A method for detecting adcp activity of a human immunoglobulin-based product in the field of infectious diseases

Abstract

The application provides a method for detecting ADCP activity of human immunoglobulin products in the field of infectious diseases, and belongs to the technical field of immunological detection. The method comprises the steps of target cell screening, effector-target ratio optimization, and test product concentration gradient optimization, and provides an accurate and intuitive biological activity detection method for antibody-dependent cell-mediated phagocytosis of immunoglobulin products, so that accurate detection of the biological activity of antibody-dependent cell-mediated phagocytosis of human immunoglobulin products is realized. The detection method is intuitive, precise and accurate, fills the blank of the detection of antibody-dependent cell-mediated phagocytosis of human immunoglobulin products, and has important significance for improving the quality control and clinical application of human immunoglobulin products.

Description

Technical Field

[0001] This invention belongs to the field of immunological detection technology, specifically relating to a method for detecting ADCP activity in human immunoglobulin products in the field of infectious diseases. Background Technology

[0002] In the field of infectious disease prevention and control, human immunoglobulin preparations, as important biotherapeutic products, are increasingly demonstrating their clinical value. These high-purity preparations, made from mixed plasma from healthy donors through a rigorous virus inactivation process, mainly include two dosage forms: intravenous immunoglobulin (IVIG) and subcutaneous immunoglobulin (SCIG). Their core active ingredient—immunoglobulin G (IgG) and its four subtypes (IgG1-IgG4)—plays an irreplaceable role in the prevention and treatment of infectious diseases due to its unique antipathogenic spectrum and bidirectional immunomodulatory mechanism, particularly showing significant efficacy against severe infections and immunodeficiency-related infections.

[0003] From a molecular perspective, IgG recognizes pathogens through its Fab segment, while its Fc segment mediates various anti-infective immune effects, including specific binding to various Fcγ receptors (FcγRI-III) and nascent Fc receptors (FcRn), thereby triggering key immune defense responses such as antibody-dependent phagocytosis (ADCP), antibody-dependent cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC). These mechanisms together constitute an important line of defense against infectious pathogens.

[0004] However, current pharmacopoeia standards (including the Chinese and European pharmacopoeias) lack quality standards for IgG, making it difficult to comprehensively assess its actual immune protective efficacy in infectious diseases.

[0005] Existing ADCP detection methods based on monoclonal antibodies (such as the reporter gene method described in patent CN 113186167A) have obvious technical defects: 1) The difference in transfection efficiency of engineered effector cells (Jurkat / NFAT / CD32a-FcεRIγ) leads to unstable detection signals; 2) Luciferase and other reporter genes are susceptible to non-specific interference; 3) Indirect detection mode cannot truly simulate the complex pathogen clearance process in infectious diseases.

[0006] Therefore, establishing a direct detection technology for ADCP activity specifically for human immunoglobulin preparations is of great significance. It can significantly improve the quality control level of infectious disease treatment products, provide key technical support for clinical evaluation of anti-infective efficacy, and improve the immunotherapy system for infectious diseases. Summary of the Invention

[0007] The purpose of this invention is to provide a method for detecting ADCP activity in human immunoglobulin products in the field of infectious diseases.

[0008] This invention provides a method for detecting ADCP activity of human immunoglobulin products in the field of infectious diseases, using human primary peripheral blood mononuclear cells as effector cells or macrophages sorted and induced from human primary peripheral blood mononuclear cells as effector cells, comprising the following steps:

[0009] (1) Human immunoglobulin products were serially diluted with buffer solution to obtain test solutions of different concentration gradients;

[0010] (2) Inoculate target cells and incubate them with the test solution from step (1); the target cells are target cells labeled with fluorescent staining reagent;

[0011] (3) Add effector cells for co-incubation according to the effector-target ratio; the effector cells are effector cells with immunophenotypic markers;

[0012] (4) Detect fluorescence, calculate cell phagocytosis rate, plot the cell phagocytosis rate on the ordinate and the logarithm of the concentration of human immunoglobulin products on the abscissa to obtain the half effective concentration.

[0013] Further, in step (1), the initial concentration of the test solution is set to 8~10 mg / ml; the gradient dilution factor is 2.5~3 times;

[0014] In step (2), the target cells are CHO-K1 cells expressing the spike protein of the novel coronavirus or PLC / PFR / 5 cells expressing the human hepatitis B surface antigen;

[0015] In step (3), the effective-to-target ratio is set to 3~5:1.

[0016] Further, in step (1), the initial concentration of the test solution is set to 10 mg / ml; the gradient dilution factor is 3 times;

[0017] In step (2), the target cells are CHO-K1 cells expressing the spike protein of the novel coronavirus;

[0018] In step (3), the effective-to-target ratio is set to 5:1.

[0019] Further, in step (1), the buffer solution is RPMI 1640 complete medium containing FBS.

[0020] Furthermore, the FBS content is 10%.

[0021] Further, in step (2), the fluorescent staining reagent is PKH26; in step (3), the immunophenotypic marker is selected from CD11b, CD14, CD45, CD64, CD163 or CD206.

[0022] Further, in step (2), the incubation conditions are: incubation at 10~40℃ for 10~50 minutes;

[0023] In step (3), the incubation conditions are: incubation at 5% CO2 and 30~40℃ for 0.5~2 hours.

[0024] Furthermore, in step (2), the incubation conditions are: incubation at 15~35℃ for 30 minutes;

[0025] In step (3), the incubation conditions are: incubation at 5% CO2 and 37°C for 1 hour.

[0026] Furthermore, the phagocytosis rate refers to the percentage of cells simultaneously labeled with fluorescent staining reagents and immunophenotypic markers among all cells labeled with fluorescent staining reagents.

[0027] Furthermore, the fitted curve is expressed mathematically as follows:

[0028] Y = Minimum response value + (Maximum response value - Minimum response value) / (1 + 10^((Log half effective concentration - X) × Hill slope))

[0029] Where X is the logarithmic value of the concentration of the human immunoglobulin product to be tested, and Y is the phagocytic rate of cells.

[0030] The present invention has achieved the following beneficial effects:

[0031] (1) This invention provides an accurate and intuitive method for detecting the biological activity of ADCP in immunoglobulin products through steps such as target cell screening, effector-to-target ratio optimization, and test sample concentration gradient optimization.

[0032] (2) This invention uses macrophages (hMDMs) obtained by sorting and inducing mononuclear cells from human primary peripheral blood mononuclear cells (hPBMCs) as effector cells and CHO-K1 cells (CHO-K1 / Spike_SARS2) that stably express the spike protein of SARS-CoV-2 as target cells; and achieves accurate detection of the biological activity of ADCP of human immunoglobulin products under appropriate effector-target ratio (5:1) and test sample concentration gradient (the initial concentration of subcutaneous human immunoglobulin (SCIG) was set at 10 mg / ml, 3-fold dilution, 8 concentration points).

[0033] (3) The detection method of the present invention is intuitive, precise and accurate, filling the current gap in ADCP detection of immunoglobulin products, and is of great significance for improving the quality control and clinical application of immunoglobulin products.

[0034] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0035] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0036] Figure 1 Results of ADCP target cell screening experiment (data in the figure are expressed as phagocytosis percentage ± SEM (n=2)).

[0037] Figure 2 The experimental results were optimized to improve the effectiveness-to-target ratio (the data in the figure are expressed as phagocytosis percentage ± SEM (n=2)).

[0038] Figure 3 The results of the initial gradient optimization for SCIG concentration are shown in the figure (data in the figure are expressed as percentage of phagocytosis ± SEM (n=3)).

[0039] Figure 4 The results were validated for precision and accuracy (the data in the figure are expressed as percentage of phagocytosis ± SEM (n=3)).

[0040] Figure 5 Results of effector cell specificity verification (data in the figure are expressed as phagocytic percentage ± SEM (n=3)). Detailed Implementation

[0041] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0042] In this invention, "room temperature" means 25±10℃ and "overnight" means 12±5 hours.

[0043] The cell resuscitation and culture steps involved in this invention are as follows:

[0044] ①hPBMCs cell resuscitation: First, preheat the complete culture medium (RPMI 1640 medium + 10% FBS) used for hPBMCs cells to 37℃. Add a certain volume (10-20 ml) of the corresponding complete culture medium to a 50 ml centrifuge tube. Then, transfer the hPBMCs cell suspension, which has been thawed in a 37℃ water bath, to the aforementioned centrifuge tube. Centrifuge at 400 g for 10 minutes. After centrifugation, discard the supernatant. Resuspend the hPBMCs cells in the corresponding complete culture medium and count them. Then, count the cells at a rate of 2E6 cells / ml (i.e., 2 × 10⁶ cells / ml). 6 hPBMCs were seeded at a density of 1 cell / ml in shake flasks or human mononuclear cell sorting kits were used to sort hMDMs into mononuclear cells for induction.

[0045] ② PLC / PRF / 5 and CHO-K1 / Spike_SARS2 cell resuscitation: First, preheat the complete culture medium used for each cell type (37℃). Add a certain volume (1-1.5 ml for a 10 cm culture dish) of the corresponding complete culture medium to a 15 ml centrifuge tube. Then, transfer the cell suspension, which has been melted in a 37℃ water bath, to the aforementioned centrifuge tube. Centrifuge at 600 rpm or 800 rpm for 5 minutes. After centrifugation, discard the supernatant, resuspend the cells in the corresponding complete culture medium, count the cells, and then seed them at an appropriate density (10^5~10^6 cells / mL) in cell culture dishes (or other consumables that can be used for cell culture).

[0046] ③ Passaging of PLC / PRF / 5 and CHO-K1 / Spike_SARS2 cells: First, preheat the complete culture medium (PLC / PRF / 5 cells: EMEM medium + 10% FBS; CHO-K1 / Spike_SARS2 cells: F-12 medium + 10% FBS), digestion solution, and other reagents used for each cell type to 37℃ or allow them to return to room temperature. PLC / PRF / 5 and CHO-K1 / Spike_SARS2 cells are adherent cells, so the culture supernatant should be discarded first. Wash the cells 1-3 times with 1× DPBS, add an appropriate amount of digestion solution, and digest them in a cell culture incubator (37℃ / 5% CO2) for a certain period of time (until large cell fragments detach). Then, add an appropriate amount of the corresponding complete culture medium to stop the digestion and collect the cell suspension into a centrifuge tube. Centrifuge the PLC / PRF / 5 and CHO-K1 / Spike_SARS2 cell suspensions at 600 rpm or 800 rpm for 5 minutes. After centrifugation, discard the supernatant, resuspend the cells in the appropriate complete culture medium, count the cells, and then seed them into cell culture dishes (or other consumables that can be used for cell culture) at an appropriate density (10^5~10^6 cells / mL).

[0047] The experimental materials used in this invention are as follows:

[0048] (1) Instruments and equipment

[0049]

[0050] (2) Experimental reagents

[0051]

[0052] (3) Experimental cells

[0053]

[0054] (4) Test sample

[0055]

[0056] Example 1: Method for detecting the biological activity of ADCP in immunoglobulin products according to the present invention

[0057] 1. Method Overview

[0058] ADCP (Advanced Dietary Therapy-Chip) occurs when the Fab fragment of IgG binds to the antigen on the surface of target cells. Its Fc fragment then interacts with immune cells (such as macrophages and monocytes) possessing Fcγ receptors (FcγRIIa, FcγRIa), prompting these immune cells to phagocytose and clear the target cells. This mechanism plays a crucial role in the body's immune defense and regulation. In flow cytometry, two different fluorescent markers are used to label effector cells and target cells respectively. Effector cells exhibit double-positive fluorescence signals after phagocytosing target cells. The proportion of cells with double-positive fluorescence signals relative to the total number of target cells characterizes the ADCP activity of the antibody.

[0059] 2. ADCP Experimental Procedure

[0060] ①-④ describe the preparation process of effector cells:

[0061] ① Fourteen days before the experiment, human primary peripheral blood mononuclear cells (hPBMCs) were resuscitated and collected, resuspended in complete culture medium (RPMI 1640 + 10% FBS), and then counted.

[0062] ② Based on the cell counting results, hPBMCs cells were collected and monocytes were sorted using a human monocyte sorting kit.

[0063] ③Monocytes were cultured for 14 days in RPMI 1640 complete medium containing 100 ng / ml M-CSF to induce them to become macrophages (hMDMs).

[0064] ④ Quality control (QC) of hMDMs or hPBMCs effector cells to assess the success of induced hMDMs: effector cells were labeled with CD11b, CD14, CD45, CD64, CD163, and CD206 by flow cytometry and incubated at 4°C for 15 minutes in the dark.

[0065] ⑤-⑧ describes the target cell preparation process:

[0066] ⑤ The day before the experiment, the target cells (CHO-K1 cells expressing the SARS-CoV-2 spike protein, i.e., CHO-K1 / Spike_SARS2 cells) were digested, centrifuged and collected, and then stained with PKH26 dye.

[0067] ⑥ Seed the PKH26-labeled target cells into new culture dishes and incubate overnight in a cell culture incubator (37℃ / 5% CO2).

[0068] ⑦ If hPBMCs are used as ADCP effector cells: they can be revived one day before the experiment and cultured overnight in RPMI 1640 complete medium.

[0069] ⑧ On the day of the experiment, PKH26-labeled and unlabeled target cells were digested, centrifuged and collected, and the cells were resuspended in ADCP experimental buffer (RPMI 1640 complete medium).

[0070] ⑨-⑲ are the specific operating procedures for ADCP experiments:

[0071] ⑨ Prepare test samples (4×) using ADCP experimental buffer: The initial concentration of the test sample, i.e., SCIG, is set at 10 mg / ml, diluted 3 times, for 8 concentration points.

[0072] ⑩ Adjust the density of PKH26-labeled target cells (200 cells / µl) and transfer the cell suspension to the corresponding wells of a 96-well experimental plate (10,000 cells / well, 50 µl / well).

[0073] ⑪ Transfer SCIG working solution to the corresponding wells of the 96-well experimental plate and incubate the experimental plate at room temperature for 30 minutes (50 µl / well).

[0074] ⑫ Digest and centrifuge to collect hMDMs or hPBMCs effector cells (centrifugation conditions: 400 g for 10 minutes) and resuspend the effector cells with ADCP experimental buffer.

[0075] ⑬ Adjust the effector cell density according to the effector-to-target ratio (5:1) and transfer the effector cell suspension to the corresponding wells of the 96-well experimental plate (100 µl / well).

[0076] ⑭ Incubate the experimental plate in a cell culture incubator (37℃ / 5% CO2) for about 1 hour.

[0077] ⑮ After incubation, remove the experimental plate, collect all cells into a new 96-well conical plate and centrifuge to obtain cell pellet.

[0078] ⑯ Prepare the CD11b flow cytometry detection antibody working solution using flow cytometry buffer (1× DPBS + 1% FBS).

[0079] ⑰ Use CD11b flow cytometry detection antibody to stain and label hMDMs or hPBMCs effector cells in the experimental plate, and incubate at 4°C for 15 minutes in the dark.

[0080] 18. After the antibody incubation for flow cytometry is completed, centrifuge to obtain cell pellet, resuspend in flow cytometry buffer and wash the cells twice.

[0081] ⑲ ADCP was detected using flow cytometry: PKH26-labeled target cells were detected via the PE fluorescence channel, and CD11b-stained effector cells were detected via the APC fluorescence channel.

[0082] 3. ADCP data analysis

[0083] The raw data from the ADCP experiment were exported using the BD FACSDiva Software system and analyzed using FlowJo, Microsoft Office Excel, and GraphPad Prism software.

[0084] In FlowJo software, ADCP data are analyzed by plotting a crosshair using a PE-APC scatter plot. The PE channel represents target cell markers, and the APC channel represents effector cell markers. Within the crosshair, PE... + APC + The number of cells in the quadrant represents PKH26. + CD11b + The number of target cells engulfed, PE + APC + Quadrants and PE + APC - The total number of cells in the quadrant represents all PKH26 cells. + Target cells.

[0085] Cell phagocytosis rate is calculated using the following formula:

[0086] % Phagocytosis by hMDMs or hPBMCs = (PKH26 + CD11b + Cells / All PKH26+ Cells) × 100%

[0087] In GraphPad Prism software, in XY Table mode, the SCIG concentration values ​​are pasted correspondingly with the obtained cell phagocytosis rate data. Then, in the Parameters: Transform module, the SCIG concentration is converted to a logarithmic concentration, i.e., X = Log(X). Next, in the Parameters: Nonlinear regression module, a log(agonist) vs. response-variable slope (four-parameter model) best-fit value is performed, and the fitted curve is generated. During the fitting, the following four-parameter equation can be used to obtain EC... 50 The relative value of Y: Y = Bottom + (Top - Bottom) / (1 + 10^((LogEC)) 50 - X)× HillSlope). Where X = Log(sample concentration), Y = cell phagocytosis rate.

[0088] The following experimental examples demonstrate the beneficial effects of the present invention.

[0089] Experimental Example 1: Optimization of the ADCP biological activity detection method of the present invention

[0090] 1. Target cell screening

[0091] (1) Experimental methods

[0092] Referring to the ADCP experimental procedure in Example 1, SCIG was diluted to 20 mg / ml using ADCP experimental buffer as the initial concentration (20, 10, 2, 0.4, 0.04, 0.008 mg / ml, a total of 6 concentration points); the effect-to-target ratio was set to 10:1, and the following cells were used as target cells: ① CHO-K1 cells expressing the SARS-CoV-2 spike protein (Spike) (CHO-K1 / Spike_SARS2) and ② PLC / PFR / 5 cells expressing human hepatitis B surface antigen (HBsAg) to explore their effects on the detection of SCIG ADCP biological activity.

[0093] (2) Experimental results

[0094] Experimental results are as follows Figure 1 As shown, SCIG cannot mediate the phagocytosis of PLC / PRF / 5 target cells by hMDMs effector cells, but it can mediate the phagocytosis of CHO-K1 / Spike_SARS2 target cells by hMDMs effector cells, and a clear dose-response curve is observed. Therefore, in subsequent assays, CHO-K1 / Spike_SARS2 cells were selected as target cells to evaluate the ADCP biological activity of SCIG.

[0095] 2. Optimization of effect-to-target ratio

[0096] (1) Experimental methods

[0097] Following the ADCP experimental procedure in Example 1, after identifying the target cells (CHO-K1 / Spike_SARS2 cells), SCIG was diluted to 20 mg / ml using ADCP experimental buffer as the initial working concentration (20, 10, 2, 0.4, 0.04, 0.008 mg / ml, a total of 6 concentration points); the effector-target ratio was set to ①3:1 or ②5:1 to investigate the effect of different effector-target ratios on the detection of ADCP biological activity of SCIG.

[0098] (2) Experimental results

[0099] Table 1. Results of ADCP effector-target ratio optimization experiment (target cells: CHO-K1 / Spike_SARS2)

[0100]

[0101] The results are as follows Figure 2 As shown in Table 1, both effect-to-target ratios exhibited clear effect curves, and the experimental window increased with increasing effect-to-target ratio. ("Experimental window" refers to the concentration range within which significant phagocytosis can be observed at a specific effect-to-target ratio. Within this concentration range, the phagocytosis rate increases significantly with increasing concentration until a plateau is reached, meaning the maximum phagocytosis rate no longer increases with increasing concentration.) Therefore, an effect-to-target ratio of 5:1 was selected for subsequent testing.

[0102] 3. Optimization of the initial gradient of SCIG concentration

[0103] (1) Experimental results

[0104] In the "Effectiveness-to-Target Ratio Optimization" experiment, this invention confirmed that SCIG can mediate the phagocytosis of CHO-K1 / Spike_SARS2 target cells by hMDMs effector cells. However, a hook effect (a phenomenon where, within a certain concentration range, the analyte concentration is directly proportional to the detection signal value; however, beyond a certain concentration threshold, the signal value decreases with increasing concentration) was also observed at higher SCIG concentrations. Therefore, based on the experimental results of the "Effectiveness-to-Target Ratio Optimization," this invention optimized the SCIG concentration gradient. The initial working concentration of SCIG was set at 8 mg / ml, with 2.5-fold and 10 mg / ml, and 3-fold dilutions, with each concentration set at eight different concentration points.

[0105] (2) Experimental results

[0106] Table 2. Results of SCIG initial concentration optimization experiment (target cells: CHO-K1 / Spike_SARS2)

[0107]

[0108] For detailed results, please see [link to results]. Figure 3 As shown in Table 2, under an effector-to-target ratio (E:T) of 5:1, SCIG at an initial concentration of 8 mg / ml with 2.5-fold serial dilutions, or at an initial concentration of 10 mg / ml with 3-fold serial dilutions, mediated the phagocytosis of CHO-K1 / Spike_SARS2 target cells by hMDMs effector cells, exhibiting a clear dose-response response with largely intact dose-response curves. However, the maximum response value was higher and the range was larger when using an initial concentration of 10 mg / ml with 3-fold serial dilutions. Therefore, subsequent experiments will be conducted with reference to this SCIG concentration gradient setting.

[0109] 4. Precision and accuracy assessment

[0110] (1) Experimental methods

[0111] Based on the experimental results of SCIG concentration gradient optimization and effect-to-target ratio optimization, this invention selects 10 mg / ml as the starting concentration of SCIG, performs 3-fold serial dilutions, and obtains a total of 8 concentration points to verify the precision and accuracy of the method.

[0112] (2) Experimental results

[0113] Table 3. Precision and accuracy validation results (target cells: CHO-K1 / Spike_SARS2)

[0114]

[0115] Table 4. Precision evaluation parameters: relative standard deviation

[0116]

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

[0118] Table 5. Accuracy Evaluation Parameters: Recovery Rate

[0119]

[0120] Note: Plate 01 is considered a 100% RS, i.e., a 100% potency standard, and its EC is... 50 The value divided by the EC values ​​of other experimental boards50 Multiplying the numerical value by 100% gives the relative valence; the mean of the relative valence can be calculated using the AVERAGE function in Excel; dividing the mean of the relative valence by 100% and then multiplying by 100% gives the recovery rate.

[0121] For detailed results, please see [link to results]. Figure 4 See Tables 3, 4, and 5. Experimental results show that this experimental system can fully characterize the ADCP biological activity of SCIG and exhibits a clear dose-response curve. In precision and accuracy experiments, hMDMs were used as target cells, and the SCIG group was analyzed in three repeated experiments. Experimental results show that under an effector-to-target ratio (E:T) of 5:1, SCIG can mediate the phagocytosis of CHO-K1 / Spike_SARS2 target cells by hMDMs effector cells. When analyzing the three sets of data, in Graphpad software, the same Top / Bottom / Hillslope values ​​were used for fitting the three sets of data (in the Parameters: Transform module, Global nonlinear regression (dose-response curves) - Bottom\Top\Hillslope 3 parameters Constraint type: shared value for all data sets, i.e., assuming consistent curve integrity for all data sets) to obtain the EC50 values ​​for each of the three sets of data. 50 Numerical results show that the dose-response curves (EC) of the three sets of data are... 50 The numerical values ​​are similar. The General Principles for the Review of Analytical Method Validation Technologies for Quality Control of Biological Products issued by the Center for Drug Evaluation state that the precision of cell assays should be less than 30%, and the relative standard deviation and recovery rate of this method are both within 30%. That is, this invention has good precision and accuracy.

[0122] 5. Validation of effector cell specificity

[0123] 1) Experimental methods

[0124] To verify the effector cell specificity of the method, this invention selected 10 mg / ml as the starting concentration of SCIG, and performed 3-fold serial dilutions to further verify the effector cell specificity of the method. Uninduced hPBMCs were used instead of hMDMs as effector cells for ADCP activity assay, and an effector-to-target ratio of 5:1 was selected.

[0125] 2) Experimental Results

[0126] Table 6. Results of effector cell specificity validation (target cells: CHO-K1 / Spike_SARS2)

[0127]

[0128] The results are as follows Figure 5 As shown in Table 6, compared with hPBMCs as effector cells, the experimental window for SCIG-mediated hMDMs effector cells to exert ADCP is more obvious, that is, hMDMs cells as effector cells have better detection results.

[0129] The above results demonstrate that this invention provides a method for detecting ADCP in human immunoglobulin products in the field of infectious diseases through steps such as target cell screening, effector-to-target ratio optimization, and test sample concentration gradient optimization. Using CHO-K1 / Spike_SARS2 cells as target cells, and sorting monocytes from hPBMCs and using monocyte-induced macrophages (hMDMs) as effector cells, the method achieves accurate detection of ADCP biological activity in human immunoglobulin products at an appropriate effector-to-target ratio (5:1) and test sample concentration gradient (SCIG initial concentration set at 10 mg / ml, 3-fold dilution, 8 concentration points).

[0130] In summary, this invention provides a method for detecting ADCP activity in human immunoglobulin products in the field of infectious diseases. Through steps such as target cell screening, effector-to-target ratio optimization, and sample concentration gradient optimization, this invention offers an accurate and intuitive method for detecting the biological activity of ADCP in immunoglobulin products, achieving accurate detection of ADCP biological activity in human immunoglobulin products. This method is intuitive, precise, and accurate, filling a gap in current ADCP detection techniques for human immunoglobulin products and is of great significance for improving the quality control and clinical application of human immunoglobulin products.

Claims

1. A method for detecting ADCP activity of human immunoglobulin products in the field of infectious diseases, characterized in that, Macrophages, sorted and induced from primary human peripheral blood mononuclear cells, were used as effector cells, including the following steps: (1) Human immunoglobulin products were serially diluted with buffer solution to obtain test solutions of different concentration gradients; (2) Inoculate target cells and incubate them with the test solution from step (1); the target cells are target cells labeled with fluorescent staining reagent; (3) Add effector cells for co-incubation according to the effector-target ratio; the effector cells are effector cells with immunophenotypic markers; (4) Detect fluorescence, calculate cell phagocytosis rate, plot the cell phagocytosis rate on the ordinate and the logarithm of the concentration of human immunoglobulin products on the abscissa to calculate the half effective concentration; In step (1), the initial concentration of the test solution is set to 10 mg / ml; the gradient dilution factor is 3 times. In step (2), the target cells are CHO-K1 cells expressing the spike protein of the novel coronavirus; In step (3), the effective-to-target ratio is set to 5:1; In step (2), the incubation conditions are: incubation at 15~35℃ for 30 minutes; In step (3), the incubation conditions are: incubation at 5% CO2 and 37°C for 1 hour.

2. The detection method according to claim 1, characterized in that, In step (1), the buffer solution is RPMI 1640 complete medium containing FBS.

3. The detection method according to claim 2, characterized in that, The FBS content is 10%.

4. The detection method according to claim 1, characterized in that, In step (2), the fluorescent staining reagent is PKH26; in step (3), the immunophenotypic marker is selected from CD11b, CD14, CD45, CD64, CD163 or CD206.

5. The detection method according to claim 1, characterized in that, The phagocytosis rate refers to the percentage of cells simultaneously labeled with fluorescent staining reagents and immunophenotypic markers among all cells labeled with fluorescent staining reagents.

6. The detection method according to claim 1, characterized in that, The fitted curve is expressed mathematically as follows: Y = Minimum response value + (Maximum response value - Minimum response value) / (1 + 10^((Log half effective concentration - X) × Hill slope)) Where X is the logarithmic value of the concentration of the human immunoglobulin product to be tested, and Y is the phagocytic rate of cells.

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