A kit, method and use for detecting complement dependent cytotoxicity

Abstract

The application discloses a kit and method for detecting complement-dependent cytotoxicity and application, and the method is used for detecting complement-dependent cytotoxicity of AQP4-IgG positive serum for non-diagnostic purposes, and comprises the following steps: obtaining HEK293T cells which transiently express AQP4 and independently express ZsGreen fluorescent protein; inoculating the HEK293T-AQP4 cells into a 96-well plate, and then culturing until the cells are confluent; after washing, incubating for 30 minutes; adding 5 μL 7-aminoactinomycin D to the cell suspension for staining, and incubating for 10 minutes at room temperature in the dark; and detecting the percentage of surviving target cells by using a flow cytometer. The application realizes high-throughput, objective and quantitative detection of serum CDC activity by using HEK293T cells which express AQP4 and ZsGreen as target cells, combining 7-AAD dead and live cell staining and flow cytometry.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a kit, method, and application for detecting complement-dependent cytotoxicity. Background Technology

[0002] Neuromyelitis optica spectrum disorder (NMOSD) is a highly disabling and even life-threatening autoimmune disease of the nervous system. Its core pathological mechanism mainly involves the binding of anti-aquaporin 4 immunoglobulin G (AQP4-IgG) to AQP4 on the surface of astrocytes, which then activates complement through the classical pathway, producing complement-dependent cytotoxicity (CDC), ultimately leading to damage to the optic nerve and spinal cord.

[0003] Currently, despite the high diagnostic specificity of AQP4-IgG, the cell-based assays (CBA) routinely used in clinical and laboratory settings have significant limitations: CBA can only detect the binding ability of AQP4-IgG to the antigen and cannot distinguish between pathogenic and non-pathogenic binding. Since the pathogenicity of AQP4-IgG positive serum depends more on the efficiency of Fc-mediated effector function than on the absolute value of antibody titer, antibody titer alone cannot accurately reflect the true severity of the disease, and the correlation between the two is poor. Some existing studies use live-cell-based assays to assess cytotoxicity by adding exogenous (human or rabbit) complement to the heat-inactivated serum of NMOSD patients. However, such methods ignore individual variations in the human endogenous complement system (such as complement gene polymorphisms) and cannot reflect the true efficiency of the immune cascade response in patients. Existing CBA methods for detecting AQP4-IgG and cytotoxicity assays with added exogenous complement are insufficient to accurately monitor and reflect the true overall serum cytotoxicity in patients.

[0004] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0005] The technical objective of this application is to address the above-mentioned shortcomings by providing a kit, method, and application for detecting complement-dependent cytotoxicity. This application uses HEK293T cells expressing AQP4 and ZsGreen as target cells, combined with 7-AAD live / dead cell staining and flow cytometry, to achieve high-throughput, objective, and quantitative detection of serum CDC activity.

[0006] To achieve the above objectives, this application provides the following technical solution: According to one aspect of this application, a method for detecting complement-dependent cytotoxicity is provided for non-diagnostic purposes, specifically for detecting complement-dependent cytotoxicity in AQP4-IgG positive serum, comprising: S100. Human full-length AQP4-M23 cDNA was cloned into the multiple cloning site of the FV115 lentiviral vector to construct the AQP4-M23 expression plasmid. The AQP4-M23 expression plasmid was transiently transfected into HEK293T cells using Lipofectamine 3000 reagent. After 6–24 hours of transfection, the medium was replaced with DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured for another 24 hours to obtain HEK293T cells that transiently expressed AQP4 and independently expressed ZsGreen fluorescent protein without fusion. These cells were designated as HEK293T-AQP4 cells. S200. Seed HEK293T-AQP4 cells at a density of 50,000 cells per well in 96-well plates and culture for 18-24 hours until the cells confluence. After washing with PBS, incubate the cells with each group of samples at room temperature for 30 minutes. Gently digest the adherent HEK293T-AQP4 cells with Accutase cell digestion solution and wash twice with PBS. Add 5 μL of 7-aminoactinomycin D to the cell suspension for staining and incubate at room temperature in the dark for 10 minutes. The samples included AQP4-IgG positive serum, negative control samples, and positive control samples. The negative control samples were serum from healthy individuals, and the positive control samples were HEK293T-AQP4 cells treated with 0.1% Triton X-100. S300. The percentage of surviving target cells was detected using flow cytometry; the cytotoxicity index was calculated using the following formula: , in, N sample : Represents the percentage of surviving target cells in the AQP4-IgG positive serum group; N neg : Represents the percentage of surviving target cells in the serum of healthy individuals; N pos : Represents the percentage of surviving target cells in the HEK293T-AQP4 cell group after 0.1% Triton X-100 treatment; The percentage of viable target cells detected by flow cytometry includes: Endogenous ZsGreen signal was detected using the fluorescein isothiocyanate channel; data acquisition was performed using a BD FACSAria II flow cytometer, ensuring at least 30,000 events were collected for each sample; data analysis was performed using FlowJo software; a gating strategy was first used to exclude cell debris and adherent cells based on forward and side scattering parameters; subsequently, 7-AAD-negative and FITC-positive cell populations were identified as viable target cells.

[0007] In some embodiments, each group of samples further includes: heat-inactivated AQP4-IgG positive serum samples and heat-inactivated AQP4-IgG positive serum supplemented with 2.5% mixed normal human complement samples; the heat inactivation is achieved by placing the serum in a 56°C water bath for 30 minutes to inactivate complement.

[0008] In some embodiments, step S200 may further include assessing the binding of AQP4-IgG using immunofluorescence.

[0009] In some embodiments, the method of assessing AQP4-IgG binding using immunofluorescence includes: fixing HEK293T-AQP4 cells with 4% paraformaldehyde for 15 minutes, then blocking them with 5% bovine serum albumin for 30 minutes; subsequently, incubating them with the test serum for 1 hour; after washing with PBS, incubating the cells with Alexa Fluor 555-labeled anti-human IgG antibody for 45 minutes; after washing again with PBS, counterstaining the cell nuclei with 4',6-diamidinyl-2-phenylindole, mounting the slides, and observing and analyzing them under a fluorescence microscope.

[0010] According to another aspect of this application, a kit for detecting complement-dependent cytotoxicity is provided, the kit comprising HEK293T cells transiently expressing AQP4 and non-fusion-independently expressing ZsGreen fluorescent protein, healthy human serum, 0.1% Triton X-100 solution, Accutase cell digestion solution, and 7-aminoactinomycin D.

[0011] According to another aspect of this application, the application of the method or the kit described herein in drug screening is provided.

[0012] In some embodiments, the drug screening is a process of screening for drugs that can salvage unresponsive patients after they have received hormone pulse therapy in the acute phase of NMOSD.

[0013] Compared with the prior art, the advantages and positive effects of this application are as follows: This application uses HEK293T cells expressing AQP4 and ZsGreen as target cells, combined with 7-AAD live and dead cell staining and flow cytometry, to achieve high-throughput, objective, and quantitative detection of serum CDC activity.

[0014] Furthermore, this application utilizes an independent ubiquitin promoter to drive ZsGreen expression, enabling the reporter gene and AQP4 to be expressed independently in a non-fusion form. This completely avoids the impact of the fusion tag on the structural integrity and biological function of the AQP4 protein, ensuring that the detection results can truly reflect AQP4-IgG-mediated complement-dependent cytotoxicity under physiological conditions.

[0015] Furthermore, by establishing a negative control of healthy human serum and a positive control of 0.1% Triton X-100, this application uses a normalization formula to calculate the cytotoxicity index (CI), which can eliminate differences between experimental batches and achieve quantifiable comparison of CDC activity at different times and in different laboratories.

[0016] Furthermore, this application includes a heat-inactivated serum group and an exogenous complement supplement group in the detection system, which can clearly distinguish whether CDC is mediated by complement and can independently assess the contributions of endogenous complement and exogenous complement, providing a reliable reference for the quality control and mechanism verification of the experimental system.

[0017] Furthermore, the kit provided in this application contains cryopreserved or ready-to-use target cells, live and dead dyes, and controls. The operating procedure is standardized and can be easily and quickly carried out in clinical laboratories or research institutions. It is suitable for various non-diagnostic purposes such as drug screening and complement activity assessment. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 A fluorescence microscope image of Embodiment 1 of this application is shown.

[0020] Figure 2 This paper illustrates a comparison of cytotoxicity indices in NMOSD patients at different disease stages in Example 2 of this application.

[0021] Figure 3 This document presents a comparison of dynamic changes in cytotoxicity index before and after IVMP treatment in NMOSD patients in the acute phase of this application, as shown in Example 2 of this application.

[0022] Figure 4 The flow cytometry analysis of the dynamic changes in serum complement-dependent cytotoxicity in patients who did not respond to PE / IA pulse therapy in Example 3 of this application is shown.

[0023] Figure 5 The flow cytometry analysis of the dynamic changes in serum complement-dependent cytotoxicity in patients who did not respond to IVIG pulse therapy in Example 3 of this application is shown.

[0024] Figure 6 The flow cytometry analysis of the dynamic changes in serum complement-dependent cytotoxicity in patients who did not respond to hormone pulse therapy and received FcRn antagonist treatment in Example 3 of this application is shown.

[0025] Figure 7 The flow cytometry analysis of the dynamic changes in serum complement-dependent cytotoxicity in patients who did not respond to hormone pulse therapy and received C5 inhibitor treatment in Example 3 of this application is shown.

[0026] Figure 8 This illustrates the changes in CI (critical blood pressure) in patients in different salvage therapy groups before and after hormone therapy and after salvage therapy in Example 3 of this application. Detailed Implementation

[0027] To better understand the above-mentioned objectives, features, and advantages of this application, the application will be further described below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

[0028] The following explains the relevant symbols: AQP4: Aquaporin-4; AQP4-IgG: Aquaporin-4 immunoglobulin G; IVMP treatment: Intravenous methylprednisolone pulse therapy; AQP4-IgG+C Endo AQP4-IgG positive serum containing active endogenous complement that has not been inactivated; AQP4-IgG+C Exo The AQP4-IgG positive serum containing endogenous complement was inactivated by heat, and an additional 2.5% of a mixed normal human complement group was added (normal human complement was purchased from QuidelOrtho, product information as follows: Name, Normal Human Serum Complement; Specification, 5.0 mL; Catalog Number, A113. This product is a homogeneous human serum complement mixture extracted from mixed plasma of healthy volunteers, mainly containing key functional proteins of the complement system; this product retains the complete function of natural complement, and CH50 activity has been verified by the manufacturer). AQP4-IgG+C Inact : Heat inactivation to inactivate endogenous complement in AQP4-IgG positive serum group; PE / IA: Plasma exchange / immunoadsorption; IVIG: Intravenous infusion of gamma globulin; NS: No significant difference; CI: Cytotoxicity Index; NMOSD: Neuromyelitis optica spectrum disorder; 7-AAD: 7-Aminoactinomycin D.

[0029] The present application will be further described below with reference to the accompanying drawings and specific embodiments.

[0030] Example 1: Complement-dependent cytotoxicity (CDC) detection.

[0031] 1. Construct a HEK293 cell line that transiently expresses AQP4.

[0032] Human full-length AQP4-M23 cDNA (GenBank ID: NM_001364286.1) was cloned into the multiple cloning site of the FV115 lentiviral vector to construct the AQP4-M23 expression plasmid. The recombinant plasmid was verified to be correct by DNA sequencing. This plasmid contains an independent ubiquitin promoter for driving the expression of ZsGreen fluorescent protein. Since ZsGreen is expressed independently of the target protein, rather than as a fusion tag, it does not interfere with the structural integrity or biological function of AQP4-M23.

[0033] The AQP4-M23 expression plasmid was transiently transfected into HEK293T cells using Lipofectamine 3000 reagent. Six to 24 hours after transfection, the medium was replaced with DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and cultured for another 24 hours to obtain HEK293T cells transiently expressing AQP4 (HEK293T-AQP4 cells). All cell culture steps were performed in a 37°C, 5% CO2 incubator. This protocol achieves a transfection efficiency of over 60%.

[0034] 2. Binding of AQP4-IgG: Before performing flow cytometry to detect CDC, it is necessary to first determine whether AQP4-IgG binding is present. CDC detection is only meaningful if AQP4-IgG binding is present. If the sample is known to be AQP4-IgG positive, this step can be omitted; however, if the sample is serum from NMOSD patients (most NMOSD patient serum is AQP4-IgG positive, a small percentage is not), this step is necessary to detect the presence of antibodies in the serum and to demonstrate that HEK293T cells successfully express the AQP4 antigen.

[0035] The binding of AQP4-IgG was assessed using a laboratory-developed immunofluorescence assay. The specific steps were as follows: First, HEK293T-AQP4 cells were fixed with 4% paraformaldehyde for 15 minutes, then blocked with 5% bovine serum albumin (BSA) for 30 minutes. Subsequently, they were incubated with the test serum for 1 hour. After washing with PBS, the cells were incubated with Alexa Fluor 555-labeled anti-human IgG antibody for 45 minutes. After washing again with PBS, the nuclei were counterstained with 4',6-diamidinyl-2-phenylindole (DAPI), mounted, and observed and analyzed under a fluorescence microscope.

[0036] Figure 1 A fluorescence microscope image of Embodiment 1 of this application is shown. Figure 1 As shown, after AQP4-IgG in the test serum binds to cells, it is specifically recognized by a secondary antibody with a red fluorescent group (displayed as red fluorescence), showing a positive signal on the surface of HEK293T-AQP4 cells. This signal merges with the ZsGreen green fluorescence expressed by the HEK293T-AQP4 cells themselves, showing obvious spatial co-localization, indicating that AQP4-IgG can specifically bind to the surface of target cells expressing AQP4. In other words, HEK293T cells successfully express AQP4 protein with native conformation and functional activity. The test serum contains an autoantibody (AQP4-IgG) that can recognize and bind to AQP4. This binding step verifies that the prerequisite for subsequent complement-dependent cytotoxicity (CDC) detection is met, namely, the target cell antigen is correctly expressed and the serum antibody has binding activity, providing a basis for the reliability of CDC detection results.

[0037] 3. Flow cytometry detection of CDC: HEK293T cells transfected for 24 hours were seeded at a density of 50,000 cells per well in 96-well plates and cultured for 18–24 hours until confluence. After washing with PBS, the cells were incubated with serum at room temperature for 30 minutes. Serum incubation included one of the following four conditions: (1) Serum group of healthy individuals; (2) Heat inactivation to inactivate endogenous complement in the AQP4-IgG positive serum group (AQP4-IgG+ C Inact ); (3) Heat inactivation was performed to inactivate AQP4-IgG positive serum containing endogenous complement, and an additional 2.5% of mixed normal complement group (AQP4-IgG+ C) was added. Exo ); (4) AQP4-IgG positive serum group containing active endogenous complement that has not been inactivated (AQP4-IgG + C Endo ).

[0038] Heat inactivation involved placing serum in a 56°C water bath for 30 minutes to inactivate complement. After the 30-minute incubation, adherent HEK293T-AQP4 cells were gently digested with Accutase and washed twice with PBS. To distinguish between live and dead cells, 5 μL of 7-aminoactinomycin D (7-AAD) was added to the cell suspension for staining, and the cells were incubated at room temperature in the dark for 10 minutes. Endogenous ZsGreen signal was detected using the FITC channel. Data acquisition was performed using a BD FACSAria II flow cytometer, ensuring at least 30,000 events were collected for each sample. Data analysis was performed using FlowJo software. A gating strategy was first used to exclude cell debris and adherent cells based on forward scattering (FSC) and side scattering (SSC) parameters. Subsequently, the 7-AAD-negative and FITC-positive cell population was identified as viable target cells. The cytotoxicity index (CI) value was calculated based on the percentage of these viable target cells using the following formula.

[0039]

[0040] in, N sample : Represents the percentage of surviving target cells in the test sample group, which is the serum group of AQP4-IgG positive patients; N neg : Represents the percentage of surviving target cells in the serum of healthy individuals; N pos : Represents the percentage of surviving target cells in the 0.1% Triton X-100 treatment group, which is the HEK293T-AQP4 cell group treated with 0.1% Triton X-100.

[0041] Example 2: Using the method of Example 1, the cytotoxicity index of NMOSD patients at different disease stages was detected.

[0042] This study was approved by the ethics committee of a hospital in Tianjin. All sample collection procedures followed the Declaration of Helsinki and relevant ethical review standards, and written informed consent was obtained from all participants. A total of 111 serum samples were obtained from 65 NMOSD patients who were confirmed to be AQP4-IgG positive. All patients met the 2015 NMOSD diagnostic criteria established by the International NMO Diagnostic Panel (IPND), specifically including: ① presence of at least one core clinical symptom; ② positive AQP4-IgG test by cell transfection immunofluorescence; ③ exclusion of other possible diagnoses. The core clinical symptoms covered the following six categories: optic neuritis, acute myelitis, zone-back syndrome (manifested as episodic hiccups, nausea, and vomiting without other identifiable cause), brainstem syndrome, symptomatic narcolepsy or acute diencephalic clinical syndrome, and cerebral syndrome. The acute phase was defined as within one month of symptom onset, and the remission phase was defined as three months or more after the most recent onset. Based on the stage of the disease, NMOSD patients were divided into an acute NMOSD group (30 cases) and a remission NMOSD group (35 cases).

[0043] Acute attacks include the initial attack and any subsequent relapse. A relapse is defined as the appearance of new neurological symptoms or a persistent worsening of existing symptoms for more than 24 hours. Relapses are typically at least 30 days after the previous attack, and other causes such as fever and infection must be ruled out. First-line treatment for acute NMOSD attacks is high-dose intravenous methylprednisolone (IVMP, 1 g daily) for 5 days, with efficacy assessed on day 7 after treatment initiation. Non-responders are defined as patients meeting any of the following criteria: Transverse myelitis (TM) flare-up: muscle strength failed to improve by at least 1 grade using the Medical Research Council Muscle Strength Rating Scale (MRC).

[0044] Optic neuritis (ON) onset: visual acuity on the near vision chart fails to improve by ≥2 lines (when baseline visual acuity is ≥0.025); or visual acuity improves by less than 1 grade (when baseline visual acuity is below the index). Other sites of infection: No significant clinical improvement was observed after evaluation by the attending physician, or the symptoms worsened.

[0045] The OSIS scale was used to objectively assess patients' clinical symptoms and serve as an important tool for monitoring clinical outcomes.

[0046] For the acute phase group, blood samples were collected before and 7 days after intravenous high-dose methylprednisolone (IVMP) treatment (i.e., before and after IVMP treatment). For those determined to be non-responsive to IVMP, an additional blood sample was collected on day 14 after the start of rescue therapy. Blood samples were processed within one hour of collection. Serum was separated by centrifugation, aliquoted, and stored at -80°C to avoid repeated freeze-thaw cycles. Heat-inactivated serum was prepared by heating in a water bath at 56°C for 30 minutes to inactivate endogenous complement.

[0047] 1. Comparison of cytotoxicity indices in NMOSD patients at different disease stages.

[0048] Figure 2 This illustrates a comparison of cytotoxicity indices in NMOSD patients at different disease stages in Example 2 of this application. Figure 2 A is a flow cytometry plot showing AQP4-IgG+ C in the remission and acute phases. Endo Representative flow cytometry images after serum incubation (target cell populations were delineated by 7-AAD negative and AQP4 / ZsGreen positive criteria). Figure 2 B represents the statistical analysis result of CI (Mann-Whitney U test, two-tailed). P < 0.05 is considered significant (GraphPad Prism 10.0). For example... Figure 2 As shown, the CI in the acute phase was significantly higher than that in the remission phase ( P = 0.0010).

[0049] 2. Comparison of dynamic changes in cytotoxicity index before and after IVMP treatment in NMOSD patients in the acute phase.

[0050] The effect of hormone pulse therapy on serum cytotoxicity was verified using flow cytometry. Figure 3 This document illustrates a comparison of dynamic changes in cytotoxicity indices before and after IVMP treatment in NMOSD patients in the acute phase of this application, as shown in Example 2 of this application. Figure 3 A shows representative flow cytometry plots of serum from patients with acute NMOSD (before and after IVMP treatment) under different complement conditions (surviving cell populations were delineated by 7-AAD negative and AQP4 / ZsGreen positive). Figure 3 B performed a quantitative comparison of the CIs of the above groups, using the Friedman test (two-tailed). P < 0.05 was considered significant. Multiple relevant inter-group comparisons were performed using GraphPad Prism 10.0, with each group compared to the CI value after salvage therapy. Figure 3 As shown, AQP4-IgG+ C before and after hormone pulse therapy Endo There was no statistically significant difference in CI values ​​between the groups. P> 0.9999), meaning that IVMP pulse therapy during an acute exacerbation of NMOSD failed to suppress the increase in CI.

[0051] Example 3: Serum samples from acute NMOSD patients who received different salvage therapies were examined using the method in Example 1 to compare the dynamic changes in cytotoxicity indices.

[0052] Patients tested were those who did not respond to steroid pulse therapy after different salvage treatments. Specifically, the method described in Example 1 was used to evaluate the changes in complement-dependent cytotoxicity in acute-phase NMOSD patients unresponsive to intravenous methylprednisolone pulse therapy after different salvage treatments. The different salvage treatments were plasma exchange / immunoadsorption (PE / IA), intravenous immunoglobulin (IVIG), neonatal Fc receptor antagonists (FcRn antagonists), or complement C5 inhibitors (C5 inhibitors). The specific treatment regimens were as follows: 4 patients received PE / IA for a total of 5 times, every other day; 3 patients received IVIG (2 g / kg, divided into 5 doses, i.e., 0.4 g / kg / day); 3 patients received the FcRn antagonist egamod (10 mg / kg, once weekly for 2 weeks); and 6 patients received the C5 inhibitor eculizumab (900 mg, once weekly for 2 weeks), along with antibiotics to prevent meningococcal infection.

[0053] Figure 4 This paper illustrates flow cytometry verification of dynamic changes in serum complement-dependent cytotoxicity in patients who did not respond to PE / IA pulse therapy in Example 3 of this application. The figure shows the results at C... Endo C Exo and C Inact Representative cell viability maps after serum incubation at different time points (before and after IVMP treatment and after PE / IA treatment) under three complement conditions. Target cell populations were delineated as 7-AAD negative and AQP4 (ZsGreen) positive.

[0054] Figure 5 This figure illustrates flow cytometry verification of dynamic changes in serum complement-dependent cytotoxicity in patients unresponsive to IVIG pulse therapy in Example 3 of this application. The figure shows the results at C... Endo C Exo and C Inact Representative cell viability maps after serum incubation at different time points (before, after IVMP treatment, and after IVIG treatment) under three complement conditions. Target cell populations were delineated as 7-AAD negative and AQP4 (ZsGreen) positive.

[0055] Figure 6This paper illustrates flow cytometry verification of dynamic changes in serum complement-dependent cytotoxicity in patients who did not respond to hormone pulse therapy after receiving FcRn antagonist treatment, as described in Example 3 of this application. The figure shows the dynamic changes in serum complement-dependent cytotoxicity in C... Endo C Exo and C Inact Representative cell viability maps after serum incubation at different time points (before and after IVMP treatment and after FcRn antagonist treatment) under three complement conditions. Target cell populations were delineated as 7-AAD negative and AQP4 (ZsGreen) positive.

[0056] Figure 7 This paper illustrates flow cytometry verification of dynamic changes in serum complement-dependent cytotoxicity in patients unresponsive to hormone pulse therapy treated with C5 inhibitors, as described in Example 3 of this application. The figure shows the changes in C5 inhibitor levels. Endo C Exo and C Inact Representative cell viability maps after incubation with patient serum at different time points (before, after IVMP treatment, and after C5 inhibitor treatment) under three complement conditions. Target cell populations were delineated as 7-AAD negative and AQP4 (ZsGreen) positive.

[0057] like Figure 4-7 As shown, flow cytometry detection of CDC can be used as a reliable means to quantitatively reflect changes in serum complement-dependent cytotoxicity before and after treatment.

[0058] After calculating the cytotoxicity index (CI) of each serum sample based on flow cytometry results, statistical analysis was performed on the serum CI values ​​of patients receiving different salvage therapies. Friedman's test was used to compare multiple relevant groups, with each group's CI value compared to its respective salvage therapies. All tests were two-tailed tests. P A value < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism 10.0 software.

[0059] Figure 8 This illustrates the changes in CI (critical blood pressure) in patients in different salvage therapy groups before and after hormone therapy and after salvage therapy in Example 3 of this application. Figure 8 Group A represents the PE / IA treatment group. Figure 8 Group B represents the IVIG treatment group. Figure 8 C represents the FcRn antagonist treatment group. Figure 8 D represents the C5 inhibitor treatment group.

[0060] like Figure 8 As shown, among different salvage treatment regimens, the PE / IA treatment group ( Figure 8 A) compared to the C5 inhibitor treatment group ( Figure 8 D) The CI levels after salvage therapy were significantly lower than those after IVMP treatment.P The values ​​were 0.0267 and 0.0452, respectively. IVIG treatment group ( Figure 8 Although the CI for B) decreased, the difference was not statistically significant. The FcRn antagonist treatment group ( Figure 8 C) CI was significantly lower than the pre-IVMP treatment level ( P = 0.0286), but there was no statistically significant difference compared to the IVMP-treated group. In other words, both PE / IA and C5 inhibitors showed significant effects in reversing serum complement-dependent cytotoxicity.

[0061] In the above methods, the test subjects are all ex vivo samples, and the test results are not directly used to determine the patient's health status or disease status.

[0062] Through the above specific embodiments, those skilled in the art can easily implement this application. However, it should be understood that this application is not limited to the specific embodiments described above. Based on the disclosed embodiments, those skilled in the art can arbitrarily combine different technical features to achieve different technical solutions.

Claims

1. A method for detecting complement-dependent cytotoxicity, characterized in that, For non-diagnostic purposes, this method is used to detect complement-dependent cytotoxicity in AQP4-IgG positive serum, including: S100. Human full-length AQP4-M23 cDNA was cloned into the multiple cloning site of the FV115 lentiviral vector to construct the AQP4-M23 expression plasmid. The AQP4-M23 expression plasmid was transiently transfected into HEK293T cells using Lipofectamine 3000 reagent. After 6–24 hours of transfection, the medium was replaced with DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured for another 24 hours to obtain HEK293T cells that transiently expressed AQP4 and independently expressed ZsGreen fluorescent protein without fusion. These cells were designated as HEK293T-AQP4 cells. S200. Seed HEK293T-AQP4 cells at a density of 50,000 cells per well in 96-well plates and culture for 18-24 hours until the cells confluence. After washing with PBS, incubate the cells with each group of samples at room temperature for 30 minutes. Gently digest the adherent HEK293T-AQP4 cells with Accutase cell digestion solution and wash twice with PBS. Add 5 μL of 7-aminoactinomycin D to the cell suspension for staining and incubate at room temperature in the dark for 10 minutes. The samples included AQP4-IgG positive serum, negative control samples, and positive control samples. The negative control samples were serum from healthy individuals, and the positive control samples were HEK293T-AQP4 cells treated with 0.1% Triton X-100. S300. The percentage of surviving target cells was detected using flow cytometry; the cytotoxicity index was calculated using the following formula: ， in, N sample : Represents the percentage of surviving target cells in the AQP4-IgG positive serum group; N neg : Represents the percentage of surviving target cells in the serum of healthy individuals; N pos : Represents the percentage of surviving target cells in the HEK293T-AQP4 cell group after 0.1% Triton X-100 treatment; The percentage of viable target cells detected by flow cytometry includes: Endogenous ZsGreen signal was detected using the fluorescein isothiocyanate channel; data acquisition was performed using a BD FACSAria II flow cytometer, ensuring at least 30,000 events were collected for each sample; data analysis was performed using FlowJo software; a gating strategy was first used to exclude cell debris and adherent cells based on forward and side scattering parameters; subsequently, 7-AAD-negative and FITC-positive cell populations were identified as viable target cells.

2. The method for detecting complement-dependent cytotoxicity according to claim 1, characterized in that, Each group of samples also includes: heat-inactivated AQP4-IgG positive serum samples and heat-inactivated AQP4-IgG positive serum supplemented with 2.5% mixed normal human complement samples; the heat inactivation is to place the serum in a 56°C water bath for 30 minutes to inactivate complement.

3. The method for detecting complement-dependent cytotoxicity according to claim 1, characterized in that, Step S200 also includes assessing the binding of AQP4-IgG using immunofluorescence.

4. The method for detecting complement-dependent cytotoxicity according to claim 3, characterized in that, The method of assessing AQP4-IgG binding using immunofluorescence includes: HEK293T-AQP4 cells were fixed with 4% paraformaldehyde for 15 minutes, then blocked with 5% bovine serum albumin for 30 minutes; subsequently, they were incubated with the test serum for 1 hour; after washing with PBS, the cells were incubated with Alexa Fluor 555-labeled anti-human IgG antibody for 45 minutes; after washing with PBS again, the nuclei were counterstained with 4',6-diamidinyl-2-phenylindole, mounted, and observed and analyzed under a fluorescence microscope.

5. A kit for detecting complement-dependent cytotoxicity, characterized in that, The kit includes HEK293T cells transiently expressing AQP4 and non-fusion independently expressing ZsGreen fluorescent protein, healthy human serum, 0.1% Triton X-100 solution, Accutase cell digestion solution, and 7-aminoactinomycin D.

6. The use of the method according to any one of claims 1-4 or the kit according to claim 5 in drug screening.

7. The application according to claim 6, characterized in that, The drug screening is the process of identifying drugs that can salvage unresponsive patients after they have received hormone pulse therapy in the acute phase of NMOSD.

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