Method for detecting antibody-dependent cell-mediated cytotoxic biological activity of human immune globulin

Abstract

The invention belongs to the field of drug detection, and particularly discloses a method for detecting antibody-dependent cell-mediated cytotoxic biological activity of human immune globulin, which comprises the following steps: 1) adding a target cell suspension into a pore plate, respectively adding human immune globulin solutions with a series of concentrations for incubation, and finally adding an effector cell suspension for incubation; (2) detecting fluorescence of the incubated pore plate in the step (1), drawing a dose effect curve and calculating half effect concentration; the target cell is a CHO-K1 cell for expressing SARS-CoV-2 Spike protein, and the effector cell is a Jurkat cell which stably expresses CD16a on a cell membrane and is stably transfected with a firefly luciferase gene which is driven to express by an NFAT response element. The detection method disclosed by the invention can be used for accurately and reliably detecting the antibody-dependent cell-mediated cytotoxic biological activity of two different human immune globulin preparations, namely IVIG and SCIG.

Description

Technical Field

[0001] The present invention belongs to the field of drug detection, and particularly relates to a method for detecting the antibody-dependent cell-mediated cytotoxicity biological activity of human immunoglobulin. Background Art

[0002] Human immunoglobulin is a glycoprotein produced by plasma cells. The main component of the glycoprotein is IgG, which can be cleaved by papain into two identical Fab fragments and one Fc fragment. Among them, the Fab fragment recognizes specific antigens, and the Fc fragment can bind to receptors on the surface of immune cells. Research shows that the biological functions of the IgG Fab fragment and Fc fragment play important roles in the immunomodulatory effect of human immunoglobulin, thus enabling human immunoglobulin to participate in the humoral immune response.

[0003] Human immunoglobulin preparations are the most widely used blood products in current clinical practice, mainly divided into subcutaneous immunoglobulin (SCIG) and intravenous immunoglobulin (IVIG). There is no product of SCIG approved for marketing in China yet, but the clinical application of foreign SCIG products shows that they have equivalent or better efficacy compared with intravenous immunoglobulin (IVIG). Such blood products as human immunoglobulin preparations are mainly used to treat diseases manifested as humoral immune deficiency or immune system dysfunction. Since their preparation raw materials are mixed plasma from thousands of people, it is determined that they contain broad-spectrum antibodies against infectious diseases and heterologous species proteins.

[0004] Antibody-dependent cell-mediated cytotoxicity (ADCC): The ADCC effect is an important mechanism of the immune system, mainly mediated by natural killer cells (NK cells). When the Fab end of the antibody binds to the antigen epitope on the surface of the target cell, its Fc end can recruit effector cells (NK cells) and bind to the Fc receptor IIIa (FcγRIIIa) on their surface, causing them to release cytotoxic substances (such as perforin and granzyme), thereby killing the target cell. The mechanism of this effect plays an important role in the immune defense and immunomodulation of the body. Therefore, detecting whether an antibody drug has ADCC biological activity and the strength of the biological activity has become a crucial step in the research and development of antibody drugs and their quality control process.

[0005] At present, a variety of methods for measuring the ADCC biological activity of antibody drugs have been established, such as the chromium-51 (51Cr) labeling / release method, the lactate dehydrogenase release method, etc. These measurement methods either have radioactive element pollution or have problems such as poor repeatability. Patent CN115537448A discloses a method for detecting hepatitis B virus by antibody-dependent cell-mediated cytotoxicity of human immunoglobulin, filling the blank of using the reporter gene method for ADCC detection of IVIG at present. However, through experimental verification, due to factors such as the types of target cells and effector cells, as well as incubation conditions and effector-to-target ratios, the detection sensitivity is not high, and the ADCC biological potency and efficacy of human immunoglobulin cannot be accurately measured. Therefore, it is necessary to provide a method with higher sensitivity and capable of accurately detecting the ADCC biological activity of human immunoglobulin drugs. Summary of the Invention

[0006] To solve the above problems, the present invention provides a method for detecting the antibody-dependent cell-mediated cytotoxic biological activity of human immunoglobulin, including the following steps: 1) Add the target cell suspension to the well plate, then add serial concentrations of human immunoglobulin solution for incubation, and finally add the effector cell suspension for incubation; 2) Take the well plate incubated in step 1) to detect fluorescence, plot the dose-effect curve with the relative light unit value as the ordinate and the logarithm of the human immunoglobulin solution concentration as the abscissa, and calculate the half-maximal effect concentration.

[0007] The target cell is CHO-K1 / SPIKE_SARS2, and the effector cell is GS-J2C / CD16A 158V.

[0008] Further, the effector-to-target ratio of effector cells to target cells in each well of the well plate is 6:1.

[0009] Further, when the human immunoglobulin solution is an intravenous injection human immunoglobulin solution, the concentration range is 2.44×10 -4 mg / ml~1mg / ml.

[0010] Furthermore, when the human immunoglobulin solution is an intravenous injection human immunoglobulin solution, 1mg / ml is used as the starting working concentration, and then diluted 6 concentrations with a 4-fold serial dilution ratio, or 0.67mg / ml is used as the starting working concentration, and then diluted 6 concentrations with a 3-fold serial dilution ratio.

[0011] Further, when the human immunoglobulin solution is a subcutaneous injection human immunoglobulin solution, the concentration range is 2×10 -4 mg / ml~2mg / ml.

[0012] Further, when the human immunoglobulin solution is a subcutaneous injection human immunoglobulin solution, the starting working concentration is 2 mg / ml, and then it is diluted 4 times at a 10-fold gradient dilution ratio.

[0013] Further, the target cell suspension, the human immunoglobulin solutions of a series of concentrations, and the effector cell suspension are prepared with RPMI 1640 containing 10% FBS.

[0014] Further, the concentration of the target cell suspension is 2.5×10 5 cells / ml, and the volume is 40 μl / well; the volume of the human immunoglobulin solutions of a series of concentrations is 20 μl / well; the concentration of the effector cell suspension is 1.5×10 6 cells / ml, and the volume is 40 μl / well.

[0015] Further, the conditions for incubating the target cell suspension with the human immunoglobulin solution are: incubating at room temperature for 30 minutes; the conditions for adding the effector cell suspension and incubating are incubating at 37°C and 5% CO2 for 4 hours.

[0016] Further, the detection fluorescence is detected using a luciferase detection kit; the luciferase detection kit is the Fire-Lumi TM luciferase detection kit.

[0017] The method for detecting the ADCC biological activity of the human immunoglobulin of the present invention can accurately and reliably detect the ADCC biological activities of two different human immunoglobulin preparations, namely intravenous injection human immunoglobulin and subcutaneous injection human immunoglobulin, through a reporter gene detection method with CHO-K1 / SPIKE_SARS2 as the target cells and GS-J2C / CD16A 158V as the effector cells in combination with a human immunoglobulin solution at a specific concentration. Compared with the similar methods disclosed in the prior art, it has higher accuracy, precision and sensitivity, and has practical popularization and application value.

[0018] Obviously, based on the above content of the present invention, according to the common general knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modification, substitution or change can also be made.

[0019] The above content of the present invention will be further described in detail below through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Description of the Drawings

[0020] Figure 1 Results of the PBMC-ADCC pre-experiment concentration exploration (LDH method); Figure 2 Experimental results after optimization of the PBMC-ADCC system (LDH method) Figure 3 Experimental results of NK92 / CD16A-vv-ADCC detection (LDH method) Figure 4 Experimental results of NK92 / CD16A-vv-ADCC detection (flow cytometry method) Figure 5 Experimental results of the ADCC dose-response experiment of SCIG on the target cell PLC / PRF / 5 Figure 6 Experimental results of the ADCC dose-response experiment of human immunoglobulin on the target cell PLC / PRF / 5 to support the exploration of human immunoglobulin concentration Figure 7 Experimental results of the ADCC dose-response experiment of human immunoglobulin on the target cell CHO-K1 / Spike_SARS2 to support the exploration of human immunoglobulin concentration Figure 8 Experimental results of the ADCC dose-response experiment of human immunoglobulin on the target cell Sp2 / 0-Ag14 to support the exploration of human immunoglobulin concentration Figure 9 Experimental results of the ADCC dose-response experiment of IVIG on the target cell CHO-K1 / Spike_SARS2; Figure 10 Experimental results of the ADCC dose-response experiment of IVIG on the target cell CHO-K1 / Spike_SARS2; Figure 11 Influence of different target cells on the method of the present invention; Figure 12 Influence of different effector cells on the method of the present invention Figure 13 Influence of different detection samples on the method of the present invention Figure 14 Experimental results of the ADCC dose-response experiment of human immunoglobulin Detailed implementation manners

[0021] The raw materials, reagents and equipment used in the specific implementation manners of the present invention are all obtained through commercial purchase.

[0022] Example 1 Detection of the ADCC biological activity of intravenous immunoglobulin IVIG of the present invention 1) Preparation of solutions Target cell suspension: Take the resuscitated CHO-K1 / SPIKE_SARS2, and prepare a cell suspension with a concentration of 2.5×10 5 cells / ml with RPMI 1640 containing 10% FBS; Effector cell suspension: Take the revived GS-J2C / CD16A 158V, and prepare a cell suspension with a concentration of 1.5×10 6 cells / ml with RPMI 1640 containing 10% FBS; IVIG solutions of a series of concentrations: Take IVIG, and prepare a solution with a concentration of 1 mg / ml as the initial working concentration solution with RPMI 1640 containing 10% FBS, and then dilute it 6 times with RPMI 1640 containing 10% FBS according to a 4-fold gradient dilution ratio. The concentrations are 2.5×10 -1 mg / ml, 6.25×10 -2 mg / ml, 1.56×10 -2 mg / ml, 3.9×10 -3 mg / ml, 9.76×10 -4 mg / ml, 2.44×10 -4 mg / ml, and a total of 7 IVIG solutions with a concentration range of 2.44×10 -4 mg / ml to 1 mg / ml are obtained; 2) Detection of ADCC biological activity Add the target cell suspension to a 96-well plate at 40 μl / well, then add the IVIG solutions of a series of concentrations to each well at 20 μl / well, incubate at room temperature for 30 minutes, and finally add the effector cell suspension to each well at 40 μl / well to make the effector-to-target ratio in each well 6:1. Then incubate at 37°C and 5% CO2 for 4 hours. Take out the 96-well plate, and add the working solution of the Fire-Lumi TM luciferase detection kit to each well at 80 μl / well and incubate for 5 - 10 minutes. Read the relative light unit value at room temperature using PHERAstar FSX; 3) Calculation Take the relative light unit value as the ordinate and the logarithm of the IVIG solution concentration as the abscissa, select a four-parameter curve equation regression model to fit the dose-effect curve of IVIG, and obtain the EC 50 value of IVIG according to the dose-effect curve.

[0023] Example 2 Detection of the ADCC biological activity of intravenous immunoglobulin (IVIG) of the present invention 1) Preparation of solutions Target cell suspension: Take the revived CHO-K1 / SPIKE_SARS2, and prepare a cell suspension with a concentration of 2.5×10 5 cells / ml with RPMI 1640 containing 10% FBS; Effector cell suspension: Take the revived GS-J2C / CD16A 158V, and prepare a cell suspension with a concentration of 1.5×10 6 cells / ml with RPMI 1640 containing 10% FBS; IVIG solutions with a series of concentrations: Take IVIG, and prepare a solution with a concentration of 0.67 mg / ml as the initial working concentration solution with RPMI 1640 containing 10% FBS, and then dilute it 6 times with RPMI 1640 containing 10% FBS according to a 3-fold gradient dilution ratio, with concentrations of 2.22×10 -1 mg / ml, 7.41×10 -2 mg / ml, 2.47×10 -2 mg / ml, 8.23×10 -3 mg / ml, 2.74×10 -3 mg / ml, 9.14×10 -4 mg / ml, to obtain a total of 7 IVIG solutions with a concentration range of 9.14×10 -4 mg / ml to 0.67 mg / ml; 2) Detection of ADCC biological activity Add the target cell suspension to a 96-well plate at 40 μl / well, then add the IVIG solutions with a series of concentrations at 20 μl / well respectively, incubate at room temperature for 30 minutes, and finally add the effector cell suspension at 40 μl / well to make the effector-to-target ratio in each well 6:1. Then incubate at 37°C and 5% CO2 for 4 hours. Take out the 96-well plate, and add the Fire-Lumi TM luciferase detection kit working solution at 80 μl / well and incubate for 5 - 10 minutes, and read the relative light unit value using PHERAstar FSX at room temperature; 3) Calculation Take the relative light unit value as the ordinate and the logarithm of the IVIG solution concentration as the abscissa, select a four-parameter curve equation regression model to fit the dose-effect curve of IVIG, and obtain the EC 50 value of IVIG according to the dose-effect curve.

[0024] Example 3 Detection of ADCC biological activity of subcutaneous immunoglobulin SCIG of the present invention 1) Preparation of solutions Target cell suspension: Take the revived CHO-K1 / SPIKE_SARS2, and prepare a cell suspension with a concentration of 2.5×10 5 cells / ml with RPMI 1640 containing 10% FBS; Effector cell suspension: Take the revived GS-J2C / CD16A 158V, and prepare a cell suspension with a concentration of 1.5×10 6 cells / ml with RPMI 1640 containing 10% FBS; SCIG solutions with a series of concentrations: Take SCIG, and prepare a solution with a concentration of 2 mg / ml as the starting working concentration solution with RPMI 1640 containing 10% FBS, and then dilute it 4 times with RPMI 1640 containing 10% FBS according to a 10-fold gradient dilution ratio, with concentrations of 2×10 -1 mg / ml, 2×10 -2 mg / ml, 2×10 -3 mg / ml, 2×10 -4 mg / ml respectively, to obtain a total of 5 concentrations of SCIG solutions with a concentration range of 2×10 -4 mg / ml to 2 mg / ml; 2) Detection of ADCC biological activity Add the target cell suspension to a 96-well plate at 40 μl / well, then add the SCIG solutions with a series of concentrations at 20 μl / well respectively, incubate at room temperature for 30 minutes, and finally add the effector cell suspension at 40 μl / well to make the effector-to-target ratio in each well 6:1. Then incubate at 37°C and 5% CO2 for 4 hours. Take out the 96-well plate, and add the Fire-Lumi TM luciferase detection kit working solution at 80 μl / well and incubate for 5 - 10 minutes. Read the relative light unit value at room temperature using PHERAstar FSX; 3) Calculation Taking the relative light unit value as the ordinate and the logarithm of the SCIG solution concentration as the abscissa, select a four-parameter curve equation regression model to fit the dose-effect curve of SCIG, and obtain the EC 50 value of SCIG according to the dose-effect curve.

[0025] The beneficial effects of the present invention are further illustrated by the following test examples: Test Example 1 Exploration of the Detection Method for the ADCC Biological Activity of Human Immunoglobulin 1. Test purpose Construct a detection method suitable for the ADCC biological activity of human immunoglobulin by trying different ADCC detection methods.

[0026] 2. Test principle Three methods were tried in this experiment to explore ADCC detection, namely (1) LDH release detection method (LDH), (2) flow cytometry detection method (FACS method), and (3) luciferase reporter gene detection method.

[0027] Methods (1) and (2) are further classified as direct killing methods. The effector cells explored are human peripheral blood mononuclear cells (hPBMCs) and NK92 / CD16A-vv (NK92 cells highly expressing CD16a). The antibody binds to the antigen on the surface of the target cell through the Fab segment, and then binds to FcyRIIa (i.e., CD16a) on the surface of the effector cell through the Fc segment, activating NK cells to release cytotoxic substances (such as perforin and granzyme) to kill the target cell. The death of the target cell is detected by flow cytometry (FACS method) or the activity of lactate dehydrogenase (LDH) released into the culture medium after the target cell is killed is directly detected (LDH release detection method) to characterize the ADCC activity of the antibody.

[0028] In the luciferase reporter gene assay, the ADCC effect is mainly mediated by FcγRIIIa. The antibody binds to FcγRIIIa on the surface of the effector cell, stimulating the intracellular NFAT response element, thereby driving the expression of firefly luciferase. The ADCC activity of the antibody can be characterized by quantitatively measuring the luciferase activity by bioluminescence.

[0029] 3. Cell lines used in the experiment 4. Human immunoglobulin information 5. Experimental design 5.1 PBMC-ADCC Assay (LDH Method) 5.2 PBMC-ADCC detection & NK92 / CD16A-vv-ADCC detection (LDH method) 5.3 NK92 / CD16A-vv-ADCC detection (flow cytometry method) 6. Experimental procedures 6.1 LDH method 6.1.1 ADCC experimental procedures 1) If the effector cells are hPBMCs, the effector cells need to be resuscitated one day before the experiment and cultured overnight with RPMI 1640 complete medium (RPMI 1640 + 10% FBS). Recombinant human interleukin 2 protein needs to be added during the culture process to make its concentration 100 IU / ml. On the day of the experiment, collect the effector cells and resuspend them with ADCC exploration experimental buffer (99% MEM α, nucleosides, no phenol red + 1% fetal bovine serum).

[0030] If the effector cells are NK92 / CD16A-vv, collect the effector cells on the day of the experiment and resuspend them in the ADCC assay buffer.

[0031] 2) Digest and centrifuge to collect the target cells and resuspend the target cells in the ADCC assay buffer.

[0032] 3) Prepare a gradient sample of human immunoglobulin in the ADCC assay buffer.

[0033] 4) Adjust the density of the target cells to 2E5 cells / ml with the ADCC assay buffer and transfer the target cell suspension to the corresponding wells of a 96-well assay plate (50 μl / well, 10000 cells / well).

[0034] 5) Transfer the gradient sample of human immunoglobulin or the ADCC assay buffer to the corresponding wells of a 96-well assay plate (50 μl / well).

[0035] 6) Incubate the assay plate at room temperature for 30 minutes.

[0036] 7) Adjust the density of the effector cells according to the E:T ratio with the ADCC assay buffer and add recombinant human interleukin-2 protein to a concentration of 200 IU / ml (so the final concentration in the assay system is 100 IU / ml), then transfer the effector cell suspension to the corresponding wells of a 96-well assay plate (100 μl / well).

[0037] 8) Incubate the assay plate in a cell culture incubator (37°C / 5% CO2) for 6 hours.

[0038] 9) After incubation, take out the 96-well assay plate, centrifuge it, carefully aspirate the incubation supernatant, and transfer it to a new 96-well assay plate (50 μl / well). Transfer the LDH assay working solution (50 μl / well, Roche, 11644793001) to the corresponding wells of the new 96-well assay plate and develop color at room temperature.

[0039] 10) Read the OD value on an ELISA reader, with the detection wavelength at 492 nm and the reference wavelength at 650 nm.

[0040] 6.1.2 ADCC Data Analysis The raw data of the ADCC assay detected by the LDH method was exported via the PHERAstar FSX system and analyzed using Microsoft Office Excel software.

[0041] The data for calculating the target cell lysis rate are all: OD 492 nm -OD 650 nm . The formula for calculating the target cell lysis rate is: %Target cell lysis = 100 × (ODSample data - OD Tumor cells plus effector cells ) / (OD Maximum release - OD Minimum release )。Among them: Maximum release is the LDH release after treating target cells with lysis buffer (1% Triton); Minimum release is the LDH release after treating target cells with ADCC exploration assay buffer; E:T (Tumor cells plus effector cells) is the LDH release after treating effector cells and target cells with ADCC exploration assay buffer; Sample group is the LDH release after treating effector cells and target cells with the sample. In Microsoft Office Excel software, the relative value of EC 50 can be obtained using the following four-parameter equation: Y = Bottom + (Top - Bottom) / (1 + 10 ^ ((LogEC 50 - X) × HillSlope)). Where X = Log (sample concentration) and Y = target cell lysis rate.

[0042] 6.2 FACS method 6.2.1 ADCC experimental procedure 1) Collect effector cells (NK92 / CD16A-vv) and resuspend them with ADCC exploration assay buffer (99% MEM α, nucleosides, no phenol red + 1% fetal bovine serum); 2) Digest and centrifuge to collect target cells and resuspend the target cells with DPBS; 3) According to the experimental requirements, label a certain amount of experimental target cells with Cell Trace Violet dye (Cell Trace Violet, C34557). After completion of labeling, resuspend the labeled target cells with ADCC exploration assay buffer; 4) Prepare a gradient sample of human immunoglobulin with ADCC exploration assay buffer; 5) Adjust the density of the Cell Trace Violet-labeled target cells to 2E5 cells / ml with ADCC exploration assay buffer and transfer the labeled target cell suspension to the corresponding wells of a 96-well experimental plate (50 μl / well, 10000 cells / well); 6) Transfer the gradient sample of human immunoglobulin or ADCC experimental buffer to the corresponding wells of a 96-well experimental plate (50 μl / well); 7) Incubate the experimental plate at room temperature for 30 minutes.

[0043] 8) According to the E:T ratio, adjust the effector cell density with the ADCC exploration experimental buffer, and add recombinant human interleukin-2 protein to make its concentration 200 IU / ml (the final concentration in the experimental system is 100 IU / ml). Transfer the effector cell suspension to the corresponding wells of a 96-well experimental plate (100 μl / well).

[0044] 9) Incubate the experimental plate in a cell incubator (37 °C / 5% CO2) for 5 hours.

[0045] 10) After incubation, take out the 96-well experimental plate, discard the supernatant after centrifugation, wash the cells with flow buffer (1× DPBS + 1% fetal bovine serum), and stain the cells with 7-AAD ten minutes before loading.

[0046] 11) Detect the ADCC effect using a flow cytometer, and detect the CellTrace Violet and 7-AAD fluorescence using the Pacific Blue and 7-AAD channels respectively.

[0047] 6.2.2 ADCC data analysis The original data of the ADCC experiment detected by the flow method is exported via the BD FACSDiva Software system and analyzed using Microsoft Office Excel and FlowJo software.

[0048] Calculate the target cell lysis rate using the following formula: % Target cell lysis = (Pacific Blue + 7-AAD + Cells / All Pacific Blue + Cells) × 100%. In Microsoft Office Excel software, the relative value of EC 50 can be obtained using the following four-parameter equation: Y = Bottom + (Top - Bottom) / (1 + 10 ^ ((LogEC 50 - X) × HillSlope)). Where, X = Log (sample concentration), Y = target cell lysis rate.

[0049] 7. Test results 7.1 LDH method - ADCC detection experimental results of PBMC effector cells 7.1.1 Preliminary experiment to confirm reasonable concentration According to the aforementioned experimental method, three effector-to-target ratio gradients were initially set: E:T = 10:1, 25:1, 50:1. Human immunoglobulin IVIG and SCIG were selected, and the ADCC effect induced by PBMC effector cells was detected using the LDH method respectively.

[0050] The experimental results are as Figure 1 shown. Under the conditions of E:T = 10:1, 25:1, 50:1, after diluting human immunoglobulin (IVIG, SCIG) appropriately and co-incubating with the corresponding effector and target cells for 6 hours, the lysis rate of experimental target cells was detected. The data in the figure are expressed as the mean ± SEM of target cell lysis rate (n = 2). From Figure 1 it can be seen that human immunoglobulin did not show obvious ADCC effects on target cells.

[0051] Based on this, optimization experiments on the concentration gradient and effector-to-target ratio of human immunoglobulin were further carried out.

[0052] 7.1.2 Optimization of the Concentration Gradient and Effector-to-Target Ratio of Human Immunoglobulin According to the experimental results of 7.1.1, the initial concentration of human immunoglobulin in the experiment was optimized and adjusted: According to the aforementioned experimental method, human immunoglobulin SCIG was selected, and two effector-to-target ratio gradients were set: E:T = 4:1, 8:1. The LDH method was used to detect the ADCC effect induced by PBMC effector cells.

[0053] The experimental results are as Figure 2 shown. Under the conditions of E:T = 4:1, 8:1, after diluting human immunoglobulin (SCIG) appropriately and co-incubating with the corresponding effector and target cells for 6 hours, the lysis rate of experimental target cells was detected. The data in the figure are expressed as the mean ± SEM of target cell lysis rate (experimental group: n = 2). From Figure 2 it can be seen that under the conditions of E:T = 4:1, 8:1, human immunoglobulin SCIG failed to mediate the killing effect of PBMC on target cells. The experimental results indicate that the optimization of the effector-to-target ratio has no significant correlation with the ADCC effect.

[0054] Based on this, the PBMC effector cells were replaced with NK92 / CD16A-vv effector cells to further carry out exploratory experiments.

[0055] 7.2 LDH Method - Experimental Results of ADCC Detection of NK92 / CD16A-vv Effector Cells According to the aforementioned experimental method, two effector-to-target ratio gradients were set: E:T = 5:1, 10:1. SCIG was selected, and the LDH method was used to detect the ADCC effect induced by NK92 / CD16A-vv effector cells.

[0056] The experimental results are as Figure 3As shown, under the conditions of E:T = 5:1 and 10:1, after diluting SCIG to a certain extent, it was co-incubated with the corresponding effector and target cells for 6 hours, and the lysis rate of the experimental target cells was detected. The data in the figure are expressed as the mean ± SEM of the target cell lysis rate (experimental group: n = 2). From Figure 3 It can be seen that under the conditions of E:T = 5:1 and 10:1, neither of the two human immunoglobulins showed obvious ADCC effects on the target cells. Based on this, the LDH method was replaced with the FACS method for further attempts.

[0057] 7.3 FACS method - ADCC detection experimental results of NK92 / CD16A-vv effector cells According to the results in 7.1 and 7.2, no killing effect of the experimental SCIG on the detected experimental target cells was observed using the LDH method. Therefore, the flow cytometry detection method was used here to evaluate the ADCC function of human immunoglobulin. According to the aforementioned experimental method, using NK92 / CD16A-vv as the effector cells, SCIG was selected, and two effector-to-target ratio gradients were set: E:T = 5:1 and 10:1. The FACS method was used to detect the ADCC effect induced by NK92 / CD16A-vv effector cells.

[0058] The experimental results are as Figure 4 shown. Under the conditions of E:T = 5:1 and 10:1, after diluting SCIG to a certain extent, it was co-incubated with the corresponding effector and target cells, namely PLC / PRF / 5 cells labeled with Cell Trace Violet dye, for 5 hours. After the co-incubation ended, the cells in the experimental wells were stained with 7-AAD, and then detected using a flow cytometer. The data in the figure are expressed as the mean ± SEM of the target cell lysis rate (experimental group: n = 2). From Figure 4 It can be seen that under the conditions of E:T = 5:1 and 10:1, SCIG failed to mediate the killing effect of NK92 / CD16A-vv on the target cells.

[0059] 8. Discussion In the study evaluating the in vitro biological activity of human immunoglobulin, two direct killing methods, namely the LDH method and the FACS method, were used to analyze the ADCC functional activity of human immunoglobulin.

[0060] In Experiment 7.1, using PBMC as the effector cells, the LDH method was used to detect the ADCC function, and the effector-to-target ratio and the initial concentration of human immunoglobulin were explored and optimized. The experimental results showed that neither IVIG nor SCIG showed ADCC function against PLC / PRF / 5 target cells, and the killing results were negative.

[0061] In Experiment 7.2, NK92 / CD16A-vv was used as effector cells, and the LDH method was used to detect ADCC function. The experimental results showed that SCIG did not exhibit ADCC function against PLC / PRF / 5 target cells, and the killing result was negative.

[0062] In Experiment 7.3, NK92 / CD16A-vv was used as effector cells, and the FACS method was used to detect ADCC function. The experimental results showed that SCIG did not exhibit ADCC function against PLC / PRF / 5 target cells, and the killing result was negative.

[0063] Based on the above experimental results, this experiment will not consider using the LDH release detection method and flow cytometry detection method as the ADCC activity detection methods. Based on this, further exploration of the luciferase reporter gene method was carried out.

[0064] 9. Preliminary Exploration of the Luciferase Reporter Gene Method Preliminarily explore the ADCC reporter gene detection method, use PLC / PRF / 5 cells expressing human hepatitis B surface antigen (HBsAg) as target cells, and use genetically engineered Jurkat cells as effector cells (GS-J2C / CD16A 158V) to detect the ADCC activity of SCIG and IVIG respectively.

[0065] The initial concentration of SCIG was 30 mg / ml, and it was diluted 4-fold with 8 concentration points. The ADCC effect induced by GS-J2C / CD16A 158V effector cells was detected using the reporter gene detection method.

[0066] The experimental results are as Figure 5 shown. Under the condition of E / T = 6:1, SCIG was serially diluted and co-incubated with target cells and effector cells. The relative light unit values in the reaction system were recorded, and the data in the figure are expressed as the mean ± SEM of relative light units (RLU) (n = 3). From Figure 5 it can be seen that SCIG can significantly induce the ADCC effect on target cells PLC / PRF / 5, and the luciferase reporter gene method is suitable as a detection method for the ADCC effect induced by SCIG on GS-J2C / CD16A 158V effector cells.

[0067] Test Example 2 Exploration and Establishment of the Luciferase Reporter Gene Method 1. Experimental Purpose This study carried out exploration on the selection of target cells for the luciferase reporter gene method, optimization of the initial concentration of IVIG / SCIG, and evaluation of the specificity, precision and accuracy of the method.

[0068] 2. Experimental Principle The ADCC effect is mainly mediated by FcγRIIIa. Antibodies bind to FcγRIIIa on the surface of effector cells, activating the NFAT response element within the effector cells, thereby driving the expression of firefly luciferase. The ADCC activity of antibodies can be characterized by quantitatively measuring luciferase activity through bioluminescence. For the ADCC reporter gene assay, CHO-K1 / Spike_SARS2 cells with overexpressed Spike protein in CHO-K1, PLC / PRF / 5 cells expressing human hepatitis B surface antigen (HBsAg), and Sp2 / 0-Ag14 cells were used as target cells; genetically engineered Jurkat cells (GS-J2C / CD16A 158V) were used as effector cells. These cells stably express CD16a (FcγRIIIa, V158 high-affinity mutant) on the cell membrane and are stably transfected with the firefly luciferase gene driven by the NFAT response element.

[0069] 3. Cell lines used in the experiment 4. Information on IVIG / SCIG and control products 5. Experimental design 6. Experimental procedures 6.1 Cell culture 6.1.1 Cell culture and subculture 6.1.1.1 Cell resuscitation: Add 4 ml of pre-warmed medium to a 15 ml centrifuge tube for standby. Quickly thaw the cryopreservation tube in a 37°C water bath, transfer the thawed cell suspension to the aforementioned centrifuge tube, centrifuge at 800 rpm for 5 minutes in a centrifuge, resuspend the cell pellet with complete medium, count the cells, and inoculate them into a cell culture dish at an appropriate density. Gently shake and mix well, then place them in a 5% CO2, 37°C incubator for culture.

[0070] 6.1.1.2 Cell subculture: 6.1.1.2.1 Cell subculture (GS-J2C / CD16A 158V): Aspirate the cells into a 15 ml centrifuge tube, centrifuge at 800 rpm for 5 minutes in a centrifuge, resuspend the cell pellet with complete medium (Probio, RD00830, RPMI 1640 + 10% FBS + 1 µg / ml Puromycin + 200 µg / ml Hygromycin B + 400 µg / ml G418), count the cells, and inoculate them into a cell culture dish at an appropriate density. Gently shake and mix well, then place them in a 5% CO2, 37°C incubator for culture.

[0071] 6.1.1.2.2 Cell passage (PLC / PRF / 5): Collect the supernatant into a 15 mL centrifuge tube, wash it once with pre-warmed DPBS, add an appropriate amount of pre-warmed Accutase digestion solution, incubate in a 37 °C incubator for a certain period of time (the specific time depends on the cell status), add an appropriate amount of complete medium (ATCC, CRL-1581, EMEM Medium + 10% FBS) to terminate the digestion, and then centrifuge at 800 rpm for 5 minutes in a centrifuge. The subsequent counting and seeding steps are the same as above.

[0072] 6.1.1.2.3 Cell passage (CHO-K1 / Spike_SARS2): Collect the supernatant into a 15 ml centrifuge tube, wash it once with pre-warmed DPBS, add an appropriate amount of pre-warmed Accutase digestion solution, incubate in a 37 °C incubator for a certain period of time (the specific time depends on the cell status), add an appropriate amount of complete medium (Probio, RD00819, F-12 + 10% FBS + 8 μg / ml Puromycin) to terminate the digestion, then count, and seed at an appropriate density in a cell culture dish. After gently shaking and mixing, place it in a 5% CO2, 37 °C incubator for culture.

[0073] 6.1.1.2.4 Cell passage (Sp2 / 0-Ag14): Aspirate the cells into a 15 ml centrifuge tube, centrifuge at 800 rpm for 5 minutes in a centrifuge, resuspend the cell pellet with complete medium (ATCC, CRL-1581, DMEM + 10% FBS), then count, and seed at an appropriate density in a cell culture dish. After gently shaking and mixing, place it in a 5% CO2, 37 °C incubator for culture.

[0074] 6.1.2 Experimental procedures for ADCC reporter gene assay 6.1.2.1 Add 2 ml of StemPro Accutase to digest the cells, centrifuge to collect the target cells, and resuspend them with 5 ml of ADCC assay buffer (RPMI 1640 + 10% FBS).

[0075] 6.1.2.2 Exploration of appropriate concentration for ADCC reporter gene assay (1) Preliminary concentration exploration experiment: According to the experimental design, prepare SCIG / IVIG to 2 mg / ml with ADCC assay buffer as the initial working concentration, and then dilute it at a 10-fold serial dilution ratio for 5 concentration points.

[0076] (2)Appropriate concentration confirmation experiment: According to the experimental design, IVIG was prepared with ADCC assay buffer to 1 mg / ml as the starting working concentration, and then diluted to 7 concentration points with a 4-fold serial dilution ratio. Also, IVIG was prepared with ADCC assay buffer to 0.67 mg / ml as the starting working concentration, and then diluted to 7 concentration points with a 3-fold serial dilution ratio.

[0077] 6.1.2.3 Adjust the target cell density (2.5E5 cells / ml) with ADCC assay buffer and transfer the target cell suspension to a 96-well assay plate (40 µl / well) according to the detection protocol.

[0078] 6.1.2.4 Transfer SCIG / IVIG concentration gradient samples or ADCC assay buffer (20 µl / well) to the corresponding wells of the 96-well plate.

[0079] 6.1.2.5 Incubate the assay plate at room temperature for 30 minutes.

[0080] 6.1.2.6 Collect effector cells and resuspend them with ADCC assay buffer.

[0081] 6.1.2.7 Adjust the effector cell density (1.5E6 cells / ml) with ADCC assay buffer according to the E / T ratio, and transfer the effector cell suspension to the corresponding wells of the 96-well plate (40 µl / well).

[0082] 6.1.2.8 Incubate the assay plate in a cell culture incubator (37℃ / 5%CO2) for 6 hours.

[0083] 6.1.2.9 After incubation, take out the 96-well assay plate and add the working solution of Fire-LumiTM luciferase detection kit (80 µl / well) to the corresponding wells, and incubate for 5 - 10 minutes.

[0084] 6.1.2.10 Read the chemiluminescence value using PHERAstar FSX at room temperature.

[0085] 6.2 ADCC data analysis The original data of the ADCC experiment was exported via the PHERAstar FSX system and analyzed using Microsoft Office Excel.

[0086] 7. Experimental results of ADCC dose response 7.1 Detection of the ADCC activity of human immunoglobulin 7.1.1 Exploration of human immunoglobulin concentration and target cells Under the condition of E / T = 6:1, according to the experimental design, SCIG / IVIG was prepared with ADCC experimental buffer to 2 mg / ml as the starting working concentration, and then diluted at a 10-fold gradient dilution ratio for 5 concentration points. The experimental results are as Figure 6 、 7 shown in Figure 8. Figure 6 Figure Figure 6 is the gradient dilution of SCIG / IVIG under the condition of E / T = 6:1, co-incubated with target cells (PLC / PRF / 5) and effector cells (GS-J2C / CD16A 158V), and the relative light unit values in the reaction system were recorded. The data in the figure are expressed as the mean ± SEM of relative light units (RLU) (n = 2); Figure 7 Figure Figure 7 is the gradient dilution of SCIG / IVIG under the condition of E / T = 6:1, co-incubated with target cells (CHO-K1 / Spike_SARS2) and effector cells (GS-J2C / CD16A 158V), and the relative light unit values in the reaction system were recorded. The data in the figure are expressed as the mean ± SEM of relative light units (RLU) (n = 2); Figure 8 Figure Figure 8 is the gradient dilution of SCIG / IVIG under the condition of E / T = 6:1, co-incubated with target cells (Sp2 / 0-Ag14) and effector cells (GS-J2C / CD16A 158V), and the relative light unit values in the reaction system were recorded. The data in the figure are expressed as the mean ± SEM of relative light units (RLU) (n = 2).

[0087] As can be seen from Figures 6 - 8 Figure Figures 6 - 8 , both human immunoglobulin IVIG and SCIG can induce the ADCC effect on target cells PLC / PRF / 5, CHO-K1 / Spike_SARS2, and Sp2 / 0-Ag14. Among the three target cells, the signal response value of human immunoglobulin for the ADCC effect on CHO-K1 / Spike_SARS2 reached the level of 10 5 RLU, but the signal response values of the other two target cells were only at the level of 10 4 RLU, indicating that target cells of different cell lines have different responses to ADCC. CHO-K1 / Spike_SARS2 is more sensitive to ADCC signal transduction, can significantly enhance the fluorescence response value of effector cells GS-J2C / CD16A 158V, making the detection dynamic range wider, thus more accurately reflecting the changes in the ADCC effect and better evaluating the activity and potency of human immunoglobulin. Therefore, the target cell determined in this study is CHO-K1 / Spike_SARS2.

[0088] In addition, when using this target cell, the concentration of SCIG in the two human immunoglobulins has shown a complete upper and lower plateau. It can be determined that 2 mg / ml is used as the starting working concentration of SCIG, and it is diluted 10-fold, with a total of 5 concentration points. Under the condition of effector-to-target ratio (E:T) of 6:1, the ADCC activity of SCIG was detected. This experimental system has an obvious dose-response relationship, and the dose-response curve is complete, which can effectively detect the ADCC activity of SCIG.

[0089] 7.1.2 Confirmation of appropriate concentration On the premise of determining CHO-K1 / Spike_SARS2 as the target cell, the starting concentration of IVIG was further adjusted and optimized, and systematic method verification was carried out.

[0090] Use IVIG, and adjust and optimize its starting concentration. Under the condition of E / T = 6:1, use the ADCC experimental buffer to prepare IVIG to 1 mg / ml as the starting working concentration, and dilute it at a 4-fold gradient dilution ratio. The experimental results of the detection system with 7 concentration points are as Figure 9 shown; use the ADCC experimental buffer to prepare IVIG to 0.67 mg / ml as the starting working concentration, and dilute it at a 3-fold gradient dilution ratio. The experimental results of the detection system with 7 concentration points are as Figure 10 shown. Figures 9 - 10 Under the condition of E / T = 6:1, IVIG was serially diluted and co-incubated with target cells and effector cells, and the relative luminescence unit values in the reaction system were recorded. The data in the figure are expressed as the mean ± SEM of relative luminescence units (RLU) (n = 2). From Figures 9 - 10 it can be seen that after the starting concentration of IVIG was optimized, the curve fitting degree was significantly improved, and obvious upper and lower platforms appeared in the effector dose curve. This step of the experiment explored the appropriate concentration range of the ADCC effect of IVIG-induced on the target cell CHO-K1 / Spike_SARS2. The exploration of the concentration of the upper and lower platforms of the system not only improved the specificity and sensitivity of the experiment, but also ensured the accuracy and reliability of the results.

[0091] 7.2 Specificity 7.2.1 Target cell specificity Try to use HUVEC cells instead of CHO-K1 / Spike_SARS2 cells as target cells, and perform ADCC activity detection according to the above method; set up a CHO-K1 / Spike_SARS2 control in parallel. The experimental results show that IVIG significantly induced the ADCC effect on the target cell (CHO-K1 / Spike_SARS2); for the target cell HUVEC group, IVIG could not induce the ADCC effect on it ( Figure 11), which reflects the specificity of the target cell CHO-K1 / Spike_SARS2 in this experimental system.

[0092] 7.2.2 Specificity of effector cells Try to use the original Jurkat-T cell line instead of the ADCC reporter cell (GS-J2C / CD16A158V) as the effector cell, and perform the ADCC activity detection according to the above method; set up a control of the ADCC reporter cell (GS-J2C / CD16A 158V) in parallel. The results are as Figure 12 shown. When using Jurkat-T (an unmodified original cell line) as the effector cell, IVIG cannot induce the ADCC effect on the target cell CHO-K1 / Spike_SARS2; while the ADCC reporter cell as the effector cell has a positive test result, which reflects the specificity of the effector cell GS-J2C / CD16A158V in this experimental system.

[0093] 7.2.3 Specificity of test samples Try to use excipients (polysorbate 80 + glycine) instead of IVIG as the test sample, and perform the ADCC activity detection according to the above method; set up a control of IVIG in parallel. The results are as Figure 13 shown. Only the IVIG group showed ADCC activity; the excipients did not show an upward trend in the detection signal and did not have ADCC activity under this experimental system. This reflects the specificity of IVIG (i.e., human immunoglobulin) in this experimental system.

[0094] 7.2.4 Summary of specificity evaluation Only the ADCC reporter cell as the effector cell has a positive test result, which reflects the specificity of the effector cell GS-J2C / CD16A 158V in this experimental system; only human immunoglobulin has a positive test result, which reflects the specificity of the test sample in this experimental system; CHO-K1 / Spike_SARS2 as the target cell has the best positive test result, which reflects the specificity of the target cell in this experimental system; the above experimental results comprehensively prove that: the method of the present invention can effectively detect the ADCC activity of human immunoglobulin and has good specificity.

[0095] 7.3 Method validation 7.3.1 Sensitivity test The initial concentration of human immunoglobulin IVIG is 1 mg / ml, diluted at a 4-fold concentration gradient, and the ADCC effect detection results at 7 concentration points are set ( Figure 9) It is shown that a complete dose standard curve can be obtained when the concentration of IVIG is in the range of 2.44×10 -4 mg / ml to 1×10 0 mg / ml; the starting concentration of IVIG is 0.67 mg / ml, diluted at a 3-fold concentration gradient, and the detection results of the ADCC effect at 7 concentration points are as follows ( Figure 10 ) It is shown that a complete dose standard curve can also be obtained when the concentration of IVIG is in the range of 9.1×10 -4 mg / ml to 6.7×10 -1 mg / ml. It can be seen that the detection limit of the method of the present invention can reach the order of magnitude of 10 -4 mg / ml, which means that the method of the present invention has a stronger ability to detect lower concentrations of IVIG or weaker ADCC effects and high sensitivity.

[0096] Next, the starting concentration of human immunoglobulin IVIG was set at 0.666666667 mg / ml, diluted at a 3-fold concentration gradient, and accuracy and precision experiments were carried out at 7 concentration points. The specific settings are as follows: Human immunoglobulin Conc. (mg / mL): 0.666666667, 0.222222222, 0.074074074, 0.024691358, 0.008230453, 0.002743484, 0.000914495.

[0097] 7.3.2 Accuracy test IVIG was used for the ADCC accuracy test study, and each sample was continuously replicated 3 times. The detection signal values corresponding to the curve concentration points were selected for linear analysis, and the results are shown in Figure 14 , Figure 14 is under the condition of E / T = 6:1, human immunoglobulin was serially diluted, co-incubated with the corresponding effector and target cells, and the relative light unit values in the reaction system were recorded. The data in the figure are expressed as the mean ± SEM (n = 3) of relative light units (RLU). The theoretical signal values were calculated according to the formula of curve fitting; the measured values and the theoretical values were linearly fitted to obtain the recovery rate, which reached 100% (Table 1), indicating that the developed method has extremely high accuracy.

[0098] Table 1 Accuracy verification of the IVIG ADCC reporter gene method (n = 3) Note: Consider Run 01 as 100% RS, that is, the standard product with 100% titer. Divide the EC50 value of Run 01 by the EC50 values of other experimental plates and then multiply by 100% to obtain the relative titer; the mean of the relative titers can be calculated using the AVERAGE function in Excel; divide the mean of the relative titers by the 100% titer and then multiply by 100% to obtain the recovery rate.

[0099] By calculating the R value three times 2 value, the obtained RSD was 0.67%. This result indicates that the degree of data dispersion is small, and the accuracy of this method is within a reasonable range.

[0100] 7.3.2 Precision test: ADCC activity detection was carried out. IVIG with an initial working concentration of 0.67 mg / ml was used for the within-day and between-day precision test studies of ADCC detection. Three independent detections were set within one day, and one 96-well cell culture plate was used for each test to detect the within-day precision. The RLU of the initial working concentration and the concentration for 50% of maximal effect (EC 50 ) were statistically analyzed to verify the precision of this method.

[0101] The experimental results are as Figure 14 shown in Table 2. By calculating the EC value three times 50 value, the obtained RSD was 1%. This result indicates that the degree of data dispersion is small, indicating that the accuracy of this method is within a reasonable range.

[0102] Table 2 Precision verification of the ADCC reporter gene method for IVIG (n = 3) 8. Conclusion Different combinations of target cells, antibodies and effector cells, as well as the conditions during detection (such as incubation time, types of target cells, and the ratio of effector cells to target cells, etc.) have a great impact on the ADCC activity detection results, increasing the complexity of the development of the ADCC activity detection method for human immunoglobulin.

[0103] This study aimed to improve the deficiencies in the existing publicly reported methods for detecting the ADCC of human immunoglobulin against hepatitis B cells in terms of target cell selection, effector cell stability, applicability of the detection method, and optimization of experimental conditions. By making attempts in three aspects: the ADCC detection method, the initial concentration of human immunoglobulin, and target cell selection, a detection method for the ADCC biological activity of human immunoglobulin with high accuracy, sensitivity and precision was obtained. The specific method steps are as follows: Human immunoglobulin products (for intravenous injection of human immunoglobulin, IVIG, the starting working concentration is 1 mg / ml, and then it is uniformly diluted at a 4-fold gradient dilution ratio to obtain 7 concentration points, or for IVIG, the starting working concentration is 0.67 mg / ml, and then it is uniformly diluted at a 3-fold gradient dilution ratio to obtain 7 concentration points; for subcutaneous injection of human immunoglobulin, SCIG, the starting working concentration is 2 mg / ml, and then it is uniformly diluted at a 10-fold gradient dilution ratio to obtain 5 concentration points); adjust the target cell density (2.5E5 cells / ml) with ADCC assay buffer (RPMI 1640 + 10% FBS), and transfer the target cell suspension to a 96-well assay plate (40 μl / well) according to the detection protocol; transfer the human immunoglobulin product to the corresponding wells of the 96-well plate and incubate the assay plate at room temperature for 30 minutes. Collect the effector cells and resuspend them with ADCC assay buffer. According to the E / T ratio of 6:1 (effector-to-target ratio, the ratio of effector cells to target cells), adjust the effector cell density (1.5E6 cells / ml) with ADCC assay buffer, and transfer the effector cell suspension to the corresponding wells of the 96-well plate (40 μl / well). Incubate the assay plate in a cell culture incubator (37°C / 5% CO2) for 6 hours. After incubation, take out the 96-well assay plate and add the working solution of the Fire-LumiTM luciferase detection kit (80 μl / well) to the corresponding wells and incubate for 5 - 10 minutes. Read the chemiluminescence value at room temperature and then perform data analysis and result collation.

[0104] In summary, the detection method for the ADCC biological activity of the present inventor's human immunoglobulin can accurately and reliably detect the ADCC biological activity of two different human immunoglobulin preparations, intravenous injection human immunoglobulin and subcutaneous injection human immunoglobulin, through the reporter gene detection method with CHO-K1 / SPIKE_SARS2 as the target cells and GS-J2C / CD16A 158V as the effector cells, in combination with a human immunoglobulin solution at a specific concentration. Compared with the similar methods disclosed in the prior art, it has higher accuracy, precision and sensitivity.

Claims

1. A method for detecting the antibody - dependent cell - mediated cytotoxicity biological activity of human immunoglobulin, characterized in that: It includes the following steps: 1) Add the target cell suspension into a well plate, then add human immunoglobulin solutions with a series of concentrations for incubation, and finally add the effector cell suspension for incubation; 2) Take the well plate incubated in step 1) to detect fluorescence. Use the relative light unit value as the ordinate and the logarithm of the human immunoglobulin solution concentration as the abscissa to plot a dose-effect curve and calculate the median effective concentration; The target cells are CHO-K1 / SPIKE_SARS2, and the effector cells are GS-J2C / CD16A 158V.

2. The detection method according to claim 1, wherein: The effector-to-target ratio of effector cells to target cells in each well of the well plate is 6:

1.

3. The detection method according to claim 1, characterized in that: When the human immunoglobulin solution is an intravenous human immunoglobulin solution, the concentration range is 2.44×10 -4 mg / ml to 1 mg / ml.

4. The detection method according to claim 3, characterized in that: When the human immunoglobulin solution is an intravenous injection human immunoglobulin solution, use 1 mg / ml as the starting working concentration and then dilute it 6-fold with a 4-fold serial dilution ratio, or use 0.67 mg / ml as the starting working concentration and then dilute it 6-fold with a 3-fold serial dilution ratio.

5. The detection method according to claim 1, wherein: When the human immunoglobulin solution is a subcutaneous injection human immunoglobulin solution, the concentration range is 2×10 -4 mg / ml to 2 mg / ml.

6. The detection method according to claim 5, characterized in that: When the human immunoglobulin solution is a subcutaneous injection human immunoglobulin solution, use 2 mg / ml as the starting working concentration and then dilute it 4-fold with a 10-fold serial dilution ratio.

7. The detection method according to claim 1, characterized in that: The target cell suspension, human immunoglobulin solutions with a series of concentrations, and effector cell suspension are prepared with RPMI 1640 containing 10% FBS.

8. The detection method according to claim 7, wherein: The concentration of the target cell suspension is 2.5×10 5 cells / mL, and the volume is 40 μL / well; the volume of the human immunoglobulin solution with a series of concentrations is 20 μL / well; the concentration of the effector cell suspension is 1.5×10 6 cells / mL, and the volume is 40 μL / well.

9. The detection method according to claim 1, characterized in that: The conditions for incubating the target cell suspension with the human immunoglobulin solution are: incubate at room temperature for 30 minutes; the conditions for incubating after adding the effector cell suspension are to incubate at 37 °C under 5% CO2 for 4 hours.

10. The detection method according to claim 1, wherein: The fluorescence detection is performed using a luciferase detection kit; the luciferase detection kit is Fire-Lumi TM Luciferase detection kit

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