Application of anti-SSTR3 autoantibody detection reagent in diagnosis of 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-SSTR3 autoantibody in diagnosis of nervous system autoimmune diseases. The serum of a patient suffering from the nervous system autoimmune disease is compared with the serum of a healthy person, and a signal existing in the serum of the patient but not existing in the serum of the healthy person is found: an autoantibody of an SSTR3 antigen is recognized, and the serum of the patient suffering from the nervous system autoimmune disease and the serum of the healthy person are detected; the invention finds that healthy people are negative in anti-SSTR3 autoantibody and part of nervous system autoimmune disease patients are positive in anti-SSTR3 autoantibody, and determines that the anti-SSTR3 autoantibody can be used as a marker for diagnosing the nervous system autoimmune disease, so that the types of biomarkers for identifying the nervous system autoimmune disease are enriched, and the application prospect is wide. The accuracy of diagnosis of the autoimmune diseases of the nervous system is improved, and particularly, auxiliary diagnosis of the autoimmune diseases of the nervous system can be realized.

Description

[0001] This application claims priority to the Chinese patent application No. CN202410964269.5, filed on July 18, 2024, and entitled "Application of reagent for detecting anti-SSTR3 autoantibody in diagnosis of nervous system autoimmune diseases", the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application belongs to the field of biological medicine, and specifically relates to the application of a reagent for detecting anti-SSTR3 autoantibody in the diagnosis of nervous system autoimmune diseases. BACKGROUND

[0003] Nervous system autoimmune diseases are an important class of diseases in the field of neurology, which can occur in the central nervous system, peripheral nervous system and neuromuscular junction. The pathological mechanism is mainly that autoimmune factors and autoantibodies attack the nervous system. Nervous system autoimmune diseases are relatively rare, have the complexity of immune diseases and the high mortality and disability characteristics of nervous system diseases, and thus have attracted high attention from clinicians and researchers.

[0004] In recent years, with the expansion of the cognition of nervous system autoantibodies and the progress of detection technology, more and more nervous system autoimmune diseases have been diagnosed. Autoantibodies are an important marker of autoimmune diseases. Each autoimmune disease is accompanied by a characteristic autoantibody profile. The presence of high-titer autoantibodies in patient serum is one of the characteristics of autoimmune diseases and an important basis for clinical diagnosis. Determining autoantibodies is helpful for the diagnosis of autoimmune diseases and has important clinical significance for judging the activity of the disease, observing the treatment effect and guiding clinical medication.

[0005] Somatostatin receptors (SSTRs) are G protein-coupled molecules encoded by five different genes (SSTR1, SSTR2, SSTR3, SSTR4 and SSTR5). SSTR1, SSTR2 and SSTR3 are constitutively expressed in the pituitary of normal humans, while SSTR4 and SSTR5 have low expression levels. Antibodies against SSTR3 can well label the morphology of neuronal primary cilia in the mouse cerebral cortex, but cannot label primary cilia in other cell types such as astrocytes, microglia and oligodendrocytes. Abnormalities in the function of neuronal primary cilia often lead to mental illness, intellectual disability and abnormal development of the nervous system, and are even closely related to the occurrence of cancer. Abnormalities in receptors and kinases specifically expressed in cilia are highly related to the occurrence of these nervous system diseases. However, the presence of anti-SSTR3 autoantibodies in patients with nervous system autoimmune diseases has not been reported. SUMMARY

[0006] The application aims to provide application of a reagent for detecting anti-SSTR3 autoantibodies in diagnosis of nervous system autoimmune diseases, specific diagnosis of nervous system autoimmune diseases, and improvement of accuracy of diagnosis of nervous system autoimmune diseases, and especially auxiliary diagnosis of nervous system autoimmune diseases.

[0007] The application provides application of a reagent for detecting anti-SSTR3 autoantibodies in preparation of a product for diagnosis of nervous system autoimmune diseases.

[0008] Preferably, the reagent for detecting anti-SSTR3 autoantibodies comprises one or more of SSTR3 protein, a cell expressing SSTR3 protein, a vector expressing SSTR3 protein, and a tissue containing SSTR3 protein.

[0009] Preferably, the amino acid sequence of the SSTR3 protein comprises any one of a), b) and c):

[0010] a) an amino acid sequence shown in SEQ ID NO. 1;

[0011] b) an amino acid sequence modified or mutated from the amino acid sequence in SEQ ID NO. 1, which can recognize anti-SSTR3 autoantibodies;

[0012] c) an amino acid sequence with a homology of ≥70% and <100% to the amino acid sequence in a) or b), which can recognize anti-SSTR3 autoantibodies.

[0013] Preferably, the amino acid sequence modified or mutated from the amino acid sequence in SEQ ID NO. 1, which can recognize anti-SSTR3 autoantibodies, is an amino acid sequence shown in SEQ ID NO. 3.

[0014] Preferably, the symptoms of the nervous system autoimmune diseases comprise one or more of mental and behavioral disorders, cognitive impairment, decreased memory of recent events, seizures, speech disorders, movement disorders, involuntary movements, decreased level of consciousness and coma, and autonomic nervous dysfunction.

[0015] Preferably, the nervous system autoimmune diseases are autoimmune encephalitis.

[0016] The application also provides a kit for diagnosis of nervous system autoimmune diseases, comprising a reagent for detecting anti-SSTR3 autoantibodies.

[0017] Preferably, the reagent for detecting anti-SSTR3 autoantibodies comprises one or more of SSTR3 protein, a cell expressing SSTR3 protein, a vector expressing SSTR3 protein, and a tissue containing SSTR3 protein.

[0018] Preferably, the amino acid sequence of the SSTR3 protein comprises any one of a) to c):

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

[0020] b) the amino acid sequence shown in SEQ ID NO. 1, which is modified or mutated and can recognize anti-SSTR3 autoantibody;

[0021] c) the amino acid sequence having an identity of ≥70% and <100% to the amino acid sequence shown in a) or b) and can recognize anti-SSTR3 autoantibody.

[0022] Preferably, the amino acid sequence shown in b) is the amino acid sequence shown in SEQ ID NO. 3.

[0023] Beneficial effects:

[0024] The present application finds that, compared with the serum of healthy people, the serum of patients with symptoms of nervous system autoimmune diseases contains anti-SSTR3 autoantibody, which can be used as a marker for diagnosing nervous system autoimmune diseases, especially as an anti-neural cell antibody for assisting in diagnosing nervous system autoimmune diseases, by comparing the serum of patients with symptoms of nervous system autoimmune diseases with the serum of healthy people.

[0025] The present application also provides a kit for diagnosing nervous system autoimmune diseases, which comprises a reagent for detecting anti-SSTR3 autoantibody. The present application first establishes a kit for diagnosing nervous system autoimmune diseases by taking a reagent for detecting anti-SSTR3 autoantibody as the main component. The kit of the present application can qualitatively or quantitatively analyze anti-SSTR3 autoantibody, and is easy to operate, thereby realizing the diagnosis of nervous system autoimmune diseases, especially the auxiliary diagnosis of autoimmune encephalitis. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows.

[0027] Figure 1 Staining results of the serum of patients and healthy people on rat brain tissue sections;

[0028] Figure 2 Co-staining results of the serum of patients and the neuronal marker Neun antibody on rat brain tissue sections;

[0029] Figure 3 Staining results of patient and healthy human serum on SSTR3 overexpressing cell slides;

[0030] Figure 4 Staining results of SSTR3 antibody on rat brain tissue sections;

[0031] Figure 5 Co-staining results of SSTR3 antibody and neuron marker β-TUBB3 on rat primary neuron cells;

[0032] Figure 6 Staining results of SSTR3 antibody on rat primary neuron cells for verifying signals detected by patient serum;

[0033] Figure 7 Staining results of SSTR3 antibody on SSTR3 overexpressing cell slides for verifying signals detected by patient serum;

[0034] Figure 8 WB results of patient serum and SSTR3 antibody for verifying SSTR3 protein overexpression;

[0035] Figure 9 Staining results of SSTR3 antibody on SSTR3 overexpressing cell slides for verifying neutralizing protein;

[0036] Figure 10 Staining results of serum neutralization experiment on SSTR3 overexpressing cell slides for verifying signals detected by patient serum;

[0037] Figure 11 Staining results of serum neutralization experiment on rat brain tissue sections for verifying signals detected by patient serum;

[0038] Figure 12 Staining results of some anti-SSTR3 antibody positive patient serum, disease control serum and healthy human serum. DETAILED DESCRIPTION

[0039] The present application provides use of a reagent for detecting anti-SSTR3 autoantibody in preparation of a product for diagnosing autoimmune diseases of nervous system.

[0040] In the present application, the reagent for detecting anti-SSTR3 autoantibody preferably comprises one or more of SSTR3 protein, cell expressing SSTR3 protein, vector expressing SSTR3 protein and tissue containing SSTR3 protein, and is further preferably SSTR3 protein.

[0041] In the present application, the cell preferably comprises a bacterial cell or a eukaryotic cell; the bacterial cell preferably comprises an E. coli cell; the eukaryotic cell preferably comprises an immortalized human cell, an insect cell or a yeast. In the specific implementation of the present application, the cell can be selected from HEK293 cell, Hela cell, CHO, Pichia pastoris, Saccharomyces cerevisiae, sf9, BL21 or Rosetta, etc. The vector of the present application preferably comprises a pTriEx vector family, a pcDNA3 family, a pET series or a pBac series. The tissue of the present application preferably is brain tissue, further preferably human, rat, primate, mouse, goat, horse, sheep or bovine brain tissue.

[0042] The SSTR3 protein of the present application preferably refers to a polypeptide having immunogenicity capable of binding to SSTR3 autoantibody. The polypeptide preferably is a polymer of ≥2 amino acids, further preferably a polymer of 2-70 amino acids, more preferably a polymer of 2, 3, 4, 5, 6, 7, 8, 10, 12, 20, 30, 40, 50, 60 or 70 amino acids. The polypeptide of the present application preferably comprises one or more epitopes derived from the protein SSTR3. The polypeptide of the present application preferably further comprises a fusion protein fused with SSTR3 and other amino acids, which are preferably connected to the N- or C-terminus, having the effect of facilitating the purification, immobilization, precipitation or identification of the polypeptide or protein, and the amino acids can constitute a tag known in the art, such as His tag, thioredoxin, maltose binding protein, glutathione-S-transferase, flag tag, myc tag or strep tag. The source of the polypeptide of the present application is not particularly limited, and both recombinant polypeptide and / or purified and isolated polypeptide can be used. The SSTR3 protein of the present application preferably is an immobilized protein, further preferably immobilized on a solid carrier.

[0043] In the present application, the amino acid sequence of the SSTR3 protein preferably comprises any one of a) to c): a) the amino acid sequence shown in SEQ ID NO. 1; b) an amino acid sequence modified or mutated from the amino acid sequence in SEQ ID NO. 1, which can recognize anti-SSTR3 autoantibody; and c) an amino acid sequence having ≥70% and <100% identity with the amino acid sequence in a) or b), which can recognize anti-SSTR3 autoantibody. The amino acid sequence modified or mutated from the amino acid sequence in SEQ ID NO. 1, which can recognize anti-SSTR3 autoantibody, preferably is the amino acid sequence shown in SEQ ID NO. 3.

[0044] In the present application, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1 preferably includes any one of 1) to 3): 1) the nucleotide sequence shown in SEQ ID NO. 2; 2) a nucleotide sequence which is mutated from the nucleotide sequence shown in SEQ ID NO. 2 and which can encode an amino acid sequence which recognizes anti-SSTR3 autoantibody; and 3) a nucleotide sequence which has an identity of ≥ 70% and < 100% to the nucleotide sequence of 1) or 2) and which can encode an amino acid sequence which recognizes anti-SSTR3 autoantibody.

[0045] The specific sequences of SEQ ID NO. 1 and SEQ ID NO. 3 of the present application are as follows:

[0046] SEQ ID NO. 1: MDMLHPSSVSTTSEPENASSAWPPDATLGNVSAGPSPAGLAVSGVLIPLVYLVVCVVGLLGNSLVIYVVLRHTASPSVTNVYILNLALADELFMLGLPFLAAQNALSYWPFGSLMCRLVMAVDGINQFTSIFCLTVMSVDRYLAVVHPTRSARWRTAPVARTVSAAVWVASAVVVLPVVVFSGVPRGMSTCHMQWPEPAAAWRAGFIIYTAALGFFGPLLVICLCYLLIVVKVRSAGRRVWAPSCQRRRRSERRVTRMVVAVVALFVLCWMPFYVLNIVNVVCPLPEEPAFFGLYFLVVALPYANSCANPILYGFLSYRFKQGFRRVLLRPSRRVRSQEPTVGPPEKTEEEDEEEEDGEESREGGKGKEMNGRVSQITQPGTSGQERPPSRVASKEQQLLPQEASTGEKSSTMRISYL.

[0047]

[0048] SEQ ID NO. 3: MDMLHPSSVSTTSEPENASSAWPPDATLGNVSAGPSPAGLAVSGVLIPLVYLVVCVVGLLGNSLVIYVVLRHTASPSVTNVYILNLALADELFMLGLPFLAAQNALSYWPFGSLMCRLVMAVDGINQFTSIFCLTVMSVDRYLAVVHPTRSARWRTAPVARTVSAAVWVASAVVVLPVVVFSGVPRGMSTCHMQWPEPAAAWYTAALGFFGPLLVICLCYLLIVVKVRSAGRRVWAPSCQRRRRSERRVTRMVVAVVALFVLCWMPFYVLNIVNVVCPLPEEPAFFGLYFLVVALPYANSCANPILYGFLSYRFKQGFRRVLLRPSRRVRSQEPTVGPPEKTEEEDEEEEDGEESREGGKGKEMNGRVSQITQPGTSGQERPPSRVASKEQQLLPQEASTGEKSSTMRISYL.

[0049] The present application carries out serological investigation on patients with symptoms of nervous system autoimmune diseases, incubates rat brain tissue slices with patient serum and healthy human serum, amplifies signals by fluorescent secondary antibodies, finds that there are autoantibodies in patient serum compared with healthy human serum, and selects signals on patient serum as autoantibodies recognizing SSTR3 antigen through biological detection chip. The co-staining of commercial antibodies and patient serum on neuron cells and SSTR3 overexpressing cells, and serum neutralization experiment verify the authenticity of the target antigen; further collect more patient serum with or without nervous system diseases and healthy human serum as control samples to screen through CBA, and verify the specificity of the target antigen.

[0050] In the present application, the symptoms of the nervous system autoimmune disease preferably include one or more of mental and behavioral disorders, cognitive impairment, decreased memory for recent events, seizures, speech disorders, movement disorders, involuntary movements, decreased level of consciousness and coma, and autonomic dysfunction, further preferably mental and behavioral disorders, cognitive impairment, decreased memory for recent events, seizures, speech disorders, movement disorders, involuntary movements, decreased level of consciousness and coma, autonomic dysfunction. The nervous system autoimmune disease of the present application is preferably autoimmune encephalitis; the patient with symptoms of the nervous system autoimmune disease is preferably a patient highly suspected by a doctor to have autoimmune encephalitis, and the serum sample is negative for autoimmune encephalitis-related autoantibodies (NMDAR, AMPA1, AMPA2, LGI1, CASPR2, GABABR, DPPX, IgLON5, GlyRα1, GABAARα1, GABAARβ3, GABAARγ2); other nervous system immune disease-related antibodies, including central nervous demyelination-related autoantibodies (AQP4, MOG, MBP, GFAP, AQP1, Flotillin1 / 2), paraneoplastic syndrome-related autoantibodies (Hu, Yo, Ri, Ma2, Ma1, CV2, Amphiphysin, Tr, Zic4, PKCγ, Recoverin, Titin, SOX1, GAD65) are also negative.

[0051] In the present application, the diagnosis is preferably auxiliary diagnosis. Anti-SSTR3 autoantibody is closely related to autoimmune diseases of nervous system, and can be used to distinguish autoimmune diseases of nervous system from other autoimmune diseases, especially to distinguish autoimmune encephalitis from other autoimmune diseases. In the field of autoimmune encephalitis detection, the diagnosis conditions of autoimmune encephalitis usually include four aspects of clinical manifestations, auxiliary examination, confirmatory test and exclusion of other causes. When the clinical manifestations and auxiliary examination of a patient are consistent with autoimmune encephalitis and other causes are excluded, the patient is diagnosed as possible autoimmune encephalitis. When the clinical manifestations and auxiliary examination of a patient are consistent with autoimmune encephalitis and other causes are excluded, and the detection of autoimmune encephalitis related markers (anti-nerve cell antibodies) is positive, the patient is diagnosed as confirmed autoimmune encephalitis. In the present application, anti-SSTR3 autoantibody is used as a marker for the diagnosis of autoimmune encephalitis. Anti-SSTR3 autoantibody is closely related to autoimmune diseases of nervous system, especially autoimmune encephalitis, and can be used for the diagnosis of autoimmune encephalitis, which belongs to the auxiliary diagnosis of autoimmune encephalitis. In the present application, anti-SSTR3 autoantibody is used as a marker. By detecting anti-SSTR3 autoantibody, a doctor can determine whether a patient has a nervous system autoimmune disease described in the present application or exclude the possibility of having another nervous system autoimmune disease according to the detection results, the clinical manifestations of the patient, auxiliary examination and detection results of disease markers, which helps the doctor to select a more promising treatment plan or treatment drug for the patient.

[0052] In the present application, the product preferably comprises a kit.

[0053] The present application also provides a kit for diagnosing autoimmune diseases of nervous system, comprising a reagent for detecting anti-SSTR3 autoantibody.

[0054] The related content of the reagent for detecting anti-SSTR3 autoantibody described in the present application has been described above and will not be repeated here. As an embodiment, the kit described in the present application can be a kit for detection based on enzyme-linked immunosorbent assay, or a kit for detection based on protein immunoblotting. The kit described in the present application preferably further comprises one or more of labeled antibodies, reaction buffer and sample diluent. The present application does not have strict requirements for the specific composition of the labeled antibodies, buffer and sample diluent, and routine selection is sufficient.

[0055] The present application takes the reagent for detecting anti-SSTR3 autoantibody as the main component, establishes a kit for diagnosing nervous system autoimmune diseases, and can qualitatively or quantitatively analyze anti-SSTR3 autoantibody, thereby diagnosing nervous system autoimmune diseases, especially having an auxiliary diagnosis function. When the kit is used for detection, there is no strict requirement for the detection method, and a method known in the art can be used, such as CBA, TBA, ELISA, immunocolloidal gold method, immunoblotting, immunospot, membrane strip method, chemiluminescence, radioimmunoassay, liquid chip method, lateral flow or flow cytometry.

[0056] The patient serum in the present application is donated by a hospital, has been agreed by the patient himself, and the healthy human serum is donated by a healthy person in a hospital physical examination center, and has been agreed by the person himself.

[0057] By including 3 patient sera diagnosed as possible autoimmune encephalitis (Antibody-negative autoimmune encephalitis, AbNAE) as biological samples, serological investigation is carried out, and the discovery process of anti-SSTR3 autoantibody is described in detail.

[0058] Inclusion criteria: (1) one or more of mental behavior abnormalities, cognitive impairment, recent memory loss, seizures, speech disorders, movement disorders, involuntary movements, decreased level of consciousness and coma, and autonomic nervous dysfunction; (2) at least 1 inflammation, such as increased white blood cells (>5×10 6 / L) in cerebrospinal fluid, increased oligoclonal bands or IgG index in cerebrospinal fluid, MRI suggesting encephalitis (brain MRI high signal limited to one side or bilateral medial temporal lobe, or involving gray matter, white matter or both in multifocal areas), brain biopsy showing lymphocyte infiltration; (3) 12 autoantibodies (NMDAR, AMPA1, AMPA2, LGI1, CASPR2, GABABR, DPPX, IgLON5, GlyR, GABAARα1, GABAARγ2, GABAARβ3) of conventional autoimmune encephalitis are negative by CBA method; (4) the presence of immune response is shown by tissue-based immunofluorescence method (TBA); (5) age ≥14 years old; (6) excluding infectious diseases (including viral encephalitis, neurosyphilis, nervous system infections caused by bacteria, fungi and parasites), metabolic and toxic encephalopathy (including hepatic encephalopathy and pulmonary encephalopathy, toxic encephalopathy caused by antibiotics, chemotherapy drugs or immunosuppressants, and radiation encephalopathy), tumors (cerebral gliomatosis, primary central nervous system lymphoma, multiple metastatic cancer), genetic diseases (mitochondrial encephalopathy, methylmalonic acidemia, adrenoleukodystrophy), and neurodegenerative diseases (Lewy body dementia, frontotemporal dementia, multiple system atrophy and hereditary cerebellar degeneration).

[0059] The information of 3 patients included in the present application is as follows:

[0060] Patient 1: male, 28 years old, with fatigue, back and leg pain for nearly half a year, less sleep, and gradually aggravated, 3 months ago with limb weakness, unsteady walking, falling when walking, slow reaction, occasional choking when drinking water, dysphagia, recent confusion, slurred speech, cerebrospinal fluid cytology showed lymphocytic inflammation, diagnosed as possible autoimmune encephalitis;

[0061] Patient 2: female, 37 years old, with seizures, specifically the patient suddenly lost consciousness without obvious inducement, trismus, tetanic convulsions of the limbs, unresponsive to calls, and relieved after about 2 minutes, accompanied by headache and fatigue after the attack. No frothing at the mouth, mental and behavioral abnormalities, MRI of the head showed right temporal lobe lesions, cerebrospinal fluid white blood cell count was slightly elevated, electroencephalogram was abnormal, diagnosed as possible autoimmune encephalitis;

[0062] Patient 3: male, 14 years old, with mental and behavioral abnormalities for nearly three months, manifested as self-talk, expressionless, self-hitting, insomnia, poor mental state at the time of diagnosis, abnormal thyroid function, increased cerebrospinal fluid white blood cells, abnormal electroencephalogram, diagnosed as possible autoimmune encephalitis.

[0063] In order to further illustrate the present application, the application of the anti-SSTR3 autoantibody provided by the present application in preparing a product for diagnosing autoimmune diseases of the nervous system is described in detail below in conjunction with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0064] Example 1

[0065] Immunofluorescence method for detecting the fluorescence signal of the serum of the patient on the rat brain tissue

[0066] 1. Preparation of rat brain tissue frozen sections:

[0067] Anesthetize adult rats, open the abdominal cavity after the limbs of the rats are hardened, expose the heart apex, perfuse PBS from the left heart apex for systemic circulation; then take out the brain tissue, fix it with methanol for 10-30 min; transfer the sample into a 30 wt.% sucrose solution for dehydration, and place it at 4℃ until the tissue block sinks; add a small amount of embedding agent OCT to the sample stage, place it in the freezing stage of the freezing microtome (manufacturer: LEICA, model: CM1950), when the tissue is slightly whitish, apply a thin layer of OCT on the surface of the sample, continue to freeze for 20 min, and then slice to obtain the rat brain tissue frozen sections.

[0068] 2. Serum incubation:

[0069] Patient serum and healthy human serum were diluted with PBST at a volume ratio of 1:10, respectively, and incubated on rat brain tissue frozen sections, incubated at room temperature for 1 hour, and washed 3 times with PBST, each time for 5 minutes; diluted FITC-labeled goat anti-human IgG secondary antibody (manufacturer: Jackson, catalog number: 109-095-170) was added, incubated at room temperature for 30 minutes, washed 3 times with PBST, each time for 5 minutes, and observed under a fluorescence microscope and photographed. The results showed that patient 1-3 serum showed positive signals in the hippocampus and cortex of rat brain tissue frozen sections, while healthy human serum did not show positive signals in these areas ( Figure 1 ), patients 1-3 had antibodies in their sera that bound to antigens in the hippocampus and cortex of rat brain tissue.

[0070] 3. Antibody Co-staining

[0071] Use neuron-specific marker NeuN antibody (manufacturer: Wuhan Sanying, catalog number: 26975-1-AP) to co-stain the slides incubated with patient serum in step 2. Dilute NeuN antibody at 1:200 with PBST and incubate on the frozen sections of rat brain tissue incubated with patient serum in step 2. Incubate at room temperature for 30 minutes, wash 3 times with PBST, 5 minutes each time; add diluted AlexaFluor 594-labeled goat anti-rabbit IgG secondary antibody (manufacturer: Jackson, catalog number: 115-585-144), incubate at room temperature for 30 minutes, wash 3 times with PBST, 5 minutes each time; DAPI is used to stain the cell nucleus, stain at room temperature for 10 minutes, wash 3 times with PBST, 5 minutes each time; observe under a fluorescence microscope and take pictures. The results showed that the signal stained by the patient serum overlapped with the signal of NeuN antibody on the frozen sections of rat brain tissue ( Figure 2 Taking patient 1 as an example), this indicates that the antigen recognized by the antibody in the patient's serum exists on neuronal cells.

[0072] Example 2

[0073] Screening and identification of target antigens

[0074] 1. Screen patient sera for more reported autoantibodies related to the nervous system. Find the target antigens of reported autoantibodies related to the nervous system, prepare cell slides that overexpress the target antigens, and assemble multiple cell slides that overexpress the target antigens into bioassay chip materials. Perform immunofluorescence staining on patient samples. The specific methods are as follows:

[0075] The 60 reported neural system autoantibody target antigens include DPPX, IgLON5, GlyR, GABAARa1, GABAARy2, GABAARb3, mGluR5, D2R, Neurexin-3a, GAD67, KCNA4 (Kv1.4), KLHL11, AK5, TRIB2, GLuR3, Gephyrin, CaVa2d (CACNA2D1), TGM2, TGM6, MUNC18-1, mGLuR1, GABAARAP, Drebrin, AGO, NAE, PDE10A, ADAM22, ROCK2, mGluR3, mGluR4, CACNB1, VAMP2, CRMP2, CACNA1A, Homer3, ATP1A3, ARHGAP26, ITPR1, septin-5, NCDN, GRID2, AP3B2, mGluR2, GRIK2 (GluR6 / GluK2), Rab6A, Rab6B, CA8 (CARPVIII), PDE10A, PLP1, NF155, NF186, CNTN1, CNTN2, CASPR1, Gliomedin, Agrin, AChR, LRP4, MuSK, MAG. The gene sequences of the 60 proteins are searched from NCBI and sent to a sequencing company for gene synthesis. Recombinant vectors corresponding to the genes are obtained, and then the recombinant vectors are transfected into 293T cells to obtain recombinant cells. The recombinant cells are prepared into a biological detection chip material overexpressing the target antigens. Whether the patient serum can react with the biological detection chip after incubation is detected by an immunofluorescence method. Whether the patient serum contains specific autoantibodies of the series of genes is explored. The specific steps are as follows:

[0076] (1) Recombinant vector construction: The gene sequences of the 60 proteins are connected to pCDNA3.1 by PCR or artificial synthesis, respectively, by a molecular cloning method to obtain 60 recombinant vectors. The constructed recombinant vectors are sequenced and correctly prepared for use.

[0077] (2) Target gene transfection: 293T cells are cultured in a 37°C, 5% CO2 cell incubator using 10% FBS-DMEM high-sugar culture medium. The 293T cells are plated on a 6 cm x 6 cm climbing sheet in a culture dish. A total of 61 dishes are used. When the cell density reaches 30%-40%, the 60 recombinant vectors corresponding to the genes and the empty pCDNA3.1 are transfected into the 293T cells using PEI transfection reagent (manufacturer: thermo, product number: BMS1003), and are labeled.

[0078] (3) Cell slide fixation: the cells grown for 48h after transfection were washed with PBS for 2 times, and then fixed with acetone for 5min, washed with PBS for 2 times, and dried at 45℃ for 30min. The cell slides were cut into 2.5mm x 2.5mm in size, and 61 kinds of 2.5mm x 2.5mm cell slides were pasted on the glass slides to prepare the bio-detection chip for screening target antigens.

[0079] (4) Immunofluorescence staining: the patient serum and the healthy human serum were diluted with PBST at a volume ratio of 1:10, and then incubated on the bio-detection chip for 1h at room temperature, washed with PBST for 3 times, each for 5min. The FITC-labeled goat anti-human IgG secondary antibody was diluted at a ratio of 1:200, and then incubated for 30min at room temperature, washed with PBST for 3 times, each for 5min. Under the fluorescence microscope, it was found that the patient serum did not have a color development reaction obviously stronger than the healthy human serum on the 60 bio-detection chips. However, according to the results of rat brain tissue section staining in Example 1, the serum of patients 1-3 had obvious staining results in the hippocampus and cortex of neurons, so it was suspected that the serum of patients 1-3 might contain new autoantibodies that could recognize neuron cells different from those reported before.

[0080] 2. 100 kinds of proteins with relatively high expression in the hippocampus and cortex of human brain were searched from the human protein atlas (https: / / www.proteinatlas.org / ), and a bio-detection chip was prepared. Whether the patient serum could have an immune reaction after incubation with the bio-detection chip was detected by immunofluorescence method, so as to explore whether the patient serum contained new autoantibodies that could recognize neuron cells different from those reported before. The specific steps were as follows:

[0081] (1) Bio-detection chip preparation: the gene sequences encoding each protein were searched from NCBI, and were sent to a sequencing company to synthesize the genes into pCDNA3.1. The synthesized recombinant vectors were transformed into the cloning bacteria TOP10 for amplification, and then the plasmids were prepared for use. The above-mentioned recombinant vectors were respectively transfected into 293T cells grown on 6cm x 6cm slides using PEI transfection reagent. After transfection for 48h, the cells were washed, fixed and dried, and then the 6cm x 6cm cell slides were cut into 2.5mm x 2.5mm in size for use. After the preparation of these cell slides, they were pasted on the glass slides for sample detection.

[0082] (2) Immunofluorescence staining: the patient serum and healthy human serum were diluted by 1:10 volume ratio using PBST, and then incubated on the bio-detection chip prepared in step (1) above, and incubated at room temperature for 1 h, washed with PBST for 3 times, each for 5 min; 1:200 diluted FITC-labeled goat anti-human IgG secondary antibody was used, and incubated at room temperature for 30 min, washed with PBST for 3 times, each for 5 min; the results were observed under a fluorescence microscope, and photographed. The results showed that the serum of patients 1-3 had obvious color reaction with an antigen on the bio-detection chip, which was stronger than that of healthy human serum, and produced a positive signal, while the healthy human serum did not produce a signal with all the antigens on the bio-detection chip Figure 3 ) After verification, the target antigen is SSTR3 (somatostatin receptor 3, accession number: NM_001051), the amino acid sequence is shown as SEQ ID NO. 1, and the nucleotide sequence encoding the protein is shown as SEQ ID NO. 2.

[0083] Example 3

[0084] Verification of expression of target antigen on rat brain tissue and neuron cells by commercial antibody

[0085] 1. Verification of expression of target antigen on rat brain tissue by immunofluorescence experiment of commercial antibody

[0086] The rat brain tissue frozen section was prepared according to the reference example 1; the commercial SSTR3 antibody was diluted by 1:200 volume ratio (manufacturer: Wuhan Three Eagle, product number: 20696-1-Ap), and then incubated on the prepared rat brain tissue frozen section, and incubated at room temperature for 1 h, washed with PBST for 3 times, each for 5 min; 1:200 diluted FITC-labeled goat anti-rabbit IgG secondary antibody was used, and incubated at room temperature for 30 min, washed with PBST for 3 times, each for 5 min; DAPI was used to stain the cell nucleus, and incubated at room temperature for 10 min, washed with PBST for 3 times, each for 5 min; the results were observed under a fluorescence microscope, and photographed. The results showed that the commercial SSTR3 antibody had staining signals in the cerebellum and hippocampus of the rat brain tissue frozen section( Figure 4 ), indicating that the SSTR3 protein was expressed on the rat brain tissue.

[0087] 2. Verification of expression of target antigen on neuron cells by immunofluorescence experiment of commercial antibody

[0088] (1) Isolation of rat primary neuron cells and preparation of cell climbing sheets: The rats were anesthetized with 10% chloral hydrate, and the brain tissues were taken out in a biological safety cabinet after 75% alcohol immersion for 3 min. The tissues were cut into a mixture with ophthalmic scissors and then digested with papain at 37°C for 30 min. After digestion, the cells were moved to a new centrifuge tube, resuspended with DMEM containing 1% BSA, centrifuged at 400 g and 4°C for 5 min, the supernatant was discarded, and the cells were resuspended with DMEM containing 1% BSA, centrifuged at 200 g and 4°C for 5 min, the supernatant was discarded, and the cells were resuspended with 20 ml of DMEM containing 10% fetal bovine serum. The cells were cultured in a cell culture dish with climbing sheets at an appropriate density, and rat primary neuron cell climbing sheets were obtained for standby use.

[0089] (2) Live cell staining: Six dishes of the above rat primary neuron cells were taken, the supernatant was discarded, and the cells were washed twice with PBS. Commercial SSTR3 antibody was diluted 1:200 with PBS, and the prepared neuron cells were incubated at room temperature for 1 h. The cells were washed three times with PBS for 5 min each time. FITC-labeled goat anti-rabbit IgG secondary antibody was diluted 1:200, and the cells were incubated at room temperature for 30 min. The cells were washed three times with PBS for 5 min each time. DAPI was used to stain the cell nuclei at room temperature for 10 min, and the cells were washed three times with PBS for 5 min each time.

[0090] (3) β-TUBB3 antibody co-staining: The neuron cells in step (1) stained with SSTR3 antibody were fixed with 0.4% paraformaldehyde at room temperature for 10 min, and washed three times with PBST for 5 min each time. Neuron cell-specific skeleton marker β-TUBB3 antibody was diluted 1:1000 with PBST, and the neuron cell climbing sheets were incubated at 4°C overnight. The cells were washed three times with PBST for 5 min each time. Alexa Fluor 594-labeled goat anti-mouse IgG secondary antibody was diluted 1:200, and the above neuron cell climbing sheets were incubated at room temperature for 30 min. The cells were washed three times with PBST for 5 min each time, observed under a fluorescence microscope, and photographed. The results showed that the signal stained by the commercial SSTR3 antibody overlapped with the signal of β-TUBB3 antibody on the primary neuron cells (Fig. 2), indicating that SSTR3 protein was expressed on the primary neuron cells. Figure 5

[0091] Example 4

[0092] Verification of signals detected by patient serum on neuron cells and SSTR3 overexpressing cell climbing sheets by commercial antibodies

[0093] 1. Verification of SSTR3 signals detected by serum on neuron cells by commercial antibodies ​

[0094] (1) Preparation of rat primary neuron cell slides: The rat primary neuron cell slides prepared in Reference Example 3 were fixed with 4% paraformaldehyde for 10 min, washed with PBS for 2 times, and terminated with 1.25 M glycine for 10 min, and washed with PBS for 2 times to obtain the rat primary neuron cell slides, which were ready for use.

[0095] (2) Serum incubation: The sera of patients 1-3 were diluted with PBST at a ratio of 1:10, and then incubated with the rat primary neuron cell slides prepared in step (1) for 1 h at room temperature, and washed with PBST for 3 times, each for 5 min; a FITC-labeled goat anti-human IgG secondary antibody was diluted at a ratio of 1:200, and then incubated with the slides for 30 min at room temperature, and washed with PBST for 3 times, each for 5 min;

[0096] (3) Antibody co-staining: A commercial SSTR3 antibody was diluted with PBST at a ratio of 1:200, and then incubated with the rat primary neuron cell slides incubated with the sera of patients 1-3 in step (2) above for 30 min at room temperature, and washed with PBST for 3 times, each for 5 min, and then a Alexa Fluor 594-labeled goat anti-rabbit IgG secondary antibody was added and diluted at a ratio of 1:200, and then incubated with the slides for 30 min at room temperature, and washed with PBST for 3 times, each for 5 min; DAPI was used to stain the cell nuclei for 10 min at room temperature, and washed with PBST for 3 times, each for 5 min; and then observed under a fluorescence microscope and photographed. The results showed that the signals of the sera of patients 1-3 on the rat primary neuron cells overlapped with the staining signals of the commercial SSTR3 antibody on the rat primary neuron cells (Fig. 2), indicating that the antibodies in the sera of patients 1-3 specifically recognized the SSTR3 protein on the rat primary neuron cells. Figure 6

[0097] 2. Verification of the SSTR3 signals detected by the commercial antibody on the SSTR3-overexpressing cell slides

[0098] (1) Preparation of cell slides: The SSTR3-overexpressing and empty pCDNA3.1 cell slides were prepared according to Reference Example 2, and ready for use.

[0099] (2) Serum incubation: The sera of patients 1-3 were diluted with PBST at a ratio of 1:10, and then incubated with the SSTR3-overexpressing cell slides for 1 h at room temperature, and washed with PBST for 3 times, each for 5 min; a FITC-labeled goat anti-human IgG secondary antibody was diluted at a ratio of 1:200, and then incubated with the slides for 30 min at room temperature, and washed with PBST for 3 times, each for 5 min;

[0100] ​(3) Antibody co-staining: The commercial SSTR3 antibody was diluted 1:200 with PBST and incubated on the SSTR3 overexpressing cell slides incubated with the serum of patients 1-3. The cells were incubated at room temperature for 30 minutes, washed 3 times with PBST, each time for 5 minutes. A 1:200 dilution of AlexaFluor 594-labeled goat anti-rabbit IgG secondary antibody was added, and the cells were incubated at room temperature for 30 minutes. The cells were washed 2 times with PBST, each time for 5 minutes. The cell nuclei were stained with DAPI at room temperature for 10 minutes, and washed 3 times with PBST, each time for 5 minutes. The cells were observed under a fluorescence microscope and photographed. The results showed that the commercial antibody had a positive signal on the SSTR3 overexpressing cell slides, and the SSTR3 protein was successfully overexpressed on 293T cells. The staining signals of the serum of patients 1-3 and the commercial SSTR3 antibody on the SSTR3 overexpressing cell slides overlapped ( Figure 7 ), indicating that the antibodies in the sera of patients 1-3 specifically recognized the SSTR3 protein on the SSTR3-overexpressing cell slides.

[0101] Example 5

[0102] Serum neutralization assay validates the signal detected in patient serum

[0103] 1. Preparation of neutralizing protein

[0104] (1) Target gene transfection: 293T cells were cultured in a 37°C, 5% CO2 cell culture incubator using 10% FBS-DMEM high glucose medium. When the cell density reached 30% to 40%, the recombinant vector SSTR3-pCDNA3.1 and the empty vector pCDNA3.1 were transfected into the 293T cells using PEI transfection reagent and labeled.

[0105] (2) Take one dish of 293T cells overexpressing SSTR3, discard the supernatant, scrape the cells into a 1.5 mL centrifuge tube with a cell scraper, centrifuge at 800 rpm at room temperature to remove the supernatant, add 200 μL PBS, and ultrasonically disrupt (disruption conditions: 10% power, disruption for 3 seconds, pause for 6 seconds, and ultrasonication for a total of 1 minute) to serve as the SSTR3 neutralizing protein; take one dish of cells transfected with empty pCDNA3.1 to prepare the control protein, and the preparation conditions and method are the same as those for the preparation of the SSTR3 neutralizing protein.

[0106] 2. Validation of Neutralizing Protein

[0107] (1) WB verification of neutralizing protein

[0108] SSTR3 neutralizing protein and empty pCDNA3.1 control protein were subjected to SDS-PAGE gel electrophoresis experiments, and the sample amount was 40μg. After the electrophoresis, the membrane was transferred under the conditions of 300mA and 90min wet transfer; 5% skim milk powder was blocked at room temperature for 1h; patient 1 serum was diluted 1:100 with TBST, and SSTR3 antibody was diluted 1:1000, and incubated at 4℃ overnight; the next day, TBST was washed 3 times, 5min each time; the corresponding HRP-labeled goat anti-human secondary antibody or HRP-labeled goat anti-rabbit secondary antibody (manufacturer: Jackson) was added, and incubated at room temperature for 1h; TBST was washed 3 times, 5min each time; chemiluminescent solution was added for color development and photography. The results showed that patient 1 serum and SSTR3 antibody had specific signals on the immunoblot of overexpressed SSTR3 protein, while there was no signal with the control protein ( Figure 8 ), indicating that the neutralizing protein was successfully prepared.

[0109] (2) Neutralization experiments with SSTR3 antibodies verify the role of neutralizing proteins

[0110] With reference to Example 2, a cell slide overexpressing SSTR3 was prepared. Two commercial SSTR3 antibodies diluted 1:200 were prepared using PBST, each containing 100 μL. 20 μL of SSTR3 neutralizing protein and 20 μL of control protein were added and incubated at room temperature for 30 minutes. The prepared cell slides overexpressing SSTR3 were incubated with the two antibodies, incubated at room temperature for 1 hour, and washed 3 times with PBST for 5 minutes each. A 1:200 diluted FITC-labeled goat anti-rabbit IgG secondary antibody was added, incubated at room temperature for 30 minutes, washed 3 times with PBST for 5 minutes each, and observed under a fluorescence microscope and photographed. The results showed that on the cell slides overexpressing SSTR3, the commercial SSTR3 antibody signal was blocked by the SSTR3 neutralizing protein, but not by the control protein ( Figure 9 ), indicating that the signal is a signal that specifically recognizes SSTR3 antigen.

[0111] 3. Serum neutralization experiment verifies the signal detected by patient serum on cell slides overexpressing SSTR3

[0112] The over-expressing SSTR3 cell slides were prepared according to the preparation method of Reference Example 2, 3 portions of 1:10 diluted serum of patient 1 were prepared with PBST, each 100 μL, 20 μL of PBST, 20 μL of SSTR3 neutralizing protein and 20 μL of control neutralizing protein were added respectively, and incubated at room temperature for 30 min. 3 portions of serum of patient 2 and 3 were prepared in the same way, and the prepared over-expressing SSTR3 cell slides were incubated with them respectively, and incubated at room temperature for 1 h, and washed with PBST for 3 times, each for 5 min. 1:200 diluted FITC labeled goat anti-human IgG secondary antibody was added, and incubated at room temperature for 30 min, and washed with PBST for 3 times, each for 5 min. The fluorescence microscope was observed, and the pictures were taken. The results showed that the serum signals of patients 1-3 were blocked by SSTR3 neutralizing protein on the over-expressing SSTR3 cell slides, while the control protein did not block the signals appeared on the over-expressing SSTR3 cell slides Figure 10 ), which indicated that the signals appeared on the over-expressing SSTR3 cell slides by serum of patients 1-3 were specific signals for recognizing SSTR3 antigen.

[0113] 4. Serum neutralization experiment on rat brain tissue slices to verify the signals detected by patient serum

[0114] The rat brain tissue frozen sections were prepared according to the preparation method of Reference Example 1; 3 portions of 1:10 diluted serum of patient 1 were prepared with PBST, each 100 μL, 20 μL of PBST, 20 μL of SSTR3 neutralizing protein and 20 μL of control neutralizing protein were added respectively, and incubated at room temperature for 30 min. 3 portions of serum of patient 2 and 3 were prepared in the same way, and the prepared rat brain tissue sections were incubated with them respectively, and incubated at room temperature for 1 h, and washed with PBST for 3 times, each for 5 min. 1:200 diluted FITC labeled goat anti-human IgG secondary antibody was added, and incubated at room temperature for 30 min, and washed with PBST for 3 times, each for 5 min. The fluorescence microscope was observed, and the pictures were taken. The results showed that the serum signals of patients 1-3 were blocked by SSTR3 neutralizing protein on the rat brain tissue sections, while the control protein did not block the signals appeared on the rat brain tissue sections Figure 11 ), which indicated that the signals appeared on the rat brain tissue sections by serum of patients 1-3 were specific signals for recognizing SSTR3 antigen.

[0115] Example 6

[0116] Verification of clinical specificity and detection rate of anti-SSTR3 autoantibody based on cell-based immunofluorescence method

[0117] 1. Summary of detection of anti-SSTR3 antibody in various diseases

[0118] To verify the clinical specificity and detection rate of anti-SSTR3 antibody, 819 serum samples in addition to the above 3 patients were collected, SSTR3 overexpressing cell slides and empty pCDNA3.1 control cell slides were prepared according to the preparation method of Example 2; SSTR3 autoantibody positive serum samples were screened using SSTR3 overexpressing cell slides according to the method of step 3 of Example 4, a total of 13 SSTR3 autoantibody positive samples were screened, and the immunofluorescence detection results are shown in Table 1, and part of the staining results are shown in Figure 12

[0119] Table 1 Immunofluorescence detection results

[0120]

[0121] According to Table 1 and Figure 12 It can be seen that among the 819 serum samples, 13 anti-SSTR3 antibody positive samples were detected; among them, 8 anti-SSTR3 antibody positive samples were detected in the serum samples of suspected autoimmune encephalitis patients, the detection rate was 3.76%, and the titer dilution detection showed that 5 samples were +++ (titer 1:1000), 1 sample was ++ (titer 1:100), and 2 samples were + (titer 1:32 and 1:10, respectively); among them, 5 anti-SSTR3 antibody positive samples were detected in the serum samples of patients with diagnosed autoimmune encephalitis, the detection rate was 3.65%, among which 4 samples were anti-NMDAR antibody positive and 1 sample was anti-GABABR antibody positive, and the titer dilution showed that 3 samples were +++ (titer 1:1000) and 2 samples were ++ (titer 1:320 and 1:100, respectively); Among the serum samples of patients with other nervous system autoimmune diseases other than autoimmune encephalitis, only 1 sample was detected to be anti-SSTR3 antibody positive, which belonged to a patient with neuromyelitis optica, and the detection rate was 0.51%, and the titer dilution showed ++ (titer 1:100); In the samples of 76 patients with other non-nervous system autoimmune diseases (including viral encephalitis, psychosis, patients without nervous system symptoms) and 204 healthy controls as controls, no anti-SSTR3 antibody positive sample was detected, and the detection rate was 0.00%.

[0122] 2. Sensitivity and specificity analysis of anti-SSTR3 antibody for assisting in the diagnosis of autoimmune encephalitis

[0123] (1) First, according to the detection results of Table 1 and Figure 12 , the sensitivity and specificity of anti-SSTR3 antibody detected by CBA method in assisting in the diagnosis of nervous system autoimmune diseases, especially autoimmune encephalitis, were systematically evaluated, and the corresponding relationship between the detection results and clinical diagnosis is shown in Table 2.

[0124] ​Table 2: correspondence between the results of the detection and the clinical diagnosis

[0125]

[0126] According to Table 2, the sensitivity, specificity, positive predictive value and negative predictive value and 95% confidence interval (95% CI) of the anti-SSTR3 antibody for assisting in the diagnosis of autoimmune encephalitis were calculated, and the results were as follows:

[0127] Sensitivity (positive coincidence rate) = 3.71%, 95% CI: 2.19%-6.25%;

[0128] Specificity (negative coincidence rate) 99.79%, 95% CI: 98.80%-99.96%;

[0129] Accuracy (total coincidence rate) = 58.73%, 95% CI: 55.33%-62.05%;

[0130] Youden's index = 0.035, 95% CI: 1.48-5.52

[0131] Positive likelihood ratio (+LR) = 17.67, 2.30-136.50

[0132] Negative likelihood ratio (-LR) = 0.965, 0.945-0.985

[0133] Positive predictive value (PPV) = 92.86%, 95% CI: 68.52%-98.73%;

[0134] Negative predictive value (NPV) = 58.14%, 95% CI: 54.70%-61.50%.

[0135] According to the above verification results, the present application provides a new antigen to be tested for binding with autoantibodies for realizing the diagnosis of autoimmune diseases of the nervous system, especially autoimmune encephalitis; the anti-SSTR3 autoantibody can be detected in patients with autoimmune diseases of the nervous system having symptoms of the nervous system, indicating that the antibody has an auxiliary role in the diagnosis of autoimmune diseases of the nervous system, especially an auxiliary diagnostic role for autoimmune encephalitis.

[0136] Example 7

[0137] Detection of SSTR3 autoantibodies in patient serum using SSTR3 mutants

[0138] In this example, one mutant of human SSTR3 gene, i.e. the mutant with deletion of amino acids 203-208 of SSTR3 protein (amino acid sequence shown as SEQ ID NO. 3), is selected, and the vector construction, preparation of cell climbing sheet overexpressing recombinant SSTR3 deletion mutant, and detection of patient serum positive to 17 anti-SSTR3 antibodies are performed according to the steps described in Example 2. The results show that the deletion mutant of SSTR3 can still recognize anti-SSTR3 antibodies in patient serum.

[0139] As can be seen from the above examples, anti-SSTR3 autoantibodies can be used as biomarkers for diagnosing autoimmune diseases of the nervous system, and the diagnosis and screening of autoimmune diseases of the nervous system can be achieved by detecting anti-SSTR3 autoantibodies, especially the auxiliary diagnosis of autoimmune diseases of the nervous system.

[0140] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. Use of a reagent for detecting anti-SSTR3 autoantibodies in the preparation of a product for diagnosing autoimmune diseases of the nervous system.

2. The use according to claim 1, characterized in that The reagent for detecting anti-SSTR3 autoantibodies comprises one or more of SSTR3 protein, cells expressing SSTR3 protein, vectors expressing SSTR3 protein, and tissues containing SSTR3 protein.

3. The use according to claim 2, characterized in that The amino acid sequence of the SSTR3 protein includes any one of a) to c): a) the amino acid sequence shown in SEQ ID NO.1; b) the amino acid sequence in SEQ ID NO. 1, after modification or mutation, can recognize the amino acid sequence of anti-SSTR3 autoantibodies; c) The amino acid sequence identity with the amino acid sequence in a) or b) is ≥70% and <100%, and can recognize the amino acid sequence of the anti-SSTR3 autoantibody.

4. The use according to claim 3, characterized in that The amino acid sequence of b) is the amino acid sequence shown in SEQ ID NO.

3.

5. The use according to claim 1, characterized in that The symptoms of the autoimmune disease of the nervous system include one or more of mental and behavioral abnormalities, cognitive impairment, recent memory loss, epileptic seizures, speech disorders, movement disorders, involuntary movements, decreased consciousness and coma, and autonomic dysfunction.

6. The use according to claim 5, characterized in that The autoimmune disease of the nervous system is autoimmune encephalitis.

7. A kit for diagnosing autoimmune diseases of the nervous system, characterized in that: Includes reagents for detecting anti-SSTR3 autoantibodies.

8. The kit according to claim 7, characterized in that The reagent for detecting anti-SSTR3 autoantibodies comprises one or more of SSTR3 protein, cells expressing SSTR3 protein, vectors expressing SSTR3 protein, and tissues containing SSTR3 protein.

9. The kit according to claim 8, characterized in that The amino acid sequence of the SSTR3 protein includes any one of a) to c): a) the amino acid sequence shown in SEQ ID NO.1; b) the amino acid sequence in SEQ ID NO. 1, after modification or mutation, can recognize the amino acid sequence of anti-SSTR3 autoantibodies; c) The amino acid sequence identity with the amino acid sequence in a) or b) is ≥70% and <100%, and can recognize the amino acid sequence of the anti-SSTR3 autoantibody.

10. The kit according to claim 9, characterized in that The amino acid sequence of b) is the amino acid sequence shown in SEQ ID NO.3.

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