A method for detecting anti-caspr1 antibody material and preparation method thereof

Abstract

This invention belongs to the field of biodetection technology, specifically relating to a material for detecting anti-Caspr1 antibodies and its preparation method. The invention constructs an expression plasmid capable of highly expressing the Caspr1 antigen, transfects it into HEK293T cells, and modifies the original antigen sequence during construction to achieve normal membrane expression. Subsequently, a modified cell culture protocol is used to obtain a material for detecting anti-Caspr1 antibodies based on cellular immunofluorescence assay. This invention, based on cellular immunofluorescence, avoids the limitations of flow cytometry, which is complex and requires expensive equipment. By modifying the protein expression mode to normal membrane expression, it avoids cell permeabilization, avoids false negatives caused by antigen conformational changes in enzyme-linked immunosorbent assay (ELISA) and Western blotting, and avoids false negatives caused by transmembrane expression due to transfection with the original Caspr1 sequence.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and specifically relates to a material for detecting anti-Caspr1 antibodies and its preparation method. Background Technology

[0002] The node of Ranvier is the structural basis for the skip conduction of myelinated fibers, comprising three regions: the nodal region, the paranodal region, and the proximal region. In the paranodal region, Caspr1 (contactin-associated protein 1), anchored to the axonome, forms a complex with contactin 1 (also located on the axonome) and fasciculus protein 155 (located in the myelin sheath), tightly connecting the loop of myelin to the axonome. This septum-like structure ensures that ion channel proteins in the nodal region and potassium ion channels in the proximal region are located in their respective positions. Clinically, the node of Ranvier is considered a key target in immune-mediated peripheral neuropathy. Literature reports the presence of IgG antibodies binding to the nodal / paranodal regions in the serum of 43% of patients with Guillain-Barré syndrome and 30% of patients with chronic inflammatory demyelinating polyradiculopathy. As research focuses on the node of Ranvier-related region, more and more target antigens are being identified, with anti-Caspr1 antibodies being a key one. Related studies and experiments have also confirmed that these autoantibodies can disrupt the adhesion between the axonome and the loop of heel, thereby affecting the transmission of nerve impulses and inducing anti-Caspr1 antibody-positive chronic inflammatory demyelinating polyradiculoneuropathy, i.e., autoimmune antibody-mediated peripheral neuropathy. Therefore, the detection of Caspr1 antibodies is undoubtedly of great importance.

[0003] Existing anti-Caspr1 antibody detection technologies and platforms mainly fall into three categories: flow cytometry (e.g., publication number WO2025211788A1: MULTIPLEX IMMUNOASSAY METHOD FOR DIAGNOSING AUTOIMMUNENODOPATHY; publication number CN116338200A: an autoantibody flow cytometry detection reagent, preparation method and detection method thereof), enzyme-linked immunosorbent assay (ELISA) / immunoblotting (BLOT) detection (e.g., publication number CN119335194A: an antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy and its preparation method thereof), and cell immunofluorescence assay (CBA) detection (e.g., publication number CN120721958A: a stabilizing agent, cell antigen dry slide and preparation method and application of the dry slide). Flow cytometry, which involves binding antigens to microspheres or magnetic beads and then detecting them with immunofluorescence, offers acceptable accuracy. However, it requires a flow cytometer, which is too demanding for routine use and involves complex procedures. Furthermore, the extracted Caspr1 antigen is difficult to preserve long-term, hindering the development of convenient diagnostic kits. ELISA, similar in principle to BLOT, involves coating antigens into the wells of an enzyme-labeled plate or on the surface of a membrane strip, followed by detection with an enzyme-labeled secondary antibody. Its drawbacks include reliance on antigen shelf life and the conformational changes inherent in the antigen coating process, which can lead to false negatives. The CBA method involves constructing an expression plasmid based on the Caspr1 antigen sequence available in the UNIPROT or NCBI database and then transfecting the cell. However, Caspr1, as a transmembrane protein, has both intracellular and extracellular segments, unlike conventional cell membrane-expressed proteins. Therefore, cells must be permeabilized before detection. However, the permeabilization step itself damages the extracellular antigen, leading to missed detections of the extracellular segment. Conversely, not permeabilizing the cell results in some missed detections of the intracellular segment. Thus, the CBA method for detecting anti-Caspr1 antibodies has a problem of missed detections. Summary of the Invention

[0004] This invention proposes a material for detecting anti-Caspr1 antibodies and its preparation method, which solves the problem of missed detection in the traditional CBA method for detecting anti-Caspr1 antibodies.

[0005] The technical solution of this invention is implemented as follows: An expression plasmid that highly expresses the Caspr1 antibody has the structure pcdna3.1-Caspr1-linker-helper-linker-×3Flag-P2A-EGFP-AmpR; wherein the antigenic sequence of Caspr1 is shown in SEQ ID NO.1, and the antigenic sequence of the helper segment is shown in SEQ ID NO.2.

[0006] The above technical solution addresses the issue that, because the Caspr1 antigen crosses the membrane multiple times, conventional methods require cell permeabilization to fully expose all antigen binding sites. However, this permeabilization step damages cells, leading to potential false negatives. The innovation of this plasmid modification lies in removing the transmembrane localization region from the original sequence, preventing the antigen from crossing the membrane multiple times. Furthermore, by adding a membrane expression localization helper segment, the antigen is linked to the cell membrane, forming a form similar to conventional membrane expression. This eliminates the need for cell permeabilization, as the antigen itself exposes the binding sites on the cell membrane, allowing it to directly bind to the antibody in the sample. This solves the false negative problem in traditional CBA methods for detecting anti-Caspr1 antibodies.

[0007] A method for preparing anti-Caspr1 antibody materials includes the following steps: S1. Place the cell carrier into a cell culture dish; S2. Add enhanced cell adhesion reagent to the cell culture dish, let it stand at room temperature for coating, and then blot dry the enhanced cell adhesion reagent. S3. Preheat the complete culture medium (DMEM+FBS+triple antibiotic mixture); S4. Take the preheated complete culture medium from step S3, add HEK293T cell suspension, and then add it to the cell culture dish from step S2. S5. Place the cell culture dish from step S4 into a cell culture incubator for cell culture. S6. Take two centrifuge tubes and label them A and B. Add opti-MEM medium to tubes A and B respectively and preheat them. S7. Add NF155 expression plasmid to tube A and add linear polyacetylimine transfection reagent to tube B; S8. Pipette the liquid from tube A into tube B to form a mixture. After mixing and letting stand, add the preheated complete culture medium from S3. S9. Take out the cell culture dish from step S5, remove the original culture medium, and add the mixture from S8 to the cell culture dish. S10. Transfer the cell culture dish from step S9 to a cell culture incubator for cell culture. S11. Remove the cell culture dish from the cell culture incubator, aspirate the original culture medium, add buffer solution to rinse, and then aspirate the buffer solution. S12. Add PFA fixative to the cell culture dish from step S11, let it stand, and then remove the fixative. S13. Add buffer solution to the cell culture dish from step S12 to rinse, and then remove the buffer solution. S14. Add blocking solution to the cell culture dish in step S13, let it stand, and then remove the blocking solution. S15. Add the preservative solution to the cell culture dish in step S14, let it stand, and then remove the preservative solution. S16. After opening the cell culture dish from step S15 and allowing it to stand to air dry, cut the cell carrier and attach it to the surface of the glass slide using UV-curable adhesive.

[0008] Through the above technical solution, in step S7, linear polyacetylimide is an auxiliary reagent that assists plasmids in entering cells through endocytosis. Compared with the lipo series transfection reagents that assist transmembrane transfection through liposomes, linear polyacetylimide has more controllable cytotoxicity, is more convenient to use, and has a lower cost.

[0009] Optionally, in step S1, the cell slide is a glass slide or a cell culture plate.

[0010] Optionally, the cell adhesion enhancer in step S2 is diluted 1:50 with 1×PBS buffer; the complete culture medium (DMEM+FBS+triple antibody mixture) in step S3 is 86% DMEM medium + 14% premium fetal bovine serum FBS + 2% penicillin-streptomycin-amphoteric acid mixture; the blocking solution in step S14 is 3% BSA dissolved in 1×PBS buffer; and the preservation solution in step S15 is 0.05% sodium thimerosal, 5% trehalose, 5% dextran-70, and 10% glycerol dissolved in 1×PBS buffer.

[0011] The above technical solution provides sufficient cell nutrition while preventing accidental microbial contamination during cell culture. The solution consists of 86% DMEM medium, 14% premium fetal bovine serum (FBS), and 2% penicillin-streptomycin-amphotericidal mixture.

[0012] The preservative solution consists of 0.05% sodium thimerosal, 5% trehalose, 5% dextran-70, and 10% glycerol dissolved in 1×PBS buffer. This invention employs a composite preservative solution system containing trehalose and dextran, followed by gentle air drying. During drying, the sugar molecules form a stable glassy protective layer on the cell surface, replacing water molecules to maintain the integrity of the cell membrane and protein structure. This significantly reduces the shrinkage and breakage damage to cells caused by fixation and drying, significantly restoring the binding capacity of antigens and antibodies, improving the antigen-binding preservation capacity of cells overexpressing antigens, and enhancing their preservation rate. By constructing a composite preservative solution containing antibacterial agents and multiple preservatives, combined with a dry solid-phase preservation strategy, the dry environment fundamentally inhibits microbial growth, and the glassy sugar matrix greatly stabilizes the conformation of biomolecules, thus synergistically achieving long-term stable preservation at room temperature and significantly extending the product's shelf life.

[0013] Optionally, the preheating temperature in step S3 is 37℃, and the culture conditions in step S5 are 37±0.5℃ and 5% CO2 for 24h; the preheating temperature in step S6 is 37℃, and the culture conditions in step S10 are 37±0.5℃ and 5% CO2 for 24h.

[0014] Optionally, the buffer in steps S11 and S13 may be 1×PBS buffer or 1×PBST buffer.

[0015] Optionally, in step S2, the coating reagent for the cell carrier can be diluted rat tail collagen type 1 or polylysine-gelatin mixture.

[0016] Through the above technical solution, this coating reagent can enhance cell adhesion and cell fixation.

[0017] Optionally, sodium thimerosal in step S15 can be replaced with sodium azide in an equal proportion, and BSA can be replaced with sheep serum in an equal proportion.

[0018] After adopting the above technical solution, the beneficial effects of the present invention are: This invention constructs an expression plasmid capable of highly expressing the Caspr1 antigen for transfection into HEK293T cells. During construction, the original antigen sequence is modified to achieve normal membrane expression. Subsequently, a modified cell culture protocol is used to obtain a material for detecting anti-Caspr1 antibodies based on the cell immunofluorescence assay (CBA). This invention avoids the false negatives caused by antigen conformational changes in ELISA and BLOT methods through the CBA approach. By modifying the protein expression mode to normal membrane expression, this invention avoids cell permeation manipulation, preventing protein damage and false negatives. This invention optimizes the detection process, requiring only secondary antibody and 1×PBS buffer as auxiliary reagents, and both primary and secondary antibody incubation only need 30 minutes at room temperature. This significantly improves the convenience and speed of using the detection material, shortens the detection time, and requires only a fluorescence microscope, avoiding the limitations of complex and expensive equipment required for flow cytometry.

[0019] The main objective of this invention is to avoid the limitations of flow cytometry, which is complex and requires expensive equipment, based on the CBA method. It also avoids the false negatives caused by changes in antigen spatial conformation in ELISA and BLOT methods, and avoids the false negatives caused by transmembrane expression due to transfection with the original Caspr1 sequence. Attached Figure Description

[0020] 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.

[0021] Figure 1 These are cell images taken under a bright-field microscope after plasmid transfection and timed culture in steps S6-S10 of the embodiment. Figure 2 These are protein fluorescence images after the preparation of the anti-Caspr1 antibody detection material in the examples; Figure 3 The image shown is a fluorescence image of the protein complex after staining with the anti-Caspr1 antibody detection material in the example. Detailed Implementation

[0022] 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.

[0023] This application discloses a material for detecting anti-Caspr1 antibodies and a method for preparing the same.

[0024] Example I. Design of plasmids and vectors for transfection 1. Vector construction and plasmid structure: pcdna3.1-target gene-linker-helper-linker-×3Flag-P2A-EGFP-AmpR; The antigen sequence of the target gene is shown in SEQ ID NO.1.

[0025] 2. Target gene: (1)Caspr1: The original antigen sequence of the target gene is shown in SEQ ID NO.3. Original antigen sequence: Molecular type: AA (DNA, RNA, AA), Organism name: Homo sapiens.

[0026] Based on the original antigen sequence of the target gene, the sequence is modified by removing bases 1-36 from the 3' segment to form a new antigen sequence, as shown in SEQ ID NO.1.

[0027] Modified antigen sequence: Molecular type: AA (DNA, RNA, AA), Organism name: Homo sapiens.

[0028] (2) Auxiliary section Original sequence: Molecular type: AA (DNA, RNA, AA), Organism name: Homo sapiens. The sequence of the auxiliary segment is shown in SEQ ID NO.2.

[0029] II. Preparation Method of Anti-Caspr1 Antibody Detection Materials 1. Calculate the required quantity according to the production batch, take out a 60mm×60mm sterile clean square glass slide (commercially purchased) 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 the complete culture medium (86 (80-90)% (v / v) DMEM medium (commercially available) + 14 (10-20)% (v / v) premium fetal bovine serum FBS (commercially available) + an additional 2 (1-3)% (v / v) penicillin-streptomycin-amphotericidal mixture (commercially available), for example, 43 mL DMEM medium + 7 mL FBS + 1 mL triple antibiotic mixture, totaling 51 mL) in a constant temperature water bath and preheat to 37°C; Take a sterile 50mL centrifuge tube from the biosafety cabinet, pour out 20mL of preheated complete culture medium, add 0.2mL of HEK293T cell suspension (Taizhen Medical Cell Bank cells, cell quantity approximately 1.5-2.0×10^6), mix well by pipetting and aspirating, then add to a culture dish containing a climbing slide, and 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 (commercially available) to each, and label them A and B respectively. At the same time, preheat the complete medium to 37°C. Add 14 (10-20) μg of Caspr1 expression plasmid to tube A and gently mix with a pipette. Add 70 (50-100, which is 5 times the number of plasmid mass) μL of linear polyacetylimide (PEI) transfection reagent (commercially available) 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 to form a mixture. Gently pipette and mix well. After standing for another 15 minutes, add 20 mL of preheated complete culture medium from step 3 and mix well by pipette. 9. Remove the cell culture dish from the cell culture incubator, remove the original culture medium in the biosafety cabinet, add the mixture from step 8 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 (commercially available) to rinse, and then remove the buffer. 12. Add 20 mL of 4% PFA fixative (commercially available), 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 buffer (3 (2-5)% (m / v) BSA dissolved in 1×PBS buffer), let stand for 60 minutes, and then remove the blocking buffer; 15. Add 20 mL of preservative solution (0.05 (0.02-0.1)% (m / v) sodium thimerosal (commercial purchase), 5 (3-10)% (m / v) trehalose (commercial purchase), 5 (3-12)% (m / v) dextran-70 (commercial purchase), 10 (5-20)% (v / v) glycerol (commercial purchase), dissolved in 1×PBS buffer), let stand for 30 minutes, and then aspirate 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 apply them to the surface of the glass slide with UV-curing adhesive (commercially available). Store at -20 degrees Celsius.

[0030] III. Methods of Using Anti-Caspr1 Antibody Detection Materials 1. Warming: Remove the glass slide with the smear, 1×PBS buffer, and AF594-labeled goat anti-human IgG (commercially available) from the refrigerator and warm them to room temperature for 10-15 minutes before staining.

[0031] 2. Cleaning: Place the glass slide in the humidified chamber, add 100uL of 1×PBS buffer to each reaction area (the slide needs to be covered), and rinse for 3 minutes.

[0032] 3. Sample serum incubation: Discard the reaction zone buffer, add 60uL of the corresponding serum sample (1:10, diluted with 1×PBS buffer) to each reaction zone, and incubate at room temperature for 30min. During the process, take care to prevent the reaction zone from drying out.

[0033] 4. Washing: Discard the incubated serum, add 100uL of 1×PBS buffer (cover the slide) to each reaction area, rinse for 3 minutes, remove the buffer, and repeat twice.

[0034] 5. Secondary antibody incubation: Add 60uL of AF594-labeled goat anti-human IgG (1:400, diluted with 1×PBS buffer), incubate at room temperature in the dark for 30 min, and take care to prevent the reaction area from drying out during the process.

[0035] Note: This step must be performed in the dark.

[0036] 6. Washing: Discard the secondary antibody, add 100uL of 1×PBS buffer (to cover the slide) to each reaction zone, rinse for 3 minutes, remove the buffer, and repeat twice.

[0037] 7. Add 60uL of 1×PBS buffer (to cover the slide), and observe the experimental results under a fluorescence inverted microscope.

[0038] The glass slides for the cell carrier in this invention can be replaced with 96-well cell culture plates; only the specific amounts of liquid added need to be changed, but the entire process remains the same. The coating reagent for the glass slides can be replaced with diluted rat tail collagen type 1, a poly-L-lysine-gelatin mixture, or other commercially available cell adhesion aids, with similar results. The slide cutting size can be customized to any size less than 60mm × 60mm without affecting the detection results; only the amounts of liquids added during use need to be changed, and the entire process remains the same. Sodium thimerosal can be replaced with an equal proportion of sodium azide, which may yield better results, but sodium azide is a highly toxic reagent, difficult to obtain, and inconvenient to use. BSA can be replaced with an equal proportion of sheep serum, with similar results, but the shelf life is reduced. In the complete plasmid vector pcdna3.1-(target gene)-linker-(helper segment)-linker-×3Flag-P2A-EGFP-AmpR, P2A can be replaced with T2A. Detection is possible even without the linker and ×3Flag sequences. EGFP can be omitted if a green fluorescent background is not required, or it can be replaced with other fluorescent protein expression genes such as mCHERRY, depending on other fluorescent background requirements. The AF594-labeled goat anti-human IgG4 secondary antibody in the instructions can be replaced with any non-green fluorescent protein-labeled anti-human IgG4 secondary antibody. The 1×PBS buffer in the instructions can be replaced with 1×PBST buffer.

[0039] Figure 1 It is a bright-field image of a cell, from Figure 1 As can be seen from the data, cells in good condition and able to fully grow indicate that the culture protocol is effective. Figure 2 In this study, the transfection efficiency can be judged from the proportion of green fluorescent cells in the total population. Figure 2 As can be seen, the green color almost fills the entire area of ​​the image, indicating high transfection efficiency and a good culture program. Figure 3 After staining, observe the staining results; it's quite obvious. Figure 3 The presence of bright red cells that stand out against the background indicates that the antigen expressed by the transfected plasmid can bind to the antibody to be tested, resulting in color development under the further binding of the secondary antibody. This demonstrates that the anti-Caspr1 antibody detection material can be practically used to detect the antibody to be tested.

[0040] Comparative Example 1 The method used is based on the prior art: Publication No. CN119335194A; Patent Title: An antibody detection kit for detecting chronic inflammatory demyelinating polyradiculoneuropathy and its preparation method; the anti-Caspr1 antibody BLOT detection reagent produced by the disclosed scheme.

[0041] Comparative Example 2 The anti-Caspr1 antibody ELISA detection reagent produced according to the prior art 1: Publication No. CN120721958A; Patent title: A stabilizing agent, cell antigen dry sheet and preparation method and application of the dry sheet; the disclosed scheme.

[0042] Comparative Example 3 The anti-Caspr1 antibody detection material obtained using the technical solution of this invention is incubated with sample serum at 37°C for 60 min, and with secondary antibody at 37°C for 45 min. The remaining operations are the same as those of the technical solution of this invention.

[0043] The difference between Comparative Example 3 and the Example is that the detection operation is simplified, mainly to demonstrate that the detection effect will not be worse after the detection operation is shortened and simplified.

[0044] Results Test (1) Testing the detection sensitivity of the example and comparative example 1 and comparative document 2: The detection results of the example and comparative example 1 and comparative example 2 using the same 20 anti-Caspr1 positive samples are compared as shown in Table 1.

[0045] Table 1. Detection results of anti-Caspr1 positive samples from the examples and Comparative Examples 1 and 2. As can be seen from the data in Table 1, the positive detection rates of Comparative Example 1 and Comparative Example 2 were 80.0% and 85%, respectively, both lower than the positive detection rate of 100% in the Example, indicating that the detection sensitivity of the reagent in the Example was higher than that of Comparative Example 1 and Comparative Example 2.

[0046] (2) Detection specificity of the test example and comparative example 1 and comparative document 2: The test results of the example and comparative example 1 and comparative example 2 using the same serum samples of 50 healthy individuals are compared as shown in Table 2.

[0047] Table 2 shows the detection results of serum samples from healthy individuals in the examples and in Comparative Examples 1 and 2. As can be seen from Table 2, the false positive rates of Comparative Example 1 and Comparative Example 2 were 12.0% and 14.0%, respectively, both higher than the false positive rate of 2.0% of the Example. Compared with Comparative Example 1 and Comparative Example 2, the Example had a lower proportion of false positive results, indicating that the detection in the Example had good specificity.

[0048] (3) Test the shelf life of the examples and comparative examples 1 and 2: Every 7 days, the same anti-Caspr1 antibody IgG (commercially purchased, diluted 1:1000 with 1×PBS buffer) was tested in the examples, comparative examples 1 and 2. The test was repeated three times within a single test. The expiration date was the day when no positive result was detected in all three tests. The results are shown in Table 3.

[0049] Table 3. Shelf life of the examples and comparative examples 1 and 2 As can be seen from Table 3, the expiration dates of Comparative Example 1 and Comparative Example 2 were 123 days and 69 days, respectively, both lower than the 204 days of the Example. This indicates that the shelf life of the reagents in the Example was significantly longer than that of Comparative Example 1 and Comparative Example 2, and that they had better stability.

[0050] (4) Test the detection sensitivity of the Example and Comparative Example 3: The detection results of the Example and Comparative Example 3 using the same 10 anti-Caspr1 positive samples are compared as shown in Table 4.

[0051] Table 4 shows the detection results of anti-Caspr1 positive samples from the examples and comparative example 3. As can be seen from the data in Table 4, the positive detection rate of both the Example and Comparative Example 3 was 100%. The cell production process of the Example was exactly the same as that of Comparative Example 3, except for the staining operation process. This test was to demonstrate that the detection sensitivity was not affected after the optimized detection process shortened the time.

[0052] (5) Detection specificity of the test example and comparative example 3: The detection results of the example and comparative example 3 using the same 20 serum samples from healthy individuals are compared, as shown in Table 5.

[0053] Table 5 shows the detection results of serum samples from the examples and comparative example 3. As can be seen from the data in Table 5, the false positive rate of both Example 1 and Comparative Example 3 was 0%. The cell production process of Example 1 was exactly the same as that of Comparative Example 3, except for the staining operation procedure. This test was to demonstrate that the detection specificity was not affected after the optimized detection procedure shortened the time.

[0054] 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. An expression plasmid that highly expresses a Caspr1 antibody, characterized in that, The structure of this plasmid is pcdna3.1-Caspr1-linker-helper-linker-×3Flag-P2A-EGFP-AmpR; The antigen sequence of Caspr1 is shown in SEQ ID NO.1, and the antigen sequence of the auxiliary segment is shown in SEQ ID NO.

2.

2. A method for preparing anti-Caspr1 antibody materials, characterized in that, Includes the following steps: S1. Place the cell carrier into a cell culture dish; S2. Add enhanced cell adhesion reagent to the cell culture dish, let it stand at room temperature for coating, and then blot dry the enhanced cell adhesion reagent. S3. Preheat the complete culture medium; S4. Take the preheated complete culture medium from step S3, add HEK293T cell suspension, and then add it to the cell culture dish from step S2. S5. Place the cell culture dish from step S4 into a cell culture incubator for cell culture. S6. Take two centrifuge tubes and label them A and B. Add opti-MEM medium to tubes A and B respectively and preheat them. S7. Add the Caspr1 expression plasmid to tube A and add the linear polyacetylimine transfection reagent to tube B; S8. Pipette the liquid from tube A into tube B to form a mixture. After mixing and letting stand, add the preheated complete culture medium from S3. S9. Take out the cell culture dish from step S5, remove the original culture medium, and add the mixture from S8 to the cell culture dish. S10. Transfer the cell culture dish from step S9 to a cell culture incubator for cell culture. S11. Remove the cell culture dish from the cell culture incubator, aspirate the original culture medium, add buffer solution to rinse, and then aspirate the buffer solution. S12. Add PFA fixative to the cell culture dish from step S11, let it stand, and then remove the fixative. S13. Add buffer solution to the cell culture dish from step S12 to rinse, and then remove the buffer solution. S14. Add blocking solution to the cell culture dish in step S13, let it stand, and then remove the blocking solution. S15. Add the preservative solution to the cell culture dish in step S14, let it stand, and then remove the preservative solution. S16. After opening the cell culture dish from step S15 and allowing it to stand to air dry, cut the cell carrier and attach it to the surface of the glass slide using UV-curable adhesive.

3. The method for preparing anti-Caspr1 antibody material according to claim 2, characterized in that, In step S1, the cell slides are glass slides or cell culture plates.

4. The method for preparing anti-Caspr1 antibody material according to claim 2, characterized in that, The cell adhesion enhancer in step S2 is diluted 1:50 with 1×PBS buffer; the complete culture medium in step S3 is 86% DMEM medium + 14% premium fetal bovine serum (FBS) + 2% penicillin-streptomycin-amphotericidal mixture; the blocking solution in step S14 is 3% BSA dissolved in 1×PBS buffer; the preservation solution in step S15 is 0.05% sodium thimerosal, 5% trehalose, 5% dextran-70, and 10% glycerol dissolved in 1×PBS buffer.

5. The method for preparing anti-Caspr1 antibody material according to claim 2, characterized in that, In step S3, the preheating temperature is 37℃, and in step S5, the culture conditions are 37±0.5℃ and 5% CO2 for 24h. In step S6, the preheating temperature is 37℃, and in step S10, the culture conditions are 37±0.5℃ and 5% CO2 for 24h.

6. The method for preparing anti-Caspr1 antibody material according to claim 2, characterized in that, The buffer in steps S11 and S13 is 1×PBS buffer or 1×PBST buffer.

7. The method for preparing anti-Caspr1 antibody material according to claim 2, characterized in that, In step S2, the coating reagent for the cell carrier can be diluted rat tail collagen type 1 or polylysine-gelatin mixture.

8. The method for preparing anti-Caspr1 antibody material according to claim 4, characterized in that, In step S15, sodium thimerosal can be replaced by sodium azide in an equal proportion, and BSA can be replaced by sheep serum in an equal proportion.

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