Kit for detecting anti-AQP4 autoantibody and application thereof

Abstract

The invention belongs to the technical field of medical immunodetection analysis, and discloses a kit for detecting an anti-AQP4 autoantibody and application of the kit. The kit comprises a cell pore plate and a cell freezing medium; reaction holes of the cell pore plate are internally coated with a coating substance, and the coating substance is one of polylysine, gelatin and collagen; the cell cryopreservation solution is a cryopreservation protection solution of cells containing overexpressed AQP4 antigens; during coating, the concentration of a coating substance is 0.5 mg / mL-3. 0 mg / mL, and the dosage of the coating substance is 40 [mu] L / hole to 60 [mu] L / hole. According to the kit for detecting the anti-AQP4 autoantibody, the detection performance can be improved, and the kit can be frozen at low temperature for a long time and then unfrozen for detection.

Description

Technical Field

[0001] This invention belongs to the field of medical immunoassay detection and analysis technology, specifically relating to a kit for detecting anti-AQP4 autoantibodies and its application. Background Technology

[0002] The myelin sheath of the central nervous system (CNS), formed by oligodendrocytes, facilitates efficient, skip-transmission of nerve electrical signals and protects normal neuronal function. CNS inflammatory demyelinating diseases are a group of diseases caused by various pathological factors leading to myelin sheath damage and loss, while nerve cells remain relatively intact. Common CNS inflammatory demyelinating diseases include neuromyelitis optic spectrum disorder (NMOSD), myelin oligodendrocyte glycoprotein-IgG associated disorders (MOGAD), multiple sclerosis (MS), and acute disseminated encephalomyelitis (ADEM). NMOSD is a spectrum of central nervous system inflammatory demyelinating diseases primarily mediated by humoral immunity through antigen-antibody interactions. These diseases primarily affect the optic nerve and spinal cord, with over 80% of patients exhibiting autoantibodies against aquaporin-4 (AQP4) (AQP4-IgG). The pathogenesis of NMOSD is mainly associated with pathogenic AQP4-IgG, which attacks astrocytes in the central nervous system, causing severe axonal damage and demyelination. As a specific immunomarker for NMOSD, AQP4-IgG possesses high specificity. The pathogenesis of NMOSD is related to AQP4-IgG, with approximately 70%-80% of patients testing positive for this antibody. Detection of this antibody is crucial for the diagnosis, differential diagnosis, and treatment of related diseases.

[0003] Currently, the primary laboratory detection method for specific AQP4-IgG corresponding to NMOSD is the cell-based indirect immunofluorescence assay (CBA), which can be further divided into fixed-cell CBA and live-cell CBA. The "Guidelines for the Diagnosis and Treatment of Neuromyelitis Optic Spectrum Disorders in China (2025 Edition)" mentions that the Live-CBA technique has higher sensitivity compared to the traditional fixed-cell method. However, Live-CBA reagents generally require the testing laboratory to establish its own reagent production system and prepare them on demand. This method requires a series of relatively complex procedures such as cell culture and transfection, demanding that the relevant laboratory be equipped with standard cell culture facilities, related equipment, and professional laboratory personnel. Furthermore, the preparation cycle for the relevant test reagents typically takes 2-3 days. Combined with the unpredictability of the sample volume, this results in significant additional resource and personnel investment and waste for testing institutions. Simultaneously, this self-built testing system also makes it difficult to standardize testing procedures and threshold interpretation among different laboratories, creating obstacles to clinical application and exhibiting significant limitations.

[0004] In the applicant's earlier invention patent application (application number 2025116114246), a cell cryopreservation solution was prepared by mixing cells overexpressing MOG antigen with cryopreservation protection solution. This solution, along with a specific coating material, was then placed in the reaction wells of a cell plate. The cell cryopreservation solution and the coated cell plate were then frozen and stored at low temperatures for extended periods. This ensured the stability of the detection reagent during long-distance cryogenic transport, enabling the kit to be used in different locations. After thawing the cell cryopreservation solution and the cell plate, the cell cryopreservation solution was added to the reaction wells of the cell plate for a short incubation to allow adhesion. The cells overexpressing MOG antigen could then be used as the detection matrix without cell fixation. The sample could be added directly while maintaining cell viability and the native state of the antigen. The antibody in the sample specifically binds to the target antigen. Then, a secondary antibody was added to form an antigen-antibody complex. The staining was observed using a fluorescence microscope, allowing for accurate detection of the presence of the corresponding antibody in the sample. The operation was simple, convenient, and highly efficient. However, when the kit was applied to the detection of AQP4-IgG, the inventors further discovered that the type of coating material used in the preparation of the cell well plates and the concentration of the solution affected the detection performance. Summary of the Invention

[0005] The purpose of this invention is to provide a kit for detecting anti-AQP4 autoantibodies, which can accurately detect antibodies based on the Live-CBA method and effectively improve the sensitivity of detection.

[0006] The following technical solutions are used to achieve the above objectives.

[0007] The first aspect of the present invention provides a kit for detecting anti-AQP4 autoantibodies, the kit comprising a cell plate and a cell cryopreservation solution;

[0008] The reaction wells of the cell plate are coated with a coating material, which is one of poly-L-lysine, gelatin, and collagen.

[0009] The cell cryopreservation solution is a cryopreservation protection solution containing cells overexpressing AQP4 antigen;

[0010] During coating, the concentration of the coating agent is 0.5 mg / mL to 3.0 mg / mL, and the amount of the coating agent used is 40 μL / well to 60 μL / well.

[0011] In some embodiments, the concentration of the coating material is 0.8 mg / mL–2.5 mg / mL, and the amount of the coating material used is 45 μL / well to 55 μL / well.

[0012] In some embodiments, the concentration of the coating material is 1 mg / mL–2 mg / mL, preferably 1.3 mg / mL to 1.6 mg / mL, and the amount of the coating material used is 48 μL / well to 52 μL / well.

[0013] In some embodiments, the concentration of the cells overexpressing the AQP4 antigen in the cryopreservation solution is 1.0 × 10⁻⁶. 6 ~6.0×10 6 Cells / mL, preferably 2.5 × 10⁻⁶. 6 ~4.0×10 6 Cells / mL.

[0014] In some embodiments, the coating is one of polylysine and gelatin, preferably polylysine.

[0015] In some embodiments, the cryopreservation solution comprises, by volume percentage: 5%~15% DMSO, 55%~65% fetal bovine serum, and 25%~35% DMEM; preferably, the cryopreservation solution comprises: 8%~12% DMSO, 58%~62% fetal bovine serum, and 28%~32% DMEM.

[0016] In some embodiments, the amount of cells overexpressing the AQP4 antigen used during detection is 1.2 × 10⁻⁶. 5 ~2.0×10⁶ per reaction well 5 The number of cells per reaction well is preferably 1.4 × 10⁻⁶. 5 ~1.6 × 10⁶ per reaction well 5 One per reaction well.

[0017] In some embodiments, the kit further includes a signal-tagged anti-human IgG secondary antibody; preferably, the signal is one of Alexa Fluor 488, DyLight 488, and fluorescein isothiocyanate.

[0018] And / or, the anti-human IgG secondary antibody is one of anti-human IgG1 secondary antibody, anti-human IgG-Fc secondary antibody, and anti-human total IgG secondary antibody.

[0019] In some embodiments, constructing the cells containing the AQP4 antigen overexpression includes the following steps:

[0020] The target gene is obtained by ligating the fluorescent protein sequence to the end of the CDS protein coding sequence of AQP4;

[0021] The obtained target gene was inserted into the overexpression plasmid vector to obtain the recombinant overexpression plasmid;

[0022] The recombinant overexpression plasmid was transfected into tool cells using a transfection reagent to obtain cells containing overexpression of AQP4 antigen.

[0023] In some embodiments, the fluorescent protein is a red fluorescent protein, preferably one of mCherry, dsRed, and TagRFP; and / or,

[0024] The overexpression plasmid vector is a eukaryotic expression vector, preferably one of pcDNA3.1, pcDNA3.4, pCDH, pLV, and pCMV; and / or,

[0025] The transfection reagent is a cationic transfection reagent, preferably one of PEI, Lipofectamine 2000, and Lipofectamine 3000; and / or,

[0026] The tool cells are one of HEK293, HEK293T, and CHO; and / or,

[0027] The CDS protein coding sequence of AQP4 is connected to the fluorescent protein sequence via a self-splicing sequence; preferably, the self-splicing sequence is one of P2A and T2A.

[0028] A second aspect of the present invention provides a method for preparing the kit for detecting anti-AQP4 autoantibodies as described above, comprising the following steps:

[0029] Preparation of cell well plates:

[0030] Add the coating solution to the reaction wells of the cell plate, incubate at room temperature for 2-5 hours, remove the solution, wash with buffer solution to obtain the cell plate, and freeze at -75°C to -85°C.

[0031] Preparation of cell cryopreservation solution:

[0032] Cells overexpressing AQP4 antigen were resuspended in cryopreservation solution and frozen at -75°C to -85°C.

[0033] A third aspect of the present invention provides the application of the kit for detecting anti-AQP4 autoantibodies as described above in the preparation of products for detecting neuromyelitis optica spectrum disorders.

[0034] A fourth aspect of the present invention provides a method for detecting anti-AQP4 autoantibodies, comprising the following steps:

[0035] Step a. After thawing the frozen cell cryopreservation solution, centrifuge to collect the cells, resuspend them in DMEM complete medium containing 8%~15% FBS, and add them to the reaction wells of the cell plate. Incubate at 36℃~38℃ for 2h~5h.

[0036] Step b. Remove the liquid from the reaction wells, add the sample to be tested into the reaction wells and incubate at room temperature for 30 min to 40 min;

[0037] Step c. Add 1%~5% paraformaldehyde solution to the reaction wells and incubate at room temperature for 10min~15min;

[0038] Step d. Add 1%~5% BSA solution to the reaction wells and incubate at room temperature for 10min~15min;

[0039] Step e. Add the signal-labeled anti-human IgG secondary antibody and incubate at room temperature for 30-40 minutes.

[0040] The inventors previously successfully constructed a detection platform for detecting anti-autoantibodies by selectively coating suitable substances in cell well plates and preparing cell cryopreservation solutions using cells overexpressing antigens and cryoprotectant. In this invention, when applying this detection platform to the detection of anti-AQP4 autoantibodies, we found that the type of coating material and the concentration of the solution during cell well plate preparation have a significant impact on the detection performance of AQP4-IgG. We also found that poly-L-lysine is more suitable as a coating material for the detection of anti-AQP4 autoantibodies. By optimizing the concentration of the coating material solution, the resulting kit for detecting anti-AQP4 autoantibodies can improve detection performance and can be stored at low temperature for a long time before thawing and detection. This is of great significance for the diagnosis, differentiation, and treatment of neuromyelitis optica spectrum disorders. Attached Figure Description

[0041] Figure 1 This is the plasmid map of the recombinant overexpression plasmid pcDNA3.4-AQP4-P2A-mCherry.

[0042] Figure 2 This is a graph showing the detection results of the kit used in Example 1 of this invention for detecting AQP4-IgG positive samples.

[0043] Figure 3 This is a graph showing the detection results of the kit used in Example 2 of this invention for detecting AQP4-IgG positive samples.

[0044] Figure 4 This is a graph showing the detection results of the kit used in Comparative Example 1 of this invention for detecting AQP4-IgG positive samples.

[0045] Figure 5 This is a graph showing the detection results of the kit used in Comparative Example 2 of this invention for detecting AQP4-IgG positive samples.

[0046] Figure 6 This is a graph showing the detection results of the kit used in Comparative Example 3 of this invention for detecting AQP4-IgG positive samples.

[0047] Figure 7 This is a graph showing the detection results of the kit used in Example 3 of this invention for detecting AQP4-IgG positive samples.

[0048] Figure 8 This is a graph showing the detection results of the kit used in Example 4 of this invention for detecting AQP4-IgG positive samples.

[0049] Figure 9 This is a graph showing the detection results of the kit used in Example 5 of this invention for detecting AQP4-IgG positive samples.

[0050] Figure 10 This is a graph showing the detection results of the kit used in Example 6 of this invention for detecting AQP4-IgG positive samples.

[0051] Figure 11 This is a graph showing the detection results of the kit used in Example 7 of this invention for detecting AQP4-IgG positive samples.

[0052] Figure 12 This is a graph showing the detection results of the kit used in Example 8 of this invention for detecting AQP4-IgG positive samples.

[0053] Figure 13 This is a graph showing the detection results of the kit used in Example 9 of this invention for detecting AQP4-IgG positive samples. Detailed Implementation

[0054] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0055] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0056] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0057] Terminology: The anti-AQP4 autoantibody described in this invention is AQP4-IgG.

[0058] In this invention, the inventors first successfully constructed a detection platform for detecting anti-autoantibodies by selectively coating suitable substances in cell well plates and preparing cell cryopreservation solutions using cells overexpressing antigens and cryoprotectant. In this invention, when applying this detection platform to the detection of anti-AQP4 autoantibodies, it was found that the type of coating material and the concentration of the solution during cell well plate preparation significantly affect the detection performance of AQP4-IgG. Furthermore, it was discovered that poly-L-lysine is particularly suitable as a coating material for the detection of anti-AQP4 autoantibodies. By optimizing the concentration of the coating material solution, the resulting kit for detecting anti-AQP4 autoantibodies improves detection performance and allows for long-term cryopreservation at low temperatures followed by thawing and detection. This has significant implications for the diagnosis, differentiation, and treatment of neuromyelitis optica spectrum disorders.

[0059] The inventors further discovered that in the detection of anti-AQP4 autoantibodies, the composition of the cryopreservation solution and the amount of cells overexpressing AQP4 antigen also affect the detection results. By optimizing the composition of the cryopreservation solution and the amount of cells overexpressing AQP4 antigen, the detection performance can be further improved.

[0060] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0061] Example 1:

[0062] This embodiment provides a method for preparing and detecting a kit for detecting anti-AQP4 autoantibodies, specifically including the following steps:

[0063] ① Plasmid Construction: The full-length CDS sequence of human AQP4 (NM_004028.5) was synthesized, and the mCherry sequence was ligated to the end by a self-splicing sequence P2A as the target gene. The target gene was inserted into the eukaryotic cell overexpression plasmid vector pcDNA3.4 to obtain the recombinant overexpression plasmid pcDNA3.4-AQP4-P2A-mCherry ( Figure 1 ).

[0064] ② Cell transfection: The recombinant overexpression plasmid pcDNA3.4-AQP4-P2A-mCherry was transfected into HEK293 cells using Lipofectamine 3000. The cells were then cultured at 37°C in a 5% CO2 cell incubator for 24 hours to obtain cells overexpressing the AQP4 antigen.

[0065] ③ Cell cryopreservation: Collect cells overexpressing AQP4 antigen by centrifugation, and cryopreserve them at a rate of 2.5 × 10⁻⁶. 6 Cells were resuspended at a concentration of 100 cells / mL using 10% DMSO + 60% fetal bovine serum + 30% DMEM cryopreservation solution, aliquoted into cryovials, placed in EVA foam cryopreservation buffer boxes, and stored at -80°C.

[0066] ④ Preparation of cell reaction wells: Add 50 μL of 1.5 mg / mL poly-L-lysine (Beyotime Biotechnology, ST509) coating solution to the reaction wells of a removable 96-well plate, incubate at room temperature for 2 hours, remove the coating, wash with 1×PBS, air dry, package, and store at -80℃.

[0067] ⑤ Cell resuscitation: Cells overexpressing AQP4 antigen were removed from the -80℃ freezer and thawed in a 37℃ water bath. After centrifugation to collect the cells, they were resuspended in DMEM complete medium containing 10% FBS at a concentration of 1.5 × 10⁻⁶ cells / mL. 5 A concentration of 1 cell / well was added to the cell reaction wells coated with poly-L-lysine and incubated at 37°C for 2 hours in a conventional electric thermostat to allow the cells to adhere to the wells for sample detection.

[0068] ⑥ Sample Detection: Discard the liquid in the reaction wells, add 50 μL of sample working solution prepared by diluting AQP4-IgG positive samples with DMEM (dilution ratio 1:10), incubate at room temperature for 30 minutes, wash with 1×PBS, add 100 μL of 4% paraformaldehyde solution, incubate at room temperature for 10 minutes, discard the liquid in the reaction wells, add 100 μL of 5% BSA solution, incubate at room temperature for 10 minutes, discard the liquid in the reaction wells, add 50 μL of Alexa Fluor 488 labeled fluorescent anti-human IgG secondary antibody working solution (Invitrogen, A-11013), incubate at room temperature for 30 minutes, wash with 1×PBS, and then observe the results using an inverted fluorescence microscope. Figure 2).

[0069] Depend on Figure 2 The results show that there are many positive signal cells, high fluorescence intensity, and normal cell morphology.

[0070] Example 2:

[0071] This embodiment provides a method for preparing and detecting a kit for detecting anti-AQP4 autoantibodies, specifically including the following steps:

[0072] ① Plasmid construction: Construct plasmids according to step ① of Example 1.

[0073] ② Cell transfection: Perform cell transfection according to step ② of Example 1.

[0074] ③ Cell cryopreservation: Perform cell cryopreservation according to step ③ of Example 1.

[0075] ④ Preparation of cell reaction wells: Prepare cell reaction wells according to step ④ of Example 1.

[0076] ⑤ Dry ice transportation: Use sufficient dry ice to fill the packaging reagents and transport them to the off-site laboratory by express delivery. The journey takes about 48 hours. After receiving the reagents, store them in a -80℃ freezer.

[0077] ⑥ Cell resuscitation: After storing the cells in a remote laboratory at -80°C for one month, perform the remaining steps according to step ⑤ of Example 1.

[0078] ⑦ Sample testing: Perform sample testing according to step ⑥ of Example 1. See the results below. Figure 3 .

[0079] Depend on Figure 3 The results show that in this embodiment, the cell cryopreservation solution and the 96-well plate coated with the reaction material were frozen and transported to a different location at low temperature using dry ice. After being frozen at low temperature for another month, the cells were tested and showed high fluorescence intensity, a large number of positive signal cells, and normal cell morphology. This indicates that the cells can remain stable after long-distance low-temperature transportation and long-term cryopreservation and can be used normally without the need for fresh preparation.

[0080] Comparative Example 1:

[0081] This comparative example uses the Fixed-CBA method to detect AQP4 antibody-positive samples. The specific detection method includes the following steps:

[0082] ① Plasmid construction: Construct plasmids according to step ① of Example 1.

[0083] ② Cell transfection: Perform cell transfection according to step ② of Example 1.

[0084] ③ Cell fixation: Add 100 μL of 4% paraformaldehyde solution to cells overexpressing AQP4 antigen, incubate at room temperature for 10 minutes, aspirate the liquid from the reaction wells, wash 3 times with 1×PBS, add 100 μL of 5% BSA solution, incubate at room temperature for 10 minutes; add 1×PBS for storage for sample detection.

[0085] ④ Sample Detection: Add 50 μL of working solution prepared by diluting the sample with DMEM (dilution ratio 1:10) (same as the sample used in Example 1), incubate at room temperature for 30 minutes, wash with 1×PBS, add 50 μL of Alexa Fluor 488-labeled fluorescent anti-human IgG secondary antibody working solution (Invitrogen, A-11013), incubate at room temperature for 30 minutes, wash with 1×PBS, and observe the results using an inverted fluorescence microscope. See [link to results]. Figure 4 .

[0086] Example 1 and Comparative Example 1 show that when cells overexpressing AQP4 antigen are prepared into a cell cryopreservation solution and combined with a kit containing a specific coating material in the reaction wells of a cell plate for the detection of anti-AQP4 autoantibodies, good fluorescence signals and a large number of cells can be obtained, thus improving the sensitivity of the detection. However, when the Fixed-CBA method is used to detect AQP4 antibody-positive samples, the fluorescence intensity is weakened and the number of positive signal cells is reduced.

[0087] Comparative Example 2:

[0088] This comparative example provides a method for preparing a kit and a detection method for detecting anti-AQP4 autoantibodies. The difference from Example 1 is that the reaction wells of the 96-well plate are not coated with any material; otherwise, they are the same as in Example 1.

[0089] The detection was performed according to the method in Example 1, and the results are shown below. Figure 5 .

[0090] Example 1 and Comparative Example 2 show that when the reaction wells of a 96-well plate are coated with a coating material, the detection of anti-AQP4 autoantibodies yields results with good fluorescence signals, abundant cell numbers, and normal cell morphology.

[0091] Without coating the reaction wells, the number of positive signal cells was significantly reduced, the fluorescence intensity was weakened, and the cell morphology was abnormal.

[0092] Comparative Example 3:

[0093] This comparative study used the standard Live-CBA method to detect AQP4 antibody-positive samples, specifically including the following steps:

[0094] ① Plasmid construction: Construct plasmids according to step ① of Example 1.

[0095] ② Cell transfection: The recombinant overexpression plasmid vector pcDNA3.4-AQP4-P2A-mCherry was transfected into HEK293 cells in cell reaction wells using Lipofectamine 3000. Cells were then cultured at 37°C in a 5% CO2 incubator for 24 hours to obtain cells overexpressing the AQP4 antigen. These cell reaction wells containing the AQP4 antigen overexpression antigen will then be used for sample detection.

[0096] ③ Sample testing: Perform sample testing according to step ⑥ of Example 1. See the results below. Figure 6 .

[0097] Examples 1 and 3 demonstrate that the kit from Example 1 achieves the same accuracy as the conventional Live-CBA method for detecting AQP4 antibody-positive samples, exhibiting a high number of positive signal cells, high fluorescence intensity, and normal cell morphology. Furthermore, the kit from Example 1 can be frozen and stored for extended periods at low temperatures, requiring only thawing before use with a simple 2-hour pre-treatment process without affecting the detection results; the operation is simple and convenient. In contrast, Comparative Example 2 requires on-site culture and transfection of the samples under laboratory cell culture conditions to obtain cells overexpressing the AQP4 antigen, necessitating complex preparation 2-3 days in advance, resulting in high hardware requirements, complex operation, and low detection efficiency.

[0098] Example 3

[0099] This embodiment provides a method for preparing and detecting an anti-AQP4 autoantibody kit. The difference from Example 1 is that the concentration of the poly-L-lysine coating solution is 6.0 mg / mL. Everything else is the same as in Example 1.

[0100] The detection was performed according to the method in Example 1, and the results are shown below. Figure 7 .

[0101] Examples 1 and 3 show that when a kit consisting of cells overexpressing AQP4 antigen and reaction wells treated with an appropriate concentration of poly-L-lysine coating solution is used for the detection of anti-AQP4 autoantibodies, it can produce results with good fluorescence signals and a large number of cells, thus improving the sensitivity of the detection. However, when the concentration of poly-L-lysine coating solution is too high, it will affect the cell survival status and significantly reduce the cell adhesion ability, resulting in weakened fluorescence brightness and a reduced number of positive signal cells after detecting AQP4 antibody positive samples.

[0102] Example 4

[0103] This embodiment provides a method for preparing and detecting an anti-AQP4 autoantibody kit. The difference from Example 1 is that the concentration of the poly-L-lysine coating solution is 0.05 mg / mL. Everything else is the same as in Example 1.

[0104] The detection was performed according to the method in Example 1, and the results are shown below. Figure 8 .

[0105] Examples 1 and 4 show that the kit, composed of cells overexpressing AQP4 antigen combined with cell reaction wells treated with an appropriate concentration of poly-L-lysine coating solution, can produce good fluorescence signals and a large number of cells when used for the detection of anti-AQP4 autoantibodies, thus improving the sensitivity of the detection. However, when the concentration of poly-L-lysine coating solution is too low, it will significantly reduce the cell adhesion ability, resulting in weakened fluorescence brightness and a reduced number of positive signal cells after detecting AQP4 antibody positive samples.

[0106] Example 5

[0107] This embodiment provides a method for preparing and detecting an anti-AQP4 autoantibody kit. The difference from Example 1 is that the coating compound, poly-L-lysine, is replaced with a matrix gel. Everything else is the same as in Example 1.

[0108] The detection was performed according to the method in Example 1, and the results are shown below. Figure 9 .

[0109] Examples 1 and 5 show that the kit composed of cells overexpressing AQP4 antigen combined with cell reaction wells treated with poly-L-lysine coating solution can produce good fluorescence signals and a large number of cells when used for the detection of anti-AQP4 autoantibodies, thus improving the detection sensitivity. However, when the coating is replaced with matrix gel, the cell adhesion ability is significantly reduced, resulting in a significant decrease in fluorescence brightness and a significant reduction in the number of positive signal cells after detecting AQP4 antibody positive samples.

[0110] Example 6

[0111] This embodiment provides a method for preparing and detecting an anti-AQP4 autoantibody kit. The difference from Example 1 is that the coating polylysine is replaced with type I collagen (Yeasen, 40125ES50). Everything else is the same as in Example 1.

[0112] The detection was performed according to the method in Example 1, and the results are shown below. Figure 10 .

[0113] Examples 1 and 6 show that when a kit consisting of cells overexpressing AQP4 antigen and cell reaction wells treated with poly-L-lysine coating solution is used to detect anti-AQP4 autoantibodies, it can produce results with good fluorescence signals and a large number of cells. However, when the coating is replaced with type I collagen, the number of positive signal cells decreases to some extent after detecting AQP4 antibody-positive samples.

[0114] Example 7

[0115] This embodiment provides a method for preparing and detecting an anti-AQP4 autoantibody kit. The difference from Example 1 is that the coating material, poly-L-lysine, is replaced with gelatin. Everything else is the same as in Example 1.

[0116] The detection was performed according to the method in Example 1, and the results are shown below. Figure 11 .

[0117] Examples 1 and 7 demonstrate that the kit, consisting of cells overexpressing AQP4 antigen combined with cell reaction wells treated with poly-L-lysine coating, yields good fluorescence signals and a large number of cells when used for the detection of anti-AQP4 autoantibodies. However, replacing the coating with gelatin slightly reduces cell adhesion, resulting in a slight decrease in the number of positive signal cells after detecting AQP4 antibody-positive samples.

[0118] Example 8

[0119] This embodiment provides a method for preparing and detecting an anti-AQP4 autoantibody kit. The difference from Example 1 is that cells overexpressing AQP4 antigen are cryopreserved using a corresponding volume of 10% DMSO + 40% fetal bovine serum + 50% DMEM cryopreservation solution. Everything else is the same as in Example 1.

[0120] The detection was performed according to the method in Example 1, and the results are shown below. Figure 12 .

[0121] Examples 1 and 8 show that the content of fetal bovine serum in the cryopreservation solution affects the detection performance. By optimizing the ratio of the components in the cryopreservation solution to 10% DMSO, 60% fetal bovine serum, and 30% DMEM, results with good fluorescence signal, high cell quantity, and normal cell morphology can be obtained. However, when using 10% DMSO + 40% fetal bovine serum + 50% DMEM as the cryopreservation solution to detect AQP4 antibody-positive samples, the cell viability is slightly affected, resulting in a slight decrease in the number of positive signal cells and abnormal morphology in some cells.

[0122] Example 9

[0123] This embodiment provides a method for preparing and detecting an anti-AQP4 autoantibody kit. The difference from Example 1 is that cells overexpressing AQP4 antigen are used at a concentration of 1.0 × 10⁻⁶. 5 A concentration of cells per well was added to the coated reaction wells. Everything else was the same as in Example 1.

[0124] The detection was performed according to the method in Example 1, and the results are shown below. Figure 13 .

[0125] Examples 1 and 9 show that the concentration of cells overexpressing AQP4 antigen added to the coated reaction wells also affects detection performance. The optimal concentration of cells overexpressing AQP4 antigen added to the coated reaction wells was 1.5 × 10⁻⁶. 5 A high concentration of cells per well yields good fluorescence signal and a large number of cells. However, changing the concentration of AQP4 antigen-overexpressing cells in the coated reaction wells to 1.0 × 10⁻⁶ cells / well results in better fluorescence intensity. 5 When cells / well are used to detect AQP4 antibody-positive samples, the cell adhesion ability is reduced, resulting in a decrease in the number of positive signal cells and a weakening of fluorescence intensity.

[0126] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0127] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A kit for detecting anti-AQP4 autoantibodies, characterized in that, The kit includes cell well plates and cell cryopreservation solution; The reaction wells of the cell plate are coated with a coating material, which is one of poly-L-lysine, gelatin, and collagen. The cell cryopreservation solution is a cryopreservation protection solution containing cells overexpressing AQP4 antigen; During coating, the concentration of the coating agent is 0.5 mg / mL to 3.0 mg / mL, and the amount of the coating agent used is 40 μL / well to 60 μL / well.

2. The kit according to claim 1, characterized in that, During coating, the concentration of the coating agent is 0.8 mg / mL to 2.5 mg / mL, and the amount of the coating agent used is 45 μL / well to 55 μL / well.

3. The kit according to claim 2, characterized in that, During coating, the concentration of the coating agent is 1 mg / mL–2 mg / mL, preferably 1.3 mg / mL to 1.6 mg / mL, and the amount of the coating agent used is 48 μL / well to 52 μL / well.

4. The kit according to any one of claims 1-3, characterized in that, The coating material is either polylysine or gelatin, preferably polylysine.

5. The kit according to any one of claims 1-3, characterized in that, The concentration of cells overexpressing AQP4 antigen in the cryopreservation solution was 1.0 × 10⁻⁶. 6 ~6.0×10 6 Cells / mL, preferably 2.5 × 10⁻⁶. 6 ~4.0×10 6 Cells / mL.

6. The kit according to any one of claims 1-3, characterized in that, The cryopreservation solution, by volume percentage, consists of the following components: 5%~15% DMSO, 55%~65% fetal bovine serum, and 25%~35% DMEM.

7. The kit according to claim 6, characterized in that, The cryopreservation solution consists of the following components: 8%~12% DMSO, 58%~62% fetal bovine serum, and 28%~32% DMEM.

8. The kit according to any one of claims 1-3, characterized in that, During the detection, the amount of cells overexpressing the AQP4 antigen used was 1.2 × 10⁻⁶. 5 ~2.0×10⁶ per reaction well 5 The number of cells per reaction well is preferably 1.4 × 10⁻⁶. 5 ~1.6 × 10⁶ per reaction well 5 One per reaction well.

9. The kit according to any one of claims 1-3, characterized in that, The kit also includes a signal-labeled anti-human IgG secondary antibody; preferably, the signal is one of Alexa Fluor 488, DyLight 488, and fluorescein isothiocyanate.

10. The use of the kit for detecting anti-AQP4 autoantibodies according to any one of claims 1-9 in the preparation of products for detecting neuromyelitis optica spectrum disorders.

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