A method for detecting antibody-dependent cell-mediated cytotoxicity of intravenous human immunoglobulin

Abstract

The application provides a method for detecting antibody-dependent cell-mediated cytotoxicity (ADCC) of intravenous immunoglobulin (IVIG), adopts liver cancer cells as target cells, and realizes accurate detection of the antibody-dependent cell-mediated cytotoxicity of IVIG by using ADCC effect reporter cells under appropriate effector-target ratio and incubation conditions; the detection method is simple in operation, high in sensitivity and accuracy, fills the blank of the current ADCC detection of IVIG, and is helpful to establishment of quality standards of IVIG products.

Description

Technical Field

[0001] This invention belongs to the field of antibody cytotoxicity detection, specifically relating to a method for detecting antibody-dependent cell-mediated cytotoxicity of intravenously injected human immunoglobulin. Background Technology

[0002] Human Immunoglobulin (pH4) for Intravenous Injection (IVIG) is produced from healthy human plasma through low-temperature ethanol protein separation or other approved separation methods, removing anticomplement activity and undergoing virus removal and inactivation treatment. The main component of IVIG is IgG, with commercially available products typically containing >95% IgG. It contains various antibodies against foreign pathogens and autoantigens and is currently widely used to treat various immunodeficiency diseases, inflammatory diseases, and autoimmune diseases. Research indicates that the IgG Fc fragment is the primary active component of IVIG. Human Fc fragment receptors include activating receptors (such as FcγRI, FcγRIIA, FcγRIIC, and FcγRIIIA) and inhibitory receptors (FcγRIIB). The Fc fragment can bind to receptors on the surface of immune cells, thereby exerting an immunomodulatory effect. Currently, the quality evaluation of IVIG in domestic and international pharmacopoeias and industry standards only involves the detection of Fc fragment activation of complement, which can only reflect the ability of Fc to activate complement, but the detection of the function of Fc fragment binding to receptor is not perfect.

[0003] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an antibody-induced cellular effect in which antibodies bind to antigenic epitopes on virus-infected or tumor cells via their Fab fragment, and their Fc fragment binds to the Fc receptors on the surface of killer cells (NK cells, macrophages, etc.), mediating the direct killing of target cells by killer cells and directly lysing pathogen-infected cells. This effect is crucial for the immune system to kill pathogen-infected cells; therefore, detecting ADCC in IVIG is of great significance.

[0004] The most classic method for detecting ADCC effector activity is the chromium-51 (51Cr) labeling / release method. Radioactive chromium-51 is incubated with target cells, allowing it to enter the cells. Excess chromium-51 is washed away, and the target cells, effector cells, and antibody are then co-incubated. If the target cells are killed, chromium-51 is released, and the presence of radioactive isotopes in the solution confirms the extent of target cell damage. This method is the gold standard for ADCC detection; however, it is rarely used due to its radioactive elements, time-consuming and labor-intensive nature, and high variability. Currently, methods such as lactate dehydrogenase release assays are more commonly used. While these methods avoid isotope contamination, they still require fresh PBMCs or NK cells from the patient as effector cells. This method not only suffers from problems such as unstable primary cell sources, cumbersome cell extraction process, large response variability due to differences in individual subjects and immune levels, and high background values ​​leading to poor experimental reproducibility, but also wastes valuable blood resources by discarding the remaining blood components after cell extraction, and has low experimental throughput and small sample size.

[0005] In recent years, many manufacturers have developed reporter gene cells for detecting ADCC effects, using genetically engineered human T cell lines to detect the early signal of ADCC induction: NFAT (activating T cell nuclear factor) activation. By measuring the bioluminescent signal produced by NFAT-dependent luciferase reporter protein, the induction level of ADCC can be displayed. This method can replace traditional detection methods that rely on NK cell cytotoxicity, greatly simplifying the detection process and enabling efficient screening of antibody drugs.

[0006] However, for IVIG, a specific target for detection, there is currently no standardized ADCC assay. Furthermore, methods using reporter gene cells for IVIG ADCC detection have not been reported. Moreover, unlike common antibody drugs, IVIG contains broad-spectrum antibodies; therefore, parameters such as the type of target cells, incubation conditions, and effector-to-target ratio can have unpredictable impacts on the sensitivity and specificity of the assay. Therefore, establishing a rapid and accurate ADCC assay for IVIG remains a worthwhile area of ​​research. Summary of the Invention

[0007] The purpose of this invention is to provide a method for rapid and accurate detection of ADCC in IVIG using reporter gene cells with ADCC effect.

[0008] This invention provides a method for detecting antibody-dependent cell-mediated cytotoxicity of intravenously injected human immunoglobulin, using liver cancer cells as target cells, and includes the following steps:

[0009] (1) The intravenous immunoglobulin to be tested was serially diluted with diluent to obtain test solutions of different concentration gradients;

[0010] (2) Inoculate target cells and incubate them with test solutions of different concentration gradients from step (1);

[0011] (3) Add effector cells for co-culture; the effector cells are Jurkat cells stably transfected with CD16 receptor, NFAT reaction element and luciferase reporter gene;

[0012] (4) Detect fluorescence, plot the dose-response curve with relative fluorescence intensity as the ordinate and the logarithm of the intravenous immunoglobulin concentration as the abscissa, and calculate the half-maximal effective concentration.

[0013] Furthermore, the ratio of effector cells to target cells is 1:(2-4).

[0014] Furthermore, the ratio of effector cells to target cells was 1:2.8.

[0015] Furthermore, the target cells are PLC / PRF / 5 cells, and the effector cells are Jurkat-NFAT-Luc-CD16 cells.

[0016] Further, the diluent in step (1) is a bovine serum albumin solution.

[0017] Furthermore, the concentration of the above bovine serum albumin solution is 2% wt.

[0018] Furthermore, the co-incubation described in step (2) is carried out for 1 to 3 hours under conditions of 5% CO2 and 37°C.

[0019] Furthermore, the incubation time mentioned above is 2 hours.

[0020] Furthermore, the co-culture described in step (3) is carried out at 5% CO2 and 37°C for 18 to 30 hours.

[0021] Furthermore, the above-mentioned cultivation time is 24 hours.

[0022] The beneficial effects of this invention are as follows: This invention uses liver cancer cells as target cells for ADCC detection of IVIG, and utilizes ADCC effect reporter gene cells to achieve accurate detection of IVIG antibody-dependent cell-mediated cytotoxicity under appropriate effector-target ratio and incubation conditions; the detection method of this invention is simple to operate, has high sensitivity and accuracy, fills the current gap in ADCC detection of IVIG, and helps to establish quality standards for IVIG products.

[0023] 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.

[0024] 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 examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0025] Figure 1 This is the ADCC dose-response curve for IVIG.

[0026] Figure 2 The results are the sensitivity test results of the method of the present invention.

[0027] Figure 3 The effect of the effective-to-target ratio on the method of the present invention.

[0028] Figure 4 The effect of incubation time after adding effector cells on the method of the present invention.

[0029] Figure 5 The effect of different target cells on the method of the present invention.

[0030] Figure 6 The effect of different target cells on the method of the present invention.

[0031] Figure 7 The results of testing different test samples using the method of this invention. Detailed Implementation

[0032] The intravenous immunoglobulin standard used in the embodiments of this invention was purchased from Shandong Taibang Biological Products Co., Ltd. (pH 4.0, batch number 202110001) (20mg / ml). The Jurkat-NFAT-Luc-CD16 cell line used in the embodiments of this invention was purchased from Novozymes Biotechnology Co., Ltd. (batch number DD1391-01). The human liver cancer Alexandrium cell line PLC / PRF / 5 cells used in the embodiments of this invention were purchased from Shanghai Fuheng Biotechnology Co., Ltd. (batch number L2226).

[0033] Unless otherwise stated, all other raw materials and equipment used in this invention are known products, obtained by purchasing commercially available products.

[0034] Effector cell culture: After resuscitation, Jurkat-NFAT-Luc-CD16 cells can be passaged for the first time after approximately 48-72 hours. When the cell density reaches 1.5-2 × 10⁻⁶ cells / year... 6cells / mL, 1 passage to 3, repeat passage every 2-3 days, do not allow the density to exceed 2×10⁻⁶. 6 The cell density is quite sensitive to cell density, so please keep the cell density within an appropriate range during culture and passage.

[0035] Target cell culture: PLC / PRF / 5 cells are adherent cells. When the confluence is about 90%, they are passaged. They are digested with trypsin for 2-3 minutes, and culture medium is added to stop the digestion. After centrifugation, the cells are collected and passaged.

[0036] Preparation of diluent: Prepare a 2% wt bovine serum albumin solution.

[0037] Example 1: Detection of antibody-dependent cell-mediated cytotoxicity of IVIG

[0038] (1) Prepare a 2% bovine serum albumin solution as a diluent; first, dilute IVIG 4 times to 5 mg / ml with the diluent, which is concentration 1, and then perform serial dilutions, successively to concentration 2, 3... up to concentration 9, for a total of 9 working concentrations, namely 5.00 mg / ml, 2.50 mg / ml, 1.25 mg / ml, 0.63 mg / ml, 0.32 mg / ml, 0.16 mg / ml, 0.08 mg / ml, 0.04 mg / ml, and 0.02 mg / ml;

[0039] (2) Target cell seeding: The target cell count was 2.8*102 6 25 μL of the solution was spread into a 96-well plate, and an equal volume of IVIG solution of different concentrations was added. Each concentration was replicated in three wells. The plate was then placed in a cell culture incubator and incubated at 37°C with 5% CO2 for 2 hours.

[0040] (3) Add effector cells: Adjust the number of effector cells Jurkat-NFAT-Luc-CD16 to 1*102 6 Inoculate 25 μL into each well and incubate in a cell culture incubator at 37°C with 5% CO2 for 24 h (effect-to-target ratio 1:2.8).

[0041] (4) Take out the 96-well plate, add 75 uL of luciferase detection reagent, and detect it with the chemiluminescence function of a multi-functional microplate reader after 3 min.

[0042] (5) Dose-response curve plotting: GraphPad Prism 8 software was used for analysis. The luciferase concentration was plotted on the ordinate, and the logarithm of the standard concentration on the abscissa. A four-parameter regression model was used to fit the dose-response curve of the standard. Figure 1 It exhibits a typical S-curve on a semi-logarithmic coordinate axis, R 2 =0.99, thus obtaining the EC of IVIG 50The concentration was 0.24 mg / ml.

[0043] The following experimental examples demonstrate the beneficial effects of the present invention.

[0044] Experimental Example 1: Accuracy, sensitivity, and specificity of the detection method of the present invention.

[0045] 1. Experimental Methods

[0046] Sensitivity:

[0047] The initial dilution concentrations of IVIG were set at 5.00 mg / ml, 6.25 mg / ml, and 8.33 mg / ml, and ADCC activity was detected according to the method in Example 1.

[0048] Precision testing:

[0049] Following the method in Example 1, IVIG with an initial working concentration of 5 mg / ml was used for intra-day and inter-day precision studies of ADCC detection. Three independent assays were performed within one day, each on a 96-well cell culture plate. Intra-day precision was assessed, and the RLU and half-maximal effect concentration (EC50) of the initial working concentration were calculated. 50 The daytime precision testing setup involved three consecutive days of testing, with one 96-well cell culture plate tested in parallel each day. The initial working concentrations of RLU and EC were statistically analyzed. 50 The value is used to verify the precision of the method.

[0050] Accuracy test:

[0051] Following the method in Example 1, IVIG at a protein concentration of 5 mg / ml was used for ADCC accuracy testing, with each sample performed in triplicate. The initial working concentration sample was used as a reference, and EC50 values ​​were calculated based on the results of each concentration sample. 50 The relative potency is calculated as follows: relative potency = reference product EC. 50 / Sample EC 50 The relative potency was multiplied by 100%; and the recovery rate was calculated as: Recovery rate = (Relative potency measured value / Relative potency theoretical value) × 100%. The accuracy of the method was verified by calculating the relative potency and recovery rate.

[0052] 2. Experimental Results

[0053] Sensitivity:

[0054] The initial working concentrations of IVIG were set at 5.00 mg / ml, 6.25 mg / ml, and 8.33 mg / ml, and then diluted in a 1:2 gradient, resulting in a total of 9 working concentrations. ADCC activity was detected according to the method in Example 1, and the results are as follows: Figure 2As can be seen, the method of the present invention can effectively detect samples in the concentration range of 0.01 mg / ml to 8.33 mg / ml, and has high sensitivity.

[0055] Precision verification:

[0056] Optimized detection conditions were used to perform intra-day and inter-day precision assays. Three independent assays were performed within a day, each on a 96-well cell culture plate, to measure intra-day precision, initial working concentrations of RLU, and EC. 50 As shown in Table 1, the relative standard deviation (RSD) is 10.86% for the initial working concentration of RLU within the day. 50 The RSD was 10.80%; the daytime precision assay was set up for three consecutive days, with one parallel experiment per day on one 96-well cell culture plate, and the RLU and EC at the initial working concentration were calculated. 50 Values, as shown in Table 3, show that the RSD of the initial working concentration of RLU is 5.13%, EC 50 With an RSD of 7.25%, it exhibits good precision in both intraday and interday detection.

[0057] Table 1. Intra-day precision verification of IVIG ADCC reporter gene assay (n=3)

[0058]

[0059] Table 2. Validation of daytime precision using the IVIG ADCC reporter gene method (n=3)

[0060]

[0061] Accuracy verification:

[0062] IVIIG was diluted with diluent to prepare 50% and 25% samples with an initial working concentration of 1.25 mg / ml. The assay was repeated three times, and the results are shown in Table 3. The average relative potency of the two groups of recovered samples were (23.50±1.69)% and (48.3±2.97)%, respectively; the corresponding recoveries were (93.5±6.3)% and (94.24±5.43)%, respectively, and the RSDs were all less than 10%, indicating good accuracy.

[0063] Table 3. Accuracy verification between IVIG ADCC reporter gene assay plates (n=3)

[0064]

[0065] Experimental Example 2: Screening of target cells, effector-to-target ratio, incubation time, and other conditions for the detection method of the present invention.

[0066] 1. Selection of effective-to-target ratio

[0067] With target-effect ratios set at 1:3 and 1:1, ADCC activity was detected according to the method in Example 1. The results are as follows: Figure 3 As shown, when the effect-to-target ratio is 1:3, the effect curve exhibits a typical S-shape. Therefore, the effect-to-target ratio is preferably around 1:3.

[0068] 2. Selection of induction time

[0069] The incubation time after adding effector cells (24 hours, 48 ​​hours) was followed by ADCC activity assay according to the method in Example 1. The results are as follows: Figure 4 As shown, greater ADCC activity was obtained after 24 hours of incubation, therefore, the preferred incubation time is 24 hours.

[0070] 3. Specificity verification

[0071] 3.1 Target Cell Specificity: K562 cells were used instead of PLC / PRF / 5 cells as target cells, and ADCC activity was detected according to the method in Example 1. The results are as follows... Figure 5 Compared to K562 cells as target cells, the dose-response curve plotted using PLC / PRF / 5 cells as target cells to detect IVIG ADCC activity showed better fit.

[0072] 3.2 Effector Cell Specificity: THP-1 cells were used instead of Jurkat-NFAT-Luc-CD16 cells as effector cells. ADCC activity was detected according to the method in Example 1. The results are as follows: Figure 6 Jurkat-NFAT-Luc-CD16 cells, as effector cells, showed better detection results.

[0073] 3.3 Antibody Specificity: Glycine was used instead of IVIG as the test sample, and ADCC activity was detected according to the method in Example 1. Results are as follows... Figure 7 IVIG has high ADCC activity, meaning that the method of this invention can effectively detect the ADCC activity of IVIG and has good specificity.

[0074] In summary, this invention provides an ADCC detection method for IVIG, using liver cancer cells as target cells and utilizing ADCC effector reporter cells under appropriate effector-target ratios and incubation conditions to accurately detect the antibody-dependent cell-mediated cytotoxicity of IVIG. The detection method of this invention is simple to operate, highly sensitive and accurate, filling the current gap in ADCC detection of IVIG and contributing to the establishment of quality standards for IVIG products.

Claims

1. A method for detecting antibody-dependent cell-mediated cytotoxicity of intravenously injected human immunoglobulin, characterized in that, Using PLC / PRF / 5 cells as target cells, the procedure includes the following steps: (1) The intravenous immunoglobulin to be tested was serially diluted with diluent to obtain test solutions of different concentration gradients; (2) Inoculate target cells and incubate them with test solutions of different concentration gradients from step (1); (3) Co-culture with effector cells; the effector cells are Jurkat-NFAT-Luc-CD16 cells; (4) Detect fluorescence, plot the dose-response curve with relative fluorescence intensity as the ordinate and the logarithm of the concentration of intravenously injected human immunoglobulin as the abscissa, and calculate the half-maximal effect concentration. The ratio of effector cells to target cells is 1:2.

8.

2. The detection method as described in claim 1, characterized in that, The diluent mentioned in step (1) is bovine serum albumin solution.

3. The detection method as described in claim 2, characterized in that, The concentration of the bovine serum albumin solution is 2% wt.

4. The detection method as described in claim 1, characterized in that, The co-incubation described in step (2) is carried out for 1 to 3 hours under conditions of 5% CO2 and 37°C.

5. The detection method as described in claim 4, characterized in that, The incubation time is 2 hours.

6. The detection method as described in claim 1, characterized in that, The co-culture described in step (3) is carried out at 5% CO2 and 37°C for 18 to 30 hours.

7. The detection method as described in claim 6, characterized in that, The incubation time is 24 hours.

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