gradient and 8 concentration points). The detection method of the present invention is intuitive to operate, has good specificity, and is highly precise and accurate. It fills a gap in the current ADCP detection of immunoglobulin products and is of great significance for improving the quality control and clinical application of immunoglobulin products.

[0037] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0038] The following is a further detailed description of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This figure shows the phagocytic rate of effector cells hMDMs on different target cells in the ADCP target cell screening experiment. The data in the figure are expressed as phagocytic rate percentage ± SEM.

[0040] Figure 2 This figure shows the phagocytic rate of Sp2 / 0-Ag14 target cells by effector cells hMDMs at different effector-target ratios in the effector-target ratio optimization experiment. The data in the figure are expressed as phagocytic rate percentage ± SEM.

[0041] Figure 3 This figure shows the phagocytic rate of Sp2 / 0-Ag14 target cells by effector cells hMDMs at an effector-target ratio of 5:1 in the test article initial concentration optimization experiment. The data in the figure are expressed as phagocytic percentage ± SEM.

[0042] Figure 4 This figure shows the precision and accuracy validation results of ADCP activity detection using Sp2 / 0-Ag14 as target cells and hMDMs as effector cells at an effector-target ratio of 5:1. The data are expressed as percentage of phagocytosis ± SEM.

[0043] Figure 5Figure 4 shows the results of the effector cell specificity validation. Data are presented as % phagocytosis ± SEM. DETAILED DESCRIPTION

[0044] The raw materials and equipment used in the present application are known products, which are obtained by purchasing commercially available products.

[0045] The main instruments and equipment used in the present application are shown in Table 1.

[0046] Table 1. Main instruments and equipment

[0047]

[0048] The reagents used in the experiments of the present application are shown in Table 2.

[0049] Table 2. Reagents used in the experiments

[0050]

[0051]

[0052] The cell lines used in the experiments of the present application are shown in Table 3.

[0053] Table 3. Cell lines used in the experiments

[0054]

[0055] The test article information of the present application is shown in Table 4.

[0056] Table 4. Test article information

[0057] Sample Supplier Stock concentration (mg / ml) SCIG Chengdu Rongseng 200

[0058] In the detection of the ADCP activity of the immunoglobulin product by the present inventor, Sp2 / 0-Ag14 cells (ATCC Cell Bank) expressing α-Gal antigen are used as target cells; hPBMCs are used as the starting material of effector cells, and monocytes are sorted from the hPBMCs and induced to become macrophages (human Monocytes-Derived Macrophages, hMDMs), which are effector cells; and a human immunoglobulin SCIG (Chengdu Rongsheng) is used. The ADCP effect is that, after the Fab segment of IgG binds to the antigen on the surface of the target cells, the Fc segment can interact with immune cells (such as macrophages and monocytes) having Fcγ receptors (FcγRIIa and FcγRIa), so as to promote the phagocytosis and clearance of the target cells by the immune cells. This mechanism plays an important role in the immune defense and immune regulation of the body. In the flow detection, two different fluorescences are used to label the effector cells and the target cells respectively; after the effector cells phagocytose the target cells, the effector cells will present a double-positive fluorescence signal, and thus the proportion of the number of cells presenting the double-positive fluorescence signal in the total number of target cells can represent the ADCP activity of the antibody.

[0059] The method of the detection method of the present application is summarized as follows:

[0060] Fourteen days before the experiment, thaw the hPBMCs cells and count, sort the monocytes using the monocyte sorting kit, then culture the monocytes in 1640 complete medium containing 100 ng / ml M-CSF for 14 days to induce the monocytes into hMDMs. One day before the experiment, digest and collect the target cells and label them with PKH26 dye, inoculate into culture dishes and incubate overnight (37°C / 5% CO2). On the day of the experiment, collect the PKH26-labeled target cells and resuspend them in ADCP experimental buffer, and prepare the test sample working solution using the ADCP experimental buffer (if the test sample is SCIG, the starting concentration of SCIG is set to 20 mg / ml, 4-fold dilution gradient, 8 concentration points). Then adjust the target cell density (200 cells / μl) and inoculate into 96-well plates, add the test sample working solution and incubate for 30 minutes. Collect the effector cells (hMDMs) and resuspend them in ADCP experimental buffer, adjust the density according to the effector-to-target ratio, then add them to the 96-well plates and incubate for 1 hour. After incubation, collect all the cells into a new 96-well V-bottom plate and centrifuge to obtain a cell pellet, add CD11b flow detection antibody working solution prepared with flow buffer (1x DPBS + 1% FBS) to the experimental plate to label and stain the effector cells, and incubate at 4°C in the dark for 15 minutes. After incubation, centrifuge to obtain a cell pellet, resuspend with flow buffer and wash twice. Finally, use a flow cytometer for detection; PKH26-labeled target cells are detected in the PE fluorescence channel, and CD11b-stained effector cells are detected in the APC fluorescence channel. The above ADCP experimental buffer is 1640 complete medium (RPMI 1640 + 10% FBS).

[0061] The cell resuscitation and culture involved in the present application are as follows:

[0062] (1) hPBMCs cell resuscitation: First, preheat the complete medium used for the cells, i.e. RPMI 1640 complete medium (RPMI 1640 + 10% FBS), in a 50 ml centrifuge tube, add a certain volume of the corresponding complete medium, then transfer the cell suspension thawed in a 37°C water bath to the aforementioned centrifuge tube, centrifuge at 400g for 10 minutes in a centrifuge, discard the supernatant after centrifugation, resuspend the cells with the corresponding complete medium and count, then inoculate them into a shake flask at an appropriate density or take a certain amount of hPBMCs to sort monocytes for inducing hMDMs.

[0063] (2) PLC / PRF / 5, Sp2 / 0-Ag14 cell recovery: First, preheat the complete medium used for each cell, and add a certain volume of the corresponding complete medium to a 15 ml centrifuge tube. Then, transfer the cell suspension melted in a 37°C water bath to the aforementioned centrifuge tube, and centrifuge at 600 rpm or 800 rpm in a centrifuge for 5 minutes. After centrifugation, discard the supernatant, resuspend the cells with the corresponding complete medium, and then count them. Finally, inoculate them in a cell culture dish (or other consumables that can be used for cell culture) at an appropriate density.

[0064] (3) PLC / PRF / 5, Sp2 / 0-Ag14 cell subculture: First, preheat or restore the complete medium, digestion solution and other reagents used for each cell to room temperature. PLC / PRF / 5 is an adherent cell, so the culture supernatant needs to be discarded, and 1x DPBS is used to rinse 1-3 times. Then, add an appropriate amount of digestion solution and incubate in a cell culture incubator (37°C / 5% CO2) for a certain period of time (when the cells are mostly detached, the digestion is stopped). Finally, collect the cell suspension into a centrifuge tube. Sp2 / 0-Ag14 cells are suspension cells, so they do not need to be digested and can be directly collected into a centrifuge tube. Centrifuge the PLC / PRF / 5 and Sp2 / 0-Ag14 cell suspensions at 600 rpm or 800 rpm in a centrifuge for 5 minutes. After centrifugation, discard the supernatant, resuspend the cells with the corresponding complete medium, and then count them. Finally, inoculate them in a cell culture dish (or other consumables that can be used for cell culture) at an appropriate density.

[0065] Example 1, Detection of the ADCP activity of the human immunoglobulin of the present application

[0066] 1. ADCP experimental procedure

[0067] (1) 14 days before the experiment, hPBMCs cells were recovered, resuspended with RPMI 1640 complete medium (RPMI 1640 + 10% FBS) and counted.

[0068] (2) According to the cell count results, a certain amount of hPBMCs cells were taken, and mononuclear cells were sorted using a mononuclear cell sorting kit.

[0069] (3) The mononuclear cells were continuously cultured for 14 days using RPMI 1640 complete medium (RPMI 1640 + 10% FBS) containing 100 ng / ml concentration of M-CSF to induce the mononuclear cells into macrophages (hMDMs).

[0070] (4) QC of hMDMs or hPBMCs effector cells for evaluation of whether the induced hMDMs are successful: the effector cells are labeled with CD11b, CD14, CD45, CD64, CD163, CD206 flow detection antibodies, incubated in a 4°C refrigerator for 15 minutes, and protected from light. Then flow cytometry is used for detection, and if CD11b, CD14, CD45, CD64, CD163, and CD206 are all positive, it indicates that the hPBMCs cells are successfully induced into hMDMs.

[0071] (5) The day before the experiment, the target cells are collected by digestion and centrifugation, and the target cells are stained and labeled with PKH26 dye.

[0072] (6) The PKH26-labeled target cells are inoculated into a new culture dish and cultured overnight in a cell incubator (37°C / 5% CO2).

[0073] (7) If hPBMCs are used as ADCP effector cells: they can be recovered the day before the experiment and cultured overnight using RPMI 1640 complete medium.

[0074] (8) On the day of the experiment, the PKH26-labeled target cells are collected by digestion and centrifugation, and the cells are resuspended with ADCP experimental buffer (1640 complete medium).

[0075] (9) The test sample is prepared with ADCP experimental buffer (RPMI 1640 + 10% FBS), i.e., SCIG working solution (since the final experimental system consists of 50 μl of target cell resuspension, 50 μl of test sample solution, and 100 μl of effector cell resuspension, the test sample solution accounts for one-fourth of the total system, and in order to ensure that the final concentration of the test sample is the desired concentration, the concentration of the test sample solution prepared in the early stage is set to 4 times the working concentration of the test sample, i.e., X4).

[0076] (10) The density of the PKH26-labeled target cells is adjusted, and the cell suspension is transferred to the corresponding wells of a 96-well experimental plate (10000 cells / well, 50 μl / well).

[0077] (11) The SCIG working solution is transferred to the corresponding wells of a 96-well experimental plate, and the experimental plate is incubated at room temperature for 30 minutes (50 μl / well).

[0078] (12) The hMDMs or hPBMCs effector cells are collected by digestion and centrifugation (centrifugation conditions: 400g for 10 minutes) and resuspended with ADCP experimental buffer.

[0079] (13) The effector cell density is adjusted according to the effector-to-target ratio, and the effector cell suspension is transferred to the corresponding wells of a 96-well experimental plate (100 μl / well).

[0080] (14) Incubate the experimental plate in the cell incubator (37°C / 5% CO2) for about 1 hour.

[0081] (15) After the incubation, remove the experimental plate and collect all the cells into a new 96-well V-bottom plate and centrifuge to obtain cell pellets.

[0082] (16) Prepare the CD11b flow detection antibody working solution with flow buffer (1x DPBS + 1% FBS).

[0083] (17) Use an excess of CD11b flow detection antibody to stain the hMDMs or hPBMCs effector cells in the experimental plate, incubate in the 4°C refrigerator for 15 minutes, avoid light.

[0084] (18) After the flow detection antibody incubation, centrifuge to obtain cell pellets, resuspend with flow buffer and wash the cells twice.

[0085] (19) Use flow cytometry to detect ADCP: PKH26-labeled target cells are detected by the PE fluorescence channel, and CD11b-stained effector cells are detected by the APC fluorescence channel.

[0086] 2. ADCP data analysis

[0087] ADCP experimental raw data is exported via the BD FACSDiva Software system and analyzed using FlowJo, Microsoft Office Excel 2021, and GraphPad Prism software.

[0088] In FlowJo software, ADCP data is analyzed by PE-APC scatter plot cross-gate, where the PE channel represents target cell labeling, and the APC channel represents effector cell labeling. Then, in the cross-quadrant, PE + APC + The number of cells in the PE + CD11b + APC quadrant represents the number of phagocytosed target cells, and the total number of cells in the PE + APC + APC quadrant represents all PKH26 + APC - target cells. +

[0089] Cell phagocytosis rate is calculated using the following formula:

[0090] Cell phagocytosis rate (%) = (PKH26 + CD11b + cell number / PKH26 + ​The concentration values of the test sample were pasted in the GraphPad Prism software in the XY Table mode to correspond to the resulting phagocytosis rate data of the cells, and then the concentration of the test sample was converted into logarithmic concentration under the Parameters: Transform module, i.e., X = Log (X), and then the log (agonist) vs. response-Variable slope (four parameters) fitting was performed under the Parameters: Nonlinear regression module, and the fitting curve was generated. In the fitting, the relative numerical value of EC50 can be obtained using the following four-parameter equation: Y = Bottom + (Top-Bottom) / (1 + 10^ ((LogEC50-X) * HillSlope)). Wherein, X = Log (sample concentration), Y = phagocytosis rate of cells. Bottom and Top represent the minimum response value and the maximum response value, respectively, and Hillslope represents the slope of the dose-response curve. When Hillslope is greater than 1, the curve is steeper, indicating that a small change in dose will result in a significant change in response. When Hillslope is less than 1, the curve is flatter, indicating that the effect of dose change on the response is smaller.

[0091] The beneficial effects of the present application are demonstrated by the following specific test examples.

[0092] The present application provides an accurate and intuitive method for detecting the ADCP biological activity of immunoglobulin products by exploring the target cell species, effector-to-target ratio and test sample concentration gradient. The method has high precision and accuracy, and is of great significance for improving the quality control and clinical application of immunoglobulin products.

[0093] Test Example 1, Target Cell Screening

[0094] The SCIG (test sample) was diluted with the ADCP experimental buffer to obtain a test sample solution, and the final starting concentration of the test sample solution in the total reaction system was 20 mg / ml. The test sample solution was diluted to 6 concentration points, i.e., 20, 10, 2, 0.4, 0.04 and 0.008 mg / ml. Sp2 / 0-Ag14 cells expressing α-Gal antigen and PLC / PFR / 5 cells expressing human hepatitis B surface antigen (HBsAg) were used as target cells to explore their effects on the detection of SCIG ADCP biological activity. At this time, the effector-to-target ratio was 3:1. ， The effector cells were hMDMs.

[0095] Then the ADCP activity detection was performed according to the method described in Example 1, and the phagocytosis rate of cells at different concentrations of the test sample under each target cell was calculated to obtain a graph of the phagocytosis rate versus concentration.

[0096] The experimental results are shown in Table 2 and Figure 2. Figure 1 Figure 1 It can be seen that when Sp2 / 0-Ag14 cells are used as target cells, a complete dose-effect curve can be obtained, while when PLC / PFR / 5 cells are used as target cells, a dose-effect curve cannot be obtained. This indicates that SCIG cannot mediate the phagocytosis of hMDMs to PLC / PRF / 5 target cells, but can mediate the phagocytosis of hMDMs to Sp2 / 0-Ag14 target cells, and has a clear dose-effect curve. Therefore, Sp2 / 0-Ag14 is selected as the target cell in the subsequent experiment to evaluate the ADCP biological activity of SCIG.

[0097] Test Example 2, Optimization of Effector-to-Target Ratio

[0098] After the target cell (Sp2 / 0-Ag14 cell) was determined, the SCIG was diluted to a final concentration of 20 mg / ml in the total system using the ADCP experimental buffer as the starting working concentration, and the SCIG concentration gradient was set to 20, 10, 2, 0.4, 0.04, 0.008 mg / ml, a total of 6 concentration points; the effector-to-target ratio was set to 3:1 or 5:1, and the effect of different effector-to-target ratios on the detection of the ADCP biological activity of SCIG was investigated. The effector cells were hMDMs 。

[0099] Then, the ADCP activity was detected according to the method described in Example 1, the cell phagocytosis rate of the different concentrations of the test sample under each effector-to-target ratio was calculated, the cell phagocytosis rate-concentration curve was obtained, and the data was analyzed using a four-parameter model.

[0100] Table 5. Results of ADCP effector-to-target ratio optimization experiment (target cell: Sp2 / 0-Ag14)

[0101]

[0102] The results are shown in Table 5 and Figure 5. Figure 2 As shown in Table 5 and Figure 5, an obvious effect curve can be seen at both effector-to-target ratios, and the experimental window increases with the increase of the effector-to-target ratio. Under the same experimental system, the maximum response value is higher and the span is larger at an effector-to-target ratio of 5:1 than at an effector-to-target ratio of 3:1, indicating that the detection sensitivity is higher and the repeatability and reliability are better at an effector-to-target ratio of 5:1. Therefore, an effector-to-target ratio of 5:1 is selected for the experiment in the subsequent detection.

[0103] Test Example 3, Optimization of Test Sample Concentration Gradient

[0104] ​In test example 2, it was confirmed that SCIG could mediate the phagocytosis of hMDMs to Sp2 / 0-Ag14 target cells, but the dose-effect curve was incomplete, lacking obvious upper and lower platforms. Therefore, in this part of the experiment, the SCIG concentration gradient was further optimized according to the experimental results of test example 2, and the initial working concentration of SCIG in the total system was set to 20 mg / ml, which was diluted by 4 times in gradient, with a total of 8 concentration points (20, 5, 1.25, 0.3125, 0.078125, 0.01953125, 0.004882813, 0.001220703 mg / ml). The effector-to-target ratio (E:T) was 5:1. The ADCP activity was detected according to the method described in Example 1. The phagocytosis rate of cells at this concentration gradient of the test sample was calculated, and a graph of the phagocytosis rate versus concentration was obtained, and a four-parameter model was used for data analysis.

[0105] The results are shown in Table 6 and Figure 2: under the condition of an effector-to-target ratio (E:T) of 5:1, SCIG could mediate the phagocytosis of hMDMs to Sp2 / 0-Ag14 target cells, and there was a significant dose-effect response, with a basically complete dose-effect curve (with a relatively complete upper and lower platform) and a larger span value. Therefore, the subsequent experiments will be carried out with reference to the setting of this test sample concentration gradient. Figure 3

[0106] Table 6. Results of test sample initial concentration optimization experiment (target cells: Sp2 / 0-Ag14)

[0107]

[0108] Test example 4: precision and accuracy investigation

[0109] According to the experimental results of test example 3, hMDMs were used as effector cells, Sp2 / 0-Ag14 cells were used as target cells, the initial working concentration of SCIG in the total system was set to 20 mg / ml, and 4 times gradient dilution was performed, with a total of 8 concentration points. The experimental results showed that the experimental system could characterize the ADCP biological activity of SCIG, and had a significant dose-effect curve, which was basically complete. Therefore, the precision and accuracy of the method were verified with this concentration setting.

[0110] In the precision and accuracy experiment, hMDMs were used as target cells, Sp2 / 0-Ag14 cells were used as target cells, the effector-to-target ratio (E:T) was 5:1, and the SCIG concentration gradient in the total system was 20, 5, 1.25, 0.3125, 0.078125, 0.01953125, 0.004882813, 0.001220703 mg / ml. The SCIG group was repeated three times for analysis. The detection was carried out according to the method described in Example 1.

[0111] ​The experimental results show that under the condition of effector-target ratio (E:T) of 5:1, the test product SCIG can mediate the phagocytosis of hMDMs on Sp2 / 0-Ag14 target cells. When analyzing the three sets of data, in the Graphpad software, the three sets of data share the same Top / Bottom / Hillslope value for fitting (in the Parameters:Transform module, set Global nonlinear regression (dose-response curves)-Bottom\Top\Hillslope 3 parameters Constraint type: shared value for all data sets, that is, it is assumed that the three sets of data curves have the same consistency) to obtain the EC50 values of the three sets of data, and the results show that the EC50 values of the three sets of data are similar, and the results are shown in Tables 6 and 7. The same sample is set for three independent detections, and each test is carried out on one 96-well cell culture plate, and three replicate holes are set on one 96-well plate for detection precision, the phagocytosis rate of the starting working concentration of cells and EC50 are shown in Table 8, and the relative standard deviation (relative standard deviation, RSD) is calculated; the recovery rate of the accuracy evaluation parameter is calculated by the relative potency of the sample, and the results are shown in Table 9. Figure 4

[0112] The RSD of the starting working concentration in the precision test is 1.82%, and the EC50 RSD is 14.02%; the recovery rate in the accuracy verification is 115.49%. According to the 9012 biological sample quantitative analysis method verification guide principle in the Pharmacopoeia 2020 edition, the precision and accuracy of the biological analysis method should be within 20%, and the precision and accuracy of the method meet the relevant requirements. That is, the present application has good precision and accuracy.

[0113] Table 7. Precision and accuracy verification results (target cells: Sp2 / 0-Ag14)

[0114]

[0115] Table 8. Precision evaluation parameters: relative standard deviation

[0116]

[0117] Note: The average value and standard deviation SD can be calculated in Excel using the relevant functions (AVERAGE function, STDEV function); the relative standard deviation RSD can be obtained by dividing the standard deviation by the corresponding average value and multiplying 100%, which can be used to analyze the precision of the results in the test detection work.

[0118] ​Table 9. Accuracy evaluation parameters: recovery rate

[0119]

[0120] Note: Plate 01 was taken as 100% RS, i.e. 100% potency standard, and its EC 50 Values divided by other experimental plate EC 50 Values multiplied by 100% to obtain relative potency; the average of relative potency can be calculated in Excel using the AVERAGE function; the average of relative potency divided by 100% potency multiplied by 100% is the recovery rate.

[0121] Test Example 5, specificity verification

[0122] Since the target cell screening test has been carried out in the early stage, only the specificity of the method to the effector cell is verified here. According to the detection method described in Test Example 4, 20 mg / ml is selected as the starting concentration, 4-fold gradient dilution, a total of 8 concentration points, and the specificity of the method to the effector cell is further verified. The specificity of the effector cell is replaced by uninduced hPBMCs cells as effector cells, and the effector target ratio is 5:1, according to the method described in Example 1, the ADCP activity detection is carried out, and the results are shown in Figure 5 and Table 10, the ADCP window of SCIG mediated hPBMCs effector cells is weak, which may be due to the complexity of hPBMCs components and multiple cell types; compared with hPBMCs as effector cells, the ADCP experiment window of SCIG mediated hMDMs effector cells is more obvious, that is, hMDMs cells as effector cells have better detection results. It can be seen that the ADCP detection method of the present application has good specificity.

[0123] Table 10. Specificity verification results of effector cells (target cells: Sp2 / 0-Ag14)

[0124]

[0125] In conclusion, the present application provides a method for detecting ADCP of human immunoglobulin products by target cell screening, optimization of effector-target ratio, and optimization of test product concentration gradient, etc., adopts Sp2 / 0-Ag14 cells as target cells, uses sorted monocytes in hPBMCs and human Monocytes-Derived Macrophages (hMDMs) induced by the monocytes as effector cells, and realizes accurate detection of ADCP biological activity of human immunoglobulin products under appropriate effector-target ratio (5:1) and test product concentration gradient (the initial concentration of SCIG is set as 20 mg / ml, 4-fold dilution gradient, and 8 concentration points). The detection method is intuitive, specific, and has high precision and accuracy, fills the blank of current detection of ADCP of immunoglobulin products, and has important significance for improving quality control and clinical application of immunoglobulin products.

Claims

1. A method for detecting ADCP activity of human immunoglobulin, characterized in that: It includes the following steps: (1) Preparation of fluorescently labeled Sp2 / 0-Ag14 cells expressing α-Gal antigen as target cells; (2) Dilute the human immunoglobulin to the initial working concentration and then perform serial dilutions; (3) adding the diluted human immunoglobulin from step (2) to the target cells and incubating; (4) adding the effector cells to the system after incubation in step (3) according to the effector-target ratio to obtain the final reaction system, and incubating; (5) After incubation in step (4), the cells are collected and incubated with flow cytometry antibodies to stain the effector cells; (6) After staining in step (5), the cells are collected and tested using a flow cytometer, and the ADCP activity of the human immunoglobulin is determined using the test data; In step (1), the fluorescent marker is a PKH26 fluorescent marker; In step (4), the effector cells are macrophages; the macrophages are macrophages induced by monocytes in primary human peripheral blood mononuclear cells; In step (4), the effector-target ratio of the effector cells to the target cells is 3:1 to 5:1; In step (4), in the final reaction system, RPMI 1640 medium containing 10% fetal bovine serum is used as the experimental buffer for dilution; In step (5), the flow cytometry detection antibody is a CD11b antibody.

2. The detection method according to claim 1, wherein: In step (3), the incubation temperature is 25-37°C, and the incubation time is 30-60 minutes.

3. The detection method according to claim 1, wherein: In step (4), the effector-target ratio of the effector cells to the target cells is 5:

1.

4. The detection method according to claim 1, wherein: In step (4), in the final reaction system, the initial concentration of human immunoglobulin is 20 mg / ml, with a 4-fold dilution gradient and a total of 8 concentration points; And / or, in step (4), the number of target cells in the final reaction system is 200 cells / μl.

5. The detection method according to claim 1, wherein: In step (4), the incubation temperature is 25-37°C and the incubation time is 1-5 hours; And / or, in step (5), the incubation is dark incubation, the incubation temperature is 0-4°C, and the incubation time is 15-30 minutes.

6. The detection method according to claim 1, wherein: In step (6), during the detection, the target cells are detected via the PE fluorescence channel, and the effector cells are detected via the APC fluorescence channel.

7. A kit for determining ADCP activity of human immunoglobulin, characterized in that: The kit includes human immunoglobulin, fluorescently labeled target cells, effector cells, flow cytometry detection antibodies and experimental buffer; The fluorescently labeled target cells are Sp2 / 0-Ag14 cells expressing α-Gal antigen and fluorescently labeled with PKH26; The effector cells are macrophages; the macrophages are macrophages induced by monocytes in primary human peripheral blood mononuclear cells; The effector-target ratio of the effector cells to the target cells is 3:1 to 5:1; The flow cytometry detection antibody is a CD11b antibody; The experimental buffer was RPMI 1640 medium containing 10% fetal bovine serum.

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