A method for detecting the biological activity of antibody-dependent cell-mediated cytotoxicity of human immunoglobulin

Abstract

The present invention belongs to the field of drug detection and specifically discloses a method for detecting the antibody-dependent cell-mediated cytotoxic biological activity of human immunoglobulin, comprising the following steps: 1) taking a target cell suspension and adding it to a well plate, then adding a series of concentrations of human immunoglobulin solutions for incubation, and finally adding an effector cell suspension for incubation; 2) taking the well plate incubated in step 1) to detect fluorescence, draw a dose-effect curve and calculate the half-maximal effective concentration; the target cells are CHO-K1 cells expressing the SARS-CoV-2 Spike protein, and the effector cells are Jurkat cells stably expressing CD16a on the cell membrane and stably transfected with the firefly luciferase gene driven by the NFAT response element. The detection method of the present invention can accurately and reliably detect the antibody-dependent cell-mediated cytotoxic biological activity of two different human immunoglobulin preparations, IVIG and SCIG.

Description

Technical Field

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

[0002] Human immunoglobulins are glycoproteins produced by plasma cells. The primary component of these glycoproteins is IgG, which can be cleaved by papain into two identical Fab segments and an Fc segment. The Fab segment recognizes specific antigens, while the Fc segment binds to receptors on the surface of immune cells. Research has shown that the biological functions of the IgG Fab and Fc segments play a crucial role in the immunomodulatory effects of human immunoglobulins, enabling them to participate in humoral immune responses.

[0003] Human immunoglobulin preparations are the most commonly used blood products in clinical practice. They are primarily divided into subcutaneous immunoglobulin (SCIG) and intravenous immunoglobulin (IVIG). While no SCIG products have been approved for marketing in China, clinical use of SCIG products abroad has demonstrated comparable or superior efficacy to intravenous immunoglobulin (IVIG). Human immunoglobulin preparations are primarily used to treat diseases manifested by humoral immune deficiency or immune system dysfunction. Because they are prepared from pooled plasma from thousands of individuals, they contain a broad spectrum of antibodies against infectious diseases and proteins from foreign species.

[0004] Antibody-dependent cell-mediated cytotoxicity (ADCC): ADCC is a key mechanism of the immune system, primarily mediated by natural killer (NK) cells. When the Fab end of an antibody binds to an antigenic epitope on the surface of a target cell, its Fc end recruits effector cells (NK cells) and binds to their surface Fc receptor IIIa (FcγRIIIa), triggering the release of cytotoxic substances (such as perforin and granzymes), thereby killing the target cell. This mechanism of action plays a crucial role in the body's immune defense and immune regulation. Therefore, testing whether an antibody drug exhibits ADCC biological activity and the strength of this activity has become a crucial step in antibody drug development and quality control.

[0005] Currently, various methods have been established to measure the ADCC biological activity of antibody drugs, such as chromium-51 (51Cr) labeling / release and lactate dehydrogenase release methods. However, these methods are subject to issues such as radioactive contamination and poor reproducibility. Patent CN115537448A discloses a method for detecting hepatitis B virus using antibody-dependent cell-mediated cytotoxicity of human immunoglobulins, filling a gap in the current ADCC detection of IVIG using reporter gene assays. However, experimental verification indicates that the assay has low sensitivity due to factors such as the types of target and effector cells, incubation conditions, and effector-target ratio, making it impossible to accurately measure the ADCC biological potency and efficacy of human immunoglobulins. Therefore, there is a need for a more sensitive and accurate method for 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 cytotoxicity biological activity of human immunoglobulin, comprising the following steps:

[0007] 1) Add the target cell suspension to the well plate, then add a series of concentrations of human immunoglobulin solution for incubation, and finally add the effector cell suspension for incubation;

[0008] 2) Detect fluorescence from the incubated well plate in step 1) and plot a dose-effect curve using the relative luminescence unit value as the ordinate and the logarithm of the human immunoglobulin solution concentration as the abscissa to calculate the median effective concentration.

[0009] The target cells are CHO-K1 / SPIKE_SARS2 and the effector cells are GS-J2C / CD16A 158V.

[0010] Furthermore, the effector-target ratio of effector cells to target cells in each well of the well plate is 6:1.

[0011] Furthermore, when the human immunoglobulin solution is an intravenous human immunoglobulin solution, the concentration range is 2.44×10 -4 mg / ml~1mg / ml.

[0012] Furthermore, when the human immunoglobulin solution is an intravenous human immunoglobulin solution, 1 mg / ml is used as the starting working concentration, and then a 4-fold gradient dilution ratio is used to dilute 6 concentrations, or 0.67 mg / ml is used as the starting working concentration, and then a 3-fold gradient dilution ratio is used to dilute 6 concentrations.

[0013] Furthermore, when the human immunoglobulin solution is a subcutaneous injection human immunoglobulin solution, the concentration range is 2×10 -4 mg / ml~2mg / ml.

[0014] Furthermore, when the human immunoglobulin solution is a subcutaneous injection human immunoglobulin solution, 2 mg / ml is used as the initial working concentration, and then diluted to 4 concentrations using a 10-fold gradient dilution ratio.

[0015] Furthermore, the target cell suspension, human immunoglobulin solution with a series of concentrations and effector cell suspension are prepared using RPMI 1640 containing 10% FBS.

[0016] Furthermore, the concentration of the target cell suspension is 2.5×10 5 cells / ml, the volume is 40 μl / well; the volume of the human immunoglobulin solution of serial concentrations is 20 μl / well; the concentration of the effector cell suspension is 1.5×10 6 cells / ml, with a volume of 40 μl / well.

[0017] Furthermore, the target cell suspension was incubated with the human immunoglobulin solution at room temperature for 30 minutes; and the effector cell suspension was added and incubated at 37° C. and 5% CO 2 for 4 hours.

[0018] Furthermore, the fluorescence detection is performed using a luciferase detection kit; the luciferase detection kit is a Fire-Lumi TM Luciferase Assay Kit.

[0019] The method for detecting the ADCC biological activity of human immunoglobulins of the present invention uses CHO-K1 / SPIKE_SARS2 as target cells and GS-J2C / CD16A 158V effector cells in combination with a human immunoglobulin solution of a specific concentration. The method can accurately and reliably detect the ADCC biological activity of two different human immunoglobulin preparations, namely, intravenous human immunoglobulin and subcutaneous human immunoglobulin, through a reporter gene detection method. Compared with similar methods disclosed in the prior art, the method has higher accuracy, precision and sensitivity, and has practical value for promotion and application.

[0020] 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.

[0021] The following further describes the above content of the present invention in detail 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1PBMC-ADCC preliminary experimental concentration exploration results (LDH method);

[0023] Figure 2 Experimental results after optimization of the PBMC-ADCC system (LDH method)

[0024] Figure 3 NK92 / CD16A-vv-ADCC assay results (LDH method)

[0025] Figure 4 NK92 / CD16A-vv-ADCC assay results (flow cytometry)

[0026] Figure 5 Results of ADCC dose-response experiments of SCIG on target cells PLC / PRF / 5

[0027] Figure 6 The results of the ADCC dose-response experiment of human immunoglobulin on the target cells PLC / PRF / 5 support the exploration of human immunoglobulin concentration.

[0028] Figure 7 The results of the ADCC dose-response experiment of human immunoglobulin on the target cells CHO-K1 / Spike_SARS2 support the exploration of human immunoglobulin concentration.

[0029] Figure 8 The results of the ADCC dose-response experiment of human immunoglobulin on target cells Sp2 / 0-Ag14 support the exploration of human immunoglobulin concentration.

[0030] Figure 9 Results of the ADCC dose-response experiment of IVIG on target cells CHO-K1 / Spike_SARS2;

[0031] Figure 10 Results of the ADCC dose-response experiment of IVIG on target cells CHO-K1 / Spike_SARS2;

[0032] Figure 11 The effects of different target cells on the methods of the present invention;

[0033] Figure 12 Effects of different effector cells on the method of the present invention

[0034] Figure 13 Effects of different test samples on the method of the present invention

[0035] Figure 14 ADCC dose-response experiment results of human immunoglobulin DETAILED DESCRIPTION

[0036] The raw materials, reagents and equipment used in the specific embodiments of the present invention are all purchased from commercial sources.

[0037] Example 1 Detection of ADCC biological activity of intravenous human immunoglobulin IVIG of the present invention

[0038] 1) Solution Preparation

[0039] Target cell suspension: Take the revived CHO-K1 / SPIKE_SARS2 and add RPMI 1640 containing 10% FBS to a concentration of 2.5×10 5 cells / mL of cell suspension;

[0040] Effector cell suspension: Take the revived GS-J2C / CD16A 158V and add RPMI 1640 containing 10% FBS to a concentration of 1.5×10 6 cells / ml of cell suspension;

[0041] IVIG solution of serial concentrations: IVIG was prepared by adding RPMI 1640 containing 10% FBS to a solution with a concentration of 1 mg / ml as the starting working concentration solution. Then, the solution was diluted with RPMI 1640 containing 10% FBS in a 4-fold gradient to obtain 6 concentrations, namely 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, the total concentration range was 2.44×10 -4 7 concentrations of IVIG solution ranging from mg / ml to 1mg / ml;

[0042] 2) ADCC biological activity assay

[0043] The target cell suspension was added to a 96-well plate at 40 μl / well, and then a series of IVIG solutions were added at 20 μl / well. The cells were incubated at room temperature for 30 minutes. Finally, the effector cell suspension was added at 40 μl / well to make the effector-target ratio of each well 6:1. The cells were then incubated at 37°C, 5% CO2 for 4 hours. The 96-well plate was removed and Fire-LumiNano was added at 80 μl / well. TM Incubate with the luciferase assay kit working solution for 5-10 minutes and read the relative luminescence units using a PHERAstar FSX at room temperature.

[0044] 3) Calculation

[0045] The relative luminescence unit value was used as the ordinate and the logarithm of the IVIG solution concentration was used as the abscissa. The dose-effect curve of IVIG was fitted using a four-parameter curve equation regression model. The EC value of IVIG was obtained based on the dose-effect curve. 50 value.

[0046] Example 2 Detection of ADCC biological activity of intravenous human immunoglobulin IVIG of the present invention

[0047] 1) Solution Preparation

[0048] Target cell suspension: Take the revived CHO-K1 / SPIKE_SARS2 and add RPMI 1640 containing 10% FBS to a concentration of 2.5×10 5 cells / ml of cell suspension;

[0049] Effector cell suspension: Take the revived GS-J2C / CD16A 158V and add RPMI 1640 containing 10% FBS to a concentration of 1.5×10 6 cells / ml of cell suspension;

[0050] IVIG solution with serial concentrations: IVIG was prepared by adding RPMI 1640 containing 10% FBS to a concentration of 0.67 mg / ml as the starting working concentration solution. The solution was then diluted with RPMI 1640 containing 10% FBS in a 3-fold serial dilution ratio to 6 concentrations, namely 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, the total concentration range was 9.14×10 -4 7 concentrations of IVIG solution ranging from 0.67 mg / ml to 0.67 mg / ml;

[0051] 2) ADCC biological activity assay

[0052] The target cell suspension was added to a 96-well plate at 40 μl / well, and then a series of IVIG solutions were added at 20 μl / well. The cells were incubated at room temperature for 30 minutes. Finally, the effector cell suspension was added at 40 μl / well to make the effector-target ratio of each well 6:1. The cells were then incubated at 37°C, 5% CO2 for 4 hours. The 96-well plate was removed and Fire-LumiNano was added at 80 μl / well. TMIncubate with the luciferase assay kit working solution for 5-10 minutes and read the relative luminescence units using a PHERAstar FSX at room temperature.

[0053] 3) Calculation

[0054] The relative luminescence unit value was used as the ordinate and the logarithm of the IVIG solution concentration was used as the abscissa. The dose-effect curve of IVIG was fitted using a four-parameter curve equation regression model. The EC value of IVIG was obtained based on the dose-effect curve. 50 value.

[0055] Example 3 Detection of ADCC biological activity of subcutaneous immunoglobulin SCIG of the present invention

[0056] 1) Solution Preparation

[0057] Target cell suspension: Take the revived CHO-K1 / SPIKE_SARS2 and add RPMI 1640 containing 10% FBS to a concentration of 2.5×10 5 cells / mL of cell suspension;

[0058] Effector cell suspension: Take the revived GS-J2C / CD16A 158V and add RPMI 1640 containing 10% FBS to a concentration of 1.5×10 6 cells / mL of cell suspension;

[0059] SCIG solution of serial concentrations: SCIG was prepared by adding RPMI 1640 containing 10% FBS to a solution with a concentration of 2 mg / ml as the starting working concentration solution, and then diluted with RPMI 1640 containing 10% FBS in a 10-fold serial dilution ratio to four concentrations, namely 2 × 10 -1 mg / ml, 2×10 -2 mg / ml, 2×10 -3 mg / ml, 2×10 -4 mg / ml, the total concentration range was 2×10 -4 SCIG solution with 5 concentrations ranging from mg / ml to 2mg / ml;

[0060] 2) ADCC biological activity assay

[0061] The target cell suspension was added to a 96-well plate at 40 μl / well, and then a series of SCIG solutions were added at 20 μl / well. The cells were incubated at room temperature for 30 minutes. Finally, the effector cell suspension was added at 40 μl / well to make the effector-target ratio of each well 6:1. The cells were then incubated at 37°C, 5% CO2 for 4 hours. The 96-well plate was removed and Fire-Lumi was added at 80 μl / well. TMIncubate with the luciferase assay kit working solution for 5-10 minutes and read the relative luminescence units using a PHERAstar FSX at room temperature.

[0062] 3) Calculation

[0063] The relative luminescence unit value was used as the ordinate and the logarithm of the SCIG solution concentration was used as the abscissa. The dose-effect curve of SCIG was fitted using a four-parameter curve equation regression model. The EC value of SCIG was obtained based on the dose-effect curve. 50 value.

[0064] The beneficial effects of the present invention are further illustrated by the following test examples:

[0065] Experimental Example 1 Exploration of the Detection Method of Human Immunoglobulin ADCC Biological Activity

[0066] 1. Purpose of the test

[0067] By trying different ADCC detection methods, a detection method suitable for ADCC biological activity of human immunoglobulin was constructed.

[0068] 2. Test principle

[0069] In this study, three methods were tried to explore ADCC detection, namely (1) LDH release assay (LDH), (2) flow cytometry (FACS) and (3) luciferase reporter gene assay.

[0070] 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 that highly express 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 the NK cell and causing it to release cytotoxic substances (such as perforin and granzyme) to kill the target cell. The ADCC activity of the antibody is characterized by flow cytometry detection of target cell death (FACS method) or direct detection of lactate dehydrogenase (LDH) activity released into the culture medium after the target cell is killed (LDH release assay method).

[0071] Luciferase reporter gene assay: The ADCC effect is mainly mediated by FcγRIIIa. Antibodies bind to FcγRIIIa on the surface of effector cells, stimulating the intracellular NFAT response element, thereby driving the expression of firefly luciferase. Quantification of luciferase activity by bioluminescence can characterize the ADCC activity of the antibody.

[0072] 3. Cell lines used in the experiment

[0073]

[0074] 4. Human immunoglobulin information

[0075]

[0076] 5. Experimental Design

[0077] 5.1PBMC-ADCC Assay (LDH Method)

[0078]

[0079] 5.2 PBMC-ADCC Assay & NK92 / CD16A-vv-ADCC Assay (LDH Method)

[0080]

[0081] 5.3 NK92 / CD16A-vv-ADCC Detection (Flow Cytometry)

[0082]

[0083] 6. Test steps

[0084] 6.1 LDH method

[0085] 6.1.1 ADCC Experimental Procedure

[0086] 1) If hPBMCs are used as effector cells, thaw the cells the day before the experiment and culture them overnight in RPMI 1640 complete medium (RPMI 1640 + 10% FBS). During the culture period, add recombinant human interleukin-2 protein to a concentration of 100 IU / ml. On the day of the experiment, harvest the effector cells and resuspend them in ADCC assay buffer (99% MEM α, nucleosides, no phenol red + 1% fetal bovine serum).

[0087] If the effector cells are NK92 / CD16A-vv, the effector cells are collected on the day of the experiment and resuspended in ADCC detection assay buffer.

[0088] 2) Digest and collect target cells by centrifugation and resuspend them in ADCC discovery assay buffer.

[0089] 3) Prepare a concentration gradient of human immunoglobulin samples using ADCC exploratory assay buffer.

[0090] 4) Adjust the target cell density to 2E5 cells / ml with ADCC discovery assay buffer and transfer the target cell suspension to the corresponding wells of a 96-well assay plate (50 μl / well, 10,000 cells / well).

[0091] 5) Transfer human immunoglobulin concentration gradient samples or ADCC scouting assay buffer to the corresponding wells of a 96-well assay plate (50 μl / well).

[0092] 6) Incubate the plate at room temperature for 30 minutes.

[0093] 7) Adjust the effector cell density based on the E:T ratio using ADCC discovery assay buffer and add recombinant human interleukin-2 protein to a concentration of 200 IU / ml (for a final concentration of 100 IU / ml in the assay system). Transfer the effector cell suspension to the corresponding wells of a 96-well assay plate (100 μl / well).

[0094] 8) Incubate the experimental plate in a cell culture incubator (37°C / 5% CO2) for 6 hours.

[0095] 9) After incubation, remove the 96-well plate, centrifuge, and carefully aspirate the supernatant. Transfer the supernatant to a new 96-well plate (50 μl / well). Transfer LDH assay working solution (50 μl / well, Roche, 11644793001) to the corresponding wells of the new 96-well plate and develop color at room temperature.

[0096] 10) Read the OD value on a microplate reader with a detection wavelength of 492 nm and a reference wavelength of 650 nm.

[0097] 6.1.2 ADCC Data Analysis

[0098] The raw data of ADCC experiments detected by LDH method were exported via PHERAstar FSX system and analyzed using Microsoft Office Excel software.

[0099] The data for calculating target cell lysis rate are: OD 492 nm -OD 650 nm The target cell lysis rate was calculated as follows: %Target cell lysis = 100 × (OD Sample data -OD Tumor cells plus effector cells ) / (OD Maximum release -OD Minimum releaseWhere: Maximum release is the LDH release after target cells were treated with lysis buffer (1% Triton); Minimum release is the LDH release after target cells were treated with ADCC discovery buffer; E:T (Tumor cells plus effector cells) is the LDH release after effector and target cells were treated with ADCC discovery buffer; Sample is the LDH release after effector and target cells were treated with sample. EC can be obtained using the following four-parameter equation in Microsoft Office Excel: 50 Relative value: Y = Bottom + (Top - Bottom) / (1 + 10 ^ ((LogEC 50 = - X) × HillSlope)). Where X = Log(Sample Concentration) and Y = Target Cell Lysis Rate.

[0100] 6.2 FACS

[0101] 6.2.1 ADCC Experimental Procedure

[0102] 1) Collect effector cells (NK92 / CD16A-vv) and resuspend in ADCC detection buffer (99% MEM α, nucleosides, no phenol red + 1% fetal bovine serum);

[0103] 2) Digest and centrifuge the target cells and resuspend them in DPBS;

[0104] 3) Label a certain number of experimental target cells with Cell Trace Violet dye (Cell Trace Violet, C34557) according to experimental requirements. After labeling, resuspend the labeled target cells in ADCC detection assay buffer;

[0105] 4) Prepare a concentration gradient of human immunoglobulin samples using ADCC exploratory assay buffer;

[0106] Adjust the density of Cell Trace Violet-labeled target cells to 2E5 cells / ml with ADCC discovery buffer and transfer the labeled target cell suspension to the corresponding wells of a 96-well assay plate (50 μl / well, 10,000 cells / well).

[0107] 6) Transfer the human immunoglobulin concentration gradient sample or ADCC assay buffer to the corresponding wells of a 96-well assay plate (50 μl / well);

[0108] 7) Incubate the plate at room temperature for 30 minutes.

[0109] 8) Adjust the effector cell density based on the E:T ratio using ADCC discovery assay buffer and add recombinant human interleukin-2 protein to a concentration of 200 IU / ml (for a final concentration of 100 IU / ml in the assay system). Transfer the effector cell suspension to the corresponding wells of a 96-well assay plate (100 μl / well).

[0110] 9) Incubate the experimental plate in a cell culture incubator (37°C / 5% CO2) for 5 hours.

[0111] 10) After incubation, remove the 96-well plate, centrifuge, and discard the supernatant. Wash the cells with flow cytometry buffer (1× DPBS + 1% fetal bovine serum). Stain the cells with 7-AAD ten minutes before loading.

[0112] 11) ADCC was detected by flow cytometry, with CellTrace Violet and 7-AAD fluorescence detected using the Pacific Blue and 7-AAD channels, respectively.

[0113] 6.2.2 ADCC Data Analysis

[0114] The raw data of ADCC experiments detected by flow cytometry were exported via BD FACSDiva Software system and analyzed using Microsoft Office Excel and FlowJo software.

[0115] The target cell lysis rate was calculated using the following formula: % Target cell lysis = (Pacific Blue + 7-AAD + Cells / All Pacific Blue + Cells) × 100%. In Microsoft Office Excel, the following four-parameter equation can be used to obtain EC 50 Relative value: Y = Bottom + (Top - Bottom) / (1 + 10 ^((LogEC 50 = - X) × HillSlope)). Where X = Log(Sample Concentration) and Y = Target Cell Lysis Rate.

[0116] 7. Test results

[0117] 7.1 LDH Assay—ADCC Assay Results of PBMC Effector Cells

[0118] 7.1.1 Preliminary experiments to confirm the appropriate concentration

[0119] According to the aforementioned experimental method, three effector-target ratio gradients were initially set up: E:T=10:1, 25:1, and 50:1. Human immunoglobulins IVIG and SCIG were selected, and the ADCC effect induced by PBMC effector cells was detected using the LDH method.

[0120] The experimental results are as follows Figure 1 As shown in the figure, human immunoglobulin (IVIG, SCIG) was diluted to a certain extent and incubated with the corresponding effector and target cells for 6 hours under the conditions of E:T=10:1, 25:1, and 50:1. The target cell lysis rate was detected. The data in the figure are expressed as the mean ± SEM (n=2) of the target cell lysis rate. Figure 1 It can be seen that human immunoglobulins do not show obvious ADCC effect on target cells.

[0121] Based on this, further optimization experiments on human immunoglobulin concentration gradient and effector-target ratio were carried out.

[0122] 7.1.2 Optimization of Human Immunoglobulin Concentration Gradient and Effector-Target Ratio

[0123] Based on the experimental results of 7.1.1, the starting concentration of the experimental human immunoglobulin was optimized and adjusted: according to the above experimental method, human immunoglobulin SCIG was selected, and two effector-target ratio gradients were set: E:T = 4:1 and 8:1. The ADCC effect induced by PBMC effector cells was detected using the LDH method.

[0124] The experimental results are as follows Figure 2 As shown in the figure, human immunoglobulin (SCIG) was diluted to a certain extent and incubated with the corresponding effector and target cells for 6 hours under the conditions of E:T=4:1 and 8:1, and the target cell lysis rate was detected. The data in the figure are expressed as the mean ± SEM of the target cell lysis rate (experimental group: n=2). Figure 2 It can be seen that under the conditions of E:T=4:1 and 8:1, human immunoglobulin SCIG failed to mediate the killing effect of PBMC on target cells. The experimental results show that the optimization of the effector-target ratio has no significant correlation with the ADCC effect.

[0125] Based on this, PBMC effector cells were replaced with NK92 / CD16A-vv effector cells to further conduct exploratory experiments.

[0126] 7.2 LDH Assay—ADCC Detection Results of NK92 / CD16A-vv Effector Cells

[0127] According to the aforementioned experimental method, two effector-target ratio gradients were set: E:T = 5:1 and 10:1, SCIG was selected, and the ADCC effect induced by NK92 / CD16A-vv effector cells was detected using the LDH method.

[0128] The experimental results are as follows Figure 3 As shown in the figure, SCIG was diluted to a certain extent and incubated with the corresponding effector and target cells for 6 hours under the conditions of E:T = 5:1 and 10:1, and the target cell lysis rate was detected. The data in the figure are expressed as the mean ± SEM of the target cell lysis rate (experimental group: n = 2). Figure 3 It can be seen that under the conditions of E:T = 5:1 and 10:1, both human immunoglobulins showed no significant ADCC effect on target cells. Based on this, the LDH method was replaced with the FACS method for further experiments.

[0129] 7.3 FACS Method - ADCC Detection Results of NK92 / CD16A-vv Effector Cells

[0130] Based on the results in 7.1 and 7.2, no cytotoxicity of the experimental SCIG against the target cells was observed using the LDH assay. Therefore, flow cytometry was used to evaluate the ADCC activity of human immunoglobulins. Following the aforementioned protocol, SCIG was selected using NK92 / CD16A-vv effector cells. Two effector-target ratio gradients (E:T = 5:1 and 10:1) were established. FACS was used to assess ADCC induced by NK92 / CD16A-vv effector cells.

[0131] The experimental results are as follows Figure 4 As shown in the figure, SCIG was diluted at different concentrations and incubated with PLC / PRF / 5 cells labeled with Cell Trace Violet dye for 5 hours at E:T ratios of 5:1 and 10:1. After incubation, the cells in the experimental wells were stained with 7-AAD and analyzed by flow cytometry. The data in the figure are expressed as mean ± SEM of target cell lysis rate (experimental group: n = 2). 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 target cells.

[0132] 8. Discussion

[0133] In the study of evaluating the in vitro biological activity of human immunoglobulins, two direct killing methods, LDH method and FACS method, were used to analyze the ADCC functional activity of human immunoglobulins.

[0134] In experiment 7.1, PBMCs were used as effector cells, and the LDH method was used to detect ADCC function. The effector-target ratio and the starting concentration of human immunoglobulin were explored and optimized. The experimental results showed that IVIG and SCIG did not exhibit ADCC function against PLC / PRF / 5 target cells, and the killing results were negative.

[0135] In experiment 7.2, NK92 / CD16A-vv was used as effector cells and ADCC function was detected using the LDH method. The experimental results showed that SCIG did not exhibit ADCC function against PLC / PRF / 5 target cells, and the killing result was negative.

[0136] In experiment 7.3, NK92 / CD16A-vv was used as effector cells and ADCC function was detected by FACS. The experimental results showed that SCIG did not exhibit ADCC function against PLC / PRF / 5 target cells, and the killing result was negative.

[0137] Based on the above experimental results, this experiment will not consider using LDH release assay and flow cytometry as ADCC activity detection methods. Based on this, we further explored the luciferase reporter gene method.

[0138] 9. Preliminary exploration of luciferase reporter gene assay

[0139] We preliminarily explored the ADCC reporter gene detection method, using PLC / PRF / 5 cells expressing human hepatitis B surface antigen (HBsAg) as target cells and genetically engineered Jurkat cells (GS-J2C / CD16A 158V) as effector cells, and performed ADCC activity detection on SCIG and IVIG, respectively.

[0140] The starting concentration of SCIG was 30 mg / ml, and the cells were serially diluted 4-fold to 8 concentration points. The ADCC effect induced by GS-J2C / CD16A 158V effector cells was detected using a reporter gene assay.

[0141] The experimental results are as follows Figure 5 As shown in the figure, SCIG was serially diluted under E / T = 6:1 and incubated with target cells and effector cells. The relative luminescence unit values ​​in the reaction system were recorded. The data in the figure are expressed as the mean relative luminescence unit (RLU) ± SEM (n = 3). Figure 5 It can be seen that SCIG can significantly induce ADCC effect on target cells PLC / PRF / 5, and the luciferase reporter gene method is suitable as a method for detecting the ADCC effect induced by SCIG on GS-J2C / CD16A 158V effector cells.

[0142] Experimental Example 2 Exploration and establishment of luciferase reporter gene assay

[0143] 1. Experimental Purpose

[0144] This study aimed to select target cells using the luciferase reporter gene method, optimize the starting concentration of IVIG / SCIG, and evaluate the specificity, precision, and accuracy of the method.

[0145] 2. Experimental Principle

[0146] The ADCC effect is primarily mediated by FcγRIIIa. Antibodies bind to FcγRIIIa on the surface of effector cells, stimulating the NFAT response element within the effector cells, thereby driving the expression of firefly luciferase. Quantifying luciferase activity through bioluminescence can characterize the ADCC activity of antibodies.

[0147] The ADCC reporter gene assay used CHO-K1 / Spike_SARS2 cells overexpressing Spike protein in CHO-K1, PLC / PRF / 5 cells expressing human hepatitis B surface antigen (HBsAg), and Sp2 / 0-Ag14 cells as target cells; genetically engineered Jurkat cells (GS-J2C / CD16A 158V) were used as effector cells. These cells stably expressed CD16a (FcγRIIIa, V158 high-affinity mutant) on the cell membrane and were stably transfected with the firefly luciferase gene driven by the NFAT response element.

[0148] 3. Cell lines used in the experiment

[0149]

[0150] 4. IVIG / SCIG and reference material information

[0151]

[0152] 5. Experimental Design

[0153]

[0154] 6. Experimental steps

[0155] 6.1 Cell culture

[0156] 6.1.1 Cell culture and passaging

[0157] 6.1.1.1 Cell Thawing: Add 4 ml of preheated culture medium to a 15 ml centrifuge tube and set aside. Thaw the cryovial rapidly in a 37°C water bath. Transfer the thawed cell suspension to the aforementioned centrifuge tube. Centrifuge at 800 rpm for 5 minutes. Resuspend the cell pellet in complete culture medium, count the cells, and seed the cells at an appropriate density in a cell culture dish. Gently shake to mix thoroughly, and then incubate in a 5% CO2, 37°C incubator.

[0158] 6.1.1.2 Cell Passaging:

[0159] 6.1.1.2.1 Cell Passaging (GS-J2C / CD16A 158V): Pipette cells into a 15 ml centrifuge tube and centrifuge at 800 rpm for 5 minutes. Resuspend the cell pellet in complete culture medium (Probio, RD00830, RPMI 1640 + 10% FBS + 1 µg / ml Puromycin + 200 µg / ml Hygromycin B + 400 µg / ml G418), count the cells, and seed the cells at an appropriate density in a cell culture dish. Gently shake to mix, and culture in a 5% CO2, 37°C incubator.

[0160] 6.1.1.2.2 Cell Passaging (PLC / PRF / 5): Collect the supernatant into a 15 mL centrifuge tube, wash once with preheated DPBS, add an appropriate amount of preheated Accutase digestion solution, and digest in a 37°C incubator for a desired time (the specific time depends on the cell status). Add an appropriate amount of complete culture medium (ATCC, CRL-1581, EMEM Medium + 10% FBS) to terminate digestion, and centrifuge at 800 rpm for 5 minutes. Subsequent counting and plating procedures are the same as above.

[0161] 6.1.1.2.3 Cell Passaging (CHO-K1 / Spike_SARS2): Collect the supernatant into a 15 ml centrifuge tube, wash once with preheated DPBS, add an appropriate amount of preheated Accutase digestion solution, and digest in a 37°C incubator for a specified time (the specific time depends on the cell status). Add an appropriate amount of complete culture medium (Probio, RD00819, F-12 + 10% FBS + 8 μg / ml Puromycin) to terminate digestion, count, and seed the cells at an appropriate density in a cell culture dish. Gently shake to mix, and then culture in a 5% CO2, 37°C incubator.

[0162] 6.1.1.2.4 Cell Passaging (Sp2 / 0-Ag14): Pipette cells into a 15 ml centrifuge tube and centrifuge at 800 rpm for 5 minutes. Resuspend the cell pellet in complete culture medium (ATCC, CRL-1581, DMEM + 10% FBS), count the cells, and seed the cells at an appropriate density in a cell culture dish. Gently shake to mix, and then culture in a 5% CO2, 37°C incubator.

[0163] 6.1.2 ADCC reporter gene assay experimental steps

[0164] 6.1.2.1 Add 2 ml of StemPro Accutase to digest the cells. Collect the target cells by centrifugation and resuspend them in 5 ml of ADCC assay buffer (RPMI 1640 + 10% FBS).

[0165] 6.1.2.2 Exploring the Suitable Concentration for ADCC Reporter Gene Assay

[0166] (1) Preliminary concentration exploration experiment: 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 to 5 concentration points using a 10-fold gradient dilution ratio.

[0167] (2) Confirmation experiment of appropriate concentration: According to the experimental design, IVIG was prepared with ADCC experimental buffer to 1 mg / ml as the starting working concentration, and then diluted to 7 concentration points with a uniform 4-fold gradient dilution ratio. IVIG was also prepared with ADCC experimental buffer to 0.67 mg / ml as the starting working concentration, and then diluted to 7 concentration points with a uniform 3-fold gradient dilution ratio.

[0168] 6.1.2.3 Adjust the target cell density (2.5E5 cells / ml) with ADCC assay buffer and transfer the target cell suspension into a 96-well assay plate (40 µl / well) according to the assay protocol.

[0169] 6.1.2.4 Transfer SCIG / IVIG concentration gradient samples or ADCC assay buffer (20 μl / well) to the corresponding wells of a 96-well plate.

[0170] 6.1.2.5 Incubate the test plate at room temperature for 30 minutes.

[0171] 6.1.2.6 Collect the effector cells and resuspend them in ADCC assay buffer.

[0172] 6.1.2.7 Adjust the effector cell density (1.5E6 cells / ml) using ADCC assay buffer according to the E / T ratio. Transfer the effector cell suspension to the corresponding wells of a 96-well assay plate (40 µl / well).

[0173] 6.1.2.8 Incubate the experimental plate in a cell culture incubator (37°C / 5% CO2) for 6 hours.

[0174] 6.1.2.9 After incubation, remove the 96-well assay plate and add Fire-LumiTM Luciferase Assay Kit working solution (80 μl / well) to the corresponding wells and incubate for 5-10 minutes.

[0175] 6.1.2.10 Read the chemiluminescence value using PHERAstar FSX at room temperature.

[0176] 6.2 ADCC Data Analysis

[0177] The raw data of ADCC experiments were exported via the PHERAstar FSX system and analyzed using Microsoft Office Excel.

[0178] 7. ADCC dose-response experimental results

[0179] 7.1 ADCC Activity Assay of Human Immunoglobulins

[0180] 7.1.1 Exploration of Human Immunoglobulin Concentration and Target Cells

[0181] Under the condition of E / T = 6:1, according to the experimental design, SCIG / IVIG was prepared with ADCC assay buffer to 2 mg / ml as the starting working concentration, and then diluted to 5 concentration points using a 10-fold serial dilution ratio. Figure 6 、 7 , as shown in 8, Figure 6 SCIG / IVIG was serially diluted at an E / T ratio of 6:1 and incubated with target cells (PLC / PRF / 5) and effector cells (GS-J2C / CD16A 158V). The relative luminescence unit (RLU) values ​​in the reaction system were recorded. Data are expressed as mean relative luminescence units (RLU) ± SEM (n = 2). Figure 7 SCIG / IVIG was serially diluted at an E / T ratio of 6:1 and incubated with target cells (CHO-K1 / Spike_SARS2) and effector cells (GS-J2C / CD16A 158V). The relative luminescence unit (RLU) values ​​in the reaction system were recorded. The data in the figure are expressed as the mean relative luminescence unit (RLU) ± SEM (n = 2). Figure 8SCIG / IVIG was serially diluted at an E / T ratio of 6:1 and incubated with target cells (Sp2 / 0-Ag14) and effector cells (GS-J2C / CD16A 158V). The relative luminescence unit (RLU) values ​​in the reaction system were recorded. The data in the figure are expressed as the mean relative luminescence unit (RLU) ± SEM (n = 2).

[0182] from Figures 6-8 It can be seen that both human immunoglobulin IVIG and SCIG can induce ADCC effects 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 ADCC effect on CHO-K1 / Spike_SARS2 reached 10 5 RLU level, but the signal response values ​​of the other two target cells were only 10 4 The RLU level indicates that target cells from different cell lines have different responsiveness to ADCC. CHO-K1 / Spike_SARS2 is more sensitive to ADCC signal transduction and can significantly enhance the fluorescence response value of the effector cell GS-J2C / CD16A 158V, thereby widening the dynamic range of detection, thereby more accurately reflecting the changes in ADCC effect and better evaluating the activity and efficacy of human immunoglobulins. Therefore, the target cells determined in this study are CHO-K1 / Spike_SARS2.

[0183] Furthermore, using this target cell, the concentrations of SCIG, one of the two human immunoglobulins, exhibited a complete upper and lower plateau. This allowed for the determination of a 2 mg / ml starting working concentration of SCIG, followed by 10-fold dilutions for a total of five concentration points. The ADCC activity of SCIG was then tested at an effector-to-target ratio (E:T) of 6:1. This experimental system exhibited a clear dose-response response and a complete dose-response curve, effectively enabling the detection of SCIG's ADCC activity.

[0184] 7.1.2 Confirmation of appropriate concentration

[0185] On the premise of determining CHO-K1 / Spike_SARS2 as the target cell, the starting concentration of IVIG was further adjusted and optimized, and a systematic method validation was carried out.

[0186] IVIG was used and its starting concentration was adjusted and optimized. Under the condition of E / T = 6:1, IVIG was prepared in ADCC assay buffer to 1 mg / ml as the starting working concentration. The experimental results of the test system with a 4-fold gradient dilution ratio and 7 dilution points were as follows: Figure 9As shown; IVIG was prepared using ADCC assay buffer to 0.67 mg / ml as the starting working concentration, with a 3-fold gradient dilution ratio. The experimental results of the test system with 7 dilution points are shown as follows Figure 10 shown. Figures 9 and 10 Under the condition of E / T=6:1, IVIG was serially diluted and incubated with target cells and effector cells. The relative luminescence unit values ​​in the reaction system were recorded. The data in the figure are expressed as the mean relative luminescence unit (RLU) ± SEM (n = 2). Figures 9 and 10 As can be seen, after optimizing the starting IVIG concentration, the curve fit was significantly improved, and distinct upper and lower plateaus emerged in the effect dose curve. This experiment explored the optimal concentration range for IVIG-induced ADCC against target cells CHO-K1 / Spike_SARS2. Exploring the concentrations of the upper and lower plateaus not only improved the specificity and sensitivity of the experiment, but also ensured the accuracy and reliability of the results.

[0187] 7.2 Specificity

[0188] 7.2.1 Target cell specificity

[0189] We attempted to use HUVEC cells instead of CHO-K1 / Spike_SARS2 cells as target cells and performed ADCC activity assays according to the above method; a CHO-K1 / Spike_SARS2 control was set up in parallel. The experimental results showed that IVIG significantly induced ADCC effects on target cells (CHO-K1 / Spike_SARS2); for the target cell HUVEC group, IVIG did not induce ADCC effects on them ( Figure 11 ), which reflects the specificity of the target cell CHO-K1 / Spike_SARS2 in this experimental system.

[0190] 7.2.2 Effector cell specificity

[0191] We tried to use Jurkat-T original cell line instead of ADCC reporter cell (GS-J2C / CD16A158V) as effector cell and performed ADCC activity detection according to the above method; ADCC reporter cell (GS-J2C / CD16A 158V) control was set up in parallel. The results are as follows Figure 12 As shown, using Jurkat-T (an original cell line without genetic modification) as effector cells, IVIG could not induce ADCC effect on target cells CHO-K1 / Spike_SARS2; while ADCC reporter cells as effector cells had positive detection results, which demonstrated the specificity of this experimental system for the effector cells GS-J2C / CD16A158V.

[0192] 7.2.3 Specificity of test samples

[0193] We tried using excipients (polysorbate 80 + glycine) instead of IVIG as the test sample and performed ADCC activity detection according to the above method; an IVIG control was set up in parallel. Figure 13 As shown, only the IVIG group exhibited ADCC activity; the excipient group showed no upward trend in the detection signal and therefore had no ADCC activity in this experimental system. This demonstrates the specificity of this experimental system for IVIG (i.e., human immunoglobulin).

[0194] 7.2.4 Summary of Specificity Assessment

[0195] Only ADCC reporter cells had positive detection results as effector cells, demonstrating the specificity of this experimental system for the effector cell GS-J2C / CD16A 158V. Only human immunoglobulin had positive detection results as a sample, demonstrating the specificity of this experimental system for the detection sample. CHO-K1 / Spike_SARS2 had the best positive detection result as a target cell, demonstrating the specificity of this experimental system for the target cell. These experimental results collectively demonstrate that the method of the present invention can effectively detect the ADCC activity of human immunoglobulin and has good specificity.

[0196] 7.3 Method Validation

[0197] 7.3.1 Sensitivity test

[0198] The initial concentration of human immunoglobulin IVIG was 1 mg / ml, and the ADCC effect test results were set at 7 concentration points with 4-fold concentration gradient dilution. Figure 9 ) showed that the concentration of IVIG was 2.44×10 -4 mg / ml~1×10 0 mg / ml can obtain a complete dose standard curve; the starting concentration of IVIG is 0.67 mg / ml, 3-fold concentration gradient dilution, and 7 concentration points are set for ADCC effect detection results ( Figure 10 ) showed that the concentration of IVIG was 9.1×10 -4 mg / ml~6.7×10 -1 mg / ml can also obtain a complete dose standard curve. It can be seen that the detection limit of the method of the present invention can reach 10 -4 The results are on the order of 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 has high sensitivity.

[0199] The starting concentration of human immunoglobulin (IVIG) was set to 0.666666667 mg / ml, and a 3-fold concentration gradient dilution was performed. The accuracy and precision experiment was conducted at 7 concentration points. The specific settings were as follows: Human immunoglobulin Conc. (mg / mL): 0.666666667, 0.222222222, 0.074074074, 0.024691358, 0.008230453, 0.002743484, 0.000914495.

[0200] 7.3.2 Accuracy test

[0201] IVIG was used in the ADCC accuracy test, and each sample was repeated three times. The detection signal values ​​corresponding to the concentration points of the curve were selected for linear analysis. The results are shown in Figure 14 , Figure 14 Human immunoglobulin was serially diluted at an E / T ratio of 6:1 and incubated with the corresponding effector and target cells. The relative luminescence units (RLU) were recorded, and data are presented as mean ± SEM (n = 3). The theoretical signal value was calculated using a curve fitting formula. A linear fit was performed between the calculated detection value and the theoretical value to determine the recovery rate, which reached 100% (Table 1), demonstrating the high accuracy of this developed method.

[0202] Table 1 Verification of the accuracy of the IVIG ADCC reporter gene assay (n=3)

[0203]

[0204] Note: Consider Run 01 as 100% RS, i.e., a 100% potency standard. Divide its EC50 value by the EC50 values ​​of the other experimental plates and multiply by 100% to obtain the relative potency. The mean relative potency can be calculated using the AVERAGE function in Excel. Divide the mean relative potency by 100% potency and multiply by 100% to obtain the recovery rate.

[0205] By calculating R three times 2 The RSD was 0.67%. This result indicates that the data dispersion is small and the accuracy of this method is within a reasonable range.

[0206] 7.3.2 Precision test:

[0207] ADCC activity was tested using IVIG at a starting working concentration of 0.67 mg / ml for intra- and inter-day precision. Three independent assays were performed per day, each using a 96-well cell culture plate. Intra-day precision was measured by calculating the RLU and half-maximal effect concentration (EC) of the starting working concentration. 50 ), verifying the accuracy of this method.

[0208] The experimental results are as follows Figure 14 As shown in Table 2, by calculating the EC 50 The RSD was 1%. This result indicates that the data dispersion is small, indicating that the accuracy of this method is within a reasonable range.

[0209] Table 2 Precision verification of IVIG ADCC reporter gene assay (n=3)

[0210]

[0211] 8. Conclusion

[0212] Different combinations of target cells, antibodies, and effector cells, as well as detection conditions (such as incubation time, target cell type, and the ratio of effector cells to target cells, etc.) have a significant impact on the ADCC activity test results, increasing the complexity of the development of human immunoglobulin ADCC activity detection methods.

[0213] This study aims to improve existing publicly reported methods for detecting ADCC hepatitis B cells with human immunoglobulin, which have many shortcomings in target cell selection, effector cell stability, assay applicability, and experimental condition optimization. By experimenting with three aspects: ADCC assay method, human immunoglobulin starting concentration, and target cell selection, a method for detecting human immunoglobulin ADCC biological activity with high accuracy, sensitivity, and precision was developed. The specific steps are as follows:

[0214] For human immunoglobulin products (IVIG: start at 1 mg / ml, then dilute using a 4-fold serial dilution to obtain 7 concentration points; IVIG: start at 0.67 mg / ml, then dilute using a 3-fold serial dilution to obtain 7 concentration points; SCIG: start at 2 mg / ml, then dilute using a 10-fold serial dilution 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 assay protocol. Transfer the human immunoglobulin product to the appropriate wells of the 96-well plate and incubate the plate at room temperature for 30 minutes. Harvest the effector cells and resuspend them in ADCC assay buffer. Adjust the effector cell density (1.5E6 cells / mL) using ADCC assay buffer, using an E / T ratio of 6:1 (effector to target ratio). Transfer the effector cell suspension to the corresponding wells of a 96-well assay plate (40 μL / well). Incubate the plate in a cell culture incubator (37°C / 5% CO2) for 6 hours. After incubation, remove the 96-well plate and add the Fire-Lumi™ Luciferase Assay Kit working solution (80 μL / well) to the corresponding wells and incubate for 5-10 minutes. Chemiluminescence readings were taken at room temperature for data analysis and results compilation.

[0215] In summary, the method for detecting the ADCC biological activity of human immunoglobulins of the present invention, using CHO-K1 / SPIKE_SARS2 as target cells and GS-J2C / CD16A 158V effector cells, in combination with a human immunoglobulin solution of a specific concentration, can accurately and reliably detect the ADCC biological activity of two different human immunoglobulin preparations, intravenous human immunoglobulin and subcutaneous human immunoglobulin, through a reporter gene detection method. Compared with similar methods disclosed in the prior art, the method 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: The steps include: 1) Add the target cell suspension to the well plate, then add a series of concentrations of human immunoglobulin solution for incubation, and finally add the effector cell suspension for incubation; 2) Detect fluorescence from the well plate incubated in step 1) and plot a dose-effect curve using the relative luminescence unit value as the ordinate and the logarithm of the human immunoglobulin solution concentration as the abscissa to calculate the median effective concentration; The target cells are CHO-K1 / SPIKE_SARS2 and the effector cells are GS-J2C / CD16A 158V; The effector-target ratio of effector cells to target cells in each well of the well plate is 6:1; When the human immunoglobulin solution is an intravenous human immunoglobulin solution, the concentration range is 2.44×10 -4 mg / ml~1mg / ml; When the human immunoglobulin solution is a subcutaneous injection human immunoglobulin solution, the concentration range is 2×10 -4 mg / ml~2mg / ml.

2. The detection method according to claim 1, wherein: When the human immunoglobulin solution is an intravenous human immunoglobulin solution, 1 mg / ml is used as the initial working concentration, and then 6 concentrations are diluted using a 4-fold gradient dilution ratio.

3. The detection method according to claim 1, wherein: When the human immunoglobulin solution is for subcutaneous injection, 2 mg / ml is used as the initial working concentration, and then diluted to 4 concentrations using a 10-fold gradient dilution ratio.

4. The detection method according to claim 1, wherein: The target cell suspension, human immunoglobulin solutions with a series of concentrations and effector cell suspension were prepared using RPMI 1640 containing 10% FBS.

5. The detection method according to claim 4, wherein: The concentration of the target cell suspension was 2.5×10 5 cells / ml, the volume is 40 μl / well; the volume of the human immunoglobulin solution of serial concentrations is 20 μl / well; the concentration of the effector cell suspension is 1.5×10 6 cells / ml, with a volume of 40 μl / well.

6. The detection method according to claim 1, wherein: The target cell suspension was incubated with the human immunoglobulin solution at room temperature for 30 minutes; the effector cell suspension was added and incubated at 37° C. and 5% CO 2 for 4 hours.

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

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