Recombinant antigen and application of transfected living cell immunofluorescence method thereof in detection of anti-myelin oligodendroglia glycoprotein IgG antibody

Abstract

The invention relates to the technical field of antibody detection, in particular to a recombinant antigen and application of a transfected living cell immunofluorescence method of the recombinant antigen in anti-myelin oligodendroglia glycoprotein IgG antibody detection. The method comprises the following steps: constructing a myelin sheath oligodendroglia glycoprotein expression plasmid, with an expression plasmid vector being pcdna3.1-target gene-linker-* 3Flag-P2A-EGFP, an amino acid sequence of the expression plasmid gene being as shown in SEQ ID NO: 1, a base sequence of the expression plasmid gene being as shown in SEQ ID NO: 2, carrying out cell transfection to make the expression plasmid enter a target cell, culturing for 16-28 h to obtain a cell immobilized with the expression plasmid, and purifying the cell to obtain the myelin sheath oligodendroglia glycoprotein. The kit can be used for detecting the anti-myelin oligodendroglia glycoprotein IgG antibody by an immunofluorescence staining method. The problems of antigen spatial conformation change, epitope shielding or cell shrinkage of cells are solved, and the detection sensitivity is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody detection, in particular to an application of a recombinant antigen and a transfected living cell immunofluorescence method thereof in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies. Background Art

[0002] Myelin sheaths in the central nervous system (CNS) are formed by oligodendrocytes, which facilitate the efficient, saltatory conduction of neural electrical signals and protect the normal function of neurons. Inflammatory demyelinating diseases of the CNS are a class of diseases in which myelin is damaged and lost due to various pathological factors, while nerve cells remain relatively intact. Common inflammatory demyelinating diseases of the CNS include multiple sclerosis (MS), neuromyelitis optica spectrum disorder (NMOSD), and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), each of which has relatively independent clinical features. Autoantibodies play a crucial role in the diagnosis and differential diagnosis of these diseases.

[0003] Anti-myelin oligodendrocyte glycoprotein IgG antibodies (anti-MOG-IgG antibodies) are autoimmune IgG antibodies directed against myelin oligodendrocyte glycoprotein (MOG) in the central nervous system and are associated with a variety of demyelinating diseases of the central nervous system, such as NMOSD and MOGAD. According to the "Expert Consensus on Antibody Detection Related to Central Nervous System Autoimmune Diseases 2022," the positive rate for MOG antibodies is 7.4%, which is of great value for the diagnosis of MOGAD. They are often found in patients with optic neuritis or myelitis but are rarely found in classic MS. Their serum titer depends on disease activity and treatment status. During long-term follow-up, the serum anti-MOG-IgG antibody titer of patients with NMOSD or MS often fluctuates around the original level and then increases again.

[0004] According to expert consensus, current methods for detecting CNS autoantibodies include cell-based immunofluorescence (CBA), indirect immunofluorescence (IIF), fluorescence immunoprecipitation assay (FIPA), and enzyme-linked immunosorbent assay (ELISA). CBA has the advantages of preserving antigen conformation and protein modifications, more accurately reflecting the characteristics of antigen-antibody binding. Its sensitivity (over 70%) and specificity (97%-100%) are both higher than those of these other methods, making it suitable for clinical adjunctive testing. Existing CBA assays for anti-MOG-IgG antibodies are mostly designed using cells after antigen fixation. These methods construct a plasmid expressing anti-myelin oligodendrocyte glycoprotein (MOG antigen) and transfect the cells. Antigen fixation is then used to provide antigen-overexpressing cells that are stable for binding to anti-MOG-IgG antibodies in the test sample. The antigen-bound anti-MOG-IgG antibodies are then labeled with a secondary antibody carrying a fluorescent protein, thereby confirming the presence of anti-MOG-IgG antibodies in the test sample.

[0005] The existing anti-MOG-IgG antibody detection method for overexpressed antigen cells is based on the design of cells after antigen fixation. The fixation method is mostly paraformaldehyde or acetone fixation, which can cause changes in the spatial conformation of the antigen, epitope blocking or cell shrinkage, affecting the sensitivity of the detection. Summary of the Invention

[0006] In order to solve the problems in the related art, the present invention provides a recombinant antigen and its transfected live cell immunofluorescence method for the detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies, which solves the problems of antigen spatial conformation changes, epitope shielding or cell shrinkage in cells, and improves the sensitivity of detection.

[0007] To solve the above problems, the following technical solutions are provided: The present invention discloses a recombinant antigen and its application in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies by live cell immunofluorescence method. A myelin oligodendrocyte glycoprotein expression plasmid 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 of the expression plasmid gene is shown in SEQ ID NO: 2. After cell transfection, the expression plasmid enters the target cells. After culturing for 16-28 hours, cells fixed with the expression plasmid are obtained, which can be used for the detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies by immunofluorescence staining.

[0008] By adopting the above scheme, a pcDNA3.1-(target gene)-linker-×3Flag-P2A-EGFP expression plasmid vector was constructed, and the promoter was used to drive the expression of the target gene (MOG antigen), ensuring efficient transcription and translation of the antigen in the transfected cells, thereby achieving high-quality expression of the antigen.

[0009] The introduction of the ×3Flag tag into the expression plasmid allows the specific recognition and binding of the antigen by the anti-Flag antibody, which not only facilitates the localization and verification of antigen expression in the cell, but can also be used for antigen purification, thereby improving the specificity and operability of the detection.

[0010] P2A self-cleaving peptide is used to connect the target gene (MOG antigen) and EGFP (enhanced green fluorescent protein), allowing them to be expressed independently in the same transcript without affecting their spatial conformation and function. EGFP is used as a reporter gene, and the cell transfection efficiency can be directly observed by fluorescence microscopy, and the expression of the expression plasmid in the cell can be monitored in real time to ensure the reliability of the detection.

[0011] This expression plasmid vector solves the problems of insufficient antigen expression and epitope exposure in fixed cell detection through multiple advantages such as an efficient expression system, tagging functional design, retention of natural antigen conformation, and easy operation. The present invention achieves high-quality and efficient expression of antigens by independently constructing expression plasmids for transfection. After transfection, the cells are not fixed and immunofluorescence staining is directly performed on living cells, which can improve detection sensitivity and effectively avoid changes in the spatial conformation of the antigen, epitope shielding, or cell shrinkage.

[0012] Cell transfection involves the following steps: Prepare solution A and solution B, where solution A contains Opti-mem, an expression plasmid, and P3000, and solution B contains Opti-mem and Lipo-3000. Add solution A to solution B, mix, and let stand to obtain a mixed transfection reagent. Mix the mixed transfection reagent with complete culture medium, add the mixture to a cell culture plate already plated with target cells, and incubate in an incubator for 16-28 hours to obtain myelin oligodendrocyte glycoprotein fixed on the cells. Among them, the volume ratio of expression plasmid, P3000 and Lipo-3000 is 1:1.5:1.5-1:3:3.

[0013] Detection of anti-MYOG IgG antibodies involves the following steps: A1: Take out the phosphate buffer and fluorescent-labeled goat anti-human IgG, let them stand and equilibrate to 22-28°C, and then dilute the fluorescent-labeled goat anti-human IgG to a ratio of 1:300-1:800. A2: Place the cells immobilized with the expression plasmid in a blank cell culture plate and allow to stand and rewarm to 22-28°C; A3: After rewarming, add 60 μL of phosphate buffer to each well of the cell culture plate. After soaking for 3-6 minutes, remove the phosphate buffer from the wells. A4: Add 60 μL of sample to each well of the cell culture plate, incubate at 35-37°C for 50-70 minutes, and then remove the sample from the well; A5: Add 60 μL of phosphate buffer to each well of the cell culture plate, wash on a horizontal shaker for 3-6 minutes, remove the phosphate buffer from the well, and repeat the wash three 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 minutes, and remove the fluorescently labeled goat anti-human IgG from the wells by aspiration. A7: Add 50-100 μL of phosphate buffer to each well of the cell culture plate, wash on a horizontal shaker for 3-6 minutes, remove the phosphate buffer from the well, and repeat the wash three times; A8: Add 60 μL of phosphate buffered saline to each well of the cell culture plate and observe the color development using a fluorescence microscope to determine whether anti-MYOG IgG antibodies are present. Fluorescence color development indicates the presence of anti-MYOG IgG antibodies in the sample; absence of fluorescence color development indicates the absence of anti-MYOG IgG antibodies in the sample. Among them, the samples are serum samples or cerebrospinal fluid samples. The serum samples need to be diluted, and the ratio of serum sample to phosphate buffer is 1:10.

[0014] Before cell transfection, cells were pretreated. The pretreatment included the following steps: S1: Add target cells to complete culture medium, collect the cell suspension and centrifuge, remove the supernatant to obtain a cell pellet, add the culture medium in complete culture medium to the cell pellet, resuspend the cell pellet until the cells are dispersed, and obtain a resuspension; S2: Add 20-25 ml of complete medium and resuspension solution to a 100 mm culture dish, shake well, and incubate in a 35-37°C, 5% CO2 incubator. S3: Add the diluted cell suspension to each well of the cell culture plate and incubate in a 35-37°C, 5% CO2 incubator to obtain a cell culture plate containing target cells.

[0015] By adopting this protocol, the complete culture medium provides sufficient nutrients to the cells and inhibits contamination, maintaining normal physiological functions in the living cell state and ensuring the correct folding and surface expression of the antigen. By omitting the fixation step used in existing techniques, immunofluorescence staining can be performed directly on living cells, avoiding cell shrinkage or epitope destruction caused by fixatives. This method maximizes the preservation of the antigen's native conformation, enabling the anti-MOG-IgG antibody to bind more fully and specifically to the target antigen, thereby improving detection sensitivity. Direct detection after transfection simplifies the operational steps, 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 is composed of DMEM medium, fetal bovine serum inactivated in a 56°C water bath for 30 min and filtered through a 0.22 μm filter membrane, and a mixture of penicillin-streptomycin-amphotericin in a volume ratio of 43:7:1-43:12:1.

[0017] The specific steps of collecting the cell suspension and centrifuging in S1 are as follows: using a dropper to collect the cell suspension into a 15 mL centrifuge tube and placing it in a centrifuge, setting the centrifuge speed to 1000 rpm, the temperature to 4° C., and the centrifugation duration to 3-5 min.

[0018] The volume requirements of the resuspension in S2 are as follows: 480-500 μL of resuspension is taken after 24 h of incubation, 200-220 μL of resuspension is taken after 48 h of incubation, and 150-180 μL of resuspension is taken after 96 h of incubation.

[0019] The above solution has the following advantages: The present invention discloses a recombinant antigen and its application in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies using a live cell immunofluorescence method. The invention constructs a pcDNA3.1-(target gene)-linker-×3Flag-P2A-EGFP expression plasmid vector and utilizes a promoter to drive the expression of the target gene (MOG antigen), thereby ensuring efficient transcription and translation of the antigen in transfected cells and achieving high-quality expression of the antigen. The ×3Flag tag introduced into the expression plasmid enables specific recognition and binding of the antigen by an anti-Flag antibody, which not only facilitates intracellular localization and verification of antigen expression but also can be used for antigen purification, thereby improving the specificity and operability of the detection. The P2A self-cleaving peptide is used to link the target gene (MOG antigen) and EGFP (enhanced green fluorescent protein), allowing the two to be independently expressed in the same transcript without affecting their spatial conformation and function. EGFP is used as a reporter gene, allowing direct observation of cell transfection efficiency using a fluorescence microscope and real-time monitoring of the expression of the expression plasmid in cells, thereby ensuring the reliability of the detection.

[0020] This expression plasmid vector has multiple advantages such as an efficient expression system, tagging function design, retention of natural antigen conformation and easy operation. The present invention achieves high-quality and high-efficiency expression of antigens by independently constructing expression plasmids for transfection. After transfection, cells are not fixed and immunofluorescence staining is directly performed on living cells, which can improve detection sensitivity and effectively avoid changes in the spatial conformation of antigens, epitope shielding or cell shrinkage in cells. DETAILED DESCRIPTION

[0021] The following will be combined with specific embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] A recombinant antigen of the present invention and its transfection live cell immunofluorescence method are used in the 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-×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 enters the cell. After culturing for 16-28 hours, cells fixed with the expression plasmid are obtained, which can be used for immunofluorescence staining to detect anti-myelin oligodendrocyte glycoprotein IgG antibodies. The ×3Flag tag is introduced into the expression plasmid, which can be specifically recognized and bound to the antigen by anti-Flag antibodies, which is not only convenient for locating and verifying antigen expression in the cell, but also can be used for antigen purification, thereby improving the specificity and operability of the detection. The P2A self-cleaving peptide is used to connect the target gene (MOG antigen) and EGFP (enhanced green fluorescent protein), allowing them to be expressed independently in the same transcript without affecting their spatial conformation and function. EGFP is used as a reporter gene, and the cell transfection efficiency can be directly observed by fluorescence microscopy. The expression of the expression plasmid in the cell can be monitored in real time to ensure the reliability of the detection.

[0023] This expression plasmid vector solves the problems of insufficient antigen expression and epitope exposure in fixed cell detection through multiple advantages such as an efficient expression system, tagging functional design, retention of natural antigen conformation, and easy operation. The present invention achieves high-quality and efficient expression of antigens by independently constructing expression plasmids for transfection. After transfection, the cells are not fixed and immunofluorescence staining is directly performed on living cells, which can improve detection sensitivity and effectively avoid changes in the spatial conformation of the antigen, epitope shielding, or cell shrinkage.

[0024] The present application embodiment is a recombinant antigen and its transfected live cell immunofluorescence method for detecting anti-myelin oligodendrocyte glycoprotein IgG antibodies; Step 1: Raw material preparation and pretreatment The enhanced cell attachment reagent and phosphate buffer solution are mixed at a volume ratio of 1:30-1:100 to obtain an attachment reagent working solution. The optimal ratio is 1:50, which has sufficient adsorption effect and is beneficial to saving reagents.

[0025] Complete culture medium was obtained by mixing DMEM medium, fetal bovine serum inactivated in a 56°C water bath for 30 min and filtered through a 0.22 μm filter membrane, and a mixture of penicillin-streptomycin-amphotericin at a volume ratio of 43:7:1 to 43:12:1. The optimal ratio was 43:7:1, which could provide sufficient nutrition for cells to maintain a reasonable growth rate and save reagents.

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

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

[0028] HEK293T human embryonic kidney cells and human cervical cancer cells are selected as target cells. This example takes HEK293T human embryonic kidney cells as an example.

[0029] For fluorescent labeling, use Alexa Fluor. Dilute Alexa Fluor-labeled goat anti-human IgG to phosphate buffer at a ratio of 1:300-1:800. A ratio higher than this may result in over-staining, resulting in high background fluorescence that affects judgment. A ratio lower than this may result in too light staining to be easily observed. The optimal ratio of Alexa Fluor-labeled goat anti-human IgG to phosphate buffer is 1:500, which provides the best observation effect under a fluorescence microscope.

[0030] The sample can be a serum sample or a cerebrospinal fluid sample. The serum sample needs to be diluted, and the ratio of serum sample to phosphate buffer is 1:10. The cerebrospinal fluid sample does not need to be diluted. This embodiment takes the serum sample as an example.

[0031] Plate coating: Spray the unopened 96-well cell culture plate with alcohol for disinfection and place it in the clean bench. Unpack it in the clean bench and pour the adhesion reagent working solution into the sample injection groove. Use a pipette to add 60μL to each well of the 96-well cell culture plate. Gently shake the plate and check to ensure that the adhesion reagent working solution can completely cover the bottom of the well. Incubate in a 35-37℃ incubator for 10-14h. Among them, 60μL is the anti-evaporation dose for multi-day storage. The actual amount of adhesion reagent added should be determined according to needs, and the wells should not be dry when used.

[0032] Pre-treating the cell culture plate with the adhesion reagent working solution enhances the adhesion of cells to the cell culture plate surface, prevents living cells from falling off during subsequent operations, and ensures the stability of the detection system.

[0033] Step 2: Cell passaging: 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, shake gently for 1.5-3 minutes to allow the trypsin digestion solution to completely contact the cell layer of HEK293T human embryonic kidney cells, collect the cell suspension with a dropper into a 15 mL centrifuge tube and place it in a centrifuge, set the centrifuge speed to 1000 rpm, the temperature to 4°C, and the centrifugation duration to 3-5 minutes, pour out the supernatant in the centrifuge tube to obtain a cell pellet, add 1 ml of culture medium in the centrifuge tube, resuspend the cell pellet, and disperse it into a uniform resuspension by pipetting; add 20-25 mL of complete culture medium to a 100 mm culture dish, then take the resuspension and add it to the 100 mm culture dish, shake well, and place it in a 35-37°C, 5% CO2 incubator for standby use; The volume requirements for the resuspension are as follows: 480-500 μL of resuspension is taken after 24 hours of incubation, 200-220 μL of resuspension is taken after 48 hours of incubation, and 150-180 μL of resuspension is taken after 96 hours of incubation.

[0034] Step 3: Cell plating: Determine the plating ratio based on cell growth and pre-plating density. If two plates are required, use a plating ratio of 1:8.75, add 2.25 plates of complete medium and cells, and evenly distribute 257 μL of the resuspension to approximately 22.5 mL of complete medium in the sample reservoir. Discard the attachment reagent working solution in the cell culture plate and add 100 μL of the diluted resuspension to each well of the cell culture plate. Incubate in a 35-37°C, 5% CO2 incubator to obtain a cell culture plate containing HEK293T human embryonic kidney cells.

[0035] Step 4: Cell transfection: After the cells were plated and incubated for 16 hours, transfection was performed. Liquid A and liquid B were prepared separately. Liquid A contained Opti-mem, expression plasmid and P3000, and liquid B contained Opti-mem and Lipo-3000. Liquid A was prepared in a microcentrifuge tube, and liquid B was prepared in a 15mL centrifuge tube. Mix them evenly, add liquid A to liquid B, mix them, and let them stand to obtain a mixed transfection reagent. The mixed transfection reagent was mixed with complete culture medium and added to a cell culture plate with HEK293T human embryonic kidney cells. The plate was placed in an incubator and incubated for 24 hours. After transfection and incubation, anti-myelin oligodendrocyte glycoprotein IgG antibodies can be detected by immunofluorescence staining. Among them, the volume ratio of expression plasmid, P3000 and Lipo-3000 is 1:1.5:1.5-1:3:3. When the volume ratio of expression plasmid, P3000 and Lipo-3000 was 1:1.5:1.5, the cell transfection efficiency was 30-40%; when the volume ratio of expression plasmid, P3000 and Lipo-3000 was 1:2:2, the cell transfection efficiency was 70-80%; when the volume ratio of expression plasmid, P3000 and Lipo-3000 was 1:3:3, the cell transfection efficiency was 30-40%; when the volume ratio of expression plasmid, P3000 and Lipo-3000 was 1:1:1, the cell transfection efficiency was 10-15%; when the volume ratio of expression plasmid, P3000 and Lipo-3000 was 1:4:4, the cell transfection efficiency was 10-12%; In summary, when the volume ratio of expression plasmid, P3000 and Lipo-3000 is 1:1.5:1.5-1:3:3, the cell transfection efficiency is high, and when the volume ratio of expression plasmid, P3000 and Lipo-3000 is 1:2:2, the cell transfection efficiency is optimal; 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 fluorescent staining detection.

[0036] Step 5: Detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies: A1: Place cells immobilized with expression plasmids into a blank 96-well cell culture plate and allow to warm to 22-28°C. A2: After rewarming, add 60 μL of phosphate buffer to each well of the cell culture plate, soak for 3-6 minutes, and then remove the phosphate buffer from the well; A3: Add 60 μL of serum sample to each well of the cell culture plate. Incubate at 35-37°C for 50-70 minutes, then remove the serum sample from the well. 60 minutes is the optimal incubation time. A4: Add 60 μL of phosphate buffer to each well of the cell culture plate, place on a horizontal shaker at an appropriate speed and wash for 3-6 minutes, remove the phosphate buffer from the well, and repeat the wash three 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 minutes, and remove the Alexa Fluor-labeled goat anti-human IgG from the wells; A6: Add 50-100 μL of phosphate buffer to each well of the cell culture plate, place on a horizontal shaker at an appropriate speed and wash for 3-6 minutes, remove the phosphate buffer from the well, and repeat the wash three times; A7: Add 60uL of phosphate buffer to each well of the cell culture plate and observe under a fluorescence microscope to check the color development and determine whether anti-MYOG IgG antibodies are present. Fluorescence color development indicates the presence of anti-MYOG IgG antibodies in the serum sample; no fluorescence color development indicates the absence of anti-MYOG IgG antibodies in the serum sample.

[0037] Obviously, the above embodiments are merely examples for clear explanation and are not limitations on the implementation methods. For ordinary technicians in the field, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation methods here, and the obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A recombinant antigen and its transfected live cell immunofluorescence method in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies, characterized in that: The following steps are involved: A myelin oligodendrocyte glycoprotein expression plasmid was constructed. The expression plasmid vector was pcDNA3.1-target gene-linker-×3Flag-P2A-EGFP. The amino acid sequence of the expression plasmid gene was shown in SEQ ID NO: 1, and the base sequence of the expression plasmid gene was shown in SEQ ID NO:

2. Cells were transfected to allow the expression plasmid to enter the target cells. After culturing for 16-28 hours, cells with the expression plasmid fixed were obtained, which could be used for detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies by immunofluorescence staining.

2. The use of a recombinant antigen and its transfected living cell immunofluorescence method in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies according to claim 1, characterized in that: Cell transfection involves the following steps: Prepare solution A and solution B, where solution A contains Opti-mem, an expression plasmid, and P3000, and solution B contains Opti-mem and Lipo-3000. Add solution A to solution B, mix, and let stand to obtain a mixed transfection reagent. Mix the mixed transfection reagent with complete culture medium, add the mixture to a cell culture plate already plated with target cells, and incubate in an incubator for 16-28 hours to obtain myelin oligodendrocyte glycoprotein fixed on the cells. Among them, the volume ratio of expression plasmid, P3000 and Lipo-3000 is 1:1.5:1.5-1:3:

3.

3. The use of a recombinant antigen and its transfected living cell immunofluorescence method in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies according to claim 1, characterized in that: Detection of anti-MYOG IgG antibodies involves the following steps: A1: Take out the phosphate buffer and fluorescent-labeled goat anti-human IgG, let them stand and equilibrate to 22-28°C, and then dilute the fluorescent-labeled goat anti-human IgG to a ratio of 1:300-1:

800. A2: Place the cells immobilized with the expression plasmid in a blank cell culture plate and allow to stand and rewarm to 22-28°C; A3: After rewarming, add 60 μL of phosphate buffer to each well of the cell culture plate. After soaking for 3-6 minutes, remove the phosphate buffer from the wells. A4: Add 60 μL of sample to each well of the cell culture plate, incubate at 35-37°C for 50-70 minutes, and then remove the sample from the well; A5: Add 60 μL of phosphate buffer to each well of the cell culture plate, wash on a horizontal shaker for 3-6 minutes, remove the phosphate buffer from the well, and repeat the wash three 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 minutes, and remove the fluorescently labeled goat anti-human IgG from the wells by aspiration. A7: Add 50-100 μL of phosphate buffer to each well of the cell culture plate, wash on a horizontal shaker for 3-6 minutes, remove the phosphate buffer from the well, and repeat the wash three times; A8: Add 60 μL of phosphate buffered saline to each well of the cell culture plate and observe the color development using a fluorescence microscope to determine whether anti-MYOG IgG antibodies are present. Fluorescence color development indicates the presence of anti-MYOG IgG antibodies in the sample; absence of fluorescence color development indicates the absence of anti-MYOG IgG antibodies in the sample. Among them, the samples are serum samples or cerebrospinal fluid samples. The serum samples need to be diluted, and the ratio of serum sample to phosphate buffer is 1:

10.

4. The use of a recombinant antigen and its transfected living cell immunofluorescence method in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies according to claim 1, characterized in that: Before cell transfection, cells were pretreated. The pretreatment included the following steps: S1: Add target cells to complete culture medium, collect the cell suspension and centrifuge, remove the supernatant to obtain a cell pellet, add the culture medium in complete culture medium to the cell pellet, resuspend the cell pellet, and pipette until the cells are dispersed to obtain a resuspension; S2: Add 20-25 ml of complete medium and resuspension solution to a 100 mm culture dish, shake well, and incubate in a 35-37°C, 5% CO2 incubator. S3: Add the diluted cell suspension to each well of the cell culture plate and incubate in a 35-37°C, 5% CO2 incubator to obtain a cell culture plate containing target cells.

5. The use of a recombinant antigen and its transfected living cell immunofluorescence method in the detection of anti-myelin oligodendrocyte glycoprotein IgG antibodies according to claim 4, characterized in that: The complete culture medium in S1 is composed of DMEM medium, fetal bovine serum inactivated in a 56°C water bath for 30 min and filtered through a 0.22 μm filter membrane, and a mixture of penicillin-streptomycin-amphotericin in a volume ratio of 43:7:1-43:12:

1.

6. The use of a recombinant antigen and its transfected living cell immunofluorescence assay as claimed in claim 4 in detecting anti-myelin oligodendrocyte glycoprotein IgG antibodies, characterized in that: The specific steps of collecting the cell suspension and centrifuging in S1 are as follows: using a dropper to collect the cell suspension into a 15 mL centrifuge tube and placing it in a centrifuge, setting the centrifuge speed to 1000 rpm, the temperature to 4° C., and the centrifugation duration to 3-5 min.

7. The use of a recombinant antigen and its transfected living cell immunofluorescence assay as claimed in claim 4 in detecting anti-myelin oligodendrocyte glycoprotein IgG antibodies, characterized in that: The volume requirements of the resuspension in S2 are as follows: 480-500 μL of resuspension is taken after 24 h of incubation, 200-220 μL of resuspension is taken after 48 h of incubation, and 150-180 μL of resuspension is taken after 96 h of incubation.