Application of reagent for detecting anti-SNAP91 autoantibody in preparation of product for detecting and / or diagnosing nervous system autoimmune diseases

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a reagent for detecting an anti-SNAP91 autoantibody in preparation of a product for detecting and / or diagnosing autoimmune diseases of a nervous system. The anti-SNAP91 autoantibody is detected in a patient suffering from the nervous system autoimmune disease for the first time, and the authenticity and specificity of the target antigen are verified, so that the SNAP91 can be used as one of the recognition antigens of the autoantibody related to the nervous system autoimmune disease; the reagent for detecting the anti-SNAP91 autoantibody can realize detection and / or diagnosis of the nervous system autoimmune disease, and especially can realize auxiliary diagnosis of the nervous system autoimmune disease. Meanwhile, the kit for detecting and / or diagnosing the autoimmune diseases of the nervous system, provided by the invention, can qualitatively or quantitatively analyze the anti-SNAP91 autoantibody, is simple and convenient to operate, and realizes the detection and / or diagnosis of the autoimmune diseases of the nervous system.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a reagent for detecting anti-SNAP91 autoantibodies in the preparation of a product for detecting and / or diagnosing autoimmune diseases of the nervous system. Background Art

[0002] Synaptosome associated protein 91 (SNAP91), also known as AP180, is an adaptor protein that regulates the endocytosis of proteins and receptors on the synaptic vesicle membrane and plays a role in the sorting and aggregation of synaptic vesicle substances (such as neurotransmitters). SNAP91 is highly expressed in neurons and plays a role in the growth of axons and dendrites of neurons. It can change the size of clathrin-coated vesicles and endosomes. SNAP91 works with CALM (clathrin-assembling lymphoid myeloid leukemia protein) in vitro to regulate the formation of vesicle-associated protein-2 (AP-2) adaptor protein complexes on lipid templates and often interacts with other endocytic pathways. SNAP91 can regulate the formation of clathrin-associated protein (CLATHRIN) grids by promoting vesicle assembly. Studies have shown that SNAP91 is associated with risk variants for schizophrenia and Alzheimer's disease, leading to functional defects in presynaptic and postsynaptic neurons. Meanwhile, SNAP91 has been proposed as one of the candidate biomarkers for glioblastoma (GBM), along with COL3A1, and reduced levels of both proteins have been found in patients with GBM and other malignant gliomas.

[0003] Autoimmune diseases of the nervous system are diseases caused by autoimmune molecules and immune cells attacking the nervous system. Common autoimmune diseases of the nervous system include autoimmune encephalitis and demyelinating diseases of the central nervous system. Autoantibodies refer to antibodies produced by the body against its own organs, cells or cell components. Disease-specific autoantibodies are the touchstone for identifying diseases and play an important role in the diagnosis and treatment of diseases. Currently, autoantibodies to the SNAP91 protein have been identified as markers for lung cancer (Chinese patent CN 110412277 B) and as biomarkers for multiple sclerosis (Bronge M, KA, Thomas OG, et al. SNAP91 ntification offour novel T cell autoantigens and personal autoreactive profiles in multiple sclerosis[J]. Science advances, 2022, 8(17):eabn1823.), but there are currently no reports on SNAP91 autoantibodies in autoimmune encephalitis. Summary of the invention

[0004] The object of the present invention is to provide a reagent for detecting anti-SNAP91 autoantibodies for use in the preparation of a product for detecting and / or diagnosing autoimmune diseases of the nervous system. The reagent for detecting anti-SNAP91 autoantibodies can specifically diagnose or detect autoimmune diseases of the nervous system, improve the diagnostic accuracy of autoimmune diseases of the nervous system, and achieve the purpose of assisting in the diagnosis of autoimmune diseases of the nervous system.

[0005] The present invention provides the use of anti-SNAP91 autoantibodies as markers in the preparation of products for detecting and / or diagnosing autoimmune diseases of the nervous system.

[0006] The present invention also provides the use of a reagent for detecting anti-SNAP91 autoantibodies in the preparation of a product for detecting and / or diagnosing autoimmune diseases of the nervous system.

[0007] Preferably, the reagent for detecting anti-SNAP91 autoantibodies comprises one or more of SNAP91 protein, a recombinant expression vector expressing SNAP91 protein, a cell expressing SNAP91 protein, a tissue expressing SNAP91 protein, and a cell lysate containing SNAP91 protein.

[0008] Preferably, the amino acid sequence of the SNAP91 protein includes any one of the following:

[0009] a): the amino acid sequence shown in SEQ ID NO.1;

[0010] b): 10% to 98% of the length of the amino acid sequence shown in SEQ ID NO.1, and the amino acid sequence that can recognize the anti-SNAP91 autoantibody;

[0011] c): The amino acid sequence in a) or b) is modified or mutated and can recognize the amino acid sequence of the anti-SNAP91 autoantibody.

[0012] Preferably, the amino acid sequence of the SNAP91 protein described in b) is as shown in SEQ ID NO.2.

[0013] Preferably, the symptoms of the autoimmune disease of the nervous system include fever, headache, mental and behavioral abnormalities, cognitive impairment, memory loss, epileptic seizures, speech disorders, movement disorders, involuntary movements, decreased consciousness and coma, autonomic dysfunction, sleep disorders, focal damage to the central nervous system, and one or more of peripheral nerve and neuromuscular junction involvement.

[0014] Preferably, the autoimmune disease of the nervous system includes autoimmune encephalitis.

[0015] Preferably, the product comprises a detection kit, which is based on immune response detection.

[0016] The present invention also provides a kit for detecting anti-SNAP91 autoantibodies, wherein the kit comprises a reagent for detecting anti-SNAP91 autoantibodies and a labeled antibody.

[0017] Preferably, the reagent for detecting anti-SNAP91 autoantibodies comprises one or more of SNAP91 protein, a recombinant expression vector expressing SNAP91 protein, a cell expressing SNAP91 protein, a tissue expressing SNAP91 protein, and a cell lysate containing SNAP91 protein.

[0018] Preferably, the amino acid sequence of the SNAP91 protein includes any one of the following:

[0019] a) the amino acid sequence shown in SEQ ID NO.1;

[0020] b) 10% to 98% of the length of the amino acid sequence shown in SEQ ID NO.1, and the amino acid sequence that can recognize the anti-SNAP91 autoantibody;

[0021] c) The amino acid sequence in a) or b) is modified or mutated and can recognize the amino acid sequence of the anti-SNAP91 autoantibody.

[0022] Preferably, the labeled antibody comprises one or more of an antibody labeled with Alexa Fluor 594, an antibody labeled with HRP and an antibody labeled with Cy3.

[0023] Preferably, the kit further comprises one or more of a positive control serum or antibody that can specifically bind to the cell slides overexpressing SNAP91, a negative control serum, a reaction buffer and a sample diluent.

[0024] Beneficial effects:

[0025] The present invention provides the use of anti-SNAP91 autoantibodies as markers in the preparation of products for detecting and / or diagnosing autoimmune diseases of the nervous system. On this basis, the present invention provides the use of reagents for detecting anti-SNAP91 autoantibodies in the preparation of products for detecting and / or diagnosing autoimmune diseases of the nervous system.

[0026] The present invention detects for the first time anti-SNAP91 autoantibodies in patients with autoimmune diseases of the nervous system. By comparing the signals of serum from patients with autoimmune diseases of the nervous system and control serum on cell slides overexpressing SNAP91 protein antigens, it is possible to clearly obtain signals present in the patient serum but not in the control serum, indicating the presence of anti-SNAP91 autoantibodies in the patient serum, verifying the authenticity and specificity of the target antigen, indicating that SNAP91 can be used as one of the recognition antigens of autoantibodies associated with autoimmune diseases of the nervous system, and the reagent for detecting anti-SNAP91 autoantibodies can realize the detection and / or diagnosis of autoimmune diseases of the nervous system, especially can realize the auxiliary diagnosis of autoimmune diseases of the nervous system.

[0027] The present invention also provides a kit for detecting and / or diagnosing autoimmune diseases of the nervous system, comprising a reagent for detecting anti-SNAP91 autoantibodies and a labeled antibody. The present invention is the first to establish a kit for detecting and / or diagnosing autoimmune diseases of the nervous system using a reagent for detecting anti-SNAP91 autoantibodies as a main component. The kit of the present invention can be used to qualitatively or quantitatively analyze anti-SNAP91 autoantibodies, is easy to operate, and can detect and / or diagnose autoimmune diseases of the nervous system, especially to achieve auxiliary diagnosis of autoimmune diseases of the nervous system. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0029] Figure 1 The staining results of the serum of patients 1-6 in Example 1 on rat brain tissue sections;

[0030] Figure 2 The staining results of the sera of healthy controls 1-3 in Example 1 on rat brain tissue sections;

[0031] Figure 3 The co-staining results of the serum of patient 1 and Synaptophysin antibody on rat brain tissue sections in Example 1;

[0032] Figure 4 The immunoprecipitation results of serum from patient 1-2 and serum from healthy control 1-3 in Example 2;

[0033] Figure 5The results of immunoblotting of the immunoprecipitates of serum from patient 1 and serum from healthy control 1 in Example 2 are shown;

[0034] Figure 6 The co-staining results of SNAP91 antibody and Synaptophysin antibody on rat brain tissue sections in Example 3, the scale bar is 200 μm;

[0035] Figure 7 The co-staining results of the serum of patients 1-3 and SNAP91 antibody on rat brain tissue sections in Example 3;

[0036] Figure 8 The co-staining results of SNAP91 antibody and β3-Tubulin antibody on neuronal cells in Example 3;

[0037] Fig. 9 The results of the co-staining experiment of the serum of patient 1-3 on neuronal cells in Example 3;

[0038] Fig.10 The staining results of the serum of patient 1-3 and healthy control 1-3 in Example 3 on the overexpressing cell slides, the scale bar is 200 μm;

[0039] Fig.11 The co-staining results of the serum of patient 1-3 and SNAP91 antibody on the overexpressing cell slides in Example 3 are shown in Figure 3. The scale bar is 200 μm.

[0040] Fig.12 The immunoblotting results of patient 1 and healthy control 1 in Example 3 are shown;

[0041] Fig.13 The staining results of the neutralized serum sample and the blood washing sample of patient 1 in Example 4 on the overexpressing cells, the scale bar is 200 μm;

[0042] Fig.14 The staining results of the neutralized serum sample and the blood washing sample of patient 1 in Example 4 on Western Blot;

[0043] Fig.15 This is the staining result of the washed blood sample of patient 1-2 in Example 5 on neuronal cells;

[0044] Fig.16 The results of the four SNAP91-positive patients screened in Example 6, the scale bar is 200 μm.

[0045] Fig.17 These are the results of 4 SNAP91 deletion mutant-positive patients screened in Example 8. The scale bar is 200 μm. DETAILED DESCRIPTION

[0046] The present invention provides the use of anti-SNAP91 autoantibodies as markers in the preparation of products for detecting and / or diagnosing autoimmune diseases of the nervous system.

[0047] On this basis, the present invention also provides the use of a reagent for detecting anti-SNAP91 autoantibodies in the preparation of a product for detecting and / or diagnosing autoimmune diseases of the nervous system.

[0048] In the present invention, as one embodiment, the reagent for detecting anti-SNAP91 autoantibodies may be one or more of SNAP91 protein, a recombinant expression vector expressing SNAP91 protein, a cell expressing SNAP91 protein, a tissue expressing SNAP91 protein, and a cell lysate containing SNAP91 protein; as another embodiment, the reagent for detecting anti-SNAP91 autoantibodies may be SNAP91 protein, a cell expressing SNAP91 protein, or a tissue expressing SNAP91 protein; as another embodiment, the reagent for detecting SNAP91 autoantibodies may be SNAP91 protein. As one embodiment, the amino acid sequence of the SNAP91 protein of the present invention includes any one of the following: a): the amino acid sequence shown in SEQ ID NO.1; b): 10% to 98% of the length of the amino acid sequence shown in SEQ ID NO.1, and the amino acid sequence that can recognize anti-SNAP91 autoantibodies; c): the amino acid sequence in a) or b) is modified or mutated, and the amino acid sequence that can recognize anti-SNAP91 autoantibodies; As another embodiment, the amino acid sequence of the SNAP91 protein is as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0049] The amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2 of the present invention are as follows:

[0050] SEQ ID NO.1:MSGQTLTDRIAAAQYSVTGSAVARAVCKATTTHEVMGPKKKHLDYLIQ *。

[0051] SEQ ID NO.2: MSGQTLTDRIAAAQYSVTGSAVARAVCCATTHEVMGPKKKHLDYLIQ ATNETNVNIPQMADTLFERATNSSWVVVFKALVTTHHLMVHGNERFIQYLASRNTLFNLSNFLDKSGSHGYDMSTFIRRYSRYNEKAFSYRQMAFDPARVKKGADGVMRTMAPEKLLKSMPILQGQIDALLEFDVHPNELTNGVYNDGVINLLEKFFEMKKGQCKDALEIYKRFLTRMTRVSEFLKVAEQVGIDKGDIPDLTQAPSSLMETLEQHLNTLEGKKPGNNEGSGAPSPLSKSPATTVTSPNSTPAKTIDTSPPVDLFATASAAVPVSTSKPSDLLDLQPDFSSGGAAAAAAPAPPPPAGGATAWGDLLGEDSLALSSVPSEAQISDPFAPETPPTTTAEIATASASASASTTTTVTAVTAEVDLFGDAFAASPGEAPAASEGAAAPATTPVAAALDACSGNDPFAPSEGSAEAAPELDLFAMKPPETSVPVVTPTASTAPPVPATAPSPAPAVAAAAAATTAATAAATTTTTTSAATATTAPPALDI FGDLFESTPEVAAAPKPDAAPSIDLFSTDAFSSPPQGASPVPESSLTADLSVDAFAAPPATTASPAKVDSSGVIDLFGDAFGSSASEPQPASQAASSSSASADLLAGFGGSFMAPSPSPVTPAQNNLLQPNFEAAFGTTPSSSSSFDPSVFDGLGDLLMPTMAPAGQPAPVSMVPPSPAMAASKALGSDLSSLASLVGNLGISGTTTKKGDLQWNAGEKKLTGGANWQPKVAPATWSAGVPPSAPLQGAVPPTSSVPPVAGAPSVGQPGAGFGMPPAGTGMMPQQPVMFAQPMMRPPFGAAAVPGTQLSPSPTPASQSPKKPPAKDPLADLNIKDFL*.

[0052] As an embodiment, the nucleotide sequence encoding the SNAP91 protein can be any one of Ⅰ) to Ⅲ): Ⅰ) the nucleotide sequence shown in SEQ ID NO.3; Ⅱ) 10% to 98% of the length of the nucleotide sequence shown in SEQ ID NO.3, and the encoded amino acid sequence can recognize anti-SNAP91 autoantibodies; Ⅲ) after the nucleotide sequence in Ⅰ) or Ⅱ) is mutated, the encoded amino acid sequence can recognize anti-SNAP91 autoantibodies.

[0053] The nucleotide sequence shown in SEQ ID NO.3 of the present invention is as follows:

[0054]

[0055] In the present invention, as an embodiment, the symptoms of the autoimmune disease of the nervous system can be fever and headache, mental and behavioral abnormalities, cognitive impairment, memory loss, epileptic seizures, speech disorders, movement disorders, involuntary movements, decreased consciousness and coma, autonomic dysfunction, sleep disorders, focal damage to the central nervous system, peripheral nerves and neuromuscular junction involvement. One or more. As another embodiment, the autoimmune disease of the nervous system can be autoimmune encephalitis. As an embodiment, the product of the present invention can be a reagent and / or a detection kit; as another embodiment, the detection kit is based on immune response detection; as another embodiment, the immune response includes but is not limited to cell-based immunofluorescence (CBA), tissue-based immunofluorescence (TBA), enzyme-linked immunosorbent assay (ELISA), immunocolloidal gold method, immunoblot, immunospot, membrane strip method, chemiluminescence, radioimmunoassay, liquid chip method, lateral flow method or flow cytometry detection technology. As an embodiment, the diagnosis of the present invention can be auxiliary diagnosis.

[0056] The present invention incubates rat brain tissue slices with serum from patients with autoimmune diseases of the nervous system and serum from healthy people, amplifies the signal by fluorescent secondary antibody, and co-stains with neuron-specific Marker antibody, and finds that compared with healthy human serum, autoantibodies are present in the patient serum, and screens out the signals on the patient serum as autoantibodies that recognize the SNAP91 antigen by immunoprecipitation and mass spectrometry identification, verifies the authenticity of the target antigen by serum neutralization experiment, and prepares experimental eluted antibodies containing SNAP91 autoantibodies and control eluted antibodies not containing SNAP91 autoantibodies by incubating the patient serum with cells overexpressing the target antigen and recovering the effective antibody components of the serum, and verifies the authenticity and specificity of the target antigen by incubating overexpressing cell slides, rat brain tissue slices, and cytotoxicity experiments.

[0057] The anti-SNAP91 autoantibodies of the present invention are closely related to autoimmune diseases of the nervous system, and can distinguish autoimmune diseases of the nervous system from other autoimmune diseases. The anti-SNAP91 autoantibodies provide a basis for the diagnosis of autoimmune diseases of the nervous system. The present invention uses anti-SNAP91 autoantibodies as markers, and by detecting anti-SNAP91 autoantibodies, according to the test results, doctors can combine the patient's clinical symptoms, physiological and biochemical test indicators, disease marker test results and other comprehensive conditions to determine whether the patient suffers from the autoimmune-related diseases of the nervous system described in the present invention or exclude the possibility of suffering from another autoimmune disease of the nervous system, which helps doctors choose treatment plans or therapeutic drugs for patients.

[0058] The present invention also provides a kit for detecting anti-SNAP91 autoantibodies, the kit comprising a reagent for detecting anti-SNAP91 autoantibodies and a labeled antibody. The reagent for detecting anti-SNAP91 autoantibodies of the present invention has been described in the above technical scheme and will not be repeated here. As an embodiment, the labeled antibody of the present invention may be one or more of FITC-labeled antibodies, Alexa Fluor 594-labeled antibodies, HRP-labeled antibodies, and Cy3-labeled antibodies. As another embodiment, the FITC-labeled antibody of the present invention may be a FITC-labeled goat anti-human IgG antibody. As an embodiment, the kit of the present invention may also include one or more of a positive control serum or antibody that can specifically bind to a cell slide overexpressing SNAP91, a negative control serum, a reaction buffer, and a sample diluent. As an embodiment, the reaction buffer may be a phosphate containing a surfactant; in a specific embodiment, the reaction buffer is a PBST buffer. As an embodiment, the sample diluent of the present invention may be a phosphate containing a surfactant. In a specific embodiment, the sample diluent may be a PBST solution.

[0059] The present invention uses a reagent for detecting anti-SNAP91 autoantibodies as a main component to establish a kit for detecting and / or diagnosing autoimmune diseases of the nervous system, which can qualitatively or quantitatively analyze anti-SNAP91 autoantibodies, thereby detecting and / or diagnosing autoimmune diseases of the nervous system, and has an auxiliary diagnosis effect. When the present invention uses the kit for detection, there is no strict requirement for the detection method used, and the well-known methods of the present invention can be used, such as cell-based immunofluorescence (CBA), tissue-based immunofluorescence (TBA), enzyme-linked immunosorbent assay (ELISA), immunocolloidal gold method, immunoblot, immunospot, membrane strip method, chemiluminescence, radioimmunoassay, liquid chip method, lateral flow method or flow cytometry detection technology.

[0060] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0061] Sample information of patients and healthy subjects in the following examples:

[0062] The patient sera in the examples were donated by the hospital and have been approved by the patient. The serum of healthy subjects came from the serum of healthy examinees donated by the hospital physical examination center and has been approved by the examinees themselves. A serological survey was conducted on 6 patients with neurological symptoms. Taking the experiment conducted on 6 patient samples as an example, 6 biological samples were included (inclusion conditions: 1. Clinical symptoms or auxiliary examinations were consistent with suspected cases of autoimmune encephalitis; 2. The results of known antibodies for autoimmune encephalitis detected using cell-based immunofluorescence (CBA) were negative; 3. The immunofluorescence (TBA) method used to test the tissue showed an immune response), and the discovery process of anti-SNAP91 autoantibodies was described in detail. The information of the 6 patients is shown in Table 1:

[0063] Table 1 Relevant information of 6 patients

[0064]

[0065] To exclude the possibility that the six patients suffered from other autoimmune diseases of the nervous system, the samples were sent to the laboratory for testing to expand the antibody spectrum. The results of 14 autoantibody tests for paraneoplastic syndrome (Ri, Hu, Yo, CV2, Ma2, Amphiphysin, Titin, Ma1, SOX1, Tr, Zic4, PKCγ, Recoverin, GAD65), 6 for central demyelinating diseases (AQP4, MBP, MOG, GFAP, AQP1, Flotillin1 / 2), 5 for peripheral neuropathy (NF-155, NF-186, CNTN1, CNTN2, CASPR1), and 5 for myasthenia gravis (AChR, LRP4, MuSK, RYR1, agrin) were all negative.

[0066] Example 1

[0067] Indirect immunofluorescence screening of patient serum

[0068] 1. Preparation of frozen slices of rat brain tissue:

[0069] Adult rats aged 10-15 weeks were selected for anesthesia. After being fully anesthetized, the abdominal cavity was opened to expose the heart, and PBS buffer was perfused from the left apex for systemic circulation. Then the brain tissue was removed, embedded in OCT and frozen in liquid nitrogen, and placed in the freezing table of a -20°C freezing microtome (manufacturer: LEICA model: CM1950) for slicing and acetone fixation to obtain frozen sections of rat brain tissue.

[0070] 2. Serum incubation:

[0071] The sera of the six patients (patients 1-6) and three healthy subjects (healthy subjects 1-3) were diluted with PBST buffer at a volume ratio of 1:10, and incubated on the frozen sections of rat brain tissue at room temperature for 1 hour, and washed 3 times with PBST buffer, each time for 5 minutes; diluted FITC-labeled goat anti-human secondary antibody (manufacturer: Jackson, catalog number 109-095-170) was added, incubated at room temperature for 30 minutes, and washed 3 times with PBST buffer, each time for 5 minutes. Under fluorescence microscope observation, it was found that the sera of patients 1-6 showed granular staining in the cerebellar granular layer on the frozen sections of rat brain tissue, and a characteristic staining pattern of filamentous choroid staining / membrane staining appeared in the hippocampus ( Figure 1 ), while the healthy control serum No. 1-3 showed no obvious positive signal in this area ( Figure 2 ).

[0072] 3. Antibody Co-staining

[0073] The neuron-specific marker Synaptophysin (SYP) antibody (manufacturer: Wuhan Mitsubishi, catalog number 17785-1-AP) was used to co-stain and co-localize the slides incubated with patient 1 serum in step 2, and AlexaFluor594-labeled goat anti-rabbit IgG (manufacturer: Jackson, catalog number 115-585-144) was used for secondary antibody incubation; DAPI was used to stain the cell nucleus, and under a microscope, it was found that the signal stained by patient 1 serum overlapped with the signal of Synaptophysin antibody in the cerebellum on the frozen section of rat brain tissue ( Figure 3 ), granular staining appeared in the cerebellar granular layer, indicating that the antigen recognized by the antibody in patient 1's serum was present on neuronal cells.

[0074] Example 2

[0075] Co-immunoprecipitation of primary cell lysates and validation and identification of target antigens

[0076] 1. Take 6 dishes of primary cultured neuronal cells of rats, discard the supernatant, wash twice with PBS buffer, fix with 2wt.% paraformaldehyde for 10min, and wash three times with 1× Hank's; dilute the serum of patients 1-2 and the serum of healthy controls 1-3 with 1× Hank's at a volume ratio of 1:200, filter with 0.22μm filter membrane, add to the fixed primary cultured neuronal cells, and incubate at 4℃ overnight; take 50μL ProteinA / G immunoprecipitation magnetic beads (manufacturer: Selleck) and add to a 2mL tube, wash three times with equilibration solution; put the incubated cells on ice, discard the supernatant, wash twice with PBS buffer, add 500μL lysis buffer (water as solvent, containing 150mM NaCl, 1mM EDTA, 100mM Tris-HCl, 0.5% sodium deoxycholate, 1% TritonX-100 and 0.1% SDS, pH7.5), collect cells, add protease inhibitor cocktail with a final concentration of 1× (manufacturer: Roche product number: 04693132001), lyse for 30 minutes, shake at intervals, centrifuge at 15000rpm for 30 minutes, take the supernatant, and measure the concentration; add the collected supernatant to the treated ProteinA / G immunoprecipitation magnetic beads, incubate with rotation at 4°C overnight; wash the incubated magnetic beads 4 times with the above lysis buffer, elute with 100μL 1× loading buffer, and collect the eluate; first add 5×SDS-PAGE loading buffer to the eluate Buffer, then add DTT with a final concentration of 0.01M, heat at 95℃ for 5min, then add iodoacetamide with a final concentration of 2wt.%, and leave at room temperature for 15min, which is the immunoprecipitation complex of patient 1-2 serum sample and healthy control 1-3 control serum sample; the prepared immune complex was subjected to SDS-PAGE electrophoresis and stained with a silver staining kit (manufacturer: thermo), and the results showed that in rat neurons, a protein of about 130KD to 180KD was detected in the immunoprecipitate captured by patient 1-2 serum, and the protein was not present in the control prepared by a similar method in healthy control serum No. 1-3 ( Figure 4 ).

[0077] 2. Western Blot Verification

[0078] Take the immune complex samples of patient 1 and healthy control 1 obtained in step 1 for SDS-PAGE electrophoresis; after the electrophoresis, use wet transfer, the transfer conditions are 300mA, 120min; then use 5% skim milk powder to block at room temperature for 1h; use TBST to dilute the serum of patient 1 and the serum of healthy person 1 control at a volume ratio of 1:200, respectively, and incubate at room temperature for 2h; wash 3 times with TBST, each time for 5min; add HRP-labeled goat anti-human IgG secondary antibody (manufacturer: Jackson, catalog number 109-035-098), and incubate at room temperature for 1h;

[0079] Wash with TBST three times, 5 min each time; add chemiluminescent solution for color development.

[0080] The results showed that the immunoprecipitate captured in primary neuronal cells with the serum of patient 1 contained a band that reacted with the autoantibodies in the serum of patient 1, and the target protein band was between 130KDa and 180KDa, while the immunoprecipitate did not contain a reactive band between 130KDa and 180KDa with the control serum of healthy person No. 1 ( Figure 5 ), demonstrating the presence of autoantibodies reactive with neuronal proteins in the serum of patient 1.

[0081] 3. Identification of target antigen by mass spectrometry

[0082] The band at 130KDa to 180KDa in gel recovery step 1 ( Figure 4 The gel strips were sent to Novogene for mass spectrometry analysis. The mass spectrometry results showed that the cut gel strips contained SNAP91 protein, as shown in Table 2:

[0083] Table 2 Mass spectrometry results

[0084]

[0085] Example 3

[0086] Commercial antibodies validate the expression of target antigens and SNAP91 signals detected in patient sera

[0087] 1. Commercial antibody validation of SNAP91 signal detected by patient serum on rat brain tissue sections

[0088] a. Commercial antibody staining: The rat brain tissue frozen sections prepared in step 1 of Example 1 were incubated with commercial SNAP91 antibody (using PBST, 1:200 dilution, red, manufacturer: Wuhan Mitsubishi and catalog number: 14093-1-AP) and commercial Synaptophysin antibody (1:200 dilution, green, manufacturer: Wuhan Mitsubishi, catalog number: 67864-1-Ig), and then incubated with AlexaFluor594-labeled goat anti-rabbit secondary antibody IgG (1:200) and FITC-labeled goat anti-mouse IgG (1:200). The cell nuclei were stained with DAPI and observed under a microscope. The results showed that the signals stained by the commercial SNAP91 antibody in the cerebellum and hippocampus on the rat brain tissue frozen sections overlapped with the signals of the Synaptophysin antibody ( Figure 6 ), granular staining appeared in the cerebellar granular layer and filamentous venous staining appeared in the hippocampus, indicating that SNAP91 protein was expressed in neuronal cells.

[0089] b. Co-staining of serum and antibodies: Rat brain tissue sections were incubated with patient 1-3 serum (PBST, 1:10 dilution, green) and SNAP91 antibody (PBST, 1:200 dilution, red), and then incubated with FITC-labeled goat anti-human IgG (PBST, 1:200 dilution) and AlexaFluor 594-labeled goat anti-rabbit IgG (PBST, 1:200 dilution). The cell nuclei were stained with DAPI and observed under a microscope.

[0090] According to the results ( Figure 7 ), it was observed that the staining signals of patient 1-3 sera on rat brain tissue sections overlapped with the staining signals of the commercial SNAP91 antibody, indicating that the antibodies in patient 1-3 sera specifically recognized the SNAP91 protein on rat brain tissue sections.

[0091] 2. Commercial antibody validation of SNAP91 signal detected by patient serum in primary neurons

[0092] a. Isolation and preparation of primary hippocampal neuron cells in rats: 10% chloral hydrate anesthetized SD pregnant mice of gestational age of 15-16 days, immersed in 75% alcohol for disinfection for 3 minutes, and the fetus was taken out of the biosafety cabinet; the fetus was rinsed twice with pre-cooled Hank's solution, the hippocampus was dissected out in pre-cooled Hank's, and immersed in pre-cooled DMEM cell culture medium; the DMEM was discarded, and the tissue blocks were cut into a minced mixture with ophthalmic scissors. Papain was added and digested at 37℃ for 30 minutes; after digestion, the cells were transferred to a new centrifuge tube, resuspended in DMEM cell culture medium, centrifuged at 400g and 4℃ for 5 minutes, the supernatant was discarded, and the DMEM cell culture medium was added again for resuspending, centrifuged at 400g and 4℃ for 5 minutes, the supernatant was discarded, and the cells were resuspended in 20ml Neurobasal (manufacturer: GIBCO, item number: 21103-049) culture medium. In subsequent experiments, OptiPrep medium (manufacturer: Sigma, catalog number: 92339-11-2) was used to separate neuronal cells from the above resuspended cells and cultured in a cell culture dish with cell slides. The rat primary cell slides were fixed with 2% paraformaldehyde (PFA) for 10 min, washed twice with Hank's, terminated with 1.25 M glycine for 10 min, and washed twice with Hank's to obtain slides.

[0093] b. Commercial SNAP91 antibody staining: The commercial SNAP91 antibody was diluted with PBS buffer at a volume ratio of 1:800, incubated on rat brain tissue primary cell slides, incubated at room temperature for 30 minutes, and washed twice with Hank's for 5 minutes each time; then diluted FITC-labeled goat anti-rabbit IgG was added, incubated at room temperature for 30 minutes, and washed twice with Hank's for 5 minutes each time; DAPI was used to stain the cell nucleus at room temperature for 10 minutes, and washed twice with Hank's for 5 minutes each time; neuron-specific β3-Tubulin antibody (manufacturer: Wuhan Mitaka, catalog number: 66375-1-Ig) was used to co-stain the slides incubated in step b, β3-Tubulin antibody was diluted 1:1000, and washed twice with Hank's for 5 minutes each time; AlexaFluor 594-labeled goat anti-mouse IgG secondary antibody was incubated at room temperature for 30 minutes, and washed twice with Hank's for 5 minutes each time; observed under a microscope.

[0094] According to the results ( Figure 8 ) It can be seen that the staining signal of the commercial SNAP91 antibody on the primary cell neurons of fetal mouse brain tissue can co-localize with the signal of the β3-Tubulin antibody on the primary cell neurons, indicating that the target antigen SNAP91 exists on neuronal cells.

[0095] c. Serum and antibody co-staining: Patients 1-3 serum (1:200 dilution, green) and commercial SNAP91 antibody (1:800 dilution, red) were incubated on primary cultured neuronal cell slides, and then incubated with FITC-labeled goat anti-human IgG (1:200) and AlexaFluor 594-labeled goat anti-rabbit IgG (1:200) and observed under a microscope.

[0096] According to the results ( Fig. 9 ), it was observed that the staining signals of patient 1-3 sera on primary cultured neuronal cell slides overlapped with the staining signals of the commercial SNAP91 antibody, indicating that the antibodies in patient 1-3 sera specifically recognized the SNAP91 protein on primary cell slides of rat brain tissue.

[0097] 3. Commercial antibody validation of SNAP91 signal detected by patient serum on overexpressing cell slides

[0098] a. Preparation of cell slides overexpressing SNAP91

[0099] By PCR or artificial synthesis, the SNAP91 gene (SEQ ID NO.1) was linked to pCDNA3.1 by molecular cloning, and the insertion site was NheⅠ / NotⅠ, to obtain the recombinant vector pCDNA3.1-SNAP91. The constructed recombinant vector pCDNA3.1-SNAP91 was sequenced correctly and then extracted for use; the recombinant vector pCDNA3.1-SNAP91 with correct sequencing was transfected into 293T cells using PEI transfection reagent (manufacturer: thermo, catalog number: BMS1003), and the cells grown for 48 hours after transfection were washed twice with PBS buffer, fixed with acetone for 30 minutes, discarded acetone, dried at 42°C for 30 minutes, and cut into 2.5 mm × 2.5 mm size, pasted for use, and the empty pCDNA3.1 slide was prepared using the same transfection and fixation method;

[0100] b. Serum from patients 1-3 and serum from healthy controls 1-3 were diluted at a ratio of 1:10 and incubated on the cell slides overexpressing SNAP91 protein. The cells were incubated at room temperature for 1 hour and washed 3 times with PBST buffer, each time for 5 minutes. FITC-labeled goat anti-human secondary antibody was used at a dilution of 1:200, incubated at room temperature for 30 minutes, and washed 3 times with PBST, each time for 5 minutes. The results were observed under a fluorescence microscope ( Fig.10 ); Use a commercial SNAP91 antibody diluted 1:1000 to incubate the SNAP91 overexpressing cell slides incubated with patient 1-3 serum, incubate at room temperature for 1 hour, wash 3 times with PBST, 5 minutes each time; Use a 1:200 dilution of AlexaFluor 594 labeled goat anti-rabbit secondary antibody, and observe the results under a fluorescence microscope ( Fig.11 ).

[0101] According to the results ( Figures 10-11 ), it was observed that the staining signals of patient 1-3 sera on the cell slides overexpressing SNAP91 overlapped with the staining signals of the commercial SNAP91 antibody, while there were no staining signals in the healthy control sera 1-3, indicating that the antibodies in patient 1-3 sera specifically recognized the SNAP91 protein on the cell slides overexpressing SNAP91.

[0102] 4. Commercial Antibody Validation of SNAP91 Signals Detected by Patient Serum in Western Blot

[0103] a. Preparation of protein samples: Take 50 mg of rat brain tissue, cut and grind them, add 500 μL of RIPA lysis buffer, and perform ultrasonic disruption. After ultrasonication, lyse on ice for 30 minutes, and shake once every 5 minutes; collect 1 dish of 293T cells overexpressing SNAP91 and 1 dish of empty pCDNA3.1 cells, add 200 μL of PBS buffer, and ultrasonically disrupt (the disruption conditions are the same as those of tissue proteins), collect all samples, centrifuge at 15000 rpm for 30 minutes, and take the supernatant to determine the protein concentration by BCA method;

[0104] b. Sample loading and transfer: 40 μg of rat whole brain tissue protein, overexpressed SNAP91 protein and pCDNA3.1 protein were loaded respectively. After electrophoresis, wet transfer was performed at 300 mA for 90 min. 5% skim milk powder was used for blocking at room temperature for 1 h.

[0105] c. Antibody incubation and color development: SNAP91 commercial antibody (1:3000 dilution), patient 1 serum (1:200 dilution) and healthy control 1 serum (1:200 dilution) were incubated on the transferred PVDF membrane, and incubated at 4°C overnight; the next day, TBST was used for washing 3 times, 5 minutes each time; HRP-labeled goat anti-rabbit / goat anti-human secondary antibody (manufacturer: Jackson) was added and incubated at room temperature for 1 hour; TBST was used for washing 3 times, 5 minutes each time; ECL chemiluminescent solution was added for color development and photography was performed. The results ( Fig.12 ) showed that SNAP91 protein was expressed in both SNAP91-overexpressing cells and rat brain tissue, and the antibodies in patient 1's serum were able to recognize SNAP91 protein in both SNAP91-overexpressing cells and rat brain tissue.

[0106] Example 4

[0107] Serum neutralization test and autoantibody recovery test verify the signal detected in patient serum

[0108] 1. Serum Neutralization

[0109] The pCDNA3.1-SNAP91 recombinant vector and empty pCDNA3.1 were transfected into 293F cells (cell density was about 3×10) using PEI transfection reagent (manufacturer: thermo, catalog number: BMS1003). 6 / mL), the two bottles of suspended cells grown for 96h after transfection were centrifuged and washed twice with PBS buffer, resuspended with 5mL PBS buffer, fixed with anhydrous ethanol for 10min, centrifuged to remove ethanol, washed twice with PBS buffer, placed in a 2mL EP tube, and resuspended with 1mL PBS buffer; 12μL of patient 1 serum was added to the fixed cells in the two tubes, incubated at 4°C overnight, and centrifuged the next day. The supernatant of the centrifuged suspension cells overexpressing SNAP91 was marked as neutralizing serum, and the supernatant of the centrifuged suspension cells without pCDNA3.1 was marked as control serum.

[0110] 2. Recovering autoantibodies

[0111] The two tubes of cells collected by centrifugation during serum neutralization in step 1 were resuspended in PBS buffer and washed 4 times, each time for 5 minutes; after washing, 500 μL of 0.1 M glycine elution solution with pH = 3 was added to each tube, and eluted on a rotating shaker at room temperature for 15 minutes. After elution, the eluate was collected by centrifugation, and 10 μL of 1 M Tris was added to the eluate to neutralize the eluate to a pH of 7.0-8.0, and 1 / 10 volume of PBS was added to obtain 2 500 μL elution samples, one of which was the experimental eluted antibody after the serum of patient 1 was combined with the overexpressed SNAP91 cells; the other was the control eluted antibody after the patient's serum was combined with the control pCDNA3.1.

[0112] 3. Immunofluorescence Experiment

[0113] The cells overexpressing SNAP91 were incubated with neutralizing serum and control serum, experimental eluted antibodies and control eluted antibodies, respectively, and incubated with FITC-labeled goat anti-human secondary antibody (1:200) and observed under a microscope.

[0114] result( Fig.13 ) showed that on the SNAP91 overexpressing cell slides, the neutralizing serum signal weakened, while the control serum signal was still obvious, indicating that the signal of the patient's serum neutralized by the SNAP91 overexpressing suspension cells was a signal that specifically recognized the SNAP91 antigen; the experimental eluted antibody had an obvious positive signal on the SNAP91 overexpressing cell slides, while the control eluted antibody did not, indicating that the experimental eluted antibody bound to the SNAP91 overexpressing suspension cells could specifically recognize the SNAP91 antigen on the overexpressing cell slides.

[0115] 4. Western Blot Experiment

[0116] Protein samples were prepared with reference to Example 3, and electrophoresis, transfer and blocking steps were performed. Neutralizing serum and control serum, experimental eluted antibodies and control eluted antibodies were incubated on the transferred PVDF membrane, respectively, and incubated overnight at 4°C; the next day, TBST was washed 3 times, 5 min each time; HRP-labeled goat anti-human secondary antibody (manufacturer: Jackson) was added and incubated at room temperature for 1 h; TBST was washed 3 times, 5 min each time; ECL chemiluminescent solution was added for color development and photography.

[0117] result( Fig.14 ) showed that in the lanes overexpressing SNAP91 protein and the brain tissue protein lanes, the neutralizing serum had a weakened band signal at 130-180KD, while the control serum still had an obvious band signal here, indicating that the signal of patient 1 serum neutralized by the SNAP91 overexpressing suspension cells was a signal that specifically recognized the SNAP91 antigen; the experimental eluted antibody had an obvious positive signal in the lanes overexpressing SNAP91 protein and the brain tissue protein lanes, while the control eluted antibody had no positive signal, indicating that the experimental eluted antibody bound to the SNAP91 overexpressing suspension cells could specifically recognize the SNAP91 antigen on the overexpressed cell proteins and brain tissue proteins.

[0118] Example 5

[0119] Studying the effects of anti-SNAP91 autoantibodies on cells at the level of primary cultured neurons

[0120] 1. Isolation and Culture of Primary Hippocampal Neurons

[0121] Referring to Example 3, primary hippocampal neuron cells were isolated and cultured, and cultured in a DMEM cell culture medium containing 1% fetal bovine serum for 24 hours for later use;

[0122] 2. Toxicity Test

[0123] The experimental eluted antibodies and control eluted antibodies recovered from the serum of patient 1-2 were prepared according to the steps of Example 4. 900 μL DMEM cell culture medium was added to each dish of cells, and then 100 μL of sterile-filtered experimental eluted antibodies as an experimental group or 100 μL of control eluted antibodies as a control group were added, and primary hippocampal neuronal cells were cultured for 24 hours;

[0124] 3. Indirect Immunofluorescence Experiment

[0125] The commercial SNAP91 antibody was diluted 1:800 and incubated with the isolated primary hippocampal neuron cells, and then incubated with FITC-labeled goat anti-rabbit secondary antibody IgG at a dilution of 1:200; the primary hippocampal neuron cells stained with SNAP91 antibody in step 2 were fixed with 0.4% paraformaldehyde, and cell slides were prepared. β3-Tubulin antibody (diluted 1:1000) was incubated with the primary hippocampal neuron cell slides, incubated overnight at 4°C, and then incubated with AlexaFluor 594-labeled goat anti-mouse secondary antibody IgG at a dilution of 1:200 on the primary hippocampal neuron cell slides, the cell nuclei were stained with DAPI, and observed under a microscope.

[0126] Observation results( Fig.15 ), the SNAP91 antibody stained obvious signals on the primary hippocampal neuron cells after 24 hours of incubation with the control eluted antibody recovered from the serum of patient 1-2, and the signal stained with the β3-Tubulin antibody was overlapped, while the signal stained with the SNAP91 antibody on the primary hippocampal neuron cells after 24 hours of incubation with the experimental eluted antibody recovered from the serum of patient 1-2 was very weak, indicating that the expression of SNAP91 protein in the primary hippocampal neuron cells after 24 hours of incubation with the experimental eluted antibody was less than that in the primary hippocampal neuron cells after 24 hours of incubation with the control eluted antibody. It can be inferred that the SNAP91 autoantibodies in the serum of patient 1-2 will lead to the downregulation of SNAP91 protein expression in primary hippocampal neuron cells. Therefore, it can be concluded that the SNAP91 autoantibodies in the sample of patient 1-2 have the effect of inhibiting the protein expression of primary hippocampal neuron cells cultured in vitro.

[0127] Example 6

[0128] Detection rate of anti-SNAP91 autoantibodies in samples suspected of autoimmune encephalitis of the nervous system

[0129] A total of 209 samples of patients with neurological diseases were selected (including 120 serum samples and 89 cerebrospinal fluid samples). The symptoms exhibited by these patients included fever and headache, mental and behavioral abnormalities, cognitive impairment, recent memory loss, epileptic seizures, speech disorders, movement disorders, involuntary movements, decreased consciousness and coma, autonomic dysfunction, sleep disorders, focal CNS damage, and involvement of peripheral nerves and neuromuscular junctions.

[0130] Using cell slides overexpressing SNAP91 and control cell slides with empty pCDNA3.1, as well as rat brain tissue sections, a total of 4 anti-SNAP91 autoantibody-positive samples (patients 7-10) were screened out, with an overall detection rate of 1.91% for anti-SNAP91 autoantibodies (2.5% for serum samples and 1.12% for cerebrospinal fluid samples). The staining results of patients 7-10 are as follows: Fig.16 The clinical symptoms of patients 7-10 are shown in Table 3. Finally, the 4 patients with anti-SNAP91 antibodies were diagnosed by clinicians as suffering from autoimmune encephalitis, of which patient 7 was complicated by central demyelinating disease. The remaining 205 patients were clinically diagnosed as not suffering from autoimmune encephalitis with SNAP91 autoantibodies as markers as described in the present invention. The doctors classified the diseases diagnosed for the remaining patients according to the "Chinese Expert Consensus on the Diagnosis and Treatment of Autoimmune Encephalitis" or other diagnostic guidelines for corresponding diseases as shown in Table 4.

[0131] Table 3 Clinical symptoms of patients 7-10

[0132]

[0133]

[0134] Table 4 Disease classification results of the remaining 205 patients

[0135]

[0136] Anti-SNAP91 autoantibodies can be detected in patients with symptoms of the autoimmune encephalitis disease of the nervous system marked by the SNAP91 autoantibodies described in the present invention, and are not detected in patients who do not suffer from the autoimmune encephalitis disease marked by the SNAP91 autoantibodies described in the present application but have other encephalitis diseases, indicating that the antibodies have an auxiliary effect on the diagnosis of autoimmune encephalitis diseases of the nervous system. The present invention provides a new antigen to be detected that binds to autoantibodies for the diagnosis of autoimmune encephalitis, and anti-SNAP91 autoantibodies can be detected in patients with symptoms of the nervous system, indicating that the autoantibodies have an auxiliary effect on the diagnosis of autoimmune diseases of the nervous system.

[0137] Example 7

[0138] Validation of the specificity of anti-SNAP91 autoantibodies by cell-based immunofluorescence assay

[0139] 40 disease control samples with other nervous system autoimmune diseases (Table 5) and 40 healthy control samples were selected, and immunofluorescence detection was performed using cell slides overexpressing SNAP91. The results showed that the selected 40 nervous system autoimmune disease samples and 40 healthy control samples did not produce cell morphology similar to that of the 4 patient samples screened out in Example 6 with the cell slides overexpressing SNAP91. The present invention provides a new antigen to be detected that binds to autoantibodies for the diagnosis of nervous system diseases.

[0140] Table 5 Information on 40 disease control samples with other autoimmune diseases of the nervous system

[0141]

[0142] Example 8

[0143] Detection of anti-SNAP91 autoantibodies in patient sera with SNAP91 mutants

[0144] A mutant of the human SNAP91 gene, i.e., a mutant lacking amino acids 193-210 of the SNAP91 protein (SEQ ID NO. 2), was selected, and the vector was constructed according to the steps described in Example 3, cell slides overexpressing the recombinant SNAP91 deletion mutant were prepared, and the sera of patients 7-10 screened out in Example 6 were tested. Results ( Fig.17 ) showed that the deletion mutant of SNAP91 could still recognize anti-SNAP91 antibodies in patient sera.

[0145] It can be concluded from the above examples that the reagent for detecting anti-SNAP91 autoantibodies of the present invention can specifically diagnose or detect autoimmune diseases of the nervous system, improve the diagnostic accuracy of autoimmune diseases of the nervous system, and achieve the purpose of assisting in the diagnosis of autoimmune diseases of the nervous system.

[0146] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of anti-SNAP91 autoantibodies as markers in the preparation of products for detecting and / or diagnosing autoimmune diseases of the nervous system.

2. Use of a reagent for detecting anti-SNAP91 autoantibodies in the preparation of a product for detecting and / or diagnosing autoimmune diseases of the nervous system.

3. The use according to claim 2, characterized in that: The reagent for detecting anti-SNAP91 autoantibodies includes one or more of SNAP91 protein, a recombinant expression vector expressing SNAP91 protein, a cell expressing SNAP91 protein, a tissue expressing SNAP91 protein, and a cell lysate containing SNAP91 protein.

4. The use according to claim 3, characterized in that: The amino acid sequence of the SNAP91 protein includes any one of the following: a): the amino acid sequence shown in SEQ ID NO.1; b): 10% to 98% of the length of the amino acid sequence shown in SEQ ID NO.1, and the amino acid sequence that can recognize the anti-SNAP91 autoantibody; c): the amino acid sequence of a) or b) is mutated and can recognize the amino acid sequence of the anti-SNAP91 autoantibody; Preferably, the amino acid sequence of the SNAP91 protein described in b) is as shown in SEQ ID NO.

2.

5. The use according to any one of claims 1 to 4, characterized in that: Symptoms of the autoimmune disease of the nervous system include one or more of fever, headache, mental and behavioral abnormalities, cognitive impairment, memory loss, epileptic seizures, speech disorders, movement disorders, involuntary movements, decreased consciousness and coma, autonomic dysfunction, sleep disorders, focal damage to the central nervous system, and involvement of peripheral nerves and neuromuscular junctions; Preferably, the autoimmune disease of the nervous system includes autoimmune encephalitis.

6. The use according to claim 2, characterized in that: The products include test kits, which are based on immune response detection.

7. A kit for detecting anti-SNAP91 autoantibodies, characterized in that: The kit comprises a reagent for detecting anti-SNAP91 autoantibodies and a labeled antibody.

8. The kit according to claim 8, characterized in that The reagent for detecting anti-SNAP91 autoantibodies includes one or more of SNAP91 protein, a recombinant expression vector expressing SNAP91 protein, a cell expressing SNAP91 protein, a tissue expressing SNAP91 protein, and a cell lysate containing SNAP91 protein; Preferably, the amino acid sequence of the SNAP91 protein includes any one of the following: a) the amino acid sequence shown in SEQ ID NO.1; b) 10% to 98% of the length of the amino acid sequence shown in SEQ ID NO.1, and the amino acid sequence that can recognize the anti-SNAP91 autoantibody; c) The amino acid sequence in a) or b) is modified or mutated and can recognize the amino acid sequence of the anti-SNAP91 autoantibody.

9. The kit according to claim 7, characterized in that The labeled antibody includes one or more of FITC-labeled antibody, Alexa Fluor 594-labeled antibody, HRP-labeled antibody and Cy3-labeled antibody.

10. The kit according to claim 7, characterized in that The kit also includes one or more of a positive control serum or antibody that can specifically bind to the cell slides overexpressing SNAP91, a negative control serum, a reaction buffer, and a sample diluent.

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