Multi-subtype antigen co-transfected cell immunofluorescence staining method and application

Abstract

The invention belongs to the technical field of antibody detection, and particularly relates to a multi-subtype antigen co-transfected cell immunofluorescence staining method and application. The method is established for the purpose of a CBA detection method, the basic principle is similar to that of an overexpression antigen cell immunofluorescence method for existing anti-GFAP-IgG antibody detection, and the difference is that three GFAP subtypes are adopted for cotransfection design, antigen subtypes are covered, and the problem of leak detection is avoided. According to the invention, the high-quality expression of the antigen is realized by self-constructing the plasmid based on the pCDH lentiviral vector. According to the invention, by optimizing the schemes of fixation, transparent preservation and the like after cell transfection culture, the dyeing performance is enhanced, and the preservation effect is improved.

Description

Technical Field

[0001] This invention belongs to the field of antibody detection technology, and specifically relates to a multi-subtype antigen co-transfection cell immunofluorescence staining method and its application. Background Technology

[0002] Anti-GFAP antibodies are autoantibodies against glial fibrillary acidic protein (GFAP), primarily associated with autoimmune diseases of the central nervous system. These antibodies are commonly used clinically to aid in the diagnosis of autoimmune glial fibrillary acidic astrocytosis, in patients who may present with symptoms such as encephalitis, myelitis, or optic neuritis.

[0003] Existing GFAP-IgG antibody (anti-glial fibrillary acidic protein IgG antibody) overexpression antigen cellular immunofluorescence assay (CBA method) products are mostly designed based on a single GFAPα antigen subtype, and the plasmids used are mostly designed using pcDNA3.1 vectors. Most existing GFAP detection reagents or protocols use the CBA method, with a few using ELISA or flow cytometry. Currently, most commercially available CBA-based GFAP antigen expression cells only transfect and express a single GFAPα antigen subtype, as it is the full-length protein among the four GFAP subtypes. Furthermore, the constructed plasmid vectors are mostly pcDNA3.1 vectors to achieve high protein expression levels and speeds. The cells used for transfection are mostly human embryonic kidney cell lines (HEK293 or 293T) or cervical cancer cell lines (HeLa). The general technical approach is as follows: transfect the constructed pcDNA3.1 antigen carrying the GFAPα expression sequence into cells, allow sufficient expression time, then fix and preserve the antigen, and finally use these cells to stain the test sample using CBA. The disadvantages of this technical approach are as follows: 1. Even with only GFAPα protein antigen, there may be situations where the antigen epitope is folded and blocked, resulting in limited detection sensitivity and a certain number of missed detections.

[0004] 2. The use of pcDNA3.1 vector may also lead to non-specific staining that is difficult to distinguish in individual samples or to all samples showing positive results in antibody tests.

[0005] 3. As a cytoplasmic protein, GFAP staining morphology is often insufficient even after permeabilization with reagents such as Triton X-100. It is common for membrane luminescence and intracytoplasmic filamentous luminescence to occur simultaneously. Summary of the Invention

[0006] This invention proposes a method that solves the problems of missed detection caused by the limited detection sensitivity due to the possibility of antigen epitope folding and obscuring of GFAPα protein antigen alone; the problem of non-specific staining that is difficult to distinguish in individual samples or the problem of all antibody tests showing positive results due to the use of pcDNA3.1 vector; and the problem that GFAP, as a cytoplasmic protein, can exhibit both membrane luminescence and intracytoplasmic filamentous luminescence simultaneously.

[0007] The technical solution of this invention is implemented as follows: A multi-subtype antigen co-transfection cell immunofluorescence staining method was developed to construct plasmids of three GFAP subtypes, followed by cell culture and transfection to obtain HEK293T cells that simultaneously expressed GFAPα, GFAPκ, and GFAPε. The three GFAP subtypes are GFAPα, GFAPκ, and GFAPε. The complete plasmid vector for GFAPα is pCDH-CMV-GFAPα-linker-X3FLAG-P2A-EGFP-AmpR. The antigenic amino acid sequence of the GFAPα plasmid is shown in SEQ ID NO:1, and the base sequence of the GFAPα plasmid is shown in SEQ ID NO:2. The complete plasmid vector for GFAPκ is pCDH-CMV-GFAPκ-linker-X3FLAG-P2A-EGFP-AmpR. The antigenic amino acid sequence of the GFAPκ plasmid is shown in SEQ ID NO:3, and the base sequence of the GFAPκ plasmid is shown in SEQ ID NO:4. The complete plasmid vector for GFAPε is pCDH-CMV-GFAPε-linker-X3FLAG-P2A-EGFP-AmpR. The antigenic amino acid sequence of the GFAPε plasmid is shown in SEQ ID NO:5, and the base sequence of the GFAPε plasmid is shown in SEQ ID NO:6.

[0008] Optional, the specific steps for cell culture are as follows: (1) Preparation of complete culture medium: DMEM medium, fetal bovine serum inactivated by 56℃ water bath and filtered through 0.22μm filter membrane, and penicillin-streptomycin-amphoteric acid mixture, the volume ratio of these three reagents is 43:7:1-43:12:1; (2) Take HEK293T 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 in the complete culture medium to the cell pellet and resuspend the cell pellet until the cells are dispersed to obtain the resuspension. (3) Add complete culture medium and resuspension to the petri dish, shake well, and incubate in a 35-37℃, 5% CO2 incubator; (4) 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 with HEK293T cells.

[0009] Optionally, the specific steps for cell transfection are as follows: Prepare transfection solution A and solution B. Solution A contains DMEM medium and three GFAP expression plasmids, and solution B contains DMEM medium and PEI transfection reagent. Mix solution A with solution B to obtain a mixed transfection reagent. Mix the mixed transfection reagent with complete medium and add it to a cell culture plate that has been seeded with HEK293T cells. Incubate the plate in an incubator to obtain HEK293T cells that simultaneously express GFAPα, GFAPκ, and GFAPε. The volume ratio of GFAPα, GFAPκ, GFAPε and PEI is 2:1:1:3-2:1.5:1.5:5.

[0010] Optionally, after cell transfection, fixation, sealing, permeabilization, and preservation procedures are required. The specific steps are as follows: (1) Prepare PFA fixative, antigen retrieval solution, blocking solution, permeation solution and preservation solution; (2) Remove the cells to be preserved and discard the original culture medium; (3) Add 1×PBS solution to wash, let stand for 2-5 minutes, and discard the PBS; (4) Add PFA fixative, let stand for 20-40 minutes, and discard the PFA; (5) Add 1×PBS solution to wash, let stand for 2-5 minutes, and discard the PBS; (6) Add antigen retrieval solution, let stand for 15-25 minutes, then discard the retrieval solution; (7) Add blocking solution, let stand for 40-70 minutes, then discard the blocking solution; (8) Add permeation solution, let stand for 15-20 minutes, then discard the permeation solution; (9) Add the preservative solution, let stand for 20-40 minutes, then discard the preservative solution; (10) After opening the lid, let it stand for 20-30 minutes to allow the reagent to air dry, then store at -20 degrees Celsius.

[0011] Optionally, the PFA fixative is 2-6% PFA dissolved in 1×PBS solution; the antigen retrieval solution is 10-30mM ammonium sulfate dissolved in 1×PBS solution; the blocking solution is 2-5% BSA; the permeabilizing solution is 0.1-0.5% Triton X-100; and the preservative solution consists of 0.02-0.1% sodium thimerosal, 3-10% trehalose, 3-12% dextran-70, and 5-20% glycerol.

[0012] Optionally, the concentration of PFA fixative is 4%, the concentration of antigen retrieval solution is 20 mM, and the concentration of blocking solution is 3%.

[0013] Optionally, the preservative solution may contain 0.05% sodium thimerosal, 5% trehalose, 5% dextran-70 and 10% glycerin.

[0014] An application of a multi-subtype antigen co-transfection cell immunofluorescence staining method is disclosed. This staining method employs a co-transfection design of three GFAP subtypes for the detection of anti-GFAP-IgG antibodies. The three GFAP subtypes are GFAPα, GFAPκ, and GFAPε.

[0015] The specific steps for detecting anti-GFAP-IgG antibodies are as follows: (1) Take out 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 1×PBS solution is 1:300-1:800. (2) Place the cells immobilized with the expression plasmid in a blank cell culture plate and allow them to warm to 22-28℃. (3) After rewarming, add phosphate buffer to each well of the cell culture plate, soak, and then remove the phosphate buffer from the wells. (4) Add the sample to each well of the cell culture plate, incubate at 35-37℃, and then remove the sample from the well; (5) Add phosphate buffer to each well of the cell culture plate, place it in a horizontal shaker to wash, remove the phosphate buffer from the well, and repeat the washing process. (6) Add diluted fluorescently labeled goat anti-human IgG to each well of the cell culture plate, incubate at 37°C in the dark, and remove the fluorescently labeled goat anti-human IgG from the well; (7) Add phosphate buffer to each well of the cell culture plate, place it in a horizontal shaker to wash, remove the phosphate buffer from the well, and repeat the washing process; (8) Add phosphate buffer to each well of the cell culture plate and observe the color development under a fluorescence microscope to determine whether there is anti-myelin oligodendrocyte glycoprotein IgG antibody. If fluorescence is developed, the sample contains anti-GFAP-IgG antibody; if no fluorescence is developed, the sample does not contain anti-GFAP-IgG antibody.

[0016] After adopting the above technical solution, the beneficial effects of the present invention are: 1. This invention is based on the CBA detection method. Its basic principle is similar to the existing anti-GFAP-IgG antibody detection method using overexpressed antigen cell immunofluorescence. The difference lies in the co-transfection design of GFAPα, GFAPκ, and GFAPε subtypes. On one hand, α, as the longest complete-sequence antigen, can meet basic detection performance requirements. On the other hand, the addition of κ and ε, two other rare pathogenic subtypes, covers the antigen subtypes and avoids missed detections in rare cases.

[0017] 2. This invention constructs a plasmid based on a pCDH lentiviral vector. First, the selection of the CMV promoter ensures basic transfection efficiency. Second, the addition of the tag protein X3FLAG facilitates sequencing verification during long-term use of the plasmid. The addition of P2A minimizes interference between the transcriptional expression of the target gene and the green fluorescent protein marker, further contributing to high transfection efficiency. Finally, EGFP, as a marker for transfection expression, helps observe whether the plasmid has been successfully transfected and expressed in cells. In summary, the vector of this invention can assist in achieving high-quality and high-efficiency expression of antigens.

[0018] 3. This invention also enhances staining performance and improves preservation effect by optimizing fixation and permeability preservation methods after cell transfection culture. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a single-subtype transfection protocol used for post-cell EGFP labeling of proteins, as shown in the green fluorescence image. Figure 2 This is a green fluorescence image of EGFP-labeled protein after the three-subtype transfection protocol in this invention is used in cells; Figure 3 This is a red fluorescence image after immunofluorescence staining of cells following a single subtype transfection protocol. Figure 4 This is a red fluorescence image of cells after immunofluorescence staining in the Sanya-type transfection protocol of this invention. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 application discloses a multi-subtype antigen co-transfection cell immunofluorescence staining method and its application.

[0023] Example I. Introduction to the Method in this Invention like Figures 1-4 As shown, a multi-subtype antigen co-transfection cell immunofluorescence staining method includes the following steps: S1. Construct plasmids for three GFAP isotypes: GFAPα, GFAPκ, and GFAPε.

[0024] (1) GFAPα Complete plasmid vector: pCDH-CMV-GFAPα-linker-X3FLAG-P2A-EGFP-AmpR The amino acid sequence of the target antigen is as follows: (AA): Homo sapiens: NP_002046.1, as shown in SEQ ID NO:1.

[0025] Target gene sequence (DNA): Homo sapiens: NM_002055.5, as shown in SEQ ID NO:2.

[0026] (2) GFAPκ Complete plasmid vector: pCDH-CMV-GFAPκ-linker-X3FLAG-P2A-EGFP-AmpR The amino acid sequence of the target antigen is as follows: (AA): Homo sapiens: NP_001229305.1, as shown in SEQ ID NO:3.

[0027] Target gene sequence (DNA): Homo sapiens: NM_001242376.3, as shown in SEQ ID NO:4.

[0028] (3) GFAPε Complete plasmid vector: pCDH-CMV-GFAPε-linker-X3FLAG-P2A-EGFP-AmpR The amino acid sequence of the target antigen is as follows: (AA): Homo sapiens: NP_001124491.1, as shown in SEQ ID NO:5.

[0029] Target gene sequence (DNA): Homo sapiens: NM_001131019.3, as shown in SEQ ID NO:6.

[0030] S2, Cell Culture S21. Preparation of complete culture medium: DMEM culture medium, fetal bovine serum that has been inactivated in a 56℃ water bath for 30 min and filtered through a 0.22μm filter membrane, and penicillin-streptomycin-amphoteric acid mixture are prepared at a volume ratio of 43:7:1-43:12:1. S22. Take the target cells (HEK293T 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. S23. 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. S24. Add the 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.

[0031] S3, Cell Transfection Prepare transfection solutions A and B. Solution A contains DMEM medium and three GFAP expression plasmids, while solution B contains DMEM medium and PEI transfection reagent. Mix solution A with solution B and let stand for 10-20 min to obtain a mixed transfection reagent. Mix the mixed transfection reagent with 10-12 mL of complete medium and add it to a cell culture plate already seeded with target cells. Incubate in an incubator for 16-28 h to obtain HEK293T cells that simultaneously express GFAPα, GFAPκ, and GFAPε. The volume ratio of GFAPα, GFAPκ, GFAPε, and PEI is 2:1:1:3-2:1.5:1.5:5.

[0032] S4. Fixed, sealed, permeable, and corrosion-resistant (1) Preparation of reagents 1) PFA fixative (2-6% PFA dissolved in 1×PBS solution, with 4% being optimal; too low a concentration results in poor antigen fixation, while too high a concentration is wasteful and toxic). 2) Antigen retrieval solution (10-30mM ammonium sulfate, dissolved in 1×PBS solution, the optimal value is 20mM, too low a value results in poor retrieval effect, too high a value will lead to excessive protein precipitation). 3) Blocking solution (mass fraction of 2-5% BSA, with 3% being optimal; too low a concentration results in poor blocking, while too high a concentration leads to high background fluorescence in the staining results). 4) Permeabilization solution (volume fraction of 0.1-0.5% Triton X-100; too low a concentration results in poor permeabilization, while too high a concentration damages cell morphology). 5) Preservative solution (0.02-0.1% sodium thimerosal by mass, 3-10% trehalose by mass, 3-12% dextran-70 by mass, and 5-20% glycerol by volume, dissolved in 1×PBS solution. The optimal ratio is 0.05% sodium thimerosal by mass, 5% trehalose by mass, 5% dextran-70 by mass, and 10% glycerol by volume. Too little sodium thimerosal will result in insufficient preservative effect, while too much will affect the staining effect. Too little trehalose and dextran will not provide sufficient adhesion and antifreeze properties, while too much will make the solution viscous, difficult to prepare, and inconvenient to use. Too little glycerol will result in poor preservation, while too much will be wasteful).

[0033] (2) Remove the cells to be preserved and discard the original culture medium; (3) Add 1×PBS solution to wash, let stand for 2-5 minutes, and discard the PBS; (4) Add PFA fixative, let stand for 20-40 minutes, and discard the PFA; (5) Add 1×PBS solution to wash, let stand for 2-5 minutes, and discard the PBS; (6) Add antigen retrieval solution, let stand for 15-25 minutes, then discard the retrieval solution; (7) Add blocking solution, let stand for 40-70 minutes, then discard the blocking solution; (8) Add permeation solution, let stand for 15-20 minutes, then discard the permeation solution; (8) Add the preservative solution, let stand for 20-40 minutes, then discard the preservative solution; (9) After opening the lid, let it stand for 20-30 minutes to allow the reagent to air dry, then store at -20 degrees Celsius.

[0034] Precautions: All steps should be completed in a biosafety cabinet; when adding liquid, it is recommended to add 100 μL per well for a 96-well plate, and determine the liquid volume for cell slides according to the culture container used. For example, add 10 mL of liquid to a 100 mm diameter culture dish.

[0035] S5. Detection of anti-GFAP-IgG antibodies, the specific steps are as follows: S51. 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 1×PBS solution is 1:300-1:800. S52. Place the cells immobilized with the expression plasmid in a blank cell culture plate and allow them to warm to 22-28℃. S53. 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 well. S54. 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. S55. 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 3 times. S56. 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. S57. Add 50-100 uL of phosphate buffer to each well of the cell culture plate, place it in a horizontal shaker and wash for 3-6 min, aspirate the phosphate buffer from the well, and repeat the washing 3 times. S58. 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-GFAP-IgG antibody in the sample; no fluorescence indicates the absence of anti-GFAP-IgG antibody in the sample.

[0036] II. Experiment This invention is based on the CBA detection method. Its basic principle is similar to the existing anti-GFAP-IgG antibody detection method using overexpressed antigen cell immunofluorescence. Compared to the CBA detection method, its advantages are: A co-transfection design using three GFAP subtypes was employed to cover antigen subtypes and avoid missed detections.

[0037] In 30 clinically diagnosed patients with autoimmune glial fibrillary acidic astrocytosis, the detection rate of this invention was 100%, and the detection rate of the single subtype-carrying pcDNA3.1 plasmid protocol was 93.3%.

[0038] High-quality antigen expression was achieved by constructing plasmids based on the pCDH lentiviral vector: such as Figure 1 and Figure 2 As shown, the cell transfection efficiency of the present invention is no less than, and even slightly better than, the single subtype carrying pcDNA3.1 plasmid scheme.

[0039] 3. Optimize fixation and permeability preservation protocols after cell transfection culture to enhance staining performance and ensure preservation effectiveness: such as... Figure 3 and Figure 4 As shown, the staining morphology detected by this invention is stronger than that of the single-subtype pcDNA3.1 plasmid scheme. The single-subtype scheme has insufficient staining contrast and exhibits simultaneous membrane luminescence and intracytoplasmic filamentous luminescence. The staining results of this invention are clear and have significant contrast, with the staining morphology being almost entirely obvious intracytoplasmic filamentous fluorescence.

[0040] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the technical solution of the present invention should be included within the protection scope of the present invention.

Claims

1. A multi-subtype antigen co-transfection cell immunofluorescence staining method, characterized in that, Three GFAP isoform plasmids were constructed, and then cell culture and transfection were performed to obtain HEK293T cells that simultaneously expressed GFAPα, GFAPκ, and GFAPε. The three GFAP subtypes are GFAPα, GFAPκ, and GFAPε. The complete plasmid vector for GFAPα is pCDH-CMV-GFAPα-linker-X3FLAG-P2A-EGFP-AmpR. The antigenic amino acid sequence of the GFAPα plasmid is shown in SEQ ID NO:1, and the base sequence of the GFAPα plasmid is shown in SEQ ID NO:

2. The complete plasmid vector for GFAPκ is pCDH-CMV-GFAPκ-linker-X3FLAG-P2A-EGFP-AmpR. The antigenic amino acid sequence of the GFAPκ plasmid is shown in SEQ ID NO:3, and the base sequence of the GFAPκ plasmid is shown in SEQ ID NO:

4. The complete plasmid vector for GFAPε is pCDH-CMV-GFAPε-linker-X3FLAG-P2A-EGFP-AmpR. The antigenic amino acid sequence of the GFAPε plasmid is shown in SEQ ID NO:5, and the base sequence of the GFAPε plasmid is shown in SEQ ID NO:

6.

2. The multi-subtype antigen co-transfection cell immunofluorescence staining method according to claim 1, characterized in that, The specific steps of cell culture are as follows: (1) Preparation of complete culture medium: DMEM medium, fetal bovine serum inactivated by 56℃ water bath and filtered through 0.22μm filter membrane, and penicillin-streptomycin-amphoteric acid mixture, the volume ratio of these three reagents is 43:7:1-43:12:1; (2) Take HEK293T 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 in the complete culture medium to the cell pellet and resuspend the cell pellet until the cells are dispersed to obtain the resuspension. (3) Add complete culture medium and resuspension to the petri dish, shake well, and incubate in a 35-37℃, 5% CO2 incubator; (4) 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 with HEK293T cells.

3. The multi-subtype antigen co-transfection cell immunofluorescence staining method according to claim 1, characterized in that, The specific steps for cell transfection are as follows: Prepare transfection solution A and solution B. Solution A contains DMEM medium and three GFAP expression plasmids, and solution B contains DMEM medium and PEI transfection reagent. Mix solution A with solution B to obtain a mixed transfection reagent. Mix the mixed transfection reagent with complete culture medium and add it to a cell culture plate already coated with HEK293T cells. Incubate the plate in an incubator to obtain HEK293T cells that simultaneously express GFAPα, GFAPκ, and GFAPε. The volume ratio of GFAPα, GFAPκ, GFAPε and PEI is 2:1:1:3-2:1.5:1.5:

5.

4. The immunofluorescence staining method for multi-subtype antigen co-transfection cells according to claim 1, characterized in that, After cell transfection, fixation, blocking, permeabilization, and preservation procedures are required. The specific steps are as follows: (1) Prepare PFA fixative, antigen retrieval solution, blocking solution, permeation solution and preservation solution; (2) Remove the cells to be preserved and discard the original culture medium; (3) Add 1×PBS solution to wash, let stand for 2-5 minutes, and discard the PBS; (4) Add PFA fixative, let stand for 20-40 minutes, and discard the PFA; (5) Add 1×PBS solution to wash, let stand for 2-5 minutes, and discard the PBS; (6) Add antigen retrieval solution, let stand for 15-25 minutes, then discard the retrieval solution; (7) Add blocking solution, let stand for 40-70 minutes, then discard the blocking solution; (8) Add permeation solution, let stand for 15-20 minutes, then discard the permeation solution; (9) Add the preservative solution, let stand for 20-40 minutes, then discard the preservative solution; (10) After opening the lid, let it stand for 20-30 minutes to allow the reagent to air dry, then store at -20 degrees Celsius.

5. The immunofluorescence staining method for multi-subtype antigen co-transfection cells according to claim 4, characterized in that, The PFA fixative is 2-6% PFA dissolved in 1×PBS solution; the antigen retrieval solution is 10-30mM ammonium sulfate dissolved in 1×PBS solution; the blocking solution is 2-5% BSA; the permeabilizing solution is 0.1-0.5% Triton X-100; the preservative solution consists of 0.02-0.1% sodium thimerosal, 3-10% trehalose, 3-12% dextran-70 and 5-20% glycerol.

6. The multi-subtype antigen co-transfection cell immunofluorescence staining method according to claim 5, characterized in that, The concentration of PFA fixative was 4%, the concentration of antigen retrieval solution was 20 mM, and the concentration of blocking solution was 3%.

7. The multi-subtype antigen co-transfection cell immunofluorescence staining method according to claim 5, characterized in that, The preservative solution contains 0.05% sodium thimerosal, 5% trehalose, 5% dextran-70, and 10% glycerin.

8. An application of a multi-subtype antigen co-transfection cell immunofluorescence staining method, characterized in that, This staining method employs a co-transfection design of three GFAP subtypes for the detection of anti-GFAP-IgG antibodies. The three GFAP subtypes are GFAPα, GFAPκ, and GFAPε.

9. The application of the multi-subtype antigen co-transfection cell immunofluorescence staining method according to claim 8, characterized in that, The specific steps for detecting anti-GFAP-IgG antibodies are as follows: (1) Take out 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 1×PBS solution is 1:300-1:

800. (2) Place the cells immobilized with the expression plasmid in a blank cell culture plate and allow them to warm to 22-28℃. (3) After rewarming, add phosphate buffer to each well of the cell culture plate, soak, and then remove the phosphate buffer from the wells. (4) Add the sample to each well of the cell culture plate, incubate at 35-37℃, and then remove the sample from the well; (5) Add phosphate buffer to each well of the cell culture plate, place it in a horizontal shaker to wash, remove the phosphate buffer from the well, and repeat the washing process. (6) Add diluted fluorescently labeled goat anti-human IgG to each well of the cell culture plate, incubate at 37°C in the dark, and remove the fluorescently labeled goat anti-human IgG from the well; (7) Add phosphate buffer to each well of the cell culture plate, place it in a horizontal shaker to wash, remove the phosphate buffer from the well, and repeat the washing process; (8) Add phosphate buffer to each well of the cell culture plate and observe the color development under a fluorescence microscope to determine whether there is anti-myelin oligodendrocyte glycoprotein IgG antibody. If fluorescence is developed, the sample contains anti-GFAP-IgG antibody; if no fluorescence is developed, the sample does not contain anti-GFAP-IgG antibody.

Images