Application of a recombinant antigen and its transfected living cell immunofluorescence method in detection of IgG antibody against myelin oligodendrocyte glycoprotein

Abstract

The application relates to the technical field of antibody detection, in particular to a recombinant antigen and application of a transfected living cell immunofluorescence method to detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies. A myelin oligodendrocyte glycoprotein expression plasmid is constructed, the expression plasmid vector is pcdna3.1-target gene-linker-x3Flag-P2A-EGFP, the amino acid sequence of the expression plasmid gene is shown as SEQ ID NO:1, the base sequence of the expression plasmid gene is shown as SEQ ID NO:2, the expression plasmid is made into target cells through cell transfection, and after 16-28h of culture, cells with the expression plasmid fixed are obtained, and the cells can be used for immunofluorescence staining method to detect anti-myelin oligodendrocyte glycoprotein IgG antibodies. The problems of antigen spatial conformation change, epitope shielding or cell shrinkage of cells are solved, and the sensitivity of detection is improved.

Description

Technical Field

[0001] This invention relates to the field of antibody detection technology, specifically to the application of a recombinant antigen and its transfection live-cell immunofluorescence assay in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies. Background Technology

[0002] In the central nervous system (CNS), myelin is formed by oligodendrocytes and facilitates efficient, skip-transmission of nerve electrical signals, protecting normal neuronal function. CNS inflammatory demyelinating diseases are a group of diseases caused by various pathological factors leading to myelin loss, while nerve cells remain relatively intact. Common CNS inflammatory demyelinating diseases include multiple sclerosis (MS), neuromyelitis optica spectrum disorders (NMOSD), and oligodendrocyte glycoprotein antibody-associated diseases (MOGAD), each with relatively independent clinical characteristics. Autoantibodies play a crucial role in the diagnosis and differential diagnosis of these diseases.

[0003] Anti-MOG-IgG antibody is an autoantibody targeting the myelin oligodendrocyte glycoprotein (MOG) in the central nervous system. It is associated with various central nervous system demyelinating diseases, such as NMOSD and MOGAD. According to the "Expert Consensus on Antibody Detection Related to Central Nervous System Autoimmune Diseases 2022," the positive rate of MOG antibody is 7.4%, which is of significant value in the diagnosis of MOGAD. It is mostly present in patients with optic neuritis or myelitis, but rarely in classic MS. Its serum titer depends on disease activity and treatment status. During long-term follow-up, the serum anti-MOG-IgG antibody titer in NMOSD or MS patients mostly fluctuates at the original level and then increases again.

[0004] According to expert consensus, current methods for detecting CNS autoantibodies include cell substrate-based immunofluorescence (CBA), indirect immunofluorescence (IIF), fluorescence immunoprecipitation assay (FIPA), and enzyme-linked immunosorbent assay (ELISA). Among these, CBA has advantages such as preserving antigen conformation and protein modifications, more accurately reflecting the characteristics of antigen-antibody binding. Furthermore, its sensitivity (above 70%) and specificity (97%–100%) are higher than the other methods mentioned above, making it suitable for clinical auxiliary detection. Existing CBA detection methods for anti-MOG-IgG antibodies are mostly based on cell design after antigen fixation. This involves constructing plasmids expressing anti-myelin oligodendrocyte glycoprotein (MOG antigen), transfecting cells, and then using antigen fixation technology to provide stable antigen-overexpressing cells that can bind to anti-MOG-IgG antibodies in the test sample. Subsequently, a secondary antibody carrying a fluorescent protein is used to label the antigen-bound anti-MOG-IgG antibody, thereby confirming the presence of anti-MOG-IgG antibodies in the test sample.

[0005] Existing immunofluorescence assays for detecting anti-MOG-IgG antibodies using overexpressed antigens are based on cells after antigen fixation. Fixation methods, such as paraformaldehyde or acetone fixation, can lead to changes in antigen spatial conformation, epitope obstruction, or cell shrinkage, thus affecting the sensitivity of the detection. Summary of the Invention

[0006] To address the problems in related technologies, this invention provides an application of recombinant antigen and its transfected live cell immunofluorescence assay in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies. This solves the problems of antigen spatial conformational changes, epitope obstruction, or cell shrinkage, thereby improving the sensitivity of detection.

[0007] To solve the above problems, the following technical solutions are provided: This invention relates to the application of a recombinant antigen and its transfection with live-cell immunofluorescence in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies. An expression plasmid for myelin oligodendrocyte glycoprotein is constructed. The expression plasmid vector is pcdna3.1-target gene-linker-×3Flag-P2A-EGFP. The amino acid sequence of the expression plasmid gene is shown in SEQ ID NO:1, and the base sequence is shown in SEQ ID NO:2. After cell transfection, the expression plasmid enters the target cells. After culturing for 16-28 hours, cells immobilized with the expression plasmid are obtained and can be used for immunofluorescence staining to detect anti-myelin oligodendrocyte glycoprotein IgG antibodies.

[0008] By adopting the above approach, the expression plasmid vector pcdna3.1-(target gene)-linker-×3Flag-P2A-EGFP is constructed, and the expression of the target gene (MOG antigen) is driven by the promoter, ensuring efficient transcription and translation of the antigen in transfected cells and achieving high-quality antigen expression.

[0009] Introducing the ×3Flag tag into the expression plasmid allows for the specific recognition and binding of antigens by anti-Flag antibodies. This not only facilitates intracellular localization and verification of antigen expression but also enables antigen purification, enhancing the specificity and operability of the detection.

[0010] The target gene (MOG antigen) and EGFP (enhanced green fluorescent protein) are linked by a P2A self-cleaving peptide, allowing both to be expressed independently in the same transcript without affecting their spatial conformation and function. EGFP serves as a reporter gene, enabling direct observation of cell transfection efficiency via fluorescence microscopy and real-time monitoring of plasmid expression in cells, ensuring the reliability of the detection.

[0011] This expression plasmid vector offers multiple advantages, including a highly efficient expression system, tagged functional design, preservation of natural antigen conformation, and ease of operation. These advantages address the problems of insufficient antigen expression and inadequate epitope exposure in immobilized cell detection. This invention achieves high-quality and high-efficiency antigen expression by constructing its own expression plasmid for transfection. After transfection, cells are not fixed; immunofluorescence staining is performed directly on live cells, improving detection sensitivity and effectively avoiding changes in antigen spatial conformation, epitope obstruction, or cell shrinkage.

[0012] Cell transfection includes the following steps: Prepare solutions A and B. Solution A contains Opti-mem, expression plasmid, and P3000, while solution B contains Opti-mem and Lipo-3000. Mix solution A with solution B and let stand to obtain a mixed transfection reagent. Mix the mixed transfection reagent with complete culture medium and add it to a cell culture plate that has been seeded with target cells. Incubate in an incubator for 16-28 hours to obtain myelin oligodendrocyte glycoprotein fixed on the cells. The volume ratio of expression plasmid, P3000 and Lipo-3000 is 1:1.5:1.5-1:3:3.

[0013] Detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies includes the following steps: A1: Take out the phosphate buffer and fluorescently labeled goat anti-human IgG, and after standing and equilibrating to 22-28℃, dilute the fluorescently labeled goat anti-human IgG. The ratio of fluorescently labeled goat anti-human IgG to phosphate buffer is 1:300-1:800. A2: Place the cells immobilized with the expression plasmid in a blank cell culture plate and allow them to warm to 22-28℃. A3: After rewarming, add 60uL of phosphate buffer to each well of the cell culture plate, soak for 3-6 minutes, and then remove the phosphate buffer from the wells. A4: Add 60uL of sample to each well of the cell culture plate, incubate at 35-37℃ for 50-70 min, and then aspirate the sample from the well. A5: Add 60 μL of phosphate buffer to each well of the cell culture plate, place it in a horizontal shaker and wash for 3-6 minutes. Remove the phosphate buffer from the wells and repeat the washing process 3 times. A6: Add 60 μL of diluted fluorescently labeled goat anti-human IgG to each well of the cell culture plate, incubate at 37°C in the dark for 30-45 min, and remove the fluorescently labeled goat anti-human IgG from the well. A7: Add 50-100uL of phosphate buffer to each well of the cell culture plate, place it in a horizontal shaker and wash for 3-6 minutes. Remove the phosphate buffer from the wells and repeat the washing process 3 times. A8: Add 60 μL of phosphate buffer to each well of the cell culture plate and observe the color development using a fluorescence microscope to determine the presence of anti-myelin oligodendrocyte glycoprotein IgG antibody. Fluorescence indicates the presence of anti-myelin oligodendrocyte glycoprotein IgG antibody in the sample; no fluorescence indicates the absence of anti-myelin oligodendrocyte glycoprotein IgG antibody in the sample. The samples used are either serum or cerebrospinal fluid. Serum samples need to be diluted, with a serum-to-phosphate buffer ratio of 1:10.

[0014] Before cell transfection, the cells are pretreated, which includes the following steps: S1: Take the target cells and add them to the complete culture medium. Collect the cell suspension and centrifuge. Remove the supernatant to obtain the cell pellet. Add the culture medium from the complete culture medium to the cell pellet and resuspend the cell pellet until the cells are dispersed to obtain the resuspension. S2: Add 20-25 ml of complete culture medium and resuspension to a 100 mm petri dish, shake well, and incubate in a 35-37℃, 5% CO2 incubator. S3: Add diluted cell suspension to each well of the cell culture plate and incubate in a 35-37℃, 5% CO2 incubator to obtain a cell culture plate containing the target cells.

[0015] By employing the above-described scheme, the complete culture medium provides ample nutrition to the cells and inhibits contamination, maintaining normal physiological functions of the cells in their live state and ensuring the correct folding and surface expression of antigens. By omitting the fixation step found in existing technologies, immunofluorescence staining is performed directly on live cells, avoiding cell shrinkage or antigen epitope destruction caused by fixatives. This maximizes the preservation of the antigen's native conformation, allowing anti-MOG-IgG antibodies to bind to the target antigen more fully and specifically, thus improving detection sensitivity. Direct detection after transfection simplifies the procedure, reduces sources of experimental error, and shortens the detection cycle, making it more suitable for rapid screening of clinical samples.

[0016] The complete culture medium in S1 consists of DMEM medium, fetal bovine serum that has been inactivated in a 56°C water bath for 30 min and filtered through a 0.22 μm filter membrane, and a penicillin-streptomycin-amphotericidal mixture in a volume ratio of 43:7:1 to 43:12:1.

[0017] The specific steps for collecting and centrifuging the cell suspension in S1 are as follows: Use a dropper to collect the cell suspension into a 15mL centrifuge tube and place it in a centrifuge. Set the centrifuge speed to 1000rpm, the temperature to 4℃, and the centrifugation time to 3-5min.

[0018] The required volume of resuspension in S2 is as follows: 480-500 μL for 24 hours of incubation, 200-220 μL for 48 hours of incubation, and 150-180 μL for 96 hours of incubation.

[0019] The above solution has the following advantages: This invention relates to the application of a recombinant antigen and its transfection live-cell immunofluorescence assay in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies. The invention constructs a pcdna3.1-(target gene)-linker-×3Flag-P2A-EGFP expression plasmid vector, utilizing a promoter to drive the expression of the target gene (MOG antigen), ensuring efficient transcription and translation of the antigen in transfected cells and achieving high-quality antigen expression. The introduction of the ×3Flag tag into the expression plasmid allows for specific recognition and binding of the antigen by anti-Flag antibodies, facilitating intracellular localization and verification of antigen expression, and also enabling antigen purification, thus improving the specificity and operability of the detection. A P2A self-cleaving peptide is used to link the target gene (MOG antigen) and EGFP (enhanced green fluorescent protein), allowing both to be expressed independently in the same transcript without affecting their spatial conformation and function. EGFP serves as a reporter gene, enabling direct observation of cell transfection efficiency via fluorescence microscopy and real-time monitoring of plasmid expression in cells, ensuring the reliability of the detection.

[0020] This expression plasmid vector offers multiple advantages, including a highly efficient expression system, tagged functional design, preservation of natural antigen conformation, and ease of operation. By constructing an expression plasmid for transfection, this invention achieves high-quality and high-efficiency antigen expression. After transfection, cells are not fixed; instead, live cells are directly used for immunofluorescence staining, which improves detection sensitivity and effectively avoids changes in antigen spatial conformation, epitope obstruction, or cell shrinkage. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] This invention relates to the application of a recombinant antigen and its transfection into a live-cell immunofluorescence assay for the detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies. An expression plasmid for myelin oligodendrocyte glycoprotein is constructed using the vector pcdna3.1-target gene-linker-×3Flag-P2A-EGFP. The amino acid sequence of the expression plasmid gene is shown in SEQ ID NO:1, and the base sequence is shown in SEQ ID NO:2. After cell transfection, the expression plasmid enters the cells, and after culturing for 16-28 hours, cells immobilized with the expression plasmid are obtained. These cells can then be used for immunofluorescence staining to detect anti-myelin oligodendrocyte glycoprotein IgG antibodies. The introduction of the ×3Flag tag into the expression plasmid allows for specific recognition and binding of the antigen by anti-Flag antibodies. This facilitates intracellular localization and verification of antigen expression and can also be used for antigen purification, improving the specificity and operability of the detection. The target gene (MOG antigen) and EGFP (enhanced green fluorescent protein) are linked by a P2A self-cleaving peptide, allowing both to be expressed independently in the same transcript without affecting their spatial conformation and function. EGFP serves as a reporter gene, enabling direct observation of cell transfection efficiency via fluorescence microscopy and real-time monitoring of plasmid expression in cells, ensuring the reliability of the detection.

[0023] This expression plasmid vector offers multiple advantages, including a highly efficient expression system, tagged functional design, preservation of natural antigen conformation, and ease of operation. These advantages address the problems of insufficient antigen expression and inadequate epitope exposure in immobilized cell detection. This invention achieves high-quality and high-efficiency antigen expression by constructing its own expression plasmid for transfection. After transfection, cells are not fixed; immunofluorescence staining is performed directly on live cells, improving detection sensitivity and effectively avoiding changes in antigen spatial conformation, epitope obstruction, or cell shrinkage.

[0024] This application describes the application of a recombinant antigen and its transfection with live cell immunofluorescence in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies; Step 1: Raw material preparation and pretreatment The enhanced cell adhesion reagent is mixed with phosphate buffer at a volume ratio of 1:30-1:100 to obtain the working solution of the adhesion reagent. The optimal ratio is 1:50, which provides sufficient adsorption and also helps to save reagent.

[0025] DMEM medium, fetal bovine serum inactivated at 56°C for 30 min and filtered through a 0.22 μm filter, and a penicillin-streptomycin-amphotericidal mixture were mixed at a volume ratio of 43:7:1-43:12:1 to obtain a complete culture medium. The optimal ratio is 43:7:1, which can provide sufficient nutrition for cells to maintain a reasonable growth rate and is also conducive to saving reagents.

[0026] A pancreatic digestion solution can be obtained by combining 0.25% ethylenediaminetetraacetic acid (EDTA) and 0.25% pancreatic enzyme.

[0027] Opti-mem is Opti-mem serum-depleted culture medium; P3000 is P3000 TM Enhancer reagent; Lipo3000 is Lipofectamine TM 3000 reagents.

[0028] The target cells were HEK293T human embryonic kidney cells and human cervical cancer cells. In this embodiment, HEK293T human embryonic kidney cells were used as an example.

[0029] Alexa Fluor was used for fluorescent labeling. The Alexa Fluor-labeled goat anti-human IgG was diluted at a ratio of 1:300 to 1:800 with phosphate buffer. A ratio higher than this may result in over-staining and high background fluorescence, affecting the judgment. A ratio lower than this may result in light staining, making it difficult to observe. The optimal ratio of Alexa Fluor-labeled goat anti-human IgG to phosphate buffer is 1:500, which provides the best observation results under a fluorescence microscope.

[0030] The sample can be a serum sample or a cerebrospinal fluid sample. The serum sample needs to be diluted. The ratio of serum sample to phosphate buffer is 1:10. The cerebrospinal fluid sample does not need to be diluted. This example uses a serum sample.

[0031] Plate coating: After disinfecting the unopened and undamaged 96-well cell culture plate with alcohol spray, place it in a laminar flow hood. Unpack the plate inside the laminar flow hood, pour the adhesion reagent working solution into the sample tank, and use a pipette to add 60 μL to each well of the 96-well cell culture plate. Gently shake and check the plate to ensure that the adhesion reagent working solution completely covers the bottom of the well. Incubate in a 35-37℃ incubator for 10-14 hours. The 60 μL is the amount to prevent volatilization for multiple days of storage. The actual amount of adhesion reagent added should be determined according to the needs, ensuring that the wells are not dry when used.

[0032] Pretreatment of cell culture plates with adherent reagent working solution enhances the adhesion between cells and the cell culture plate surface, preventing live cells from detaching during subsequent operations and ensuring the stability of the detection system.

[0033] Step 2: Cell passage: Take the culture medium containing HEK293T human embryonic kidney cells, rinse with phosphate buffer and discard the original culture medium. Add 1 ml of trypsin digestion solution and gently shake for 1.5-3 min to ensure the trypsin digestion solution completely contacts the cell layer of HEK293T human embryonic kidney cells. Collect the cell suspension into a 15 mL centrifuge tube and place it in a centrifuge. Set the centrifuge speed to 1000 rpm, the temperature to 4℃, and the centrifugation time to 3-5 min. Discard the supernatant in the centrifuge tube to obtain the cell pellet. Add 1 ml of culture medium from the complete culture medium to the centrifuge tube to resuspend the cell pellet. Disperse it into a uniform resuspension by pipetting. Add 20-25 mL of complete culture medium to a 100 mm culture dish, then add the resuspension to the 100 mm culture dish, shake well, and place in a 35-37℃, 5% CO2 incubator for later use. The required volume of the resuspended solution is as follows: 480-500 μL for 24 hours of incubation, 200-220 μL for 48 hours of incubation, and 150-180 μL for 96 hours of incubation.

[0034] Step 3: Cell plating: Determine the plating ratio based on cell growth and pre-plating density. If plating two plates is required, use a 1:8.75 plating ratio, adding 2.25 plates of complete culture medium and cell volume. Add approximately 22.5 mL of complete culture medium and 257 μL of resuspension to the sample loading well, discard the adhesion reagent working solution in the cell culture plate, add 100 μL of diluted resuspension to each well of the cell culture plate, and incubate at 35-37℃ in a 5% CO2 incubator to obtain a cell culture plate coated with HEK293T human embryonic kidney cells.

[0035] Step 4: Cell transfection After 16 hours of cell seeding and incubation, transfection was performed. Two solutions, A and B, were prepared: Solution A contained Opti-mem, the expression plasmid, and P3000; Solution B contained Opti-mem and Lipo-3000. Solution A was prepared in microcentrifuge tubes, and Solution B in 15 mL centrifuge tubes. The solutions were mixed, and Solution A was added to Solution B and allowed to stand to obtain a mixed transfection reagent. This mixed transfection reagent was then mixed with complete culture medium and added to cell culture plates already seeded with HEK293T human embryonic kidney cells. The plates were incubated for 24 hours. After transfection, immunofluorescence staining was used to detect anti-myelin oligodendrocyte glycoprotein IgG antibodies. The volume ratio of the expression plasmid, P3000, and Lipo-3000 was 1:1.5:1.5–1:3:3. When the volume ratio of expression plasmid, P3000, and Lipo-3000 is 1:1.5:1.5, the cell transfection efficiency is 30-40%; when the volume ratio is 1:2:2, the cell transfection efficiency is 70-80%; when the volume ratio is 1:3:3, the cell transfection efficiency is 30-40%; when the volume ratio is 1:1:1, the cell transfection efficiency is 10-15%; and when the volume ratio is 1:4:4, the cell transfection efficiency is 10-12%. In summary, the cell transfection efficiency is relatively high when the volume ratio of expression plasmid, P3000, and Lipo-3000 is 1:1.5:1.5-1:3:3, and the cell transfection efficiency is optimal when the volume ratio is 1:2:2. When the volume ratio is greater than or less than this range, the cell transfection efficiency will be greatly reduced, resulting in the failure of subsequent fluorescence staining detection.

[0036] Step 5: Detect anti-myelin oligodendrocyte glycoprotein IgG antibody: A1: Place the cells immobilized with the expression plasmid in a blank 96-well cell culture plate and allow them to warm to 22-28℃. A2: After rewarming, add 60uL of phosphate buffer to each well of the cell culture plate, soak for 3-6 minutes, and then remove the phosphate buffer from the wells. A3: Add 60 μL of serum sample to each well of the cell culture plate, incubate at 35-37°C for 50-70 min, and then aspirate the serum sample from the well; the incubation time of 60 min is optimal. A4: Add 60uL of phosphate buffer to each well of the cell culture plate, place it on a horizontal shaker at an appropriate speed and wash for 3-6 minutes. Remove the phosphate buffer from the wells and repeat the washing process 3 times. A5: Add 60 μL of diluted Alexa Fluor-labeled goat anti-human IgG to each well of the cell culture plate, incubate at 37°C in the dark for 30-45 min, and aspirate the Alexa Fluor-labeled goat anti-human IgG from the wells; A6: Add 50-100 uL of phosphate buffer to each well of the cell culture plate, place it on a horizontal shaker at an appropriate speed and wash for 3-6 minutes. Remove the phosphate buffer from the wells and repeat the washing process 3 times. A7: Add 60 μL of phosphate buffer to each well of the cell culture plate and observe the color development using a fluorescence microscope to determine the presence of anti-myelin oligodendrocyte glycoprotein IgG antibodies. Fluorescent color development indicates the presence of anti-myelin oligodendrocyte glycoprotein IgG antibodies in the serum sample; no fluorescent color development indicates the absence of anti-myelin oligodendrocyte glycoprotein IgG antibodies in the serum sample.

[0037] Obviously, the above embodiments are merely examples for clear illustration and are not intended to limit the implementation. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all implementation methods here, and the obvious variations or modifications derived therefrom are still within the protection scope of this invention.

Claims

1. The application of a recombinant antigen transfection live-cell immunofluorescence assay in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies, characterized in that, Includes the following steps: A plasmid for expressing myelin oligodendrocyte glycoprotein was constructed. The expression plasmid vector was pcdna3.1-target gene-linker-×3Flag-P2A-EGFP. The amino acid sequence of the expression plasmid gene is shown in SEQ ID NO:1, and the base sequence of the expression plasmid gene is shown in SEQ ID NO:

2. After cell transfection, the expression plasmid entered the target cells. After culturing for 16-28 hours, cells with the expression plasmid fixed were obtained and could be used for immunofluorescence staining to detect anti-myelin oligodendrocyte glycoprotein IgG antibodies. Cell transfection includes the following steps: Prepare solutions A and B. Solution A contains Opti-mem, expression plasmid, and P3000, while solution B contains Opti-mem and Lipo-3000. Mix solution A with solution B and let stand to obtain a mixed transfection reagent. Mix the mixed transfection reagent with complete culture medium and add it to a cell culture plate that has been seeded with target cells. Incubate in an incubator for 16-28 hours to obtain myelin oligodendrocyte glycoprotein fixed on the cells. The volume ratio of expression plasmid, P3000 and Lipo-3000 was 1:1.5:1.5-1:3:3; The application is based on non-diagnostic purposes.

2. The application of the recombinant antigen transfection live-cell immunofluorescence assay as described in claim 1 in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibody, characterized in that, The detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies includes the following steps: A1: Take out the phosphate buffer and fluorescently labeled goat anti-human IgG, and after standing and equilibrating to 22-28℃, dilute the fluorescently labeled goat anti-human IgG. The ratio of fluorescently labeled goat anti-human IgG to phosphate buffer is 1:300-1:

800. A2: Place the cells immobilized with the expression plasmid in a blank cell culture plate and allow them to warm to 22-28℃. A3: After rewarming, add 60uL of phosphate buffer to each well of the cell culture plate, soak for 3-6 minutes, and then remove the phosphate buffer from the wells. A4: Add 60uL of sample to each well of the cell culture plate, incubate at 35-37℃ for 50-70 min, and then aspirate the sample from the well. A5: Add 60 μL of phosphate buffer to each well of the cell culture plate, place it in a horizontal shaker and wash for 3-6 minutes. Remove the phosphate buffer from the wells and repeat the washing process 3 times. A6: Add 60 μL of diluted fluorescently labeled goat anti-human IgG to each well of the cell culture plate, incubate at 37°C in the dark for 30-45 min, and remove the fluorescently labeled goat anti-human IgG from the well. A7: Add 50-100uL of phosphate buffer to each well of the cell culture plate, place it in a horizontal shaker and wash for 3-6 minutes. Remove the phosphate buffer from the wells and repeat the washing process 3 times. A8: Add 60 μL of phosphate buffer to each well of the cell culture plate and observe the color development using a fluorescence microscope to determine the presence of anti-myelin oligodendrocyte glycoprotein IgG antibody. Fluorescence indicates the presence of anti-myelin oligodendrocyte glycoprotein IgG antibody in the sample; no fluorescence indicates the absence of anti-myelin oligodendrocyte glycoprotein IgG antibody in the sample. The samples used are either serum or cerebrospinal fluid. Serum samples need to be diluted, with a serum-to-phosphate buffer ratio of 1:

10.

3. The application of the recombinant antigen transfection live-cell immunofluorescence assay as described in claim 1 in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibody, characterized in that, Before cell transfection, the cells are pretreated, which includes the following steps: S1: Add the target cells to the complete culture medium, collect the cell suspension and centrifuge, remove the supernatant to obtain the cell pellet, add the culture medium in the complete culture medium to the cell pellet, resuspend the cell pellet, and blow and aspirate until the cells are dispersed to obtain the resuspension. S2: Add 20-25 ml of complete culture medium and resuspension to a 100 mm petri dish, shake well, and incubate in a 35-37℃, 5% CO2 incubator. S3: Add diluted cell suspension to each well of the cell culture plate and incubate in a 35-37℃, 5% CO2 incubator to obtain a cell culture plate containing the target cells.

4. The application of the recombinant antigen transfection live-cell immunofluorescence assay as described in claim 3 in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibody, characterized in that, The complete culture medium in S1 consists of DMEM medium, fetal bovine serum that has been inactivated in a 56°C water bath for 30 min and filtered through a 0.22 μm filter membrane, and a penicillin-streptomycin-amphotericidal mixture in a volume ratio of 43:7:1 to 43:12:

1.

5. The application of the recombinant antigen transfection live-cell immunofluorescence assay as described in claim 3 in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibody, characterized in that, The specific steps for collecting and centrifuging the cell suspension in S1 are as follows: Use a dropper to collect the cell suspension into a 15mL centrifuge tube and place it in a centrifuge. Set the centrifuge speed to 1000rpm, the temperature to 4℃, and the centrifugation time to 3-5min.

6. The application of the recombinant antigen transfection live-cell immunofluorescence assay as described in claim 3 in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibody, characterized in that, The required volume of resuspension in S2 is as follows: 480-500 μL for 24 hours of incubation, 200-220 μL for 48 hours of incubation, and 150-180 μL for 96 hours of incubation.