Screening method and application of Asian human NMOSD high-pathogenicity antibody

Abstract

The invention discloses a screening method and application of an Asian human NMOSD high-pathogenicity antibody, and belongs to the technical field of autoimmune disease diagnosis and treatment. The screening method comprises the following steps: screening an Asian human NMOSD patient with poor glucocorticoid treatment prognosis, extracting serum AQP4-IgG, carrying out specific capture through M23 type AQP4 transfection cells, combining with primary astrocyte C3 inducibility verification, and screening to obtain the high-pathogenicity antibody. The antibody can mediate construction of a glucocorticoid treatment resistant NMOSD animal model, and is applied to disease pathogenic mechanism research and targeted drug screening. The antibody is screened on the basis of clinical prognosis phenotype and functional characteristics, sequence identification is not needed, the technical blank of Asian human NMOSD high-pathogenicity antibody screening is filled, and the application value is remarkable.

Description

A screening method for highly pathogenic NMOSD antibodies in Asian populations and its application Technical Field

[0001] This invention belongs to the field of diagnostic and therapeutic technology for autoimmune diseases, specifically involving a method and application for screening highly pathogenic antibodies against NMOSD in Asian populations. It is particularly applicable to the construction of animal models of neuromyelitis optica spectrum disease (NMOSD), the analysis of pathogenic mechanisms, and the development of targeted drugs. It can be widely used in related research and translational applications by biopharmaceutical companies, research institutes, and clinical medical institutions. Background Technology

[0002] Neuromyelitis optica spectrum disorders (NMOSD) are a group of autoimmune diseases characterized by inflammatory demyelination of the central nervous system and axonal damage. Their incidence rate in Asian populations is as high as 0.3-0.5 per 100,000, and their disability rate is 2-3 times higher than that of Caucasians, seriously threatening the health and quality of life of Asian populations. In clinical practice, glucocorticoid pulse therapy is the first-line standard treatment for acute NMOSD, but approximately 30% of Asian patients do not respond well to this treatment. The relapse rate within 12 months after treatment exceeds 60%, and it is often accompanied by serious sequelae such as irreversible visual impairment and motor dysfunction, requiring further reliance on second-line treatments such as plasma exchange and immunosuppressants. This not only significantly increases the medical burden but may also exacerbate permanent neurological damage due to delayed treatment.

[0003] Existing technology has clearly established that the autoantibody of aquaporin 4 (AQP4) (AQP4-IgG) is the core pathogenic factor of NMOSD. After binding to the AQP4 protein on the surface of astrocytes, this antibody initiates a cascade of pathological reactions, including complement activation, inflammatory cell infiltration, and glial cell activation, ultimately leading to neuronal apoptosis and demyelination. However, studies have found no significant correlation between AQP4-IgG titer and patient prognosis. Some high-titer patients have a good prognosis after treatment, while low-titer patients may experience persistent relapses. This phenomenon suggests significant molecular heterogeneity of AQP4-IgG, with different molecular subtypes of antibodies exhibiting fundamental differences in pathogenicity and treatment sensitivity.

[0004] Currently, existing technologies only report pathogenic AQP4 antibodies (such as rAb-53 monoclonal antibody) targeting Caucasians. However, due to differences in genetic background (such as HLA gene polymorphism) and immunophenotype (such as inflammatory cytokine secretion profile) resulting from racial differences, the molecular structure (such as antigen-binding epitopes and glycosylation modification patterns) and pathogenic characteristics (such as complement activation efficiency and glial cell activation capacity) of these antibodies differ significantly from the natural antibodies in Asian NMOSD patients. Clinical data show that diagnostic tools and therapeutic targets developed based on Caucasian antibodies experience a decrease in accuracy and efficacy of more than 30% when applied to Asian patients, failing to meet the needs for precision diagnosis and treatment of NMOSD in Asian populations.

[0005] Furthermore, existing screening methods for AQP4-IgG suffer from several technical shortcomings: First, the screening strategies lack targeting, often employing non-directional separation techniques that enrich antibodies solely based on their binding activity with the AQP4 protein, without considering the patient's clinical prognostic phenotype (such as treatment response). This results in the screened antibodies lacking clear clinical relevance characteristics, exhibiting insufficient pathogenicity and specificity, making them unsuitable for prognostic prediction and treatment guidance. Second, they rely on complex sequence identification techniques. Existing methods require cumbersome steps such as mass spectrometry sequencing and gene cloning to identify antibody sequences, leading to complex procedures, high experimental costs (over 5,000 RMB per sample), and a cycle of 2-3 weeks, limiting their large-scale application in clinical and research settings. Third, the functional validation system is incomplete. Existing methods mostly only validate the binding activity of antibodies with AQP4, failing to validate the function of NMOSD core pathogenic pathways (such as the complement activation pathway), thus failing to accurately assess the actual pathogenicity of the antibodies.

[0006] Studies on the pathogenesis of NMOSD based on astrocyte-microglia interactions have confirmed that complement C3 protein is the core signaling molecule in this interaction. The ability of AQP4-IgG to induce astrocytes to produce C3 protein directly determines the severity of the disease and the treatment response. Clinical follow-up data show that patients with high C3 protein expression have a 4.2 times higher resistance rate to glucocorticoid therapy and a 3.8 times higher risk of relapse compared to patients with low expression. Therefore, developing a screening method for highly pathogenic antibodies against NMOSD based on clinical prognostic phenotypes and core pathogenic pathway functional characteristics, without relying on sequence identification, and specifically targeting the Asian population, has crucial clinical value and application prospects for filling the technological gap in precision medicine for NMOSD in Asian populations and promoting the development of targeted drugs. Summary of the Invention

[0007] To address the following core shortcomings in existing technologies:

[0008] 1. There is a lack of screening methods for highly pathogenic NMOSD antibodies specifically for Asian populations. Existing methods are mostly designed based on the characteristics of antibodies from Caucasian populations, which cannot match the genetic background, immune phenotype, and pathogenic characteristics of antibodies in Asian patients, resulting in poor clinical relevance of the screened antibodies.

[0009] 2. Existing screening methods do not take into account the patient's clinical prognostic phenotype, but rely solely on the binding activity of antibodies to AQP4 for separation. The pathogenicity of the screened antibodies lacks a clear correlation with the treatment response, and they rely on complex technologies such as mass spectrometry sequencing, which are cumbersome and costly.

[0010] 3. The existing screening methods have an incomplete functional validation system and have not been targeted for validation of the core pathogenic pathway of NMOSD (complement activation pathway), making it impossible to accurately assess the actual pathogenicity of the antibody;

[0011] 4. The lack of standardized application scenarios to match the screening methods means that the specific uses of the screened antibodies in model building and drug development are not clearly defined, resulting in limited technology transfer value.

[0012] The purpose of this invention is to provide a method and application for screening highly pathogenic antibodies against NMOSD in Asian populations. Through an innovative process of "clinical prognostic phenotype-directed screening + subtype-specific capture + core pathway function verification", highly pathogenic antibodies with clear therapeutic resistance correlations are obtained. At the same time, a standardized animal model application system is established to address the shortcomings of existing technologies and promote the development of precision medicine for NMOSD in Asian populations.

[0013] To achieve the above objectives, this invention adopts the following technical solution, with its core innovation lying in the end-to-end design of "targeted screening - specific capture - functional verification - application implementation":

[0014] 1. Screening methods for highly pathogenic NMOSD antibodies in Asian populations:

[0015] The screening method of this invention is "guided by clinical prognostic phenotype, based on subtype specificity, and centered on core pathway function," and does not rely on sequence identification. The specific technical design is as follows:

[0016] (1) Targeted screening criteria: Focusing on Asian patients with poor prognosis after glucocorticoid treatment, whose antibodies naturally exhibit high pathogenicity and treatment resistance. Clinical follow-up data validated that antibody-mediated complement activation efficiency in these patients was more than 3.5 times higher than in patients with good prognosis, and glial cell activation was significantly increased, ensuring the targeted nature and clinical relevance of the screening. The NMOSD animal model constructed using this antibody-mediated approach still showed a high relapse rate (≥80%) and severe neurological impairment (behavioral score ≥3 points) after glucocorticoid treatment, fully demonstrating treatment resistance characteristics and providing a precise clinical simulation model for subsequent drug screening.

[0017] (2) Specific capture strategy: Immunoaffinity capture was performed using 293T cells transfected with M23 type AQP4. M23 type AQP4 is the major functional subtype of AQP4 protein, and its orthogonal array structure is the main binding target of natural antibodies in NMOSD patients. Its binding affinity to AQP4-IgG is more than 2.8 times higher than that of other subtypes (such as M1 type). This strategy can effectively eliminate the interference of non-specific IgG (such as antinuclear antibodies, antithyroid antibodies, etc.), so that the purity of the AQP4-IgG obtained by screening is ≥95%, which is significantly better than existing methods (the purity is usually 70%-80%).

[0018] (3) Functional validation criteria: The core validation indicator was "the ability to induce astrocytes to produce C3 protein," and a quantitative threshold of "more than 1.5 times that of rAb-53 monoclonal antibody" was set. As the core signaling molecule of the complement pathway, the expression level of C3 protein directly reflects the pathogenicity of the antibody. This quantitative standard was validated through more than 100 clinical samples, and the predictive sensitivity for treatment resistance in Asian NMOSD patients reached 92.3%, and the specificity reached 88.6%, ensuring that the selected antibody has clear and stable high pathogenicity. At the same time, an rAb-53 monoclonal antibody control group (pathogenic antibody of Caucasian race) and a PBS blank control group were set up. Through horizontal comparison, the unique pathogenic characteristics of highly pathogenic antibodies in Asian race were further clarified.

[0019] 2. Application protocols for highly pathogenic antibodies:

[0020] This invention clearly defines the core application scenario of the highly pathogenic antibodies obtained through screening as "construction of NMOSD animal models" and establishes a standardized application system. The specific technical solution is as follows:

[0021] (1) Optimization of modeling method: The atlanto-occipital myelin sheath insertion technique was used to deliver the antibody directly to the high-incidence area of ​​spinal cord lesions (the area corresponding to the first lumbar vertebra). This area is the main area affected by clinical lesions in NMOSD patients. The local antibody concentration is more than 10 times higher than that of systemic administration, avoiding antibody loss and non-specific tissue damage caused by systemic administration. This increases the success rate of model construction from 60%-70% of the existing methods to more than 90%, and the model repeatability is significantly improved (the coefficient of variation of behavioral scores is ≤8%).

[0022] (2) Clear model characteristics: The constructed model is "glucocorticoid therapy resistant type", which is highly consistent with the pathological characteristics of refractory NMOSD patients in clinical practice. After 14 days of glucocorticoid (dexamethasone) treatment by gavage at a dose of 30 mg / kg, the behavioral score of the model rats was still ≥3 points, the relapse rate was ≥80%, and the expression level of complement C3 protein decreased by less than 10%. In contrast, the model constructed based on Caucasian antibodies showed a score decrease of ≥50% after treatment, which fully reflects the unique characteristics of NMOSD treatment resistance in Asians.

[0023] (3) Extended Application Value: This model can be used to screen targeted drugs for refractory NMOSD and establish a three-dimensional quantitative evaluation system of "behavioral improvement + molecular pathological repair + tissue damage reversal". Behavioral improvement is defined as a score improvement of ≥30%; molecular pathological repair is defined as a downregulation of C3 protein expression of ≥40% and a downregulation of glial cell activation marker (GFAP) expression of ≥35%; and tissue damage reversal is defined as an upregulation of myelin marker (MBP) expression of ≥30% and a reduction in neuronal apoptosis rate of ≥45%, providing standardized tools and objective evaluation indicators for drug development. This model has been successfully applied to the screening of targeted drugs such as C3aR inhibitors and complement pathway antagonists, verifying its practicality and reliability in drug development.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] 1. An innovative screening method for highly pathogenic NMOSD antibodies specifically targeting Asian populations was established. This method, for the first time, combines "clinical prognostic phenotype" with "core pathway functional characteristics," identifying antibodies with clear therapeutic resistance relevance and filling a technological gap in screening highly pathogenic antibodies specific to Asian populations. This method eliminates the need for complex techniques such as mass spectrometry sequencing to identify antibody sequences, simplifies the process (reducing the cycle to 7-10 days), and lowers experimental costs by over 60%, making it suitable for large-scale application in clinical laboratories and research institutions.

[0026] 2. The screening process adopts a triple guarantee system of "clinical prognostic phenotype-oriented screening + M23 subtype-specific capture + C3 function verification" to ensure that the obtained antibodies have high specificity (purity ≥95%), high pathogenicity (C3 induction ability ≥1.5 times that of rAb-53 monoclonal antibody) and high clinical relevance (treatment resistance prediction accuracy ≥90%). This solves the core defects of existing screening methods that "only look at binding activity and not clinical relevance" and "only look at presence and not functional strength".

[0027] 3. The standardized application of highly pathogenic antibodies in the construction of NMOSD animal models has been clarified. The constructed models can accurately simulate the pathological characteristics of refractory NMOSD in Asians (treatment resistance, high relapse rate, and severe neurological damage). Compared with existing models based on Caucasian antibodies, they are more in line with the clinical reality of Asian patients. This provides a standardized tool for the study of the pathogenesis mechanism of this type of disease (such as the analysis of ethnic-specific pathogenic pathways) and the screening of targeted drugs (such as the development of drugs specifically for Asians).

[0028] 4. It has a wide range of applications. The screening method can be used for the rapid isolation of highly pathogenic antibodies in clinical samples, prognostic risk stratification, and treatment optimization. The constructed model can be used for the screening and evaluation of drugs with different mechanisms of action (complement inhibitors, glial cell regulators, neuroprotective agents, etc.). At the same time, it can provide standardized antigens for the development of NMOSD diagnostic kits, and promote the development of precision medicine for NMOSD from diagnosis, prognosis to treatment. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can understand and implement it.

[0030] Example 1: Screening for highly pathogenic NMOSD antibodies in Asian populations.

[0031] 1. Patient screening:

[0032] Ten Asian patients (all Han Chinese) meeting the 2015 international NMOSD diagnostic criteria were selected. All patients met the poor prognostic criteria of relapse within 12 months after glucocorticoid pulse therapy. There were 3 males and 7 females, aged 28-56 years (mean age 42.3 years). All patients were confirmed to be AQP4-IgG positive by cell transfection indirect immunofluorescence assay (CBA), and patients with other autoimmune diseases (such as multiple sclerosis, systemic lupus erythematosus, etc.), infectious diseases (such as viral encephalitis, tuberculous meningitis, etc.) and neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease, etc.) were excluded. A 5ml blood sample was collected from each patient before treatment, centrifuged at 3000 rpm for 10 min to separate serum, and the serum from all 10 patients was mixed thoroughly and stored at -80℃ for later use.

[0033] 2. Serum AQP4-IgG extraction:

[0034] (1) Take 2 ml of column material containing Protein A and pack it into a 10 ml filter column. Wash it 3 times with PBS balanced salt solution at pH=7.0, with a volume of 5 ml each time and a flow rate of 1 ml / min to remove residual ethanol from the column material.

[0035] (2) Dilute the thawed mixed serum with PBS to 20 ml, add it to the filter column in two portions, and let it stand for 15 min after each addition. Repeat the column pass three times to ensure that the IgG in the serum fully binds to Protein A.

[0036] (3) Wash the column material three times with PBS at pH 7.0, 5 ml each time, to remove non-specific bound proteins (such as albumin, transferrin and non-specific antibodies).

[0037] (4) Slowly add 4 ml of glycine-hydrochloric acid buffer (acidic elution buffer) with pH=3.0 to adhere to the wall, collect the elution buffer, and then immediately add 1 mol / L Tris-HCl solution to adjust the pH to 7.2 to avoid damage to antibody activity;

[0038] (5) The pH-adjusted eluent was transferred to a 100KD ultrafiltration tube and centrifuged at 4000r / min to concentrate it to 500μl to obtain crude IgG extract. The IgG concentration was determined by BCA method to be 1.2-1.5μg / μl.

[0039] 3. Specific capture:

[0040] (1) 293T cells transfected with M23 type AQP4-GFP were cultured in advance. After the cell confluence reached 80%, they were seeded into 6-well plates with 2 ml of culture system (DMEM medium containing 10% fetal bovine serum) per well.

[0041] (2) Add 100 μl of crude IgG extract to each well and incubate at 37°C and 5% CO2 for 2 h. Gently shake the culture plate once every 30 min during the incubation period to ensure that the antibody binds fully to the cells.

[0042] (3) Wash the cells three times with PBS, 5 ml each time, gently blowing to avoid cell detachment and remove unbound IgG;

[0043] (4) Add 1 ml of glycine-hydrochloric acid buffer at pH 3.0 to each well and incubate at room temperature for 10 min to dissociate the specifically bound AQP4-IgG;

[0044] (5) Collect the dissociation solution and adjust the pH to 7.2 with 1 mol / L Tris-HCl solution to obtain a specific AQP4-IgG solution. SDS-PAGE electrophoresis confirmed that only two specific bands appeared in the solution: the IgG heavy chain (approximately 55 kDa) and the light chain (approximately 24 kDa), with no other bands and a purity ≥95%. The transmembrane structure of M23 AQP4 makes it the main binding target of AQP4-IgG, ensuring specific capture. Simultaneously, the antigen-binding region (CDR region) of the IgG molecule precisely matches the antigenic epitope of AQP4, guaranteeing the targeted binding activity of the antibody to AQP4.

[0045] 4. Verification of high pathogenicity:

[0046] (1) Primary astrocyte culture: Lewis rats within 48 hours of birth were harvested, and brain tissue was isolated under sterile conditions. The cells were digested with 0.125% trypsin at 37°C for 15 min, and digestion was terminated by adding DMEM medium containing 20% ​​fetal bovine serum. The cells were centrifuged at 1500 r / min for 5 min, resuspended to form a cell suspension, and seeded into culture flasks. The cells were then incubated at 37°C. After being cultured until they fused into sheets, the cells were identified using GFAP antibody immunofluorescence assay. The proportion of positive cells was ≥90%, which met the experimental requirements.

[0047] (2) Co-incubation treatment: The specific AQP4-IgG solution was diluted to 1.0 μg / ml and co-incubated with primary astrocytes. An rAb-53 monoclonal antibody control group (same concentration) and a PBS blank control group were set up, with 3 replicates for each group. The incubation was carried out at 37℃. Incubate for 24 hours;

[0048] (3) C3 protein detection: Total protein was extracted from cells in each group and quantified using the BCA method. 20 μg of protein was then subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, and C3 protein-specific primary antibody (dilution ratio 1:1000) and HRP-labeled secondary antibody (dilution ratio 1:5000) were added. ECL color development was performed, and the gray values ​​of the bands were quantitatively analyzed using ImageJ software. The relative expression level was calculated using GAPDH as an internal reference.

[0049] (4) Result determination: The AQP4-IgG induced C3 protein expression level in this embodiment was 1.8 times that of the rAb-53 monoclonal antibody group, which met the ≥1.5 times criterion and was determined to be a highly pathogenic antibody; the astrocyte activation rate (GFAP positive cell ratio) induced by the antibody was 78.5%, which was significantly higher than that of the rAb-53 monoclonal antibody group (52.3%) and the PBS blank control group (12.6%), confirming that it has a stronger glial cell activation ability; this verification result is consistent with the pathogenesis mechanism of NMOSD (complement activation-glial cell activation-neuron / myelin damage) and the pathological pathway mediated by C3 protein, confirming that the high pathogenicity of the antibody is consistent with the clinical pathological mechanism.

[0050] Example 2: Application of highly pathogenic antibodies in the construction of NMOSD animal models.

[0051] 1. Pretreatment of laboratory animals:

[0052] Thirty female Lewis rats aged 6 - 8 weeks with a body weight of 120 - 180 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the animal license number was SCXK (Beijing) 2021 - 0006. After 1 week of adaptive feeding, the behavior of the rats was observed daily through three criteria: "tail tension test", "gait coordination observation", and "limb mobility assessment". After confirming no neurological function abnormalities such as tail weakness or abnormal gait, they were used for modeling. After the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3.5 ml / kg), they were fixed prone on the operating table. The neck was depilated and disinfected. The skin was incised along the posterior midline of the neck (about 1.5 cm in length), and the subcutaneous tissue and muscles were bluntly dissected to expose the area between the atlas (C1) and the occipital bone. The fascia and fat on its surface were gently scraped off with a cotton swab to expose the atlanto-occipital membrane; a PE10 catheter with a length of 10.0 cm was slowly inserted into the subarachnoid space, with an insertion depth of 7.0 cm, ensuring that the inner mouth was positioned at the first lumbar vertebra position; the catheter was fixed with medical sutures, and the muscles and skin were sutured layer by layer. After the operation, penicillin sodium (100,000 U / rat) was injected intramuscularly to prevent infection. After 2 days of adaptive feeding, after observing that the rats had no abnormalities such as fever, catheter detachment, or limb paralysis, they entered the subsequent steps.

[0053] Modeling and drug administration:

[0054] The 30 rats were randomly divided into 3 groups (10 rats in each group): model group, rAb-53 monoclonal antibody control group, and PBS blank control group;

[0055] (1) Model group: Inject the highly pathogenic antibody screened in Example 1 into the subarachnoid space through the catheter, with an injection dose of 8 μL / rat (concentration 1 μg / μL);

[0056] (2) rAb-53 monoclonal antibody control group: Inject the same dose of rAb-53 monoclonal antibody (concentration 1 μg / μL);

[0057] (3) PBS blank control group: Inject an equal amount of PBS;

[0058] Each group was administered once a day for 5 consecutive days. The administration time was fixed at 9:00 - 10:00 in the morning. After administration, the rats were gently fixed for 10 minutes to avoid drug reflux.

[0059] Model verification:

[0060] (1) Behavioral scoring: 7-10 days after modeling, behavioral scores were scored daily using a 0-5 scale (0: no abnormality; 1: tail weakness; 2: hind limb weakness but able to stand; 3: hind limb paralysis and unable to stand; 4: limb weakness; 5: near death or death). The results showed that the behavioral score of the model group rats was 3.2±0.3, the rAb-53 monoclonal antibody control group was 1.4±0.2, and the PBS blank control group was 0.2±0.1. The score of the model group was significantly higher than that of the control group (P<0.001).

[0061] (2) Immunohistochemical detection: On day 10 after modeling, rats were sacrificed, and spinal cord tissue (corresponding to the first lumbar vertebra) was collected, fixed in 4% paraformaldehyde, embedded in paraffin, and serially sectioned (5 μm thick). Immunohistochemical staining was used to detect the expression of C3 protein and MBP. The results showed that the positive expression rate of C3 protein in the model group was 78.5±6.2%, which was significantly higher than that in the rAb-53 monoclonal antibody control group (35.2±4.8%) and the PBS blank control group (12.3±2.1%); the proportion of demyelinated area of ​​MBP was 42.6±5.3%, which was significantly higher than that in the control group (P<0.001), which was consistent with the pathological characteristics of NMOSD.

[0062] (3) Verification of treatment resistance: Starting on the 7th day after modeling, the model group and the rAb-53 monoclonal antibody control group were treated by gavage with glucocorticoids (dexamethasone) at a dose of 30 mg / kg once a day for 14 consecutive days; after the treatment, the behavioral score of the model group was 3.1±0.2 points, and that of the rAb-53 monoclonal antibody control group was 1.2±0.1 points, which confirmed that the model group had clear glucocorticoid treatment resistance characteristics, which is consistent with the pathological phenotype of refractory NMOSD;

[0063] (4) Molecular pathological detection: Total protein was extracted from spinal cord tissue, and the expression of GFAP (astrocyte activation marker) and NeuN (neuronal injury marker) was detected by Western Blot. The results showed that the relative expression level of GFAP protein in the model group was 2.3±0.2 times that of the rAb-53 monoclonal antibody control group, and the relative expression level of NeuN protein was 0.5±0.1 times that of the control group, further confirming that the pathological characteristics of the model were highly consistent with those of clinically refractory NMOSD patients.

[0064] The foregoing embodiments describe in detail the implementation process of the screening method and application of the present invention. Its core innovation lies in obtaining highly pathogenic NMOSD antibodies against Asians without sequence identification through a three-dimensional technical system of "clinical prognostic phenotype-oriented screening + M23 type AQP4 specific capture + C3 induction ability functional verification", and applying them to the construction of therapeutic resistant animal models.

[0065] The patient screening process strictly limited ethnicity and prognostic phenotypes to ensure the clinical relevance and targeting of the antibody. Specific capture utilized the subtype-dominant binding characteristics of M23 AQP4 to effectively exclude non-specific proteins, achieving antibody purity of over 95%. Functional validation used C3 protein expression level as the core quantitative indicator, combined with clinical follow-up data to establish clear judgment criteria, ensuring the antibody's high pathogenicity and stability. Model construction, through precise catheter positioning and standardized dosing protocols, ensured a high success rate (≥90%) and repeatability (behavioral score coefficient of variation ≤8%). Treatment resistance validation further confirmed the model's clinical simulation value, providing a precise tool for targeted drug screening.

[0066] The technical solution of this invention has been validated through multi-center clinical samples and optimized through repeated experiments, ensuring its scientific validity and reliability. Those skilled in the art can appropriately adjust parameters such as the number of patient samples, antibody concentration, and incubation time according to actual experimental conditions. As long as these adjustments do not deviate from the core technical solution of this invention (clinical prognostic phenotype guidance, M23 subtype specific capture, and C3 functional verification), they all fall within the scope of protection of this invention.

Claims

1. A method for screening highly pathogenic NMOSD antibodies in Asian populations, characterized in that: Includes the following steps: Step Step 1: Patient Screening: Asian patients who meet the 2015 international NMOSD diagnostic criteria and have experienced disease relapse, worsening visual impairment, or decreased motor function within 12 months of glucocorticoid pulse therapy are screened for AQP4-IgG positivity. Step 2: Serum AQP4-IgG Extraction: Blood samples are collected from patients before treatment, serum is separated by centrifugation, and total IgG is extracted from the serum using Protein A column chromatography. The crude IgG extract is obtained by ultrafiltration concentration. Step 3: Specific Capture: The crude IgG extract is co-incubated with 293T cells transfected with M23 type AQP4-GFP at 37°C. After incubation for 2 hours under the specified conditions, and washing to remove unbound antibodies, the specifically bound AQP4-IgG was dissociated with acidic buffer, and the dissociation solution was collected and adjusted to neutral pH. Step 4: Pathogenicity verification: The specifically captured AQP4-IgG was co-incubated with primary astrocytes for 24-48 hours, with an rAb-53 monoclonal antibody control group and a PBS blank control group set up. The expression level of C3 protein in astrocytes was quantitatively detected by Western blotting. If the C3 protein expression level induced by the target antibody was more than 1.5 times that of the rAb-53 monoclonal antibody group, it was determined to be a highly pathogenic antibody.

2. The screening method according to claim 1, characterized in that: In step 1, the number of patients screened should be no less than 10, and the serum samples should be mixed before proceeding with the subsequent IgG extraction process.

3. The screening method according to claim 1, characterized in that: In step 3, the acidic buffer solution is a glycine-hydrochloric acid buffer solution with pH=3.

0. After dissociation, the pH is adjusted to 7.2 with 1 mol / L Tris-HCl solution.

4. The application of the highly pathogenic NMOSD antibody obtained by the screening method according to claim 1 in the construction of an NMOSD animal model, characterized in that: The procedure includes the following steps: Step 1: Animal pretreatment: Select 6-8 week old female Lewis rats, acclimatize them for 1 week, and after confirming the absence of neurological dysfunction, surgically implant a PE10 catheter into the subarachnoid space of the spinal cord; Step 2: Modeling and drug administration: Inject the highly pathogenic antibody obtained in claim 1 into the subarachnoid space through the catheter at a dose of 5-10 μL / rat (concentration 1 μg / μL), once daily for 5 consecutive days; Step 3: Model validation: 7-10 days after modeling, use a 0-5 scale for behavioral scoring, and simultaneously detect the expression of C3 protein and the degree of MBP demyelination in the spinal cord tissue by immunohistochemistry. A behavioral score ≥3 points and conforming to the pathological characteristics of NMOSD is considered a qualified model.

5. The application according to claim 4, characterized in that: The NMOSD animal model was glucocorticoid-resistant; after 14 days of glucocorticoid gavage treatment at a dose of 30 mg / kg, the behavioral score was still ≥3.