Detection of anti-nf155 antibody material and methods of making same

Abstract

This invention belongs to the field of biodetection technology, specifically relating to a material for detecting anti-NF155 antibodies and its preparation method. This invention constructs an expression plasmid capable of highly expressing the NF155 antigen, transfects it into HEK293T cells, and modifies the pCDH vector with enhancers during construction. Subsequently, a modified cell culture protocol is used to obtain a material for detecting anti-NF155 antibodies based on a cell immunofluorescence methodology. This invention uses a cell immunofluorescence methodology to maximize the preservation of the antigen's spatial conformation through cell expression and antigen immobilization, preventing false negatives due to changes in binding sites. This invention selects the lentiviral vector pCDH to avoid the problem of non-specific binding that is difficult to distinguish, as the proteins expressed by common target gene vectors such as pcDNA3.1 may themselves contain antibody binding sites. This improves detection specificity and solves the problem of false negatives.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and specifically relates to a material for detecting anti-NF155 antibodies and its preparation method. Background Technology

[0002] Neurofascin (NF) is a member of the immunoglobulin superfamily, exhibiting highly specific cellular localization: primarily distributed in the neuronal axon membrane and nodes of Ranvier. In the axon membrane, it participates in intercellular adhesion and signal transduction; in the nodes of Ranvier, it maintains axonal structural stability and ensures efficient nerve signal transmission through interaction with ankyrin G. Through selective splicing, it produces subtypes such as NF155, NF166, NF180, and NF186, with a structure comprising six immunoglobulin domains, five fibronectin type III repeat units, a transmembrane domain, and a cytoplasmic domain. NF155 is particularly involved in axonal initiation stability and myelination in the mature nervous system. It maintains myelin structure by binding to the Caspr / Contactin complex, while its deficiency leads to impaired nerve conduction. Studies have found that anti-NF155 specific antibodies are among the most common nodular and paranodular antibodies, accounting for 4% to 18% of all chronic demyelinating polyneuropathy (CIDP) cases; therefore, its detection has significant clinical implications.

[0003] Existing anti-NF155 antibody detection technologies and platforms almost entirely rely on enzyme-linked immunosorbent assay (ELISA). The standard procedure involves designing an NF155 sequence vector and expression plasmid, expressing it in bacteria, then isolating, purifying, and coating the plasmid onto an ELISA plate. However, the isolation and purification process inherently damages the antigen protein, and the protein coated on the plate loses its original spatial conformation. Therefore, to address the issue of false negatives, a newer approach has been to increase the number of NF155 antigen clones, hoping to bind antibodies capable of binding to any of the clones. However, even with five NF155 antigens added, false negatives still occur in ELISA detection because the problem of altered binding sites due to changes in protein spatial conformation remains unresolved. Summary of the Invention

[0004] This invention proposes a material for detecting anti-NF155 antibodies and its preparation method, which solves the problem of missed detection caused by changes in binding sites due to changes in protein spatial conformation.

[0005] The technical solution of this invention is implemented as follows: An expression plasmid that highly expresses NF155 antibody has the structure pCDH-CMVenhancer-CMVpromoter-NF155-linker-×3Flag-P2A-EGFP-AmpR, wherein the antigen sequence of NF155 is shown in SEQ ID NO.1.

[0006] Through the above technical solution, the advantage of plasmid construction in this invention is the selection of the pCDH vector. This is because the most commonly used pcDNA3.1 vector for plasmid construction has a small probability of causing false positives due to unknown antibodies in the sample attacking the protein expressed by the plasmid vector itself. The pCDH vector does not have this problem. However, using the pCDH vector can lead to a lower expression level of the target gene. Therefore, this invention adds an additional CMV enhancer after the original CMV promoter in the vector structure to enhance the expression ability of the pCDH vector.

[0007] A method for preparing materials for detecting anti-NF155 antibodies, using a cell immunofluorescence assay to prepare materials for detecting anti-NF155 antibodies.

[0008] The above-described technical solution utilizes cellular immunofluorescence methodology to overcome the deficiency caused by altered antigen spatial conformation. Both ELISA and BLOT methods rely on extracting or synthesizing antigens and immobilizing them on a carrier surface. The extraction and synthesis processes themselves disrupt the antigen's spatial conformation, and the immobilization and preservation process on a planar carrier further disrupts it. This invention employs the CBA method, where the antigen does not need to be extracted; it is synthesized by cells and preserved within them. The immobilization process also occurs within the cell, where the antigen is immobilized along with the cell itself onto the carrier surface. Therefore, the antigen's spatial conformation is preserved to the greatest extent among all methodologies. This optimal preservation of antigen conformation, combined with the expression plasmid designed in this invention that highly expresses the NF155 antibody, solves the problem of missed detection caused by altered binding sites due to changes in protein spatial conformation in existing technologies.

[0009] A method for preparing anti-NF155 antibody materials, characterized by comprising 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, mix well, let stand, and then add the preheated complete culture medium from step S3. S9. Take out the cell culture dish from step S5, remove the original culture medium, and add the liquid mixture from tube A and tube B obtained in step 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.

[0010] 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.

[0011] Optionally, in step S1, the cell slide is a glass slide or a cell culture plate.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] Optionally, the buffer in steps S11 and S13 may be 1×PBS buffer or 1×PBST buffer.

[0017] Optionally, in step S2, the coating reagent for the cell carrier can be diluted rat tail collagen type 1 or polylysine-gelatin mixture.

[0018] Through the above technical solution, this coating reagent can enhance cell adhesion and cell fixation.

[0019] 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.

[0020] 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 NF155 antigen for transfection into HEK293T cells. During construction, the pCDH vector is modified with an enhancer. Subsequently, a modified cell culture protocol yields a material for detecting anti-NF155 antibodies based on the cell immunofluorescence assay (CBA). This invention replaces the existing enzyme-linked immunosorbent assay (ELISA) with the cell immunofluorescence assay (CBA), maximizing the preservation of the antigen's spatial conformation through cell expression and antigen immobilization, preventing false negatives due to changes in binding sites. This invention selects the lentiviral vector pCDH to avoid the problem of non-specific binding that may be difficult to distinguish due to the presence of antibody binding sites in proteins expressed by common target gene vectors such as pcDNA3.1, thus improving detection specificity. This invention modifies the pCDH vector by adding an enhancer to the CMV promoter, enabling cells to express the antigen efficiently, improving detection sensitivity, and solving the problem of false negatives, thereby maintaining detection sensitivity without reducing detection specificity. Attached Figure Description

[0021] 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.

[0022] 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-NF155 antibody detection material in the examples; Figure 3 The image shown is a fluorescence image of the protein complex after staining with the anti-NF155 antibody detection material in the example. Detailed Implementation

[0023] 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.

[0024] This application discloses a material for detecting anti-NF155 antibodies and a method for preparing the same.

[0025] Example I. Design of plasmids and vectors for transfection 1. Vector construction and plasmid structure: pCDH-CMVenhancer-CMVpromoter-target gene-linker-×3Flag-P2A-EGFP-AmpR; 2. Target gene Target gene: NF155; the original antigen sequence of the target gene is shown in SEQ ID NO.1; molecular type: AA (DNA, RNA, AA); organism name: Homo sapiens.

[0026] II. Preparation of Anti-NF155 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.

[0027] 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.

[0028] 3. Place the complete culture medium (86 (80-90)% (v / v) DMEM medium (commercial purchase) + 14 (10-20)% (v / v) premium fetal bovine serum FBS (commercial purchase) + an additional 2 (1-3)% (v / v) penicillin-streptomycin-amphotericidal mixture (commercial purchase). For example, 43 mL of DMEM medium + 7 mL of FBS + 1 mL of triple antibiotic mixture, for a total of 51 mL) in a constant temperature water bath and preheat to 37°C.

[0029] Take a sterile 50mL centrifuge tube from the biosafety cabinet, pour 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 then add it to a culture dish containing a climbing slide. Transfer the culture dish to a cell culture incubator.

[0030] 5. Incubate at 37±0.5℃ and 5% CO2 for 24 hours in a cell culture incubator.

[0031] 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.

[0032] Add 12 (10-20) μg of NF155 expression plasmid to tube A and gently mix with a pipette. Add 60 (50-100, which is 5 times the number of plasmids) μL of linear polyacetylimide (PEI) transfection reagent (commercially available) to tube B and gently mix with a pipette.

[0033] 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 from step 3 above, and mix with a pipette.

[0034] 9. Remove the cell culture dish from the cell culture incubator, remove the original culture medium in the biosafety cabinet, add the liquid mixture from tube A and tube B in step 8 above to the culture dish, and transfer the culture dish to the cell culture incubator.

[0035] 10. Incubate at 37±0.5℃ and 5% CO2 for 24 hours in a cell culture incubator.

[0036] 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.

[0037] 12. Add 20 mL of 4% PFA fixative (commercially available), let stand for 30 minutes, and then remove the fixative.

[0038] 13. Add 10 mL of 1×PBS buffer to rinse, then remove the buffer.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] III. Methods of Using Anti-NF155 Antibody Detection Materials 1. Warming: Remove the glass slide with the smear, 1×PBS buffer, and AF555-labeled goat anti-human IgG4 (commercially available) from the refrigerator and warm to room temperature for 10-15 minutes before staining.

[0043] 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.

[0044] 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 37℃ for 40min.

[0045] 4. Washing: Discard the incubated serum, add 100uL of 1×PBS buffer (to cover the slide) to each reaction area, rinse for 3 minutes, remove the buffer, and repeat 3 times.

[0046] 5. Secondary antibody incubation: Add 60 μL of AF555-labeled goat anti-human IgG4 (1:400, diluted with 1×PBS buffer) and incubate at 37°C for 30 min. Note: This step requires protection from light at the beginning.

[0047] 6. Washing: Discard the secondary antibody, add 100uL of 1×PBS buffer to each reaction zone (the slide needs to be covered), rinse for 3 minutes, remove the buffer, and repeat 3 times.

[0048] 7. Add 60 μL of 1×PBS buffer and observe the experimental results under a fluorescence inverted microscope.

[0049] The glass slides for the cell vector in this invention can be replaced with 96-well cell culture plates, requiring only a change in the specific liquid amounts, while the entire process remains unchanged. The coating reagents for the glass slides can be replaced with diluted rat tail collagen type 1, poly-L-lysine-gelatin mixture, or other commercially available cell adhesion aids. The slide cutting size can be customized to any size less than 60mm × 60mm without affecting the detection results; only the liquid amounts added during use need to be changed, while the entire process remains the same. Sodium thimerosal can be replaced with an equal proportion of sodium azide. BSA can be replaced with an equal proportion of sheep serum. In the complete plasmid vector pCDH-CMVenhancer-CMVpromoter-target gene-linker-×3Flag-P2A-EGFP-AmpR, P2A can be replaced with T2A. Detection is possible even without the linker and ×3Flag sequences. If a green fluorescent background is not required, EGFP can be omitted, or it can be replaced with other fluorescent protein expression genes such as mCHERRY, depending on other fluorescent background requirements. The AF555-labeled goat anti-human IgG4 secondary antibody used in the instructions can be replaced with any non-green light band fluorescein-labeled anti-human IgG4 secondary antibody. The 1×PBS buffer used in the instructions can be replaced with 1×PBST buffer.

[0050] Figure 1 It is a bright-field image of a cell, from Figure 1 As can be seen, good cell condition and complete confluence indicate a good culture protocol. 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, green 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 are significantly brighter than the cell background (green arrows indicate positive cell signals) indicates that the antigen expressed by the transfected plasmid in the cell can bind to the antibody to be tested, thus developing color under the further binding of the secondary antibody. This demonstrates that the anti-NF155 antibody detection material can be practically used to detect the antibody to be tested.

[0051] Comparative Example The ELISA reagents are produced according to the scheme disclosed in the prior art: "A detection method combining anti-NF155 antibody detection and enzyme-linked immunosorbent assay", publication number CN120089402A.

[0052] Results Test (1) The detection performance of the methods in the examples and the comparative examples was compared. The detection results of the same 15 anti-NF155 positive samples used in the examples and the comparative examples are shown in Table 1.

[0053] Table 1 Comparison of the detection results of the methods in the examples and comparative examples. As can be seen from Table 1, the positive detection rate of the comparative example was 86.7%, which was 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 the comparative example.

[0054] (2) The shelf life of the methods in the examples and comparative examples was compared. The same anti-NF155 antibody IgG (commercially purchased, diluted 1×PBS buffer 1:1000) was used for testing every 7 days. The test was repeated three times within a single test. The day when no positive result was detected in all three tests was taken as the expiration date. The results are shown in Table 2.

[0055] Table 2 Comparison of shelf life detected by the method in the examples and comparative examples. As can be seen from Table 2, the expiration date of the comparative example was 98 days, which is lower than the 287 days in the example, indicating that the shelf life of the reagents in the example is significantly longer than that of the comparative example, and the stability is better.

[0056] 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 NF155 antibody, characterized in that, The structure of the plasmid is pCDH-CMVenhancer-CMVpromoter-NF155-linker-×3Flag-P2A-EGFP-AmpR, wherein the antigen sequence of NF155 is shown in SEQ ID NO.

1.

2. A method for preparing materials for detecting anti-NF155 antibodies, characterized in that, Materials for detecting anti-NF155 antibodies were prepared using a cell immunofluorescence assay.

3. The method for preparing anti-NF155 antibody material according to claim 2, 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 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, mix well, let stand, and then add the preheated complete culture medium from step S3. S9. Take out the cell culture dish from step S5, remove the original culture medium, and add the liquid mixture from tube A and tube B obtained in step 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.

4. The method for preparing anti-NF155 antibody material according to claim 3, characterized in that, In step S1, the cell slides are glass slides or cell culture plates.

5. The method for preparing anti-NF155 antibody material according to claim 3, 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.

6. The method for preparing anti-NF155 antibody material according to claim 3, 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.

7. The method for preparing anti-NF155 antibody material according to claim 3, characterized in that, The buffer in steps S11 and S13 is 1×PBS buffer or 1×PBST buffer.

8. The method for preparing anti-NF155 antibody material according to claim 3, 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.

9. The method for preparing anti-NF155 antibody material according to claim 5, 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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