Combination of reagents for detecting ADCP activity of human immune globulin to non-human species cells, detection kit and detection method thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a reagent combination for detecting ADCP activity of human immune globulin to non-human species cells, a detection kit and a detection method thereof. The conditions such as target cells, effector cells, the efficiency-target ratio and the sample concentration gradient in the detection kit and the detection method thereof are determined through an early-stage screening experiment. According to the present invention, the Sp2 / 0-Ag14 cell is adopted as the target cell, the THP-1-NF-kB cell is adopted as the effector cell, and the in vitro detection of the ADCP activity of the non-human species cell by using the human immune globulin is achieved under the optimal sample concentration gradient; the detection method is simple, rapid and high in precision and accuracy, fills the blank of the detection method of the non-human species immunoglobulin ADCP, provides a method support for scientific research and early research and development of antibody drugs, saves cost and time, and has important significance for quality control of the antibody drugs.

Description

Technical Field

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

[0002] The ADCP effect (Antibody-Dependent Cellular Phagocytosis) is that after the Fab segment of an antibody binds to an antigen on the surface of a target cell (a virus-infected cell or a tumor cell), the Fc segment of the antibody can interact with immune cells (such as monocytes, macrophages, neutrophils and dendritic cells) with FcγRIIa (CD32a) or FcγRI (CD64) receptors, prompting these immune cells to phagocytose and eliminate the target cell. This mechanism plays an important role in the body's immune defense and immune regulation. Based on this mechanism, developers have developed monoclonal antibody drugs that recognize different molecular targets. The clinical efficacy of these antibody drugs usually depends on the combination of Fab- and Fc-mediated biological functions. During the development of antibody drugs, it is necessary to characterize the critical quality attributes (CQAs) of candidate antibody drugs, including the strength of the binding of candidate antibody drugs to the target and the degree of binding to the patient's immune system, to induce the ADCP effect and thereby reduce the risks during drug use. Therefore, detecting whether an antibody drug has the biological activity of the ADCP effect and the strength of this biological activity has become a crucial step in the research and development of antibody drugs and their quality control process.

[0003] At present, a variety of methods for measuring the ADCP biological activity of antibody drugs have been established, such as detection methods based on peripheral blood mononuclear cells (PBMC) and biological detection methods based on cell reporter genes. These traditional measurement methods highly rely on donor primary cells, are complex, time-consuming, laborious, expensive, and have poor repeatability. The research on the ADCP biological activity measurement of human immunoglobulin drugs is still relatively limited. In addition, the detection field of immunoglobulin ADCP in non-human species (mice) is still blank. In scientific research, in order to deeply understand the mechanism of action of antibody drugs and simulate the in-vivo environment, non-human species cells or animals are often used as experimental models to detect the ADCP activity of antibody drugs; in the field of antibody drug R & D, in order to screen and optimize antibodies, and at the same time reduce costs and time, non-human species cells or animals are also often used as models for early R & D; in order to establish a standardized ADCP detection method, in the production and quality control process of antibody drugs, strict activity detection needs to be carried out on each batch of products. Developing an ADCP activity detection method for non-human species (such as mice) cells can be used as an effective quality control means to ensure the efficacy and safety of antibody drugs.

[0004] Human immunoglobulin is the most widely used blood product in clinical practice. Since the immunomodulatory and anti-inflammatory properties of immunoglobulin were elucidated, the application of immunoglobulin as an antibody drug has increased rapidly and has become a valuable tool for treating many diseases characterized by humoral immunodeficiency or leading to immune system dysfunction. According to the injection method, immunoglobulin antibody drugs can be divided into subcutaneous immunoglobulin (SCIG), intramuscular immunoglobulin (IMIG), and intravenous immunoglobulin (IVIG). With the use of portable syringes and the development of drug propulsion accuracy technology, the use of SCIG has become more and more widespread. SCIG has equivalent or better efficacy compared with intravenous immunoglobulin (IVIG) and exhibits a series of advantages that IVIG does not have, such as being able to be used at home, self-administered, having low side effects, and being able to maintain a more stable physiological level of IgG concentration. Given the advantages such as the good efficacy demonstrated by immunoglobulin antibody drugs in treating various diseases, their R & D and production have shown a booming trend in recent years.

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

[0006] Aiming at the defects 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 against non-human species cells, aiming 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 species cells. The combination of detection reagents includes: 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, Cloudman S91 cells, and the effector cells are selected from THP-1-NF-κB cells and GS-J2B-FCGR2A cells. The number ratio of effector cells to target cells is 4-2:1-3.

[0008] Preferably, the effector cell is THP-1-NF-κB cell.

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

[0010] The present invention provides a detection kit for the ADCP activity of human immunoglobulin against non-human species cells. The detection kit includes: the combination of detection reagents as described in any one of the above, an ADCP assay buffer, and a working solution of a luciferase detection kit.

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

[0012] The present invention provides a detection method for non-diagnostic purposes using the combination of detection reagents as described in any one of the above and the detection kit as described in any one of the above. The detection method includes the following steps: Step 1, mixing and incubating the human immunoglobulin to be tested with the target cells and effector cells. Step 2, adding the working solution of the luciferase detection kit, and detecting the fluorescence signal after incubation.

[0013] Preferably, in step 1, the mixing is the mixing of 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 with the ADCP assay buffer.

[0014] Preferably, the concentration of the human immunoglobulin solution to be tested is the concentration of 6-8 concentration points within the range of 0.0006-2.5 mg / ml.

[0015] Preferably, in step 1, the concentration of the target cell suspension is 4×10 5 -6×10 5cells / 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.

[0016] Preferably, in step 1, the process of mixing and incubating 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 injection immunoglobulin, intramuscular injection immunoglobulin, and intravenous injection immunoglobulin; and / or, in step 2, the dosage ratio of the working solution of the luciferase detection kit to the effector cells is 70-90 μL: 2.4-3.6×10 5 cells; and / or, in step 2, the incubation time is 5-10 minutes.

[0017] In the present invention, the "effector-to-target ratio" refers to the number ratio of effector cells (Effector Cells) to target cells (Target Cells).

[0018] The present invention provides a combination of detection reagents, a detection kit, and a detection method for the ADCP activity of human immunoglobulin against non-human species cells. Through preliminary screening experiments, the present invention has determined conditions such as target cells, effector cells, effector-to-target ratio, and sample concentration gradient in the detection method. The present invention uses Sp2 / 0-Ag14 cells as target cells and THP-1-NF-kB cells as effector cells, and realizes the in vitro detection of the ADCP activity of human immunoglobulin against non-human species (mouse) cells under the preferred effector-to-target ratio and sample concentration gradient. The detection method is simple, rapid, with high precision and accuracy, fills the blank of the detection method for the ADCP of immunoglobulin of non-human species (mouse), provides method support for scientific research and early R & D of antibody drugs, saves costs and time, and has important significance for the quality control of antibody drugs. The detection method of the present invention has good application prospects.

[0019] 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.

[0020] The following is a further detailed description of the above content of the present invention in the form of specific embodiments by way 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

[0021] Figure 1 It is a graph of the experimental results for the screening of ADCP effector cells. The data in the graph are expressed as the mean relative light units (RLU) ± SEM (n = 3), and SEM represents the standard error of the mean.

[0022] Figure 2 It is a graph of the experimental results for the optimization of the initial concentration gradient of SCIG. The data in the graph are expressed as the mean relative light units (RLU) ± SEM (n = 3), and SEM represents the standard error of the mean.

[0023] Figure 3 It is a graph of the experimental results for the verification of target cell specificity.

[0024] Figure 4 It is a graph of the experimental results for the verification of effector cell specificity.

[0025] Figure 5 It is a graph of the experimental results for the verification of sample specificity.

[0026] Figure 6 It is a graph of the experimental results for the verification of precision and accuracy. The data in the graph are expressed as the mean relative light units (RLU) ± SEM (n = 3), and SEM represents the standard error of the mean.

[0027] Figure 7 It is a graph of the experimental results for the application of the ADCP detection method to sample IVIG. The data in the graph are expressed as the mean relative light units (RLU) ± SEM (n = 3), and SEM represents the standard error of the mean. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] In the following examples and experimental examples, the reagents and raw materials not specifically described are commercially available products.

[0029] The present invention uses a reporter gene assay to evaluate the antibody-dependent cellular phagocytosis (ADCP) activity of human immunoglobulin. ADCP occurs when the Fab segment of an antibody binds to an antigen on the surface of a target cell (virus-infected cell or tumor cell), and its Fc segment can interact with immune cells (such as monocytes, macrophages, neutrophils, and dendritic cells) with FcγRIIa (CD32a) and / or FcγRIa (CD64a) receptors, prompting these immune cells to phagocytose and eliminate the target cell.

[0030] The information of the samples and control products used in the present invention is shown in Table 1.

[0031] Table 1 Example 1 Kit for Detecting the ADCP Activity of Human Immunoglobulin Against Non-Human Species Cells and Its Detection Method I. Detection Kit This example provides a kit and a detection method for detecting the ADCP activity of human immunoglobulin against mouse cells. The detection kit includes target cells, effector cells, ADCP assay buffer, and Fire-LumiTM luciferase detection kit working solution. Among them, Sp2 / 0-Ag14 cells (mouse myeloma cells) are used as target cells. Sp2 / 0-Ag14 cells are derived from the mouse spleen and are obtained by fusing spleen cells of BALB / c mice immunized with sheep red blood cells and P3X63Ag8 myeloma cells; genetically engineered THP-1 cells (THP-1-NF-κB, human leukemia monocytes) are used as effector cells. These cells naturally express CD32A (FcγRIIA) and CD64 (FcγRI), and are stably transfected with NF-κB response elements and the firefly luciferase gene.

[0032] II. Detection Method The detection method of this embodiment is to use Sp2 / 0-Ag14 cells as target cells. Sp2 / 0-Ag14 cells are derived from the spleen of mice and are obtained by fusing splenocytes of BALB / c mice immunized with sheep red blood cells and P3X63Ag8 myeloma cells; genetically engineered THP-1 cells (THP-1-NF-κB, human leukemia monocytes) are used as effector cells. These cells naturally express CD32a (FcγRIIa) and CD64a (FcγRIa), and are stably transfected with NF-κB response elements and the firefly luciferase gene. The antibody binds to FcγRIIa or FcγRIa on the surface of effector cells, activates the NF-κB response element in THP-1-NF-κB cells, and then drives the expression of firefly luciferase. The activity of luciferase is quantified by bioluminescence to characterize the ADCP activity of the antibody.

[0033] 1. Cell culture and subculture Cell resuscitation: Add 4 ml of pre-warmed medium (Probio, RD00941, RPMI 1640 + 10% FBS) 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 (Probio, RD00941, RPMI 1640 + 10% FBS + 400 μg / ml Hygromycin B), count the cells, and inoculate them into a cell culture dish at an appropriate density. After gently shaking and mixing, place them in a 5% CO2, 37°C incubator for culture.

[0034] Cell subculture: (1) Subculture of effector cells THP-1-NF-kB: 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 (90% RPMI 1640 + 10% FBS + 400 μg / ml Hygromycin B), count the cells, and inoculate them into a cell culture dish at an appropriate density. After gently shaking and mixing, place them in a 5% CO2, 37°C incubator for culture.

[0035] (2) Subculture of target cells Sp2 / 0-Ag14: 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, and digest it in a 37°C incubator. When most of the cell morphology becomes round, add an appropriate amount of complete medium (DMEM + 10% FBS) to terminate the digestion, then centrifuge at 800 rpm for 5 minutes in a centrifuge. After adding an appropriate amount of complete medium, count the cells and inoculate them into a cell culture dish at an appropriate density. After gently shaking and mixing, place them in a 5% CO2, 37°C incubator for culture.

[0036] 2. ADCP Reporter Gene Experimental Procedures (1)Centrifuge to collect target cells Sp2 / 0-Ag14 (purchased from ATCC) and resuspend with 1 ml of ADCP assay buffer (RPMI1640 + 10% FBS).

[0037] (2)Using 2.5 mg / ml as the starting working concentration, serially dilute the test human immunoglobulin (SCIG) with ADCP assay buffer at a 4-fold dilution ratio for 7 concentration points to prepare the test sample solutions with concentrations of 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.

[0038] (3)Adjust the target cell density with ADCP assay buffer (5E5 cells / ml) and transfer the target cell suspension to a 96-well assay plate (40 μl / well).

[0039] (4)Transfer the test sample solutions to the corresponding wells of the 96-well plate at 20 μl / well respectively.

[0040] (5)Incubate the assay plate at room temperature for 30 minutes.

[0041] (6)Collect effector cells (THP-1-NF-κB, purchased from Probio) and resuspend with ADCP assay buffer.

[0042] (7)According to an E / T (effector-to-target ratio, the ratio of effector cells to target cells) of 3:2, adjust the effector cell density with ADCP assay buffer (7.5E5 cells / ml) and transfer the effector cell suspension to the corresponding wells of the 96-well assay plate (40 μl / well).

[0043] (8)Incubate the assay plate in a cell culture incubator (37°C / 5% CO2) for 4 hours.

[0044] (9)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.

[0045] (10)Read the chemiluminescence value using a PHERAstar FSX at room temperature.

[0046] 3. ADCP Data Analysis After exporting the original data of the ADCP experiment, analyze it using Microsoft Office Excel.

[0047] Taking the relative luminescence unit value as the ordinate and the logarithm concentration of the SCIG solution as the abscissa, a four-parameter curve equation regression model was used to fit the dose-effect curve of SCIG.

[0048] Example 2 Human Immunoglobulin Anti-non-human Species Cell ADCP Activity Detection Kit and Its Detection Method The detection kit was prepared and detected according to the method of Example 1, with the difference that: the human immunoglobulin sample to be detected was replaced by IVIG instead of SCIG.

[0049] The technical solution of the present invention will be further described below through experiments.

[0050] Experimental Example 1 Selection of Effector Cells in the Detection of ADCP Activity of Human Immunoglobulin I. Experimental Method According to the detection method of Example 1, the ADCP effector cells were screened. The sources and gene modification information of the selected effector cells are as follows: (1) THP-1-NF-κB, this cell naturally expresses CD32a (FcγRIIa) and CD64a (FcγRIa), and stably transfected with the NF-κB-Luc luciferase reporter gene; (2) GS-J2B-FCGR2A (Jurkat / FCGR2A / NFAT-Luc), purchased from Probio, a genetically engineered Jurkat cell, and stably transfected with FCGR2A and NFAT-Luc luciferase reporter genes, FCGR2A is used to encode FcγRIIa; (3) GS-J8 (Jurkat / NF-κB-Luc), purchased from Probio, a genetically engineered Jurkat cell, and stably transfected with the NF-κB-Luc luciferase reporter gene.

[0051] Sp2 / 0-Ag14 was selected as the target cell and SCIG as the sample. Under the condition of E / T = 3:2, SCIG was used as the starting working concentration at 1.333 mg / ml, and SCIG was serially diluted. Using a 6-fold serial dilution ratio, 6 concentration points were diluted. Using the luciferase reporter gene method in Example 1, the ADCP effect detection experiments of SCIG on the target cell Sp2 / 0-Ag14 were carried out on three different effector cells (THP-1-NF-κB, GS-J8, GS-J2B-FCGR2A) respectively. The experimental conditions are shown in Table 2.

[0052] Table 2 Effector Cell Screening Experiment II. Experimental Results The experimental results are as Figure 1As shown, when the initial working concentration of SCIG was 1.333 mg / ml, SCIG could induce the ADCP effect of two effector cells (THP-1-NF-κB, GS-J2B-FCGR2A) on the target cell Sp2 / 0-Ag14, and the killing result was positive. However, the response curve of the GS-J8 effector cell did not show an obvious dose-effect relationship, indicating that SCIG could not induce its ADCP effect on the target cell. The THP-1-NF-κB and GS-J2B-FCGR2A effector cells showed an obvious S-shaped dose-effect curve. As the SCIG dose increased, the ADCP effect became gradually obvious and showed exponential growth. The presence or absence of the dose-effect curve was closely related to the reliability and effectiveness of the research method. Therefore, the GS-J8 was not selected as the effector cell in the detection method of the present invention.

[0053] Within the linear interval concentration range of the S-shaped dose-effect curve, the change in the drug dose and the change in the effect will show a relatively stable proportional relationship. When using GS-J2B-FCGR2A as the effector cell to detect the ADCP effect of SCIG, the effective detection concentration range corresponding to the linear interval of the SCIG sample was only 0.00617284 - 0.2222222 mg / ml (determined according to the concentration at which the upper and lower platforms began to appear in the curve), including only 3 effective concentration points. When using THP-1-NF-κB as the effector cell to detect the ADCP effect of SCIG, the effective detection concentration range corresponding to the linear interval of the SCIG sample included at least 0.000171468 - 1.333333 mg / ml (because the upper platform had not yet appeared in the curve), and included at least 6 effective concentration points. A wider effective concentration range can reflect that the method has lower requirements for the sample volume, can include more different samples for detection using this method, and has advantages such as better sensitivity. Therefore, in this experimental example, THP-1-NF-κB was preferably used as the effector cell for the subsequent establishment of the system method.

[0054] Experimental Example 2 Optimization of the Detection Concentration of Samples in the ADCP Activity Detection of Human Immunoglobulin I. Experimental Method Due to the poor design of the initial concentration and gradient of SCIG in the experimental system in Experimental Example 1, the ADCP dose-effect curve did not have an upper platform (see Figure 1 ), so this experimental example carried out an optimization experiment on the initial concentration of SCIG. Under the condition of E / T = 3:2, with 2.5 mg / ml as the initial working concentration of SCIG, SCIG was gradient-diluted. When diluting 7 concentration points at a 4-fold gradient dilution ratio, the ADCP dose-response effect of human immunoglobulin on the target cell Sp2 / 0-AG14 on the effector cell THP-1-NF-κB was detected. The experimental conditions are shown in Table 3.

[0055] Table 3 Screening Experiment on Initial Concentration of Samples to be Tested II. Experimental Results The detection results after improving the initial concentration are as Figure 2 shown. SCIG can induce the ADCP effect of effector cell THP-1-NF-κB on target cell Sp2 / 0-Ag14, the killing result is positive, the dose-effect curve fitting degree is good, and the expected saturation state (i.e., having upper and lower platforms) is achieved. Therefore, in this experimental example, 2.5 mg / ml is preferably used as the initial working concentration of subcutaneous human immunoglobulin for further verification experiments on the specificity, accuracy, and precision of the detection method for SCIG samples as the best concentration gradient for carrying out ADCP activity detection.

[0056] Experiment Example 3 Specificity Experiment of Detection Method I. Experimental Method The concentrations of human immunoglobulin SCIG samples (mg / mL) are set as: 2.5 mg / ml, 0.625 mg / ml, 0.15625 mg / ml, 0.0390625 mg / ml, 0.009765625 mg / ml, 0.002441406 mg / ml, 0.000610352 mg / ml. According to the method of Example 1, SCIG is used for the ADCP specificity test study, and 3 replicates are made continuously for each sample.

[0057] (1) Target Cell Specificity Use 293T cells instead of Sp2 / 0-Ag14 cells as target cells, set the 293T cell line as a control, and perform ADCP activity detection according to the method of Example 1.

[0058] (2) Effector Cell Specificity Use the THP-1 original cell line (THP-1) as effector cells, set the THP-1-NF-κB cells as a control, and perform ADCP activity detection according to the method of Example 1.

[0059] (3) Specificity of Detection Samples Use excipients (polysorbate 80 + maltose, the negative control in Table 1) instead of SCIG as the detection sample, and perform ADCP activity detection according to the method of Example 1.

[0060] II. Experimental Results The results of target cell specificity are as Figure 3 shown. Using Sp2 / 0-Ag14 cells as target cells to detect the ADCP activity of human immunoglobulin, compared with 293T, it has significantly better fluorescence response values, indicating that 293T cells are not suitable for detecting the ADCP effect of human immunoglobulin as target cells.

[0061] The effector cell specificity results are as Figure 4 shown. Using THP-1-NF-κB cells as effector cells to detect the ADCP activity of human immunoglobulin, compared with THP-1 cells, it has a significantly better fluorescence response value, indicating that THP-1 cells are not suitable as effector cells for detecting the ADCP effect of human immunoglobulin.

[0062] The test sample specificity results are as Figure 5 shown. Only SCIG showed ADCP activity, and the negative control group did not show an upward trend in the detection signal and did not have ADCP activity in this experimental system, indicating that the method of the present invention can specifically and effectively detect the ADCP activity of SCIG.

[0063] The above results comprehensively show that Sp2 / 0-Ag14 as the target cell has the best positive detection result, reflecting the specificity of the target cell in this experimental system; only THP-1-NF-kB cells as effector cells have positive detection results, reflecting the specificity of effector cell THP-1-NF-kB in this experimental system; only human immunoglobulin samples have positive detection results, reflecting the specificity of the test samples in this experimental system. Therefore, the method of the present invention can effectively detect the ADCP activity of human immunoglobulin and has good specificity.

[0064] Experimental Example 4 Accuracy and Precision of the Detection Method I. Accuracy Test 1. Experimental Method The concentrations (mg / mL) of the human immunoglobulin SCIG samples were set as: 2.5 mg / ml, 0.625 mg / ml, 0.15625 mg / ml, 0.0390625 mg / ml, 0.009765625 mg / ml, 0.002441406 mg / ml, 0.000610352 mg / ml. According to the method of Example 1, SCIG was used for the ADCP accuracy test study, and each sample was repeated 3 times continuously.

[0065] The data analysis method is as follows: Select the detection signal values corresponding to the curve concentration points for linear analysis, calculate the theoretical signal values according to the formula of curve fitting, perform linear fitting on the calculated detection values and theoretical values to obtain the R 2 value, and then compare the RSD of the R 2 values of the 3 repetitions.

[0066] 2. Experimental Results The results are as Figure 6As shown in Table 4, the relative standard deviation of the three repeated detection signal values of this method is within 15% (the accuracy meets the standard: the deviation is within the range of ±20%), and it has good accuracy.

[0067] Table 4 Accuracy Verification of the ADCP Reporter Gene Method II. Precision Test 1. Experimental Method The SCIG with an initial working concentration of 2.5 mg / ml was used for the within-day precision test study of ADCP detection. The SCIG was serially diluted, and when diluting 7 concentration points with a 4-fold serial dilution ratio, according to the method of Example 1, 3 independent detections were set within one day, and 1 96-well cell culture plate was used for each test to detect the within-day precision, and the RLU of the initial working concentration was statistically analyzed to verify the precision of this method.

[0068] 2. Experimental Results The results are as Figure 6 As shown in Table 5, the relative standard deviation of the detection signal values of 3 plates of this method is within 30% (the precision meets the standard: the deviation is within the range of ±30%), and it has good precision and can be used for the ADCP detection of SCIG.

[0069] Table 5 Precision Verification of the ADCP Reporter Gene Method The above results show that the detection method of the present invention has good accuracy and precision.

[0070] Experimental Example 5 Application of the ADCP Detection Method to IVIG Samples I. Experimental Method In order to explore whether the ADCP detection method can also be applied to IVIG samples, this experimental example further carried out the ADCP dose-response experimental effect of human immunoglobulin IVIG on the target cell Sp2 / 0-Ag14 in effector cells THP-1-NF-κB. Under the condition of E / T = 3:2, IVIG with an initial working concentration of 2.5 mg / ml was serially diluted, and 7 concentration points were diluted with a 4-fold serial dilution ratio. According to the method of Example 1, IVIG was used for the ADCP effect detection experiment, and 3 replicates were continuously made for each sample.

[0071] II. Experimental Results As Figure 7As shown, the above system is used for the detection of the ADCP effect of IVIG. The results show that IVIG can also induce the ADCP effect of effector cells THP-1-NF-κB on target cells Sp2 / 0-Ag14. The killing result is positive, the dose-effect curve has a good fitting degree, and it reaches the expected saturation state (i.e., there are upper and lower platforms).

[0072] 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 immunoglobulin on mouse cells. The present invention provides a method for detecting the ADCP of human immunoglobulin products in the field of infectious diseases through steps such as effector cell screening and sample concentration gradient optimization. The selection of target cells and effector cells is very important for this detection method, and the setting of the concentration gradient of the human immunoglobulin to be tested also has a significant impact on the detection results.

[0073] Under preferred conditions, using SCIG or IVIG as the detection sample, the ADCP effect of effector cells THP-1-NF-κB (human leukemia monocytes that naturally express CD32a (FcγRIIa) and CD64a (FcγRIa) and have stably transfected the NF-κB-Luc luciferase reporter gene) on target cells Sp2 / 0-Ag14 was measured. The 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 (the starting concentrations of SCIG and IVIG were set at 2.5 mg / ml, diluted 4-fold, with 7 concentration points). The detection method of the present invention has target cell specificity, effector cell specificity, and detection sample specificity, and has high sensitivity, accuracy, and precision, and is applicable to the detection of the ADCP activity of various human immunoglobulins (IVIG, SCIG).

[0074] The above results comprehensively show that the detection method of the present invention is simple, fast, has high precision and accuracy, fills the blank of the detection method for the ADCP activity of human immunoglobulin on non-human species (mouse) cells, provides method support for scientific research and early R & D of antibody drugs, saves costs and time, and has important significance for the quality control of antibody drugs. The detection method of the present invention has good application prospects.

Claims

1. A combination of detection reagents for the ADCP activity of human immunoglobulin against non-human species cells, characterized in that: The combination of the detection reagents includes: 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, Cloudman S91 cells, and the effector cells are selected from THP-1-NF-κB cells, GS-J2B-FCGR2A cells, and the number ratio of effector cells to target cells is 4-2:1-3.

2. The combination of detection reagents for the ADCP activity of human immunoglobulin against non-human species cells according to claim 1, characterized in that: The effector cell is THP-1-NF-κB cell.

3. The combination of detection reagents for the ADCP activity of human immunoglobulin against non-human species cells according to claim 1, characterized in that: The target cell is Sp2 / 0-Ag14 cell; and / or, the number ratio of effector cells to target cells is 3:

2.

4. A detection kit for the ADCP activity of human immunoglobulin against non-human species cells, characterized in that: The detection kit includes: the combination of the detection reagents for the ADCP activity of human immunoglobulin against non-human species cells described in claim 1, ADCP assay buffer, and luciferase detection kit working solution.

5. The kit for detecting the ADCP activity of human immunoglobulin against non-human species cells according to claim 4, characterized in that: The ADCP experimental buffer is selected from RPMI 1640 containing 8-12% animal serum; and / or, the working solution of the luciferase detection kit is Fire-Lumi TM Working solution of the luciferase detection kit.

6. A method for non-diagnostic detection using a combination of detection reagents for the ADCP activity of human immunoglobulin according to any one of claims 1 to 3 against non-human species cells or a detection kit for the ADCP activity of human immunoglobulin according to claim 4 or 5 against non-human species cells, characterized in that, The detection method includes the following steps: Step 1, mix and incubate the test human immunoglobulin with target cells and effector cells. Step 2, add the luciferase detection kit working solution, and detect the fluorescence signal after incubation.

7. A method for non-diagnostic purposes of detecting using the combination of detection reagents for the ADCP activity of human immunoglobulin against non-human species cells and the kit for detecting the ADCP activity of human immunoglobulin against non-human species cells according to claim 6, characterized in that: In step 1, the mixing is the mixing of the test human immunoglobulin solution with the target cell suspension and the effector cell suspension, and the test human immunoglobulin solution, the target cell suspension and the effector cell suspension are prepared with ADCP assay buffer.

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

9. A method for non-diagnostic purposes of detection using the combination of detection reagents for the ADCP activity of human immunoglobulin against non-human species cells and the detection kit for the ADCP activity of human immunoglobulin against non-human species cells according to claim 7, 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.

10. A method for non-diagnostic purposes using the combination of the detection reagent for the ADCP activity of human immunoglobulin against non-human species cells and the detection kit for the ADCP activity of human immunoglobulin against non-human species cells according to claim 6, characterized in that: In step 1, the process of the mixing and incubation includes: incubating the test human immunoglobulin with target cells at 18-37 °C for 25-35 minutes, and then adding effector cells and incubating at 35-37 °C for 3.8-4.2 hours. and / or, the test human immunoglobulin is selected from subcutaneous injection immunoglobulin, intramuscular injection immunoglobulin, intravenous injection immunoglobulin; And / or, in step 2, the dosage ratio of the luciferase detection kit working solution to the effector cells is 70-90 μL: 2.4-3.6×10 5 cells; and / or, in step 2, the incubation time is 5-10 minutes.

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