A processing method of a cell solid carrier glass slide for a central nervous system demyelination antibody spectrum (IgG) detection kit (cellular immunofluorescence method) and application thereof

Abstract

This invention belongs to the fields of cell immobilization and cell culture technology or antibody detection technology, and relates to a method for processing cell-immobilized carrier slides for a central nervous system demyelinating antibody profile (IgG) detection kit (cell immunofluorescence assay) and its application. The method involves sequentially soaking the slides in an acid bath, activating them in an alkaline bath, treating them with alcohol, and coating them with poly-L-lysine and a coating solution, each solution being prepared at a specific concentration. The prepared carrier slides exhibit strong cell adhesion, preventing cell detachment and edge effects. When the prepared cell-covered slides are used for central nervous system demyelinating antibody profile detection, they can efficiently and accurately achieve simultaneous detection of multiple specific antibodies, including MOG, AQP4, and GFAP, with high sensitivity, good specificity, and stable results, demonstrating significant clinical application value and promising prospects for industrial promotion.

Description

Technical Field

[0001] This invention belongs to the fields of cell immobilization and cell culture technology or antibody detection technology, specifically relating to a method for processing cell immobilization carrier slides for a central nervous system demyelinating antibody spectrum (IgG) detection kit (cell immunofluorescence assay) and its application. Background Technology

[0002] Central nervous system demyelinating diseases are a class of neurological disorders characterized primarily by the destruction or loss of myelin sheaths. These diseases encompass various subtypes, including neuromyelitis optica spectrum disorders, oligodendrocyte glycoprotein antibody-associated diseases, multiple sclerosis, and acute disseminated encephalomyelitis. These diseases are characterized by insidious onset and rapid progression, easily leading to neurological dysfunction, and in severe cases, limb paralysis, visual impairment, and even death. Therefore, early and accurate diagnosis is crucial for disease intervention and prognosis improvement. Currently, the clinical diagnosis of these diseases heavily relies on the detection of specific antibodies. Cell slides, as the core carrier for antibody detection, directly determine the accuracy, stability, and repeatability of antibody test results, serving as a vital foundation for accurate disease screening and diagnosis. In clinical practice and laboratory testing, the preparation of cell slides relies on high-quality cell-immobilized carrier slides. The surface properties and processing technology of the carrier slides directly affect cell adhesion, growth status, and antigen expression levels, thereby influencing the efficiency of subsequent antibody-antigen specific binding.

[0003] However, existing methods for processing cell-immobilized carrier slides still have several technical challenges that urgently need to be addressed: First, insufficient cell adhesion. Most processing methods rely solely on simple washing or single modification, failing to form stable hydrophilic groups and adhesion sites on the slide surface. This leads to cell detachment after inoculation and culture, especially during subsequent antibody incubation and washing, where cell detachment directly causes signal loss and false negatives, severely impacting detection reliability. Second, uneven cell distribution. Inconsistent surface modification results in cell aggregation and edge effects after inoculation, with some areas having excessively high cell density while others lack cell attachment. This compromises the representativeness of the detection area and reduces the reproducibility of results. Third, decreased detection performance after long-term storage. Carrier slides without proper modification have poor surface stability. During refrigeration at 4°C or storage at room temperature, surface-active groups are prone to degradation and oxidation, further reducing cell adhesion and failing to meet the needs of clinical batch preparation and long-term storage and transportation, increasing detection costs and operational complexity.

[0004] The aforementioned technical issues directly lead to a low success rate in cell slide preparation, resulting in problems such as insufficient sensitivity, poor specificity, and large fluctuations in results during subsequent antibody testing, making it difficult to meet the actual needs of clinical batch testing and early accurate diagnosis.

[0005] Therefore, developing a cell-solidified carrier slide processing method with excellent cell adhesion stability, standardized preparation process, scalable production capability, and adaptability for efficient detection of central nervous system demyelinating antibody profiles is crucial. This method would address the core pain points of existing technologies, such as weak cell adhesion, uneven distribution, and poor storage stability. It would achieve standardized preparation of cell-climbing slides, improve the sensitivity, specificity, and repeatability of antibody detection, not only fill existing technological gaps but also provide technical support for the early and accurate diagnosis and disease monitoring of central nervous system demyelinating diseases, reduce clinical testing costs, and has significant clinical application value, academic value, and industrial application prospects. Summary of the Invention

[0006] To address the aforementioned shortcomings, this invention provides a method for processing cell-immobilized carrier slides for a central nervous system demyelinating antibody profile (IgG) detection kit (cell immunofluorescence assay) and its application.

[0007] the term: The term "demyelinating diseases of the central nervous system" as used in this invention refers to a class of nervous system diseases characterized by the destruction or loss of nerve myelin sheaths, including various types such as neuromyelitis optica spectrum disorders, oligodendrocyte glycoprotein antibody-associated diseases, acute disseminated encephalomyelitis, multiple sclerosis, optic neuritis, myelitis, and brainstem encephalitis.

[0008] The term "central nervous system demyelinating antibody spectrum" as used in this invention refers to a collection of specific antibodies associated with central nervous system demyelinating diseases, including any one or more antibodies from MOG, AQP4, and GFAP.

[0009] The term "MOG" used in this invention is an abbreviation for Myelin Oligodendrocyte Glycoprotein, which is a core antigen related to myelin in the central nervous system. Its specific antibodies are important biomarkers for demyelinating diseases of the central nervous system.

[0010] The term "AQP4" used in this invention is an abbreviation for aquaporin 4, which is an important antigen associated with demyelinating diseases of the central nervous system. Its specific antibodies are characteristic markers of neuromyelitis optica spectrum disorders and other related diseases.

[0011] The term "GFAP" used in this invention is an abbreviation for Glial Fibrillary Acidic Protein, which is a specific marker of astrocytes. Its related antibodies are closely related to the pathological process of demyelinating diseases of the central nervous system and can be used as an auxiliary marker for disease detection.

[0012] The term "mCherry" used in this invention refers to a monomeric red fluorescent protein, which is also a widely used red fluorescent reporter tag in the fields of life sciences and medical diagnostics. In the mCherry-MOG plasmid, mCherry-AQP4 plasmid, and mCherry-GFAP plasmid of this invention, mCherry is expressed independently of the target antigens MOG, AQP4, and GFAP. mCherry serves as a positive transfection reporter tag; the appearance of red fluorescence in cells indicates that the plasmid has been successfully transfected into the cells and is being expressed normally, providing a direct assessment of the overall transfection efficiency. The green fluorescent signal (Alexa Fluor 488-labeled secondary antibody) corresponds to the specific binding site between the patient's antibody and the target antigen. The interpretation range is the transfected red fluorescent cell region; the appearance of a specific green fluorescent signal within the red fluorescent cell region indicates a positive result, while the absence of a corresponding green fluorescent signal within the red fluorescent cell region indicates a negative result. This design is crucial for ensuring the accuracy and excellent specificity of the CBA method (cell immunofluorescence assay).

[0013] The mCherry-MOG, mCherry-AQP4, and mCherry-GFAP plasmids used in this invention are not particularly limited in their source. They can be obtained through conventional commercial channels or constructed using conventional gene synthesis and molecular cloning methods in the art. As is known in the art, dual-expression recombinant plasmids that can simultaneously express fluorescent reporter proteins and target antigens are conventionally constructed vectors in this field. Even without detailed descriptions of their specific sources, those skilled in the art can clearly understand their intended use and implement them based on existing technology and the functions and uses described in this invention. Any recombinant expression vector that can stably express the corresponding target antigen in eukaryotic cells, normally produce mCherry red fluorescent signals to indicate the overall extent of transfected positive cells, and can be used for cell transfection, cell slide preparation, and specific recognition, binding, and immunofluorescence detection of central nervous system demyelination-related antibodies, regardless of its specific construction method, backbone vector, or source of preparation, falls within the protection scope of this invention.

[0014] In this invention, the antibodies used for secondary antibody incubation are also purchased from commercially available sources. Different brands and catalog numbers can be selected according to the species of the primary antibody and the detection requirements. Examples of selections are as follows: if the primary antibody is a rabbit polyclonal antibody, goat anti-rabbit IgG secondary antibody can be used; if the primary antibody is a mouse monoclonal antibody, goat anti-mouse IgG secondary antibody can be used. The antibody used in this embodiment of the invention was purchased from ThermoFisher Scientific, catalog number A11013; the working solution used is phosphate buffer; and the main component of the mounting medium used is glycerol. Conventional working solutions or mounting medium types in the art can be used. The working solution or mounting medium can be routinely prepared or commercially purchased according to the type of secondary antibody and incubation conditions, and its specific components, concentrations, and sources are not limited.

[0015] The term "cell crawler" used in this invention refers to a product obtained by inoculating and culturing host cells (such as HEK293 cells) transfected with the recombinant plasmid of this invention on a pretreated glass crawler as a solidification carrier, followed by transfection incubation, fixation, and blocking. It can be used for immunofluorescence detection of demyelination antibody profiles of the central nervous system.

[0016] The term "CBA method" or "cellular immunofluorescence assay" used in this invention refers to a method for qualitative detection of specific autoantibodies in a test sample using mammalian cells transfected with a specific recombinant plasmid as the antigen matrix. In this invention, the method is characterized by: utilizing mCherry red fluorescence as an endogenous antigen localization tag, eliminating the need for an additional red fluorescent primary antibody; indicating antibody binding via an Alexa Fluor 488 green fluorescent secondary antibody; and determining a positive result only when the red and green fluorescence completely overlap. This method significantly reduces non-specific background caused by differences in cell state and fixation procedures, improving the accuracy of antibody detection related to central nervous system demyelinating diseases.

[0017] The term "biochip" as used in this invention refers to a microchip device that integrates functions such as sample introduction, reaction, and detection. Its main body is equipped with a reaction tank, sample introduction port, sample aspiration port, and labeling area, which can realize the simultaneous detection of multiple targets of the central nervous system demyelination antibody spectrum. It has the characteristics of small sample volume, simple operation, and high detection efficiency.

[0018] The technical solution of this invention is as follows: On one hand, the present invention provides a method for processing cell immobilization support slides, the method comprising the following steps: S1. Clean the glass slide substrate, dry it, and then immerse it in an acid bath solution. After immersion, rinse it to obtain glass slide 1. S2. Slide 1 is immersed in an alkaline solution. After immersion, it is rinsed to obtain slide 2. S3 and glass slide 2 are immersed in an alcohol solution. After immersion, they are rinsed to obtain an activated glass slide. S4. After sterilization of the activated glass slides, mount them, add poly-L-lysine solution for soaking, remove excess poly-L-lysine solution from the culture wells after soaking, and dry to obtain pre-coated glass slides. S5. Add the coating composite solution to the culture wells, incubate, remove excess liquid from the culture wells, wash, and obtain cell-immobilized carrier slides.

[0019] Specifically, the acid tank solution mentioned in step S1 is a potassium dichromate cleaning solution, which is composed of potassium dichromate, concentrated sulfuric acid and water.

[0020] More specifically, the ratio of potassium dichromate, concentrated sulfuric acid, and water is 100-125g: 125-150mL: 1L.

[0021] More specifically, the ratio of potassium dichromate, concentrated sulfuric acid, and water used is (100-101, 101-102, 102-103, 103-104, 104-105, 105-106, 106-107, 107-108, 108-109, 109-110, 110-111, 111-112, 112-113, 113-114, 114-115, 115-116, 116-117, 117-118, 118-119, 119-120, 120-121, 121-122, 122-123, 123-124). 124-125g): (125-126, 126-127, 127-128, 128-129, 129-130, 130-131, 131-132, 132-133, 133-134, 134-135, 135-136, 136-137, 137-138, 138-139, 139-140, 140-141, 141-142, 142-143, 143-144, 144-145, 145-146, 146-147, 147-148, 148-149 or 149-150mL): 1L.

[0022] Preferably, the ratio of potassium dichromate, concentrated sulfuric acid, and water is (100-101, 101-102, 102-103, 103-104, 104-105, 105-106, 106-107, 107-108, 108-109, 109-110, 110-111, 111-112 or 112-113 g): (125-126, 126-127, 127-128, 128-129, 129-130, 130-131, 131-132, 132-133, 133-134, 134-135, 135-136, 136-137 or 137-138 mL): 1 L.

[0023] More preferably, the ratio of potassium dichromate, concentrated sulfuric acid and water is 100-112.5g: 125-137.5mL: 1L.

[0024] More preferably, the ratio of potassium dichromate, concentrated sulfuric acid, and water is 100g:125mL:1L.

[0025] Specifically, the slide substrate used in step S1 includes any one or more of the following: rectangular slide substrate, square slide substrate, and circular slide substrate.

[0026] The present invention does not impose specific limitations on the shape and size of the glass slide substrate, and can be flexibly selected according to actual experimental needs; in some specific embodiments of the present invention, a square glass slide substrate with a diameter of 24 mm is used as an example. This embodiment is only for illustration and is not a limitation on the scope of protection of the present invention.

[0027] Specifically, the soaking time described in step S1 is 24-48 hours.

[0028] More specifically, the soaking time in step S1 is 24-25, 25-26, 26-27, 27-28, 28-29, 29-30, 30-31, 31-32, 32-33, 33-34, 34-35, 35-36, 36-37, 37-38, 38-39, 39-40, 40-41, 41-42, 42-43, 43-44, 44-45, 45-46, 46-47 or 47-48 hours.

[0029] Preferably, the soaking time in step S1 is 24 hours.

[0030] Specifically, the alkaline solution mentioned in step S2 is an aqueous solution containing 35-40 g / L NaOH.

[0031] More specifically, the alkaline solution mentioned in step S2 is an aqueous solution containing 35-36, 36-37, 37-38, 38-39 or 39-40 g / L NaOH.

[0032] Preferably, the alkaline solution in step S2 is an aqueous solution containing 35-36, 36-37, or 37-38 g / L NaOH.

[0033] More preferably, the alkaline solution in step S2 is an aqueous solution containing 37.5 g / L NaOH.

[0034] Specifically, the soaking time in step S2 is 30-45 minutes.

[0035] More specifically, the soaking time in step S2 is 30-31, 31-32, 32-33, 33-34, 34-35, 35-36, 36-37, 37-38, 38-39, 39-40, 40-41, 41-42, 42-43, 43-44 or 44-45 minutes.

[0036] Preferably, the soaking time in step S2 is 30 minutes.

[0037] Specifically, the alcohol solution mentioned in step S3 is a 65%-75% v / v aqueous ethanol solution.

[0038] More specifically, the alcohol solution mentioned in step S3 is a 65%-66%, 66%-67%, 67%-68%, 68%-69%, 69%-70%, 70%-71%, 71%-72%, 72%-73%, 73%-74%, or 74%-75% v / v aqueous ethanol solution.

[0039] Preferably, it is a 65%-66%, 66%-67%, 67%-68%, 68%-69%, or 69%-70% v / v aqueous ethanol solution.

[0040] Specifically, the soaking time described in step S3 is 1-2 hours.

[0041] More specifically, the soaking time in step S3 is 1.0-1.5h or 1.5-2.0h.

[0042] Preferably, the soaking time in step S3 is 1 hour.

[0043] Specifically, the sterilization described in step S4 includes any one or more of the following: autoclaving, dry heat sterilization, and ultraviolet sterilization.

[0044] In some specific embodiments of the present invention, the sterilization in step S4 is performed using a high-pressure steam sterilizer; the sterilization temperature is 110-130℃, the pressure is 0.05-0.20 MPa, and the time is 10-30 min; preferably 121℃, 0.1 MPa, and 20 min.

[0045] Specifically, the patching step in step S4 includes: placing the sterilized glass slide into the culture well so that the glass slide adheres to the bottom of the well.

[0046] In some specific embodiments of this invention, the culture wells are 6-well culture plates, and the pore size of the 6-well culture plate is adapted to a square glass slide substrate with a diameter of 24 mm. This invention does not strictly limit the specifications of the culture plate and the shape and size of the glass slide substrate, and can flexibly select them according to actual experimental needs.

[0047] Specifically, the polylysine solution mentioned in step S4 is a solution containing 80-120 μg / mL polylysine.

[0048] More specifically, the polylysine solution mentioned in step S4 is a solution containing 80-90, 90-100, 100-110, or 110-120 μg / mL of polylysine.

[0049] Preferably, the polylysine solution in step S4 is a solution containing 80-90 or 90-100 μg / mL of polylysine.

[0050] More preferably, the polylysine solution in step S4 is a solution containing 100 μg / mL polylysine.

[0051] Specifically, the soaking time described in step S4 is 10-30 minutes.

[0052] More specifically, the soaking time in step S4 is 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20, 20-21, 21-22, 22-23, 23-24, 24-25, 25-26, 26-27, 27-28, 28-29 or 29-30 minutes.

[0053] Preferably, the soaking time in step S4 is 20 minutes.

[0054] Specifically, the coating complex solution mentioned in step S5 is a solution containing fibronectin and laminin.

[0055] More specifically, the mass ratio of fibronectin to laminin is 1:1.5-2.

[0056] More specifically, the mass ratio of fibronectin to laminin is 1: (1.50-1.55, 1.55-1.60, 1.60-1.65, 1.65-1.70, 1.70-1.75, 1.75-1.80, 1.80-1.85, 1.85-1.90, 1.90-1.95 or 1.95-2.00).

[0057] Preferably, the mass ratio of fibronectin to laminin is 1:(1.50-1.55, 1.55-1.60, 1.60-1.65, 1.65-1.70 or 1.70-1.75).

[0058] More preferably, the mass ratio of fibronectin to laminin is 1:1.5.

[0059] Specifically, the coating complex solution mentioned in step S5 is a solution containing 8-12 μg / mL fibronectin and 15-21 μg / mL laminin.

[0060] More specifically, the coating complex solution mentioned in step S5 is a solution containing 8-9, 9-10, 10-11, 11-12 μg / mL fibronectin and 15-16, 16-17, 17-18, 18-19, 19-20, 20-21 μg / mL laminin.

[0061] Preferably, the coating complex solution in step S5 is a solution containing 10 μg / mL fibronectin and 15 μg / mL laminin.

[0062] Specifically, the incubation time in step S5 is 1-3 hours, and the incubation temperature is 35-39°C.

[0063] More specifically, the incubation time in step S5 is 1-2 or 2-3 hours, and the incubation temperature is 35-36, 36-37, 37-38 or 38-39°C.

[0064] Preferably, the incubation time in step S5 is 2 hours, and the incubation temperature is 37°C.

[0065] In another aspect, the present invention provides cell-solidified carrier slides prepared by any of the above-mentioned processing methods.

[0066] In another aspect, the present invention provides the application of cell-immobilized carrier slides prepared by any of the above processing methods in the preparation of cell-crawling slides or antibody detection products based on cell-crawling slides.

[0067] Specifically, the method for preparing the cell climbing slide includes: seeding target cells onto a cell immobilization carrier slide, culturing the cells to allow them to adhere and grow, thereby obtaining the cell climbing slide.

[0068] More specifically, the target cells include mammalian eukaryotic host cells that can be stably transfected with exogenous recombinant plasmids and express target antigens related to demyelinating diseases of the central nervous system.

[0069] Preferably, the target cells include, but are not limited to, any one or more of the following: HEK293 cells, HEK293T cells, HEK293F cells, CHO cells, COS7 cells, and HeLa cells.

[0070] In certain specific embodiments of the present invention, HEK293 cells are selected as target cells. The HEK293 cells can be efficiently transfected with mCherry-MOG, mCherry-AQP4, and mCherry-GFAP fluorescent plasmids, stably expressing MOG, AQP4, and GFAP specific antigens, and simultaneously expressing mCherry red fluorescent reporter protein for antigen localization and transfection efficiency verification. This is suitable for the adherent growth characteristics of the cell immobilization carrier slide of the present invention and is applicable to the simultaneous detection of central nervous system demyelination antibody spectrum cell immunofluorescence.

[0071] Specifically, the antibody detection products include any one or more of the following: central nervous system demyelinating antibody spectrum detection products and central nervous system demyelinating disease diagnostic products.

[0072] Preferably, the antibody detection product contains a biochip, the biochip comprising a chip body; the chip body is provided with a reaction groove, a sample inlet, a sample aspiration hole and a labeling area; and a cell slide containing target cells is fixed in the reaction groove.

[0073] Preferably, the central nervous system demyelinating antibody includes any one or more of MOG antibody, AQP4 antibody, and GFAP antibody.

[0074] Preferably, the central nervous system demyelinating diseases include any one or more of the following: neuromyelitis optica spectrum disorders, myelin oligodendrocyte glycoprotein antibody-associated diseases, acute disseminated encephalomyelitis, multiple sclerosis, optic neuritis, myelitis, and brainstem encephalitis.

[0075] The beneficial effects of this invention are as follows: The cell-immobilized carrier slide prepared by this invention exhibits strong cell adhesion and excellent stability, effectively preventing cell detachment and edge effects, and significantly improving the success rate and uniformity of cell slide preparation. Cell slides based on this cell-immobilized carrier slide can efficiently and accurately detect multiple specific antibodies such as MOG, AQP4, and GFAP simultaneously, with high sensitivity, good specificity, and stable results. It can be widely used in the early diagnosis, disease monitoring, and screening of demyelinating diseases of the central nervous system, filling existing technological gaps, reducing detection costs, and possessing significant clinical application value and promising prospects for industrial promotion. Attached Figure Description

[0076] Figure 1 The number of cells per single smear is shown in the figure. Compared with Example 1, ns represents no significant difference, * represents p<0.05, *** represents p<0.001, and **** represents p<0.0001.

[0077] Figure 2 The figure shows the results of the detachment rate determination; compared with Example 1, ns represents no significant difference, * represents p<0.05, ** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001.

[0078] Figure 3 For example, in a sample containing anti-AQP4 antibody, the green fluorescent signal and the red fluorescent signal overlap in position and have essentially the same characteristics.

[0079] Figure 4 For an example sample that does not contain anti-AQP4 antibody, the location of the red fluorescent signal is where there is no fluorescent signal in the green fluorescent signal area. Detailed Implementation

[0080] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0081] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0082] Example 1: A method for processing cell-immobilized support slides 1. Preparation of modifying reagents (1) Preparation of acid tank solution: 400g of potassium dichromate was added to 4L of purified water and heated and stirred at 45℃ to obtain potassium dichromate solution. After returning to room temperature, 500mL of concentrated sulfuric acid was slowly added to potassium dichromate solution and mixed thoroughly to obtain acid tank solution.

[0083] (2) Preparation of alkaline solution: 150g NaOH was added to 4L of purified water and stirred to dissolve, thus obtaining alkaline solution.

[0084] (3) Preparation of alcohol solution: 70% v / v ethanol-water solution.

[0085] (4) Preparation of polylysine solution: 100 μg / mL polylysine aqueous solution.

[0086] (5) Preparation of coating complex solution: sterile PBS containing 10 μg / mL fibronectin and 15 μg / mL laminin.

[0087] 2. Cleaning and activation of the glass slide substrate (1) Take a square glass slide with a diameter of 24 mm as the slide substrate (select the specification according to the experimental requirements), rinse it with purified water 3-5 times to remove surface impurities, and dry it; immerse the rinsed glass slide in the prepared acid tank solution for 24 hours; after soaking, take the glass slide out of the acid tank with tweezers and rinse it repeatedly with purified water 8-10 times.

[0088] (2) Immerse the cleaned glass slide in the alkaline solution for 30 minutes. After immersion, remove the glass slide with tweezers and rinse it repeatedly with purified water 8-10 times.

[0089] (3) Immerse the cleaned glass slides in an alcohol solution for 1 hour; remove the glass slides after immersion in alcohol and rinse them repeatedly with purified water 8-10 times. Place the glass slides in a high-pressure steam sterilizer (121℃, 0.1MPa) for 20 minutes to sterilize. After sterilization, remove the slides and place them in a clean bench to cool to room temperature to obtain sterile glass slides.

[0090] (4) Perform the mounting operation in the clean bench: Place the sterile glass slides stably on the bottom of the wells of the sterile culture plate (6-well plate, select the size according to the experimental requirements), ensuring that the glass slides are in close contact with the bottom of the wells, and place one glass slide in each culture well.

[0091] (5) Add polylysine solution to each culture well where a glass slide is placed, ensuring that the solution completely covers the surface of the glass slide. Soak for 20 minutes to allow the polylysine to be evenly adsorbed on the surface of the glass slide. After soaking, use a sterile pipette to remove the excess polylysine solution from the well. Rinse twice with sterile purified water. Place the culture plate in a clean bench, open the culture plate cover, and allow it to air dry naturally until the surface of the glass slide is completely dry.

[0092] (6) Add coating composite solution to each culture well to ensure that the solution completely covers the surface of the slide. Place the culture plate in a constant temperature incubator at 37°C and incubate for 2 hours. After incubation, use a sterile pipette to remove the excess coating composite solution from the well. Wash twice with sterile PBS to obtain cell solidification carrier slides, which can be used directly for cell seeding experiments or sealed and stored at 4°C for later use.

[0093] Example 2: A method for processing cell-solidification carrier slides 1. Preparation of modifying reagents (1) Preparation of acid tank solution: 450g of potassium dichromate was added to 4L of purified water and heated and stirred at 45℃ to obtain potassium dichromate solution. After returning to room temperature, 550mL of concentrated sulfuric acid was slowly added to potassium dichromate solution and mixed thoroughly to obtain acid tank solution.

[0094] (2) Preparation of alkaline solution: 140g NaOH was added to 4L of purified water and stirred to dissolve, thus obtaining alkaline solution.

[0095] (3) Preparation of alcohol solution: 65% v / v ethanol-water solution.

[0096] (4) Preparation of polylysine solution: 80 μg / mL polylysine aqueous solution.

[0097] (5) Preparation of coating complex solution: sterile PBS containing 12 μg / mL fibronectin and 21 μg / mL laminin.

[0098] 2. Cleaning and activation of the glass slide substrate The cleaning and activation steps of the slide substrate in Example 2 differ from those in Example 1 only in that steps (2) and (3) are different. The specific process is as follows: (2) Immerse the cleaned glass slide in the alkaline solution for 45 minutes. After immersion, remove the glass slide with tweezers and rinse it repeatedly with purified water 8-10 times.

[0099] (3) Immerse the cleaned glass slides in an alcohol solution for 2 hours; remove the glass slides after immersion in alcohol and rinse them repeatedly with purified water 8-10 times. Place the glass slides in a high-pressure steam sterilizer (121℃, 0.1MPa) for 20 minutes to sterilize. After sterilization, remove the slides and place them in a clean bench to cool to room temperature to obtain sterile glass slides.

[0100] Example 3: A method for processing cell-solidification carrier slides 1. Preparation of modifying reagents (1) Preparation of acid tank solution: 500g of potassium dichromate was added to 4L of purified water and heated and stirred at 45℃ to obtain potassium dichromate solution. After returning to room temperature, 600mL of concentrated sulfuric acid was slowly added to potassium dichromate solution and mixed thoroughly to obtain acid tank solution.

[0101] (2) Preparation of alkaline solution: 160g NaOH was added to 4L of purified water and stirred to dissolve, thus obtaining alkaline solution.

[0102] (3) Preparation of alcohol solution: 75% v / v ethanol-water solution.

[0103] (4) Preparation of polylysine solution: 120 μg / mL polylysine aqueous solution.

[0104] (5) Preparation of coating complex solution: sterile PBS containing 8 μg / mL fibronectin and 16 μg / mL laminin.

[0105] 2. Cleaning and activation of the glass slide substrate The cleaning and activation steps for the glass slide substrate in Example 3 are the same as those in Example 1.

[0106] Comparative Example 1: A method for processing a cell-immobilized carrier slide. 1. Preparation of modifying reagents (1) Preparation of acid tank solution: concentrated sulfuric acid.

[0107] (2) Preparation of alcohol solution: anhydrous ethanol.

[0108] (3) Preparation of polylysine solution: 100 μg / mL polylysine aqueous solution.

[0109] 2. Cleaning and activation of the glass slide substrate (1) Take a square glass slide with a diameter of 24 mm as the slide substrate (select the specification according to the experimental requirements), rinse it with purified water 3-5 times to remove surface impurities, and dry it; immerse the rinsed glass slide in the prepared acid tank solution for 24 hours; after soaking, take the glass slide out of the acid tank with tweezers and rinse it repeatedly with purified water 8-10 times.

[0110] (2) Immerse the cleaned glass slides in an alcohol solution for 6 hours; remove the glass slides after immersion in alcohol and rinse them repeatedly with purified water 8-10 times. Place the glass slides in a high-pressure steam sterilizer (121℃, 0.1MPa) for 20 minutes to sterilize. After sterilization, remove the slides and place them in a clean bench to cool to room temperature to obtain sterile glass slides.

[0111] (3) Perform the mounting operation in the clean bench: Place the sterile glass slides stably on the bottom of the wells of the sterile culture plate (6-well plate, select the size according to the experimental requirements), ensuring that the glass slides are in close contact with the bottom of the wells, and place one glass slide in each culture well.

[0112] (4) Add polylysine solution to each culture well where a glass slide is placed, ensuring that the solution completely covers the surface of the glass slide. Soak for 20 minutes to allow polylysine to be evenly adsorbed on the surface of the glass slide. After soaking, use a sterile pipette to remove excess polylysine solution from the well. Rinse twice with sterile purified water. Place the culture plate in a clean bench, open the culture plate cover, and allow it to air dry naturally until the surface of the glass slide is completely dry. This is the prepared cell solidification carrier glass slide, which can be used directly for cell inoculation experiments or sealed and stored at 4°C for later use.

[0113] Comparative Example 2: A method for processing a cell-immobilized carrier slide. The only difference between Comparative Example 2 and Example 1 is the preparation of the acid tank solution (1). The specific steps are as follows: (1) Preparation of acid tank solution: 400g of potassium dichromate was added to 4L of purified water and heated and stirred at 45℃ to obtain potassium dichromate solution. After returning to room temperature, 400mL of concentrated sulfuric acid was slowly added to potassium dichromate solution and mixed thoroughly to obtain acid tank solution.

[0114] Comparative Example 3: A method for processing cell-immobilized support slides The only difference between Comparative Example 3 and Example 1 is the preparation of the acid tank solution (1). The specific steps are as follows: (1) Preparation of acid tank solution: 480g of potassium dichromate was added to 4L of purified water and heated and stirred at 45℃ to obtain potassium dichromate solution. After returning to room temperature, 800mL of concentrated sulfuric acid was slowly added to potassium dichromate solution and mixed thoroughly to obtain acid tank solution.

[0115] Comparative Example 4: A method for processing a cell-immobilized support slide. The only difference between Comparative Example 4 and Example 1 is in "(4) Preparation of poly-L-lysine solution" and "(5) Preparation of coating composite solution". The specific steps are as follows: (4) Preparation of polylysine solution: 200 μg / mL polylysine aqueous solution.

[0116] (5) Preparation of coating complex solution: sterile PBS containing 20 μg / mL fibronectin and 20 μg / mL laminin.

[0117] Comparative Example 5: A method for processing a cell-immobilized support slide. The only difference between Comparative Example 5 and Example 1 is the preparation of the coating composite liquid (5). The specific steps are as follows: (5) Preparation of coating complex solution: sterile PBS containing 25 μg / mL fibronectin.

[0118] Comparative Example 6: A method for processing a cell-immobilized support slide. The only difference between Comparative Example 6 and Example 1 is the preparation of the coating composite liquid (5). The specific steps are as follows: (5) Preparation of coating complex solution: sterile PBS containing 25 μg / mL laminin.

[0119] Comparative Example 7: A method for processing a cell-immobilized support slide. The only difference between Comparative Example 7 and Example 1 is that the coating composite liquid treatment step is not included.

[0120] 1. Preparation of modifying reagents The preparation steps of the modified reagent in Comparative Example 7 are the same as those in Example 1.

[0121] 2. Cleaning and activation of the glass slide substrate Steps (1) to (4) are the same as in Example 1.

[0122] (5) Add poly-L-lysine solution to each culture well where a glass slide is placed, ensuring that the solution completely covers the surface of the glass slide. Soak for 20 minutes to allow poly-L-lysine to be evenly adsorbed on the surface of the glass slide. After soaking, use a sterile pipette to remove excess poly-L-lysine solution from the well. Rinse twice with sterile purified water. Place the culture plate in a clean bench, open the culture plate cover, and allow it to air dry naturally until the surface of the glass slide is completely dry. This is the cell solidification carrier glass slide, which can be used directly for cell inoculation experiments or sealed and stored at 4°C for later use.

[0123] Experiment Example 1: Application of Cell Immobilization Support Slides 1. Cell Culture HEK293 cells were cultured to the logarithmic growth phase. The old culture medium was discarded, and the cell surface was washed twice with PBS buffer. 0.25% trypsin digestion solution was added, and the cells were incubated at 37°C for 3 minutes. After observing the cells becoming rounded and detaching from the culture vessel under an inverted microscope, DMEM medium containing 10% FBS was added to terminate the digestion. The cells were gently pipetted to prepare a single-cell suspension, and the cell concentration was adjusted to 1×10⁶ cells / mL. 5 per mL.

[0124] 2. Cell inoculation Add 2 mL of single-cell suspension of adjusted concentration to each well of a 6-well culture plate containing cell-immobilized carrier slides of Examples 1-3 and Comparative Examples 1-7. Gently shake the culture plate to distribute the cells evenly on the surface of the slides. Place the plate in a 37°C, 5% CO2 incubator and incubate for 24 hours for subsequent detection.

[0125] 3. Uniform cell distribution and morphological observation After 24 hours of cell culture, the 6-well culture plates were removed, the culture medium was aspirated, and the cells were gently washed three times with PBS buffer (pH 7.4) to remove non-adherent cells. Each well was fixed with 4% paraformaldehyde for 15 minutes, washed three times with PBS, stained with hematoxylin for 8 minutes, rinsed with tap water, separated by 1% hydrochloric acid ethanol for a few seconds, rinsed with tap water, stained with eosin for 2 minutes, rinsed with tap water, and mounted with neutral resin after air drying. Cell morphology was observed under a light microscope, and the number of cells on each slide was counted and statistically analyzed. The statistical analysis results are shown in Table 1 and... Figure 1 As shown: Table 1. Cell count per slice (n=3)

[0126] The number of cells per single climbing slide in Examples 1-3 was significantly higher than that in all comparative examples, indicating that the cell immobilization carrier slides in these examples can promote the adhesion and growth of more HEK293 cells and have better cell adhesion performance.

[0127] 4. Cell detachment test After 24 hours of cell seeding and culture, the cell immobilization carrier slides from Examples 1-3 and Comparative Examples 1-7 were removed and placed into new sterile 24-well cell culture plates. 1 mL of sterile PBS buffer (pH=7.4) was added to each well to ensure complete wetting of the slide surface and prevent any areas not in contact with the buffer from affecting the experimental results. The culture plate was placed in a constant-temperature shaker, and the rotation speed was adjusted to 100 rpm. Shaking was performed at room temperature for 10 minutes to simulate the slight disturbance during in vitro cell culture and to examine the adhesion of cells to the carrier slide surface. After shaking, each group of slides was removed and gently rinsed twice with sterile PBS buffer for 1 minute each time, maintaining consistent pressure to avoid cell detachment. Another group of slides cultured in parallel for 24 hours without shaking was used as a blank control group, with three replicates per group.

[0128] The slides of the shaking group and the control group were fixed, stained and mounted using the HE staining method (hematoxylin-eosin staining) as described above. They were then observed under an optical microscope, and the number of cells on each slide was counted and statistically analyzed.

[0129] The detachment rate (%) = (number of cells per smear in the control group - number of cells per smear in the oscillation group) / number of cells per smear in the control group × 100%.

[0130] The measurement results are shown in Table 2 and Figure 2 As shown: Table 2 Results of sheet removal rate measurement (n=3)

[0131] The cell detachment rate of the cell solidification carrier slides prepared in Examples 1-3 was less than 7.5%, indicating excellent cell adhesion stability. However, the cell detachment rate of the cell solidification carrier slides prepared in Comparative Examples 1-7 was significantly higher than that of the groups in Examples 1-3, indicating that their cell adhesion was poor and cell detachment was more likely to occur.

[0132] Experiment Example 2: Cell-solidified carrier slides used for the preparation of cell-crawling smears with different antigens. 1. Host cell inoculation and culture HEK293 cells were cultured to the logarithmic growth phase. The old culture medium was discarded, and the cell surface was washed twice with PBS buffer. 0.25% trypsin digestion solution was added, and the cells were incubated at 37°C for 3 minutes. After observing the cells becoming rounded and detaching from the culture vessel under an inverted microscope, DMEM medium containing 10% FBS was added to terminate the digestion. The cells were gently pipetted to prepare a single-cell suspension, and the cell concentration was adjusted to 1×10⁶ cells / mL. 5 per mL.

[0133] Add 2 mL of single-cell suspension of adjusted concentration to each well of a 6-well culture plate containing cell immobilization carrier slides from Example 1. Gently shake the culture plate to distribute the cells evenly on the surface of the slides. Place the plate in a 37°C, 5% CO2 incubator and incubate for 24 hours for subsequent transfection.

[0134] 2. Recombinant plasmid transfection and incubation The target plasmids were three mCherry-labeled plasmids (mCherry-MOG plasmid, mCherry-AQP4 plasmid, and mCherry-GFAP plasmid).

[0135] Transfection was performed using PEI transfection reagent. Two EP tubes were prepared, each containing 125 μL of Opti-MEM medium. In one EP tube (tube ①), 3 μg of the target plasmid was added, and in the other EP tube (tube ②), 12 μL of PEI transfection reagent was added. The mixture was thoroughly mixed by pipetting and allowed to stand for 10 min. The liquid from tube ① was then added to tube ②, thoroughly mixed by pipetting, and allowed to stand for 5 min to obtain the transfection complex. The transfection complex was then added dropwise to a 6-well plate containing cell spreaders, gently mixed, and incubated at 37°C in a 5% CO2 incubator for 48 h.

[0136] After incubation, the culture medium was discarded, and the cell slides were rinsed three times with sterile PBS buffer for 5 min each time. 1 mL of 4% paraformaldehyde fixative was added to each well, and the slides were fixed at room temperature for 30 min. The fixative was discarded, and the slides were rinsed three times with sterile PBS buffer for 5 min each time. 1 mL of 5% BSA blocking solution was added to each well, and the slides were incubated at 37°C for 1 h. After blocking, the slides were rinsed three times with sterile PBS buffer for 5 min each time. The coverslips were removed with sterile forceps, placed on sterile filter paper, and the edges were blotted dry. The slides were then dried to obtain cell slides for antibody detection, namely HEK293-MOG slides, HEK293-AQP4 slides, and HEK293-GFAP slides.

[0137] Experiment Example 3: Assembly and Use of Biochips 1. Composition of a biochip The biochip is a long, strip-shaped cell antigen plate with a clearly defined structural layout, divided into a front and a back panel. The front panel includes a sample inlet, aspiration port, a QR code identification label, and a mixing chamber, all integrated on the same plane for easy sample loading, aspiration, and traceability. The back panel features a reaction chamber for fixing the cell antigen plates. HEK293-MOG, HEK293-AQP4, and HEK293-GFAP climbing plates, along with a blank control antigen plate, are arranged sequentially from left to right within the reaction chamber, enabling parallel detection of multiple antibodies.

[0138] 2. Cell Antigen Plate Assembly Process (1) Antigen patching: The antigen patches of each indicator cell and the blank control antigen patch are attached to the reaction tank in the set order from left to right; (2) Sealing and encapsulation: The reaction area is covered with a regular glass slide, and the reaction area with the antigen slide is sealed and encapsulated with UV adhesive. (3) Curing and fixing: Use a wafer baking machine to heat and cure the UV glue encapsulation area to firmly bond the glass slide to the chip body and ensure that the reaction area is sealed and leak-proof; (4) Identification and coding: A QR code and corresponding identification are printed on the label area using a marking machine to complete the chip identification marking; (5) Finished product packaging: The assembled and solidified cell antigen plates are packed into aluminum foil bags and sealed. They are stored in a dark and moisture-proof environment for easy storage, transportation and clinical use.

[0139] This chip integrates sample mixing, multi-channel reaction, QR code traceability, and sealed packaging, enabling simultaneous detection of three antibodies related to central nervous system demyelination.

[0140] 3. Instructions for use of biochips It is recommended to conduct the experiment at room temperature to ensure the accuracy of the results.

[0141] (1) Warming and washing: Take out the biochip and place it in a humidified box (self-provided) to equilibrate at room temperature for 10 minutes. Add 70 μL of working solution to each reaction zone and soak for 10 minutes, then aspirate the working solution.

[0142] (2) Sample incubation: Add 70 μL of the sample to be tested to each reaction zone and incubate at room temperature for 1 hour.

[0143] (3) Cleaning: Remove the sample to be tested, clean the biochip reaction area with working solution, add 70 μL of working solution to each reaction area, clean the reaction area quickly twice, add 70 μL of working solution for the third time, soak for 5 min and then remove the working solution.

[0144] (4) Secondary antibody incubation: Add 70 μL of diluted fluorescent secondary antibody (fluorescently labeled goat anti-human IgG) to each reaction zone and incubate at room temperature in the dark for 30 minutes. (From this step onwards, keep the entire process in the dark).

[0145] (5) Cleaning: Remove the fluorescent secondary antibody (fluorescently labeled goat anti-human IgG), clean the biochip reaction area with working solution, add 70 μL of working solution to each reaction area, clean the reaction area quickly twice, add 70 μL of working solution for the third time, soak for 5 min and then remove the working solution.

[0146] (6) Result interpretation: Add 70 μL of mounting medium to each reaction zone, observe the fluorescence signal under a 20x objective lens, and analyze the detection results.

[0147] (7) Interpretation criteria Taking AQP4 as an example: 1) Use an inverted fluorescence microscope to observe the emitted fluorescence signals at wavelengths of 480-500nm and 540-565nm, respectively, and observe whether green and red fluorescence signals are emitted. 2) Compare the location and characteristics of the green fluorescent signal with the location of the red fluorescent signal to confirm whether they overlap; 3) If the location emitting the green fluorescence signal overlaps with the location emitting the red fluorescence signal, and the characteristics are basically the same, then it can be determined that the sample contains anti-AQP4 antibody; 4) If no green fluorescent signal is emitted; or the location of the green fluorescent signal does not overlap with the location of the red fluorescent signal; or the location of the green fluorescent signal overlaps with the location of the red fluorescent signal, but the two have significant differences in characteristics; then the sample is determined to be free of anti-AQP4 antibody. Figure 3 The green fluorescence signal and the red fluorescence signal overlap in position and have basically the same characteristics, indicating that the sample contains anti-AQP4 antibody; Figure 4The absence of a red fluorescent signal at the location of the green fluorescent signal indicates that the sample does not contain anti-AQP4 antibody.

[0148] Similarly, in addition to AQP4 transfected cells labeled with fluorescent protein, the reagent also includes one or more of MOG and GFAP transfected cells labeled with fluorescent protein, thus enabling the simultaneous detection and interpretation of multiple antibodies in the sample. If any one or more of MOG, AQP4, and GFAP are determined to be positive, the sample is diagnosed as a central nervous system demyelinating disease sample.

[0149] 4. Stability test The packaged chip was stored at 4℃ and removed at 0d, 1d, 3d, 5d and 7d respectively to determine the test compliance rate.

[0150] Serum samples were collected from patients clinically diagnosed with central nervous system demyelinating diseases and who were antibody-positive, including: 3 cases of MOG antibody-positive serum, 3 cases of AQP4 antibody-positive serum, and 3 cases of GFAP antibody-positive serum. After collection, the serum samples were frozen at -20°C and thawed at 4°C before use. The samples were then centrifuged (3000 rpm, 10 min) to remove the precipitate, and the supernatant was collected for later use. The prepared clinical serum samples were diluted 1:10 with sterile PBS buffer, thoroughly mixed, and stored on ice for later use.

[0151] The chips taken out at each time point were operated according to the steps of rewarming, washing, sample incubation, cleaning, secondary antibody incubation, cleaning, and fluorescence observation in the instructions for use of the biochip. All antigen plates in the reaction chamber of each chip were observed using a fluorescence microscope with a magnification of 200x. The experimental results of each antigen plate were observed, and images were acquired and recorded using image analysis software to determine its detection stability.

[0152] The measurement results are shown in Table 3: Table 3 Stability Testing

[0153] Note: In the table, "+" indicates a positive test result, and "-" indicates a negative test result.

[0154] The test results show that the biochip prepared by this invention can maintain 100% stable detection of three antibodies, MOG, AQP4 and GFAP, when stored at 4°C for the first 5 days. By the 7th day of storage, except for one MOG antibody positive sample which was detected as negative, other positive samples can still be accurately detected. The overall stability is excellent and can meet the needs of clinical batch preparation, storage and transportation and routine testing.

[0155] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method of processing a cell immobilization support glass slide, characterized by, The processing method includes the following steps: S1. Clean the glass slide substrate, dry it, and then immerse it in an acid bath solution. After immersion, rinse it to obtain glass slide 1. S2. Slide 1 is immersed in an alkaline solution. After immersion, it is rinsed to obtain slide 2. S3 and glass slide 2 are immersed in an alcohol solution. After immersion, they are rinsed to obtain an activated glass slide. S4. After sterilization of the activated glass slides, mount them, add poly-L-lysine solution for soaking, remove excess poly-L-lysine solution from the culture wells after soaking, and dry to obtain pre-coated glass slides. S5. Add coating composite solution to culture wells, incubate, remove excess liquid from culture wells, wash, and obtain cell immobilization carrier slides. The acid tank solution mentioned in step S1 is a potassium dichromate cleaning solution, which is composed of potassium dichromate, concentrated sulfuric acid and water; the ratio of potassium dichromate, concentrated sulfuric acid and water is 100-125g: 125-150mL: 1L. The polylysine solution mentioned in step S4 is a solution containing 80-120 μg / mL polylysine; The coating complex solution mentioned in step S5 is a solution containing fibronectin and laminin, wherein the mass ratio of fibronectin to laminin is 1:1.5-2.

2. The treatment method according to claim 1, characterized in that, The acid tank solution mentioned in step S1 is a potassium dichromate cleaning solution, which is composed of potassium dichromate, concentrated sulfuric acid and water; the ratio of potassium dichromate, concentrated sulfuric acid and water is 100-112.5g: 125-137.5mL: 1L. The polylysine solution mentioned in step S4 is a solution containing 80-100 μg / mL polylysine; The coating complex solution mentioned in step S5 is a solution containing fibronectin and laminin, wherein the mass ratio of fibronectin to laminin is 1:1.5-1.

75.

3. The processing method according to claim 1, characterized in that, The coating complex solution mentioned in step S5 is a solution containing 8-12 μg / mL fibronectin and 15-21 μg / mL laminin.

4. The processing method according to claim 1, characterized in that, The alkaline solution mentioned in step S2 is an aqueous solution containing 35-40 g / L NaOH; the alcohol solution mentioned in step S3 is a 65%-75% v / v ethanol aqueous solution.

5. The processing method according to claim 1, characterized in that, The soaking time in step S1 is 24-48 hours; or the soaking time in step S2 is 30-45 minutes; or the soaking time in step S3 is 1-2 hours; or the soaking time in step S4 is 10-30 minutes; or the incubation time in step S5 is 1-3 hours, and the incubation temperature is 35-39°C.

6. The processing method according to claim 1, characterized in that, The sterilization described in step S4 includes any one or more of the following: autoclaving, dry heat sterilization, and ultraviolet sterilization.

7. The processing method according to claim 1, characterized in that, The patching step described in step S4 includes: placing the sterilized glass slide into the culture well so that the glass slide adheres to the bottom of the well.

8. Cell-solidified carrier slides prepared by the processing method according to any one of claims 1-7.

9. The use of the cell-immobilized carrier slide prepared by the processing method according to any one of claims 1-7 in the preparation of cell-spreading slides or antibody detection products based on cell-spreading slides.

10. The application according to claim 9, characterized in that, The method for preparing the cell climbing slide includes: seeding target cells onto a cell immobilization carrier slide, culturing the cells to allow them to adhere and grow, thereby obtaining the cell climbing slide.

11. The application according to claim 10, characterized in that, The antibody detection products include any one or more of the following: central nervous system demyelinating antibody spectrum detection products and central nervous system demyelinating disease diagnostic products.

12. The application according to claim 11, characterized in that, The antibody detection product contains a biochip, which includes a chip body; the biochip body is provided with a reaction groove, a sample inlet, a sample aspiration hole and a labeling area; and a cell slide containing target cells is fixed in the reaction groove.

13. The application according to claim 11, characterized in that, The central nervous system demyelinating antibodies include any one or more of MOG antibodies, AQP4 antibodies, and GFAP antibodies.

14. The application according to claim 11, characterized in that, The aforementioned demyelinating diseases of the central nervous system include any one or more of the following: neuromyelitis optica spectrum disorders, oligodendrocyte glycoprotein antibody-associated diseases, acute disseminated encephalomyelitis, multiple sclerosis, optic neuritis, myelitis, and brainstem encephalitis.

Images