A material for detecting anti-lgli1 antibodies, methods of making and methods of use thereof
Patent Information
- Application Number
- CN202611509202.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-09-28
- Publication Date
- 2026-11-03
AI Technical Summary
Existing anti-LGI1 antibody detection technologies have a problem of false negatives, mainly due to the masking of antigen epitopes or differences in expression efficiency caused by LGI1 secreted proteins, which existing methods cannot effectively solve.
By constructing three expression plasmids, LGI1, ADAM22, and ADAM23, and co-transfecting them, two binding antigens, LGI1-ADAM22 and LGI1-ADAM23, are formed in the cells. ADAM22 and ADAM23 compete for binding to LGI1, avoiding antigen epitope masking. Furthermore, by modifying fragments of the original LGI1 antigen sequence, the spatial conformation of the antigen is kept stable.
It significantly improved the detection rate of anti-LGI1 antibodies and the stability of the detection system, reduced the false negative rate, and extended the shelf life of the materials.
Abstract
Description
Technical Field
[0001] This invention relates to the field of biological detection technology, specifically to a material, preparation method, and usage method for detecting anti-LGI1 antibodies. Background Technology
[0002] Leucine-rich glioma-inactivated 1 (LGI1) is a secretory neuronal protein primarily expressed in the hippocampus. It is a ligand for two epilepsy-associated proteins, ADAM22 and ADAM23. When antibodies bind to LGI1, they disrupt presynaptic and postsynaptic signaling, leading to neuronal overexcitation. Mutations in the LGI1 gene are associated with autosomal dominant partial epilepsy with auditory features. Furthermore, there is a class of diseases primarily caused by autoimmune attack on the LGI1 protein, known as anti-LGI1 encephalitis. Anti-LGI1 encephalitis mainly affects the central nervous system, with typical symptoms including facial-brachial dystonia, cognitive decline, epilepsy, and hyponatremia. Therefore, the detection of autoantibodies against LGI1 is crucial for the diagnosis and treatment of anti-LGI1-related diseases.
[0003] Existing anti-LGI1 antibody detection technologies and platforms almost entirely rely on cell immunofluorescence assays (CBA). However, because LGI1 is a secreted protein, without modification or treatment, the antigen will be secreted extracellularly after normal expression and cannot be collected. To address this issue, the design scheme for antigen expression in existing CBA technologies and reagents for detecting anti-LGI1 antibodies is as follows: Option 1 uses two plasmids expressing LGI1 and ADAM23 respectively for co-transfection, so that ADAM23 binds to LGI1 and keeps it in the cell; Option 2 uses a single plasmid that simultaneously expresses LGI1 and ADAM23 (the vector is constructed using P2A or T2A separation dual-target gene technology) for transfection, and the principle is the same as that of Option 1; Scheme 3 splits the original LGI1 antigen amino acid sequence into two functional regions, 1-233 and 224-557, constructs expression plasmids for each region, and co-transfects them to retain the secreted protein in the cell by altering its structure.
[0004] In Scheme 1, although ADAM23 is the natural ligand of LGI1, its binding still covers part of the binding site on the LGI1 protein surface. This may prevent some anti-LGI1 antibody subtypes that would normally bind to the blocked sites from binding properly, resulting in missed detections. In Scheme 2, although separating the two target genes using P2A or T2A self-cleaving sequences is a commonly used antigen expression technique in CBA development, unlike conventional antigen subtype coverage which only requires the presence of both target antigens, anti-LGI1 antibody detection requires LGI1 to form a binding antigen with ADAM23. P2A self-cleaving leads to a significant difference in transcriptional expression efficiency between the two target gene segments, thus significantly reducing binding efficiency. This scheme not only shares the disadvantages of Scheme 1 but is also inferior to Scheme 1 in terms of effectiveness. Scheme 3, while avoiding the antigen epitope blocking problem of Schemes 1 and 2, significantly alters the original spatial conformation of the LGI protein, which can also lead to some missed detections. In summary, the common drawback of the existing schemes is that while addressing the LGI1 secretion problem, they can also lead to missed detections for various reasons. Summary of the Invention
[0005] In order to solve the problems in related technologies, the present invention provides a material, preparation method and usage method for detecting anti-LGI1 antibodies, which solves the problem of missed detection when LGI1 is secreted.
[0006] To solve the above problems, the following technical solutions are provided: The present invention provides a material for detecting anti-LGI1 antibodies, which involves co-transfection of three expression plasmids: LGI1, ADAM22, and ADAM23.
[0007] By employing the above technical approach, three expression plasmids, LGI1, ADAM22, and ADAM23, were co-transfected, enabling the simultaneous formation of two binding antigens, LGI1-ADAM22 and LGI1-ADAM23, in the cells. ADAM22 and ADAM23 can competitively bind to LGI1, allowing the antigenic epitope that was blocked by one ligand to be normally exposed in the other binding antigen and available for the antibody to be detected, thus preventing false negatives caused by antigenic epitope blocking and solving the problem of false negatives during LGI1 secretion. Furthermore, each gene is expressed independently, avoiding the expression efficiency differences caused by the self-cleavage of single plasmid P2A and T2A, and significantly improving the detection rate while maintaining specificity.
[0008] Furthermore, the LGI1 expression plasmid vector is pcdna3.1-target gene-linker-×3Flag-P2A-EGFP-AmpR, and the ADAM22 and ADAM23 expression plasmid vectors are pcdna3.1-target gene-linker-×3Flag-P2A-AmpR.
[0009] By adopting the above technical solution, the LGI1 expression plasmid is pcdna3.1-target gene-linker-×3Flag-P2A-EGFP-AmpR, and the ADAM22 and ADAM23 expression plasmids are pcdna3.1-target gene-linker-×3Flag-P2A-AmpR. This approach is beneficial for efficient expression in cells. The linker improves the flexibility of the fusion protein, the ×3Flag facilitates expression identification and quality control, P2A-EGFP can be used to trace transfection and expression status, and AmpR facilitates plasmid screening. The ADAM22 and ADAM23 plasmids do not contain EGFP, which helps reduce interference with subsequent fluorescence detection and improves the stability of the detection system.
[0010] Furthermore, the original LGI1 antigen sequence was modified into a fragment. The amino acid sequence of the modified LGI1 is shown in SEQ ID NO.1, the amino acid sequence of ADAM22 is shown in SEQ ID NO.2, and the amino acid sequence of ADAM23 is shown in SEQ ID NO.3. The co-transfection was performed using the modified LGI1, ADAM22, and ADAM23.
[0011] By employing the above technical solution, the original LGI1 antigen sequence is fragment-modified by mutating amino acids 35-37 from KKP to LSL and amino acids 71-72 from SL to NS, thereby reducing the secretion tendency of LGI1, allowing the antigen to remain in the cell and reducing antigen loss during culture. Furthermore, the LGI1 functional region is not split, avoiding significant changes to the original spatial conformation of the antigen, thus reducing false negatives caused by conformational changes. ADAM22 and ADAM23 form two complex antigens with LGI1, which can compete with each other to expose epitopes, further reducing the false negative rate.
[0012] Furthermore, a method for preparing a material for detecting anti-LGI1 antibodies includes the following steps: S1: Place the cell carrier into a cell culture dish and add cell adhesion reagent to the culture dish for coating; S2: Preheat the complete culture medium; S3: Add the cells to the preheated complete culture medium, mix well, transfer to the coated cell culture dish, and place the cell culture dish in a cell culture incubator for cell culture. S4: Take two centrifuge tubes, label them tube A and tube B, add opti-MEM medium to tube A and tube B respectively, and preheat them; S5: Add LGI1, ADAM22, and ADAM23 expression plasmids to tube A, add Lipo6000™ transfection reagent to tube B, mix well and let stand, then add the liquid from tube A to tube B, mix well and let stand. S6: Add the preheated complete culture medium to tube B and mix well to obtain the mixture; S7: Remove the original culture medium from the cell culture dish in S3, add the mixture obtained in S6, and place it in a cell culture incubator for cell culture. S8: Rinse the cell culture medium after cell culture. After rinsing, open the cell culture dish and let it stand to allow the surface of the cell carrier to air dry, and the anti-LGI1 antibody material can be obtained.
[0013] Using the above technical solution, cell slides based on cell immunofluorescence are obtained through steps such as cell vector coating, cell culture, plasmid co-transfection, fixation and air drying. This method has a clear operation process, good reproducibility, is conducive to the expression and retention of antigens in cells, and is convenient for subsequent preservation, transportation and detection.
[0014] Furthermore, the complete culture medium in S2 was prepared by mixing 43 parts DMEM medium, 7 parts fetal bovine serum (FBS) and 1 part penicillin-streptomycin-amphotericidal mycotoxin.
[0015] Furthermore, in tube S5, 4 μg of LGI1, 4 μg of ADAM22 and 4 μg of ADAM23 expression plasmids were added to tube A, and 24 μL of Lipo6000™ transfection reagent was added to tube B.
[0016] Furthermore, the rinsing process in S8 includes the following steps: remove the cell culture dish, aspirate the original culture medium, add buffer solution for rinsing, aspirate the buffer solution after rinsing, add fixative, let stand, and then aspirate the fixative solution; add buffer solution for rinsing, and then aspirate the buffer solution; add blocking solution, let stand, and then aspirate the blocking solution; add preservative solution, let stand, and then aspirate the preservative solution.
[0017] Furthermore, a method for using a material for detecting anti-LGI1 antibodies includes the following steps: A1: Add serum sample to each reaction area of the slide and incubate. After incubation, remove the serum sample and rinse. A2: Add AF555-labeled goat anti-human IgG secondary antibody working solution for a second incubation. After incubation, remove and rinse. A3: Finally, add 1×PBS buffer and observe using a fluorescence inverted microscope.
[0018] By adopting the above technical solution, this method is based on cell immunofluorescence, which is simple to operate and provides intuitive results. It can detect anti-LGI1 IgG antibodies in human serum and is suitable for clinical auxiliary diagnosis and scientific research testing of anti-LGI1 encephalitis and other related diseases.
[0019] Furthermore, the serum sample is prepared by diluting 1 part serum with 10 parts 1×PBS buffer, and the working solution of goat anti-human IgG secondary antibody is prepared by diluting 1 part AF555-labeled goat anti-human IgG with 400 parts 1×PBS buffer.
[0020] Furthermore, both A1 and A2 were rinsed with 1×PBS buffer.
[0021] The above solution has the following advantages: 1. Using the preparation method of this invention, in the process of preparing a material for detecting anti-LGI1 antibodies, three expression plasmids, LGI1, ADAM22, and ADAM23, are co-transfected, so that two binding antigens, LGI1-ADAM22 and LGI1-ADAM23, are formed simultaneously in the cells. ADAM22 and ADAM23 can competitively bind to LGI1, so that the antigen epitope blocked by one ligand binding is normally exposed in the other binding antigen and can be bound by the antibody to be detected, preventing false negatives caused by antigen epitope blocking, thus solving the problem of false negatives during LGI1 secretion; and each gene is expressed independently, avoiding the expression efficiency differences caused by the self-cleavage of single plasmid P2A and T2A, and significantly improving the detection rate while maintaining specificity.
[0022] 2. The LGI1 expression plasmid is pcdna3.1-target gene-linker-×3Flag-P2A-EGFP-AmpR, and the ADAM22 and ADAM23 expression plasmids are pcdna3.1-target gene-linker-×3Flag-P2A-AmpR. This combination is beneficial for efficient expression in cells. The linker improves the flexibility of the fusion protein, the ×3Flag facilitates expression identification and quality control, P2A-EGFP can be used to trace transfection and expression status, and AmpR facilitates plasmid screening. The ADAM22 and ADAM23 plasmids do not contain EGFP, which helps reduce interference with subsequent fluorescence detection and improves the stability of the detection system. Detailed Implementation
[0023] 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.
[0024] This application provides an embodiment of a material, preparation method, and usage method for detecting anti-LGI1 antibodies.
[0025] I. Design of plasmids and vectors for transfection 1. Vector construction and plasmid structure: The LGI1 expression plasmid vector is pcdna3.1-target gene-linker-×3Flag-P2A-EGFP-AmpR. The expression plasmid vectors for ADAM22 and ADAM23 are pcdna3.1-target gene-linker-×3Flag-P2A-AmpR.
[0026] 2. Target gene: (1) LGI1: The original antigen sequence is shown in SEQ ID NO.4. Original antigen sequence: molecular type: AA (DNA, RNA, AA), organism name: Homo sapiens.
[0027] The original LGI1 antigen sequence was modified by mutating amino acids 35-37 from KKP to LSL and amino acids 71-72 from SL to NS to form a new antigen sequence, as shown in SEQ ID NO.1.
[0028] Modified antigen sequence: Molecular type: AA (DNA, RNA, AA), Organism name: Homo sapiens.
[0029] (2) ADAM22: Original antigen sequence: Molecular type: AA (DNA, RNA, AA), organism name: Homo sapiens, the sequence of ADAM22 is shown in SEQ ID NO.2.
[0030] (3) ADAM23: Original antigen sequence: Molecular type: AA (DNA, RNA, AA), organism name: Homo sapiens, the sequence of ADAM23 is shown in SEQ ID NO.3.
[0031] II. Preparation method of materials used for detecting anti-LGI1 antibodies 1. Calculate the required quantity according to the production batch, take out a 60mm×60mm sterile clean square glass slide from the biosafety cabinet and place it into a 100mm cell culture dish; 2. Add 20 mL of enhanced cell adhesion reagent (commercially available, diluted 1:50 with 1×PBS buffer) to the culture dish, let it stand at room temperature for 60 minutes to coat the cells, and then blot off the adhesion reagent. 3. Place 43 mL of 80-90% DMEM culture medium, 7 mL of 10-20% premium fetal bovine serum (FBS) and 1 mL of 1-3% penicillin-streptomycin-amphoteric acid mixture in a constant temperature water bath and preheat to 37°C. 4. Take a sterile 50mL centrifuge tube in the biosafety cabinet, pour 20mL of preheated complete culture medium, add 0.2mL of HEK293T cell suspension (cell volume approximately 1.5-2.0×10^6), mix well by pipetting and aspirating, and then add it to a culture dish containing a climbing slide. Transfer the culture dish to a cell culture incubator. 5. Incubate at 37±0.5℃ and 5% CO2 for 24 hours in a cell culture incubator; 6. Take out two sterile 15mL centrifuge tubes from the biosafety cabinet, add 1mL of opti-MEM medium to each, and label them tube A and tube B. At the same time, preheat the complete medium to 37°C. 7. Add 4 μg LGI1, 4 μg ADAM23, and 4 μg ADAM22 expression plasmids to tube A and gently mix with a pipette. Add 24 μL Lipo6000™ transfection reagent to tube B and gently mix with a pipette. 8. After standing at room temperature for 5 minutes, gently add the liquid from tube A to tube B, mix gently with a pipette, let stand for another 15 minutes, then add 20 mL of preheated complete culture medium and mix with a pipette. 9. Remove the cell culture dish from the cell culture incubator, remove the original culture medium in the biosafety cabinet, add the above mixture to the culture dish, and transfer the culture dish to the cell culture incubator. 10. Incubate at 37±0.5℃ and 5% CO2 for 24 hours in a cell culture incubator; 11. Remove the cell culture dish containing the inoculated cells from the cell culture incubator, remove the original culture medium in the biosafety cabinet, add 3 mL of 1×PBS buffer to rinse, and then remove the buffer. 12. Add 20 mL of 4% PFA fixative, let stand for 30 minutes, and then remove the fixative. 13. Add 10 mL of 1×PBS buffer to rinse, then aspirate the buffer. 14. Add 20 mL of blocking solution prepared by dissolving 2-5% BSA in 1×PBS buffer, let stand for 60 minutes, and then remove the blocking solution. 15. Add 20 mL of the preservative solution prepared by dissolving 0.02-0.1% (m / v) sodium thimerosal, 3-10% (m / v) trehalose, 3-12% (m / v) dextran-70, and 5-20% (v / v) glycerol in 1×PBS buffer, let stand for 30 minutes, and then remove the preservative solution. 16. After opening the lid and letting it stand for 20 minutes to allow the surface of the slide to air dry, cut it into 3mm×3mm pieces with a cutting machine. Take the required number of individual pieces and attach them to the surface of the glass slide with UV-curing adhesive. Store at -20℃.
[0032] III. Methods of Using Materials for Detecting Anti-LGI1 Antibodies 1. Warming: Remove the glass slide with the smear, 1×PBS buffer, and AF555-labeled goat anti-human IgG from the refrigerator and warm it to room temperature for 10-15 minutes before staining.
[0033] 2. Cleaning: Place the slide in the humidified chamber, add 60 μL of 1×PBS buffer to each reaction area, cover the slide, and rinse for 3 minutes.
[0034] 3. Sample serum incubation: Discard the reaction zone buffer, add 60 μL of the corresponding serum sample (diluted from 1 part serum and 10 parts 1×PBS buffer) to each reaction zone, and incubate at 37°C for 40 min.
[0035] 4. Washing: Discard the incubated serum, add 60ul of 1×PBS buffer to each reaction area, cover the slide, rinse for 3 minutes, remove the buffer, and repeat 3 times.
[0036] 5. Secondary antibody incubation: Add 60 μL of AF555-labeled goat anti-human IgG working solution (diluted with 1 part AF555-labeled goat anti-human IgG and 400 parts 1×PBS buffer), and incubate at 37°C for 30 min.
[0037] Note: This step requires protection from light.
[0038] 6. Washing: Discard the secondary antibody, add 60 μL of 1×PBS buffer to each reaction zone, cover the slide, rinse for 3 min, remove the buffer, and repeat 3 times.
[0039] 7. Add 60 μL of 1×PBS buffer and observe the experimental results under a fluorescence inverted microscope.
[0040] This invention utilizes co-transfection with three expression plasmids (LGI1, ADAM22, and ADAM23) to simultaneously generate two binding antigens, LGI1-ADAM22 and LGI1-ADAM23, in cells. ADAM22 and ADAM23 can competitively bind to LGI1, ensuring that antigenic epitopes blocked by one ligand are normally exposed in the other binding antigen and available for antibody binding, preventing false negatives due to epitope obstruction. Furthermore, each gene is expressed independently, avoiding the expression efficiency differences caused by P2A and T2A self-splicing of single plasmids, significantly improving the detection rate while maintaining specificity. The LGI1 expression plasmid is pcdna3.1. The target gene-linker-×3Flag-P2A-EGFP-AmpR expression plasmids for ADAM22 and ADAM23 are pcdna3.1-. This structure facilitates efficient expression in cells. The linker enhances the flexibility of the fusion protein, the ×3Flag facilitates expression identification and quality control, P2A-EGFP can be used to trace transfection and expression status, and AmpR facilitates plasmid selection. The ADAM22 and ADAM23 plasmids do not contain EGFP, which helps reduce interference with subsequent fluorescence detection and improves the stability of the detection system.
[0041] Comparative Example 1: The plasmid used is also a single plasmid that simultaneously expresses LGI1 and ADAM23 dual antigens, and no sequence modification is performed. The other production and usage methods are the same as in the example. Comparative Example 2: The plasmids used were replaced with two plasmids expressing LGI1 and ADAM23 respectively. No sequence modification was performed. Co-transfection was carried out at a total ratio of 1:1 with a total amount of 10 μg. The other production and usage methods were the same as in the example. Comparative Example 3: The plasmids used were replaced with two plasmids expressing the EPTP and LRR functional regions of LGI1, respectively. No sequence modification was performed. Co-transfection was carried out at a total ratio of 1:1 with a total amount of 10 μg. The other production and usage methods were the same as in the example. The detection performance of the embodiments of the present invention was tested compared with that of Comparative Examples 1, 2, and 3: The results of this invention embodiment are compared with those of Comparative Examples 1, 2, and 3, which used the same 30 anti-LGI1 positive samples: Embodiments of the present invention Comparative Example 1 Comparative Example 2 Comparative Example 3 Number of positive cases detected 30 23 27 22 Detection positive rate 100% 76.7% 90% 73.3% The shelf life of the embodiments of the present invention and comparative examples 1, 2, and 3 was tested: Every 7 days, the same three anti-LGI1 positive samples were tested using the embodiments of the present invention, Comparative Example 1, Comparative Example 2, and Comparative Example 3. The day when all three anti-LGI1 positive samples could no longer be detected was taken as the final expiration date. The results were compared: Embodiments of the present invention Comparative Example 1 Comparative Example 2 Comparative Example 3 Sample 1 Expiration Date 357 350 357 294 Sample 2 Expiration Date 399 350 350 308 Sample 3 Expiration Date 392 381 357 322 Final expiration date 399 381 357 322 The invention embodiment has the highest detection rate of anti-LGI1 positive samples, reaching 100%. By co-transfecting with three expression plasmids, LGI1, ADAM22, and ADAM23, and modifying the LGI1 sequence, it is possible to effectively reduce false negatives and improve the positive detection rate.
[0042] The embodiments of the present invention are significantly superior to Comparative Example 1, indicating that the scheme of simultaneously expressing LGI1 and ADAM23 with a single plasmid without sequence modification has a significant problem of missed detection.
[0043] The embodiments of the present invention are superior to Comparative Example 2, demonstrating that although co-transfection with only LGI1 and ADAM23 plasmids is better than the single plasmid scheme, it is still not as good as the scheme that simultaneously adds ADAM22 and ADAM23 to form two competitive binding antigens.
[0044] The lowest detection rate was observed in Comparative Example 3, indicating that splitting LGI1 into two functional regions for separate expression, while addressing the secretion issue, would lead to more missed detections due to significant alterations to the original spatial conformation of LGI1.
[0045] Therefore, this invention, through co-transfection with three plasmids and modification of the LGI1 sequence, can maximize the compensation for missed detections caused by antigen epitope obstruction and conformational changes, thereby improving detection sensitivity.
[0046] The final expiration date of this embodiment of the invention is 399 days, which is the longest shelf life. Compared with Comparative Example 1, the final expiration date of this embodiment of the invention is extended by about 18 days; compared with Comparative Example 2, it is extended by about 42 days; and compared with Comparative Example 3, it is extended by about 77 days.
[0047] Comparative Example 3 had the shortest final failure date, only 322 days, indicating that although segmented expression of LGI1 may solve the secretion problem, the material has poor stability.
[0048] Therefore, by modifying the original LGI1 antigen sequence, using a three-plasmid co-transfection system, and combining subsequent fixation, blocking, and preservation treatments, this invention can significantly improve the preservation stability of anti-LGI1 antibody detection materials and extend their shelf life.
[0049] In terms of detection performance, the detection rate of the present invention in 30 anti-LGI1 positive samples reached 100%, which was significantly higher than that of comparative examples 1, 2 and 3, indicating that co-transfection of the three expression plasmids and modification of the LGI1 sequence can effectively reduce false negatives. In terms of preservation stability, the final expiration date of the present invention reached 399 days, which was significantly longer than that of comparative examples 1-3, indicating that the material has better shelf life and stability.
[0050] 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 material for detecting anti-LGI1 antibodies, characterized in that, Three expression plasmids, LGI1, ADAM22, and ADAM23, were constructed and co-transfected.
2. The material for detecting anti-LGI1 antibodies as described in claim 1, characterized in that, The LGI1 expression plasmid vector is pcdna3.1-target gene-linker-×3Flag-P2A-EGFP-AmpR, and the ADAM22 and ADAM23 expression plasmid vectors are pcdna3.1-target gene-linker-×3Flag-P2A-AmpR.
3. The material for detecting anti-LGI1 antibodies as described in claim 1, characterized in that, The original LGI1 antigen sequence was modified into a fragment, and the amino acid sequence of the modified LGI1 is shown in SEQ ID NO.
1. The co-transfection was performed using the modified LGI1, ADAM22, and ADAM23.
4. A method for preparing a material for detecting anti-LGI1 antibodies, using the material with the antibody as described in claim 1, characterized in that, Includes the following steps: S1: Place the cell carrier into a cell culture dish and add cell adhesion reagent to the culture dish for coating; S2: Preheat the complete culture medium; S3: Add the cells to the preheated complete culture medium, mix well, transfer to the coated cell culture dish, and place the cell culture dish in a cell culture incubator for cell culture. S4: Take two centrifuge tubes, label them tube A and tube B, add opti-MEM medium to tube A and tube B respectively, and preheat them; S5: Add LGI1, ADAM22, and ADAM23 expression plasmids to tube A, add transfection reagent to tube B, mix well and let stand, then add the liquid from tube A to tube B, mix well and let stand. S6: Add the preheated complete culture medium to tube B and mix well to obtain the mixture; S7: Remove the original culture medium from the cell culture dish in S3, add the mixture obtained in S6, and place it in a cell culture incubator for cell culture. S8: Rinse the cell culture medium after cell culture. After rinsing, open the cell culture dish and let it stand to allow the surface of the cell carrier to air dry, and the anti-LGI1 antibody material can be obtained.
5. A method for preparing a material for detecting anti-LGI1 antibodies as described in claim 4, characterized in that, The complete culture medium in S2 was prepared by mixing 43 parts of DMEM medium, 7 parts of fetal bovine serum (FBS) and 1 part of penicillin-streptomycin-amphotericidal mycotoxin.
6. A method for preparing a material for detecting anti-LGI1 antibodies as described in claim 4, characterized in that, In tube S5, add 4 μg of LGI1, 4 μg of ADAM22 and 4 μg of ADAM23 expression plasmids to tube A, and add 24 μL of Lipo6000™ transfection reagent to tube B.
7. A method for preparing a material for detecting anti-LGI1 antibodies as described in claim 4, characterized in that, The S8 intermediate washing process includes the following steps: Remove the cell culture dish, aspirate the original culture medium, add buffer solution to rinse, aspirate the buffer solution after rinsing, add fixative, let stand, aspirate the fixative solution; add buffer solution to rinse, aspirate the buffer solution after rinsing; add blocking solution, let stand, aspirate the blocking solution; add preservative solution, let stand, aspirate the preservative solution.
8. A method of using a material for detecting anti-LGI1 antibodies, comprising using the material with the antibody as described in claim 1, characterized in that, Includes the following steps: A1: Add serum sample to each reaction area of the slide and incubate. After incubation, remove the serum sample and rinse. A2: Add AF555-labeled goat anti-human IgG secondary antibody working solution for a second incubation. After incubation, remove and rinse. A3: Finally, add 1×PBS buffer and observe using a fluorescence inverted microscope.
9. The method of using the material for detecting anti-LGI1 antibodies as described in claim 8, characterized in that, The serum sample was prepared by diluting 1 part serum with 10 parts 1×PBS buffer, and the working solution of goat anti-human IgG secondary antibody was prepared by diluting 1 part AF555-labeled goat anti-human IgG with 400 parts 1×PBS buffer.
10. The method of using the material for detecting anti-LGI1 antibody as described in claim 8, characterized in that, Both A1 and A2 were rinsed with 1×PBS buffer.