A combination of detection reagents for detecting ADCP activity of human immunoglobulin on non-human cells, a detection kit and a detection method thereof

Abstract

The present invention belongs to the field of biological detection technology, and in particular to a combination of detection reagents for the ADCP activity of human immunoglobulin on non-human cells, a detection kit and a detection method thereof. The present invention determines the target cells, effector cells, effect-target ratio, sample concentration gradient and other conditions in the detection kit and its detection method through preliminary screening experiments. The present invention adopts Sp2 / 0-Ag14 cells as target cells and THP-1-NF-kB cells as effector cells, and realizes in vitro detection of the ADCP activity of human immunoglobulin on non-human cells under a preferred sample concentration gradient. The detection method is simple, rapid, and has high precision and accuracy, filling the gap in the detection method of immunoglobulin ADCP of non-human species, providing method support for scientific research and early development of antibody drugs, saving cost and time, and having important significance for the quality control of antibody drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to a combination of detection reagents for detecting ADCP activity of human immunoglobulin on non-human cells, a detection kit and a detection method thereof. Background Art

[0002] The ADCP effect (Antibody-Dependent Cellular Phagocytosis, ADCP) occurs when the antibody Fab fragment binds to antigens on the surface of target cells (virus-infected cells or tumor cells). The antibody Fc fragment then interacts with immune cells (such as monocytes, macrophages, neutrophils, and dendritic cells) that possess FcγRIIa (CD32a) or FcγRI (CD64) receptors, prompting these cells to phagocytose and eliminate the target cells. This mechanism of action plays a crucial role in the body's immune defense and immune regulation. Based on this mechanism, researchers have developed monoclonal antibody drugs that recognize various molecular targets. The clinical efficacy of these antibody drugs often depends on the combination of Fab and Fc-mediated biological functions. During the antibody drug development process, it is essential to characterize the critical quality attributes (CQAs) of the candidate antibody drug, including the strength of the candidate antibody binding to the target and the degree of binding to the patient's immune system, to trigger the ADCP effect and thereby mitigate the risks of the drug during use. Therefore, detecting whether an antibody drug has ADCP biological activity and the strength of this biological activity has become a crucial step in the development and quality control of antibody drugs.

[0003] Currently, a variety of assays for ADCP bioactivity of antibody drugs have been established, including peripheral blood mononuclear cell (PBMC)-based assays and cell-based reporter gene-based bioassays. These traditional assays are highly dependent on donor primary cells, are complex, time-consuming, labor-intensive, expensive, and have poor reproducibility. Consequently, research on ADCP bioactivity assays for human immunoglobulin drugs remains limited. Furthermore, ADCP assays for immunoglobulins in non-human species (e.g., mice) remain largely unexplored. In scientific research, non-human cell or animal models are often used as experimental models to assess ADCP activity of antibody drugs to gain a deeper understanding of their mechanisms of action and to mimic the in vivo environment. In antibody drug development, these models are also often used in early-stage research to screen and optimize antibodies while reducing costs and time. To establish standardized ADCP assays, rigorous activity testing is required for each batch of antibody drug during production and quality control. Developing ADCP activity assays specific to non-human cell species (e.g., mice) can serve as an effective quality control tool to ensure the efficacy and safety of antibody drugs.

[0004] Human immunoglobulin (HIG) is the most widely used blood product in clinical practice. Since the identification of its immunomodulatory and anti-inflammatory properties, its use as an antibody drug has rapidly increased, becoming a valuable tool for treating many diseases characterized by humoral immune deficiency or immune system dysfunction. Based on the route of administration, HIG antibody drugs can be categorized as subcutaneous HIG (SCIG), intramuscular HIG (IMIG), and intravenous HIG (IVIG). With the advent of portable injectors and advances in accurate drug delivery technology, SCIG has become increasingly widespread. SCIG offers comparable or superior efficacy to intravenous HIG (IVIG) and a number of advantages not found in IVIG, including home use, self-administration, reduced side effects, and more stable maintenance of physiological IgG concentrations. Given their proven efficacy in treating a wide range of diseases, the research, development, and production of HIG antibody drugs has flourished in recent years.

[0005] Therefore, there is an urgent need to develop a rapid and sensitive method for detecting the ADCP activity of human immunoglobulins against non-human (mouse) cells. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention provides a combination of detection reagents, a detection kit and a detection method for the ADCP activity of human immunoglobulin on non-human cells, the purpose of which is to evaluate the in vitro ADCP biological activity of human immunoglobulin.

[0007] The present invention provides a combination of detection reagents for the ADCP activity of human immunoglobulin against non-human cells, the combination of detection reagents comprising: target cells and effector cells; wherein the target cells are selected from Sp2 / 0-Ag14 cells, MM3MG-HER2Δ16 cells, 4T1 cell line, E0771 cells, B16-F10 cells, and Cloudman S91 cells; the effector cells are selected from THP-1-NF-κB cells and GS-J2B-FCGR2A cells; and the ratio of the number of effector cells to target cells is 4-2:1-3.

[0008] Preferably, the effector cells are THP-1-NF-κB cells.

[0009] Preferably, the target cells are Sp2 / 0-Ag14 cells; and / or the ratio of the number of effector cells to target cells is 3:2.

[0010] The present invention provides a detection kit for the ADCP activity of human immunoglobulin on non-human cells, comprising: a combination of the detection reagents described above, an ADCP assay buffer, and a luciferase detection kit working solution.

[0011] Preferably, the ADCP assay buffer is selected from RPMI 1640 containing 8-12% animal serum; and / or the luciferase assay kit working solution is Fire-Lumi TM Luciferase Assay Kit Working Solution.

[0012] The present invention provides a method for performing a non-diagnostic detection using a combination of the detection reagents described in any one of the above items and the detection kit described in any one of the above items, the detection method comprising the following steps:

[0013] Step 1: Incubate the human immunoglobulin to be tested with target cells and effector cells.

[0014] Step 2: Add the luciferase detection kit working solution and detect the fluorescence signal after incubation.

[0015] Preferably, in step 1, the mixing is mixing the human immunoglobulin solution to be tested with the target cell suspension and the effector cell suspension, and the human immunoglobulin solution to be tested, the target cell suspension and the effector cell suspension are prepared using ADCP assay buffer.

[0016] Preferably, the concentration of the human immunoglobulin solution to be tested is 6-8 concentration points within the range of 0.0006-2.5 mg / ml.

[0017] Preferably, in step 1, the concentration of the target cell suspension is 4×10 5 -6×105 cells / ml; and / or, the concentration of the effector cell suspension is 6×10 5 -9×10 5 cells / ml; the volume ratio of the human immunoglobulin solution to be tested, the target cell suspension, and the effector cell suspension is 19-21:39-41: 39-41.

[0018] Preferably, in step 1, the mixed incubation process comprises: incubating the human immunoglobulin to be tested with the target cells at 18-37° C. for 25-35 minutes, and then adding the effector cells and incubating at 35-37° C. for 3.8-4.2 hours;

[0019] And / or, the human immunoglobulin to be tested is selected from subcutaneous immunoglobulin, intramuscular immunoglobulin, and intravenous immunoglobulin;

[0020] And / or, in step 2, the ratio of the luciferase detection kit working solution to the effector cells is 70-90 μl: 2.4-3.6×10 5 cell;

[0021] And / or, in step 2, the incubation time is 5-10 minutes.

[0022] In the present invention, "effector-target ratio" refers to the ratio of the number of effector cells to target cells.

[0023] The present invention provides a combination of reagents, a detection kit, and a detection method for detecting the ADCP activity of human immunoglobulins against non-human cells. Through preliminary screening experiments, the present invention determined the target cells, effector cells, effector-target ratio, and sample concentration gradient conditions used in the detection method. Using Sp2 / 0-Ag14 cells as target cells and THP-1-NF-κB cells as effector cells, the present invention achieved in vitro detection of the ADCP activity of human immunoglobulins against non-human (mouse) cells at an optimized effector-target ratio and sample concentration gradient. This detection method is simple, rapid, and highly precise and accurate, filling a gap in the detection of ADCP activity of immunoglobulins from non-human (mouse) species. It provides methodological support for scientific research and early-stage antibody drug development, saving costs and time. It is of great significance for the quality control of antibody drugs and has promising application prospects.

[0024] 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.

[0025] 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

[0026] Figure 1 The figure shows the experimental results of ADCP effector cell screening. The data are expressed as relative luminescence units (RLU) mean ± SEM (n=3), and SEM represents the standard error of the mean.

[0027] Figure 2 The figure shows the experimental results of the optimization of the starting gradient of SCIG concentration. The data in the figure are expressed as the mean ± SEM of relative luminescence units (RLU) (n=3), and SEM represents the standard error of the mean.

[0028] Figure 3 Figure 2 is the experimental result for the verification of target cell specificity.

[0029] Figure 4 This figure shows the experimental results for the validation of effector cell specificity.

[0030] Figure 5 This is the experimental result diagram for sample specificity verification.

[0031] Figure 6 This figure shows the experimental results of precision and accuracy verification. The data in the figure are expressed as the mean ± SEM of relative luminescence units (RLU) (n=3), and SEM represents the standard error of the mean.

[0032] Figure 7 This figure shows the experimental results of the application of the ADCP detection method on the IVIG sample. The data in the figure are expressed as the mean ± SEM of the relative luminescence unit (RLU) (n=3), and SEM represents the standard error of the mean. DETAILED DESCRIPTION

[0033] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available.

[0034] The present invention uses a reporter gene assay to evaluate the antibody-dependent cellular phagocytosis (ADCP) activity of human immunoglobulins. ADCP is a process in which the Fab segment of an antibody binds to antigens on the surface of target cells (virus-infected cells or tumor cells). The Fc segment of the antibody then interacts with immune cells (such as monocytes, macrophages, neutrophils, and dendritic cells) that have FcγRIIa (CD32a) and / or FcγRIa (CD64a) receptors, prompting these immune cells to phagocytose and eliminate the target cells.

[0035] The information of the samples and reference substances used in the present invention is shown in Table 1.

[0036] Table 1

[0037]

[0038] Example 1 Human immunoglobulin anti-non-human cell ADCP activity detection kit and detection method

[0039] 1. Detection Kit

[0040] This example provides a kit and method for detecting the ADCP activity of human immunoglobulins against mouse cells. The kit includes target cells, effector cells, ADCP assay buffer, and Fire-Lumi™ luciferase assay kit working solution. Sp2 / 0-Ag14 cells (mouse myeloma cells) are used as target cells. Sp2 / 0-Ag14 cells are derived from mouse spleens and are fused with P3X63Ag8 myeloma cells and BALB / c mouse splenocytes immunized with sheep red blood cells. Genetically engineered THP-1 cells (THP-1-NF-κB, human leukemic mononuclear cells) are used as effector cells. These cells naturally express CD32A (FcγRIIA) and CD64 (FcγRI) and are stably transfected with the NF-κB response element and the firefly luciferase gene.

[0041] 2. Detection Method

[0042] The detection method in this example uses Sp2 / 0-Ag14 cells as target cells. Sp2 / 0-Ag14 cells are derived from the spleen of BALB / c mice immunized with sheep red blood cells and fused with P3X63Ag8 myeloma cells. Genetically engineered THP-1 cells (THP-1-NF-κB, human leukemic mononuclear cells) are used as effector cells. These cells naturally express CD32a (FcγRIIa) and CD64a (FcγRIa) and are stably transfected with the NF-κB response element and the firefly luciferase gene. The antibody binds to FcγRIIa or FcγRIa on the effector cells, activating the NF-κB response element in the THP-1-NF-κB cells and driving the expression of firefly luciferase. Luciferase activity is quantified by bioluminescence, thereby characterizing the antibody's ADCP activity.

[0043] 1. Cell culture and passaging

[0044] Cell Thawing: Add 4 ml of prewarmed culture medium (Probio, RD00941, RPMI 1640 + 10% FBS) to a 15 ml centrifuge tube. 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 (Probio, RD00941, RPMI 1640 + 10% FBS + 400 μg / ml Hygromycin B). Count the cells, seed the cells at an appropriate density in a cell culture dish, gently shake to mix, and incubate in a 5% CO2, 37°C incubator.

[0045] Cell passaging: (1) Passaging of effector THP-1-NF-kB cells: Pipette the cells into a 15 ml centrifuge tube and centrifuge at 800 rpm for 5 minutes. Resuspend the cell pellet in complete culture medium (90% RPMI 1640 + 10% FBS + 400 μg / ml Hygromycin B) and count the cells. Then, inoculate the cells into a cell culture dish at an appropriate density, shake gently to mix, and place in a 5% CO2, 37°C incubator for culture.

[0046] (2) Passaging of target cells Sp2 / 0-Ag14: Collect the supernatant in 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. When most cells become round, add an appropriate amount of complete culture medium (DMEM + 10% FBS) to terminate the digestion and centrifuge at 800 rpm for 5 minutes. Add an appropriate amount of complete culture medium and count the cells. Inoculate the cells at an appropriate density in a cell culture dish, shake gently to mix, and place in a 5% CO2, 37 °C incubator for culture.

[0047] 2. ADCP reporter gene experimental steps

[0048] (1) Collect target cells Sp2 / 0-Ag14 (purchased from ATCC) by centrifugation and resuspend in 1 ml of ADCP assay buffer (RPMI1640 + 10% FBS).

[0049] (2) Using 2.5 mg / ml as the starting working concentration, the human immunoglobulin (SCIG) to be tested was graded diluted with ADCP assay buffer at a 4-fold graded dilution ratio through 7 concentration points to prepare the test sample solution. The concentrations of the test sample solution were 2.5 mg / ml, 0.625 mg / ml, 0.15625 mg / ml, 0.0390625 mg / ml, 0.009765625 mg / ml, 0.002441406 mg / ml, and 0.000610352 mg / ml, respectively.

[0050] (3) Adjust the target cell density (5E5 cells / ml) with ADCP assay buffer and transfer the target cell suspension into a 96-well assay plate (40 μl / well).

[0051] (4) Transfer the sample solution to be tested into the corresponding wells of a 96-well plate at 20 μl / well.

[0052] (5) Incubate the test plate at room temperature for 30 minutes.

[0053] (6) Collect effector cells (THP-1-NF-κB, purchased from Probio) and resuspend them in ADCP assay buffer.

[0054] (7) Based on an E / T (effector-target ratio, the ratio of effector cells to target cells) of 3:2, adjust the effector cell density (7.5E5 cells / ml) with ADCP assay buffer and transfer the effector cell suspension to the corresponding wells of a 96-well assay plate (40 μl / well).

[0055] (8) Incubate the experimental plate in a cell culture incubator (37°C / 5% CO2) for 4 hours.

[0056] (9) After the incubation is completed, remove the 96-well experimental plate and add the Fire-LumiTM luciferase assay kit working solution (80 μl / well) to the corresponding wells and incubate for 5-10 minutes.

[0057] (10) Read the chemiluminescence value using PHERAstar FSX at room temperature.

[0058] 3. ADCP data analysis

[0059] The raw data of ADCP experiments were exported and analyzed using Microsoft Office Excel.

[0060] The dose-effect curve of SCIG was fitted using a four-parameter curve equation regression model, with the relative luminescence unit value as the ordinate and the logarithmic concentration of the SCIG solution as the abscissa.

[0061] Example 2 Human immunoglobulin anti-non-human cell ADCP activity detection kit and detection method

[0062] The detection kit was prepared and the detection was carried out according to the method of Example 1, except that the human immunoglobulin sample to be tested was replaced by IVIG instead of SCIG.

[0063] The technical solution of the present invention is further illustrated by experiments below.

[0064] Experimental Example 1 Selection of effector cells in ADCP activity assay of human immunoglobulin

[0065] 1. Experimental Methods

[0066] ADCP effector cells were screened according to the detection method of Example 1. The sources and genetic modification information of the selected effector cells are as follows: (1) THP-1-NF-κB, which naturally expresses CD32a (FcγRIIa) and CD64a (FcγRIa) and is stably transfected with the NF-κB-Luc luciferase reporter gene; (2) GS-J2B-FCGR2A (Jurkat / FCGR2A / NFAT-Luc), purchased from Probio, is a genetically engineered Jurkat cell stably transfected with the FCGR2A and NFAT-Luc luciferase reporter genes, with FCGR2A encoding FcγRIIa; (3) GS-J8 (Jurkat / NF-κB-Luc), purchased from Probio, is a genetically engineered Jurkat cell stably transfected with the NF-κB-Luc luciferase reporter gene.

[0067] Sp2 / 0-Ag14 cells were selected as target cells and SCIG was used as the sample. SCIG was serially diluted using a 6-fold dilution ratio at a starting working concentration of 1.333 mg / ml under an E / T ratio of 3:2. The ADCP effect of SCIG on the target cell Sp2 / 0-Ag14 was detected using the luciferase reporter gene assay described in Example 1 on three different effector cells (THP-1-NF-κB, GS-J8, and GS-J2B-FCGR2A). The experimental conditions are shown in Table 2.

[0068] Table 2 Effector cell screening experiment

[0069]

[0070] 2. Experimental Results

[0071] The experimental results are as follows Figure 1 As shown, at a starting working concentration of 1.333 mg / ml, SCIG induced ADCP in both effector cell types (THP-1-NF-κB and GS-J2B-FCGR2A) against target Sp2 / 0-Ag14 cells, resulting in positive killing results. However, the response curves for GS-J8 effector cells showed no clear dose-response relationship, indicating that SCIG was ineffective in inducing ADCP against target cells. THP-1-NF-κB and GS-J2B-FCGR2A effector cells exhibited distinct S-shaped dose-response curves, with the ADCP effect becoming increasingly pronounced and increasing exponentially with increasing SCIG dose. The presence or absence of a dose-response curve is closely related to the reliability and validity of the research method, and therefore GS-J8 was not selected as an effector cell in the detection method of this invention.

[0072] Within the linear concentration range of a sigmoidal dose-response curve, changes in drug dose and effect exhibit a relatively stable proportional relationship. When using GS-J2B-FCGR2A cells as effector cells to detect the ADCP effect of SCIG, the linear range for SCIG samples only covered a concentration range of 0.00617284 to 0.2222222 mg / ml (determined by the concentrations at which the curve plateaus), encompassing only three effective concentration points. In contrast, when using THP-1-NF-κB cells as effector cells to detect the ADCP effect of SCIG, the linear range for SCIG samples extended from 0.000171468 to 1.333333 mg / ml (because the curve still did not plateau), encompassing at least six effective concentration points. This wider effective concentration range reflects the advantages of this method, including lower sample requirements, the ability to accommodate a wider range of samples, and superior sensitivity. Therefore, THP-1-NF-κB cells were selected as the effector cells for subsequent system development in this experiment.

[0073] Experimental Example 2 Optimization of Sample Concentration in ADCP Activity Assay of Human Immunoglobulin

[0074] 1. Experimental Methods

[0075] Due to the poor initial SCIG concentration and gradient design of the experimental system in Experimental Example 1, the ADCP dose-effect curve failed to have an upper platform (see Figure 1Therefore, this experiment involved optimizing the starting concentration of SCIG. Under an E / T ratio of 3:2, SCIG was diluted at a starting working concentration of 2.5 mg / ml. SCIG was serially diluted through seven concentration points using a 4-fold dilution ratio to test the dose-response of human immunoglobulin (ADCP) on effector THP-1-NF-κB cells against target Sp2 / 0-AG14 cells. The experimental conditions are shown in Table 3.

[0076] Table 3 Screening experiment of starting concentration of test samples

[0077]

[0078] 2. Experimental Results

[0079] The test results after improving the starting concentration are as follows Figure 2 As shown, SCIG can induce the ADCP effect of effector cells THP-1-NF-κB on target cells Sp2 / 0-Ag14, and the killing result is positive. The dose-effect curve has a good fit and reaches the expected saturation state (i.e., it has upper and lower platforms). Therefore, this experimental example preferably uses 2.5 mg / ml as the starting working concentration of subcutaneous injection of human immunoglobulin as the optimal concentration gradient for ADCP activity detection, and further conducts verification experiments on the specificity, accuracy, and precision of the SCIG sample detection method.

[0080] Experimental Example 3 Specificity experiment of detection method

[0081] 1. Experimental Methods

[0082] The sample concentrations (mg / mL) of human immunoglobulin (SCIG) were set as follows: 2.5 mg / ml, 0.625 mg / ml, 0.15625 mg / ml, 0.0390625 mg / ml, 0.009765625 mg / ml, 0.002441406 mg / ml, and 0.000610352 mg / ml. SCIG was used in ADCP-specificity studies according to the method of Example 1, with three replicates for each sample.

[0083] (1) Target cell specificity

[0084] 293T cells were used as target cells instead of Sp2 / 0-Ag14 cells, and 293T cell lines were set as controls. ADCP activity was detected according to the method of Example 1.

[0085] (2) Effector cell specificity

[0086] ADCP activity was detected according to the method of Example 1 using the THP-1 original cell line (THP-1) as effector cells and THP-1-NF-κB cells as controls.

[0087] (3) Specificity of test samples

[0088] ADCP activity was detected according to the method of Example 1, using excipients (polysorbate 80 + maltose, negative control in Table 1) instead of SCIG as the test sample.

[0089] 2. Experimental Results

[0090] Target cell specificity results such as Figure 3 As shown, Sp2 / 0-Ag14 cells were used as target cells to detect the ADCP activity of human immunoglobulin, and had significantly better fluorescence response values ​​than 293T cells, indicating that 293T cells were not suitable for detecting the ADCP effect of human immunoglobulin as target cells.

[0091] Effector cell specificity results such as Figure 4 As shown, THP-1-NF-κB cells used as effector cells to detect the ADCP activity of human immunoglobulin have significantly better fluorescence response values ​​than THP-1 cells, indicating that THP-1 cells are not suitable for detecting the ADCP effect of human immunoglobulin as effector cells.

[0092] The results of the sample specificity test are as follows Figure 5 As shown, only SCIG showed ADCP activity, and the negative control group did not show an increasing trend in the detection signal, and had no ADCP activity under this experimental system, indicating that the method of the present invention can specifically and effectively detect the ADCP activity of SCIG.

[0093] The above results demonstrate that Sp2 / 0-Ag14 cells, as target cells, exhibit the best positive detection results, demonstrating the target cell specificity of this experimental system. Only THP-1-NF-κB cells, as effector cells, exhibit positive detection results, demonstrating the specificity of this experimental system for THP-1-NF-κB effector cells. Furthermore, only human immunoglobulin samples exhibit positive detection results, demonstrating the sample specificity of this experimental system. Therefore, the method of the present invention can effectively detect ADCP activity of human immunoglobulins and exhibits good specificity.

[0094] Experimental Example 4 Accuracy and Precision of the Detection Method

[0095] 1. Accuracy test

[0096] 1. Experimental methods

[0097] The concentrations (mg / mL) of human immunoglobulin (SCIG) samples were set as follows: 2.5 mg / ml, 0.625 mg / ml, 0.15625 mg / ml, 0.0390625 mg / ml, 0.009765625 mg / ml, 0.002441406 mg / ml, and 0.000610352 mg / ml. SCIG was used in an ADCP accuracy test according to the method of Example 1, with three replicates for each sample.

[0098] The data analysis method is as follows: select the detection signal value corresponding to the concentration point of the curve for linear analysis, calculate the theoretical signal value according to the curve fitting formula, perform linear fitting on the calculated detection value and the theoretical value, and obtain R 2 The values ​​were then compared for the three replicates R 2 The RSD of the values.

[0099] 2. Experimental results

[0100] The results are as follows Figure 6 As shown in Table 4 , the relative standard deviation of the signal values ​​of three repeated detections of this method was within 15% (accuracy met the standard: deviation was within the range of ±20%), which showed good accuracy.

[0101] Table 4 Verification of the accuracy of the ADCP reporter gene assay

[0102]

[0103] 2. Precision Test

[0104] 1. Experimental methods

[0105] SCIG with an initial working concentration of 2.5 mg / ml was used in a daily precision test for ADCP detection. SCIG was serially diluted to 7 concentration points using a 4-fold serial dilution ratio. According to the method of Example 1, three independent tests were set up within one day, with one 96-well cell culture plate tested each time. The intra-day precision was tested and the RLU of the initial working concentration was counted to verify the precision of the method.

[0106] 2. Experimental results

[0107] The results are as follows Figure 6 As shown in Table 5, the relative standard deviation of the detection signal values ​​of the three plates of this method is within 30% (precision meets the standard: deviation is within the range of ±30%), which has good precision and can be used for ADCP detection of SCIG.

[0108] Table 5 Precision verification of ADCP reporter gene assay

[0109]

[0110] The above results show that the detection method of the present invention has good accuracy and precision.

[0111] Experimental Example 5 Application of ADCP Detection Method on IVIG Samples

[0112] 1. Experimental Methods

[0113] To explore the applicability of the ADCP assay to IVIG samples, this study further investigated the ADCP effect of human immunoglobulin (IVIG) on THP-1-NF-κB effector cells against Sp2 / 0-Ag14 target cells. Using an E / T ratio of 3:2, IVIG was serially diluted using a 4-fold dilution ratio through seven concentration points at a starting concentration of 2.5 mg / ml. The ADCP assay was performed using the method described in Example 1, with three replicates performed for each sample.

[0114] 2. Experimental Results

[0115] like Figure 7 As shown, the above system was used to detect the ADCP effect of IVIG. The results showed that IVIG could also induce the ADCP effect of effector cells THP-1-NF-κB on target cells Sp2 / 0-Ag14. The killing result was positive, the dose-effect curve had a good fit, and reached the expected saturation state (i.e., it had upper and lower platforms).

[0116] The results of the above experimental examples show that the detection method of the present invention can be used to detect the ADCP activity of human immunoglobulins against mouse cells. The present invention provides an ADCP detection method for human immunoglobulin products in the field of infectious diseases through steps such as effector cell screening and sample concentration gradient optimization. This detection method is very important for the selection of target cells and effector cells. The setting of the concentration gradient of the human immunoglobulin to be tested also has a significant impact on the detection results.

[0117] Under optimized conditions, using SCIG or IVIG as test samples, the ADCP activity of THP-1-NF-κB effector cells (human leukemic monocytic cells naturally expressing CD32a (FcγRIIa) and CD64a (FcγRIa) and stably transfected with the NF-κB-Luc luciferase reporter gene) against Sp2 / 0-Ag14 target cells was measured. Accurate detection of the ADCP biological activity of human immunoglobulin products was achieved at an effector-to-target ratio of 3:2 and a sample concentration gradient (SCIG and IVIG starting at 2.5 mg / ml, four-fold dilution, and seven concentration points). The detection method of the present invention exhibits target cell specificity, effector cell specificity, and test sample specificity, with high sensitivity, accuracy, and precision, making it suitable for detecting the ADCP activity of a variety of human immunoglobulins (IVIG and SCIG).

[0118] The above results collectively demonstrate that the detection method of the present invention is simple, rapid, and highly precise and accurate, filling the gap in methods for detecting ADCP activity of human immunoglobulins on non-human (mouse) cells. It provides methodological support for scientific research and early development of antibody drugs, saves costs and time, and is of great significance for the quality control of antibody drugs. The detection method of the present invention has good application prospects.

Claims

1. A combination of reagents for detecting ADCP activity of human immunoglobulin against non-human cells, characterized in that: The combination of the detection reagents includes: target cells and effector cells; wherein the target cells are Sp2 / 0-Ag14 cells, the effector cells are THP-1-NF-κB cells, and the ratio of the number of effector cells to target cells is 4-2:1-3.

2. The combination of reagents for detecting ADCP activity of human immunoglobulin on non-human species cells according to claim 1, characterized in that: The ratio of the effector cells to the target cells is 3:

2.

3. A kit for detecting ADCP activity of human immunoglobulin against non-human cells, characterized in that: The detection kit comprises: a combination of detection reagents for the ADCP activity of human immunoglobulin on non-human cells according to claim 1, ADCP experimental buffer, and luciferase detection kit working solution.

4. The kit for detecting ADCP activity of human immunoglobulin on non-human cells according to claim 3, characterized in that: The ADCP assay buffer is selected from RPMI 1640 containing 8-12% animal serum; and / or the luciferase assay kit working solution is Fire-Lumi TM Luciferase Assay Kit Working Solution.

5. A method for detecting ADCP activity of human immunoglobulin on non-human cells for non-diagnostic purposes using a combination of a reagent for detecting ADCP activity of human immunoglobulin on non-human cells according to claim 1 or 2 and a kit for detecting ADCP activity of human immunoglobulin on non-human cells according to claim 3 or 4, characterized in that: The detection method comprises the following steps: Step 1: Incubate the human immunoglobulin to be tested with target cells and effector cells. Step 2: Add the luciferase detection kit working solution and detect the fluorescence signal after incubation.

6. The method for detecting ADCP activity of human immunoglobulin on non-human cells according to claim 5, wherein the method comprises: In step 1, the mixing is mixing the human immunoglobulin solution to be tested with the target cell suspension and the effector cell suspension. The human immunoglobulin solution to be tested, the target cell suspension and the effector cell suspension are prepared using ADCP assay buffer.

7. The method for detecting ADCP activity of human immunoglobulin on non-human cells for non-diagnostic purposes using the combination of the detection reagents and the detection kit for ADCP activity of human immunoglobulin on non-human cells according to claim 6, characterized in that: The concentration of the human immunoglobulin solution to be tested is 6-8 concentration points within the range of 0.0006-2.5 mg / ml.

8. The method for detecting ADCP activity of human immunoglobulin on non-human cells for non-diagnostic purposes using the combination of the detection reagents and the detection kit for ADCP activity of human immunoglobulin on non-human cells according to claim 6, characterized in that: In step 1, the concentration of the target cell suspension is 4×10 5 -6×10 5 cells / ml; and / or, the concentration of the effector cell suspension is 6×10 5 -9×10 5 cells / ml; the volume ratio of the human immunoglobulin solution to be tested, the target cell suspension, and the effector cell suspension is 19-21:39-41: 39-41.

9. The method for detecting ADCP activity of human immunoglobulin on non-human cells for non-diagnostic purposes using the combination of the detection reagents and the detection kit for ADCP activity of human immunoglobulin on non-human cells according to claim 5, characterized in that: In step 1, the mixed incubation process includes: incubating the human immunoglobulin to be tested with the target cells at 18-37° C. for 25-35 minutes, and then adding the effector cells and incubating at 35-37° C. for 3.8-4.2 hours; And / or, the human immunoglobulin to be tested is selected from subcutaneous immunoglobulin, intramuscular immunoglobulin, and intravenous immunoglobulin; And / or, in step 2, the ratio of the luciferase detection kit working solution to the effector cells is 70-90 μl: 2.4-3.6×10 5 cell; And / or, in step 2, the incubation time is 5-10 minutes.

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