Application of reagent for detecting anti-NCAM2 autoantibody in detection and / or 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-NCAM2 autoantibody to detection and / or diagnosis of nervous system autoimmune diseases. The serum of a patient with nervous system autoimmune diseases and the serum of a healthy person are used for incubating rat primary neuronal cells, and it is found that compared with the serum of the healthy person, an autoantibody capable of recognizing the NCAM2 antigen exists in the serum of the patient. The NCAM2 can be used as one of the recognition antigens of the autoantibody related to the nervous system autoimmune disease, the kit is established by detecting the anti-NCAM2 autoantibody and a reagent for detecting the anti-NCAM2 autoantibody, and the detection and / or diagnosis of the nervous system autoimmune disease can be realized by qualitatively or quantitatively analyzing the anti-NCAM2 autoantibody, so that the nervous system autoimmune disease can be improved. Particularly, the accuracy of autoimmune encephalitis detection and / or diagnosis is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the use of a reagent for detecting anti-NCAM2 autoantibodies in detecting and / or diagnosing autoimmune diseases of the nervous system. Background Art

[0002] Autoimmune diseases of the nervous system are a major category of important diseases in the field of neurology. They are characterized by the complexity of immune diseases and the high mortality and disabling nature of autoimmune diseases of the nervous system. Among them, autoimmune encephalitis and central inflammatory demyelinating diseases are the more common autoimmune diseases mediated by autoantibodies in the nervous system. The detection and screening of autoantibodies play a very important role in the diagnosis and differential diagnosis of the disease.

[0003] With advances in science and technology and the exploration of neurological autoimmune diseases, progress has been made in identifying autoantibody-mediated immune mechanisms in patients with these diseases. However, due to the complex clinical manifestations and diverse pathogenic mechanisms, some patients presenting with neurological autoimmune diseases still have negative serological test results. Finding and identifying new autoantibodies for neurological autoimmune diseases and improving existing diagnostic and treatment capabilities remains a daunting task.

[0004] Neural cell adhesion molecules (NCAMs) are primarily classified into two subtypes: NCAM1 and NCAM2. In humans, the NCAM2 gene is located on chromosome 21 (region 21q21.1) and comprises 25 exons. Its protein is primarily expressed in numerous regions of the central nervous system, including the cerebral cortex, hippocampus, and olfactory system. NCAM2 regulates neuronal morphogenesis and axonal structure, controlling important neuron-specific processes such as neuronal differentiation, synaptogenesis, and memory formation. In the adult brain, NCAM2 is closely associated with the organization of axons and dendrites in the olfactory system located in the temporal lobe. NCAM2 is highly expressed in dendritic spines and regulates synaptic plasticity and learning processes, highlighting the importance of NCAM2 in nervous system development. Currently, the presence of anti-NCAM2 autoantibodies in patients with neurological autoimmune diseases has not been reported. Summary of the Invention

[0005] The purpose of the present invention is to provide new markers for the detection and / or diagnosis of autoimmune diseases of the nervous system, especially autoimmune encephalitis, to detect and / or diagnose autoimmune diseases of the nervous system, and to improve the accuracy of detection and / or diagnosis of autoimmune diseases of the nervous system, especially autoimmune encephalitis.

[0006] To achieve the above objectives, the present invention provides the use of a reagent for detecting anti-NCAM2 autoantibodies in the preparation of a product for detecting and / or diagnosing autoimmune diseases of the nervous system.

[0007] Preferably, the product comprises a kit.

[0008] Preferably, the reagent for detecting anti-NCAM2 autoantibodies comprises one or more of NCAM2 protein, a lysate containing NCAM2 protein, and a biological material expressing NCAM2 protein; the biological material comprises one or more of a recombinant vector, a cell, and a tissue;

[0009] The lysate includes cell lysate and / or tissue lysate.

[0010] Preferably, the amino acid sequence of the NCAM2 protein includes any one of a) to c):

[0011] a) the amino acid sequence shown in SEQ ID NO: 1;

[0012] b) an amino acid sequence that is ≥70% and <100% identical to the amino acid sequence of SEQ ID NO: 1 and that can recognize an NCAM2 autoantibody;

[0013] c) The amino acid sequence in a) or b) is modified or mutated to be able to recognize the amino acid sequence of the NCAM2 autoantibody.

[0014] Preferably, after modification or mutation, the amino acid sequence in a) or b) can recognize the amino acid sequence of the NCAM2 autoantibody as shown in SEQ ID NO: 3.

[0015] Preferably, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 includes any one of I) to III):

[0016] Ⅰ) the nucleotide sequence shown in SEQ ID NO: 2;

[0017] II) having an identity of ≥70% and <100% with the nucleotide sequence of SEQ ID NO: 2, and capable of expressing an amino acid sequence that recognizes an NCAM2 autoantibody;

[0018] III) After the nucleotide sequence in I) or II) is mutated, the amino acid sequence of the autoantibody that recognizes NCAM2 can be expressed.

[0019] Preferably, 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 level of consciousness, coma and autonomic dysfunction.

[0020] Preferably, the autoimmune disease of the nervous system is autoimmune encephalitis.

[0021] The present invention also provides a kit for detecting and / or diagnosing autoimmune diseases of the nervous system, comprising a labeled antibody and a reagent for detecting anti-NCAM2 autoantibodies.

[0022] Preferably, the reagent for detecting anti-NCAM2 autoantibodies comprises one or more of NCAM2 protein, a lysate containing NCAM2 protein, and a biological material expressing NCAM2 protein; the biological material comprises one or more of a recombinant vector, a cell, and a tissue;

[0023] The lysate includes cell lysate and / or tissue lysate.

[0024] Beneficial effects:

[0025] The present invention uses serum from patients with autoimmune diseases of the nervous system and serum from healthy individuals to incubate primary rat neurons. By amplifying the signal with a fluorescent secondary antibody and co-staining with a neuron-specific marker antibody, it is found that compared with healthy human serum, the patient serum contains autospecific signals. By immunoprecipitation and mass spectrometry identification, the signals on the patient serum are screened as autoantibodies that recognize the NCAM2 antigen. The authenticity of the target antigen is verified by serum neutralization experiments, incubation of primary rat neurons, overexpressed cell slides, and rat brain tissue proteins / primary neuronal proteins, indicating that the NCAM2 protein is expressed in both rat brain tissue and rat neurons and that a clear signal is present. NCAM2 can be used as one of the recognition antigens for autoantibodies associated with autoimmune diseases of the nervous system. By detecting anti-NCAM2 autoantibodies, it is possible to detect and / or diagnose autoimmune diseases of the nervous system, especially to achieve auxiliary diagnosis of autoimmune encephalitis of the nervous system.

[0026] Furthermore, the present invention uses a reagent for detecting anti-NCAM2 autoantibodies as a main component to establish a kit for detecting and / or diagnosing nervous system-related diseases. The kit can qualitatively or quantitatively analyze anti-NCAM2 autoantibodies, is easy to operate, and can detect and / or diagnose autoimmune diseases of the nervous system, thereby improving the accuracy of detection and / or diagnosis of autoimmune diseases of the nervous system, especially autoimmune encephalitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0028] Figure 1The staining results of the sera of patients 1 to 4 and the sera of controls 1 to 3 on rat primary neurons are shown; the scale bar is 200 μm;

[0029] Figure 2 The co-staining results of patients 1 to 3 sera and MAP2 antibodies on rat primary neuronal cells; the scale bar is 200 μm;

[0030] Figure 3 These are the results of SDS electrophoresis of immunoprecipitates of sera from patients 1 to 4 and sera from controls 1 to 3;

[0031] Figure 4 These are the WB results of the immunoprecipitates of sera from patients 1 to 4 and sera from controls 1 to 3;

[0032] Figure 5 Western blot results of the commercial NCAM2 antibody and the IP elution sample of primary neurons from patient 1;

[0033] Figure 6 The co-staining results of NCAM2 commercial antibody and sera from patients 1 to 4 on overexpressing cells are shown; the scale bar is 200 μm;

[0034] Figure 7 Validate the WB results of NCAM2 neutralizing proteins for NCAM2 commercial antibodies;

[0035] Figure 8 The staining results of the serum neutralization experiment of patients 1 to 4 on primary neuronal cells are shown in Figure 1. The scale bar is 200 μm.

[0036] Figure 9 The staining results of the serum neutralization experiment of patients 1 to 4 on the cell slides overexpressing NCAM2 are shown; the scale bar is 200 μm;

[0037] Figure 10 These are the Western blot results of the serum neutralization experiment with patient 1 on rat brain tissue proteins and primary neuronal proteins;

[0038] Figure 11 The staining results are shown for sera from three patients with positive anti-NCAM2 antibodies, disease control sera, and healthy controls; the scale bar is 200 μm. DETAILED DESCRIPTION

[0039] The present invention provides use of a reagent for detecting anti-NCAM2 autoantibodies in preparing a product for detecting and / or diagnosing autoimmune diseases of the nervous system.

[0040] As an embodiment, the product of the present invention includes a kit.

[0041] As an embodiment, the reagent for detecting anti-NCAM2 autoantibodies of the present invention comprises one or more of NCAM2 protein, a lysate containing NCAM2 protein, and a biological material expressing NCAM2 protein; the biological material comprises one or more of a recombinant vector, cells, and tissues; and the lysate comprises a cell lysate and / or a tissue lysate.

[0042] In one embodiment, the NCAM2 protein described herein is obtained by inserting a gene encoding the NCAM2 protein into a starting vector, expressing the resulting recombinant vector in an expression system, and purifying the protein. The present invention does not strictly require the type of the base vector; a conventionally selected one can be used, for example, pCDNA3.1. In one embodiment, the expression system described herein preferably includes a prokaryotic expression system, a yeast expression system, a baculovirus expression system, or a mammalian cell expression system. In one embodiment, the purification described herein includes affinity chromatography, molecular sieve chromatography, ion exchange chromatography, or hydrophobic chromatography. The present invention does not strictly require the specific purification method; a conventionally selected one can be used. In one embodiment, the tissue described herein includes mammalian tissue. In one embodiment, the mammal described herein includes one or more of humans, primates, and mice. In one embodiment, the tissue described herein includes brain tissue. In one embodiment, the cells described herein include one or more of neurons, HEK293, HeLa, 293T cells, and CHO cells.

[0043] In one embodiment, the amino acid sequence of the NCAM2 protein of the present invention comprises any one of a) to c): a) the amino acid sequence set forth in SEQ ID NO: 1; b) an amino acid sequence having ≥70% and <100% identity to the amino acid sequence set forth in SEQ ID NO: 1 and capable of recognizing an NCAM2 autoantibody; c) an amino acid sequence in a) or b) that, after modification or mutation, can recognize an NCAM2 autoantibody. In one embodiment, the amino acid sequence in a) or b) that has ≥70% and <100% identity to the amino acid sequence set forth in SEQ ID NO: 1 can be 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the amino acid sequence in a) or b) that, after modification or mutation, can recognize an NCAM2 autoantibody is as set forth in SEQ ID NO: 3.

[0044] In one embodiment, the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1 comprises any one of I) to III): I) the nucleotide sequence set forth in SEQ ID NO: 2; II) an amino acid sequence having ≥70% and <100% identity to the nucleotide sequence set forth in SEQ ID NO: 2 and capable of expressing an amino acid sequence that recognizes an NCAM2 autoantibody; III) a nucleotide sequence in I) or II) that, after mutation, can express an amino acid sequence that recognizes an NCAM2 autoantibody. In one embodiment, the nucleotide sequence having ≥70% and <100% identity to the nucleotide sequence set forth in SEQ ID NO: 2 can be 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 1.

[0045] The sequence information of SEQ ID NO: 1 to SEQ ID NO: 3 of the present invention is as follows:

[0046] SEQ ID NO:1: MSLLLSFYLLGLLVSSGQALLQVTISLSKVELSVGESKFFTCTAIGEPESIDWYNPQGEKIISTQRVVVQKEGVRSRLTIYNANIEDAGIYRCQATDAKGQTQEATVVLEIYQKLTFREVVSPQEFKQGEDAEVVCRVSSSPAPAVSWLYHNEEVTTISDNRFAMLANNNLQILNINKSDEGIYRCEGRVEARGEIDFRDIIVIVNVPPAISMPQKSFNATAERGEEMTFSCRASGSPEPAISWFRNGKLIEENEKYILKGSNTELTVRNIINSDGGPYVCRATNKAGEDEKQAFLQVFVQPHIIQLKNETTYENGQVTLVCDAEGEPIPEITWKRAVDGFTFTEGDKSLDGRIEVKGQHGSSSLHIKDVKLSDSGRYDCEAASRIGGHQKSMYLDIEYAPKFISNQTIYYSWEGNPINISCDVKSNPPASIHWRRDKLVLPAKNTTNLKTYSTGRKMILEIAPTSDNDFGRYNCTATNHIGTRFQEYILALADVPSSPYGVKIIELSQTTAKVSFNKPDSHGGVPIHHYQVDVKEVASEIWKIVRSHGVQTMVVLNNLEPNTTYEIRVAAVNGKGQGDYSKIEIFQTLPVREPSPPSIHGQPSSGKSFKLSITKQDDGGAPILEYIVKYRSKDKEDQWLEKKVQGNKDHIILEHLQWTMGYEVQITAANRLGYSEPTVYEFSMPPKPNIIKDTLFNGLGLGAVIGLGVAALLLILVVTDVSCFFIRQCGLLMCITRRMCGKKSGSSGKSKELEEGKAAYLKDGSKEPIVEMRTEDERVTNHEDGSPVNEPNETTPLTEPEKLPLKEEDGKEALNPETIEIKVSNDIIQSKEDDSKA*; Among them, the bold part is the different part of the amino acid sequences described in SEQ ID NO:1 and SEQ ID NO:3.

[0047]

[0048] SEQ ID NO: 3: *;

[0049] As an embodiment, the symptoms of the autoimmune disease of the nervous system described in the present invention include one or more of mental and behavioral abnormalities, cognitive impairment, recent memory loss, epileptic seizures, speech disorders, movement disorders, involuntary movements, decreased level of consciousness, coma and autonomic dysfunction.

[0050] As an embodiment, the autoimmune disease of the nervous system described in the present invention is autoimmune encephalitis.

[0051] The present invention uses the serum of patients with autoimmune diseases of the nervous system, screens the antibody spectrum of autoimmune encephalitis, and excludes other diseases by clinicians, preliminarily determines the research subjects, and uses the serum of patients and healthy people to incubate rat primary neuronal cells, amplifies the signal by fluorescent secondary antibody, and co-stains with neuron-specific marker antibodies, and finds that compared with healthy people's serum, the patient's serum has autospecific signals, and the signals on the patient's serum are screened out as autoantibodies that recognize NCAM2 antigen by immunoprecipitation and mass spectrometry identification. Through serum neutralization experiments, rat primary neuronal cells, overexpression cell slides, and rat brain tissue protein / primary neuronal protein tests are carried out. The authenticity of the target antigen was confirmed, indicating that NCAM2 protein was expressed in mouse brain tissue and rat neurons with obvious signals. Further sera from patients with and without neurological autoimmune diseases and healthy subjects were collected as control samples. Samples with staining patterns similar to those of patient sera on primary neuronal cells were screened by overexpressing NCAM2 cell slides to verify the specificity of the target antigen. This showed that NCAM2 can serve as one of the recognition antigens for autoantibodies associated with neurological autoimmune diseases. Reagents for detecting anti-NCAM2 autoantibodies can realize the detection and / or diagnosis of neurological autoimmune diseases, especially the auxiliary diagnosis of neurological autoimmune encephalitis.

[0052] The present invention also provides a kit for detecting and / or diagnosing autoimmune diseases of the nervous system, comprising a labeled antibody and a reagent for detecting anti-NCAM2 autoantibodies.

[0053] As an embodiment, the reagent for detecting anti-NCAM2 autoantibodies of the present invention comprises one or more of NCAM2 protein, a lysate containing NCAM2 protein, and a biological material expressing NCAM2 protein; the biological material comprises one or more of a recombinant vector, cells, and tissues; and the lysate comprises a cell lysate and / or a tissue lysate.

[0054] As an embodiment, the labeled antibody of the present invention includes one or more of FITC-labeled goat anti-human IgG antibody, AlexaFluor 594-labeled goat anti-rabbit IgG antibody, HRP-labeled goat anti-human IgG antibody and HRP-labeled goat anti-rabbit IgG antibody.

[0055] In one embodiment, the kit of the present invention further comprises one or more of a solid phase carrier, a buffer, and a washing solution. In one embodiment, the solid phase carrier of the present invention comprises a polyethylene plate, a membrane, a glass slide, magnetic beads, a chromatography filler, a microfluidic channel, or a polyacrylamide gel. In one embodiment, the membrane of the present invention comprises a nylon membrane, a nitrocellulose membrane, or a PVDF membrane. In the specific operation process of the present invention, the corresponding labeled antibody, solid phase carrier, buffer, and washing solution are selected according to the specific detection method of the kit.

[0056] The present invention utilizes a reagent for detecting anti-NCAM2 autoantibodies as a primary component to develop a kit for detecting and / or diagnosing autoimmune diseases of the nervous system. The kit can qualitatively or quantitatively analyze anti-NCAM2 autoantibodies, thereby detecting and / or diagnosing autoimmune diseases of the nervous system, and is particularly useful as an auxiliary diagnostic tool. When using the kit of the present invention for detection, there are no strict requirements for the detection method employed; well-known methods can be employed, such as cell-based immunofluorescence assay (CBA), tissue-based immunofluorescence assay (TBA), enzyme-linked immunosorbent assay (ELISA), immunocolloidal gold assay, immunoblotting, immunospot, membrane strip assay, chemiluminescence, radioimmunoassay, liquid phase chip assay, lateral flow assay, or flow cytometry.

[0057] To further illustrate the present invention, the application of the reagent for detecting anti-NCAM2 autoantibodies provided by the present invention in detecting and / or diagnosing autoimmune diseases of the nervous system is described in detail below with reference to the accompanying drawings and examples. However, these figures should not be construed as limiting the scope of protection of the present invention.

[0058] The patient serum used in this invention was donated by the hospital and has been approved by the patient. The serum of healthy people (healthy control serum) was donated by the hospital physical examination center and has been approved by the patient. All sera were screened for the following 56 autoantibodies: anti-NMDAR, AMPAR, LGI1, GABABR, CASPR2, DPPX, IgLON5, GlyRα1, GABAAR, GABARAP, mGluR1, mGluR2, mGluR5, D2R, Neurexin-3α, AK5, GFAP, ANNA3, MAP1b, PDE10A, ARHGAP26, KLHL11, LUZP4, Homer3, MUNC18-1, NCDN, ITPR, C A8, GRID2, AP3B2, Gephyrin, ATP1A3, CACNA2d1, SEZ6L2, GRIK2, GluR3, KCTD16, TGM2, TGM6, KCNA4, TRIB2, ROCK2, SYN1, RGS8, GAD65 / 67, Hu (ANNA-1, HuB / C / D), Ri (ANNA-2, Nova1 / 2), Yo, CV2, Ma1, Ma2, Amphiphysin, PKCγ, Zic4, SOX1 and Tr antibodies.

[0059] The patient information of the present invention having symptoms of autoimmune diseases of the nervous system is as follows:

[0060] Patient 1: A 69-year-old male had limb convulsions with unconsciousness in the past 3 months, which resolved on their own. He could not recall the course of the seizures afterwards. The seizures were accompanied by epileptic seizures or urinary and bowel disorders. MRI showed abnormal high signals in the medial temporal lobes of both sides, abnormal leukocytosis in the cerebrospinal fluid, and brain biopsy showed inflammatory infiltration. The above 56 autoantibody tests were negative. The clinician diagnosed him with possible autoimmune encephalitis (possible autoimmune encephalitis is antibody-negative autoimmune encephalitis. Reference: Guan Hongzhi, Cui Liying. Standardization and individualization of diagnosis and treatment of autoimmune encephalitis [J]. Chinese Journal of Neurology, 2020, 53(1): 5-7).

[0061] Patient 2: A 56-year-old female patient presented with memory loss in the past month, accompanied by depression, gradual behavioral abnormalities, incoherent speech, and mild cognitive impairment, with a gradual loss of ability to perform daily activities, memory impairment, and epileptic seizures. The patient's cerebrospinal fluid showed positive oligoclonal bands, MRI showed abnormal high signals in the bilateral medial temporal lobes, and the electroencephalogram showed abnormalities. Screening revealed concomitant small cell lung cancer. The above 56 autoantibody tests were negative, and the clinician diagnosed her with possible autoimmune encephalitis.

[0062] Patient 3: A 42-year-old male developed mental and behavioral abnormalities without obvious causes, accompanied by nausea and vomiting, memory loss, poor spirits, and normal bilateral visual fields. One week later, he developed epileptic seizures and impaired consciousness, oral and facial movement disorders, central hypoventilation, cerebrospinal fluid cytology showed lymphocytic inflammation, MRI abnormalities suggested autoimmune encephalitis, and brain biopsy showed inflammatory infiltration. The above 56 autoantibody tests were negative, and the clinician diagnosed him with possible autoimmune encephalitis.

[0063] Patient 4: A 38-year-old male with subacute onset of the disease. He developed slow reaction and unsteady gait in the past six months. He developed slurred speech in the past week, accompanied by convulsions and epilepsy. He also had cognitive impairment, neuropsychiatric disorders, depression and delusions. MRI of the head showed lesions in the left temporal lobe and an elevated IgG index in the cerebrospinal fluid. Tests for the above 56 autoantibodies were negative, and the clinician diagnosed him with possible autoimmune encephalitis.

[0064] The above four patients all had subacute onset and encephalitis symptoms, abnormal head MRI, epileptic seizures and varying degrees of consciousness / cognitive disorders. Two of them (patients 3 and 4) also had mental disorders, and one had small cell lung cancer (patient 2). The condition of these four patients showed significant improvement after immunotherapy.

[0065] Example 1

[0066] Exclusion of other diseases and screening of autoimmune encephalitis antibody spectrum

[0067] 1. Differential diagnosis of diseases

[0068] Based on the clinical symptoms of the four patients, the doctors diagnosed them as possible autoimmune encephalitis patients. Through the disease differential diagnosis criteria of autoimmune encephalitis, the following diseases have been excluded in the four patients: 1) central nervous system infection and infectious encephalopathy, such as viral encephalitis; 2) metabolic encephalopathy and toxic encephalopathy; 3) Hashimoto's encephalopathy; 4) central nervous system tumors, such as glioma and lymphoma; 5) genetic diseases, such as mitochondrial encephalopathy; 6) nervous system lesions involving rheumatic diseases; 7) neuromuscular diseases, such as myasthenia gravis; 8) other autoimmune diseases of the nervous system, such as central demyelinating disease, peripheral neuropathy, etc.

[0069] 2. Screening of the antibody spectrum for autoimmune encephalitis

[0070] Using a biodetection chip, we screened the expanded antibody spectrum of autoimmune encephalitis in samples from four patients, and used the sera of five healthy individuals as controls. We found 56 target antigens of antibodies related to autoimmune encephalitis (including NMDAR encephalitis, limbic encephalitis, encephalomyelitis, brainstem encephalitis, cerebellar ataxia, etc.) that have been reported in the literature, including NMDAR, AMPAR, LGI1, GABABR, CASPR2, DPPX, IgLON5, GlyRα1, GABAAR, GABARAP, mGluR1, mGluR2, mGluR5, D2R, Neurexin-3α, AK5, GFAP, ANNA3, MAP1b, PDE10A, AR HGAP26, KLHL11, LUZP4, Homer3, MUNC18-1, NCDN, ITPR, CA8, GRID2, AP3B2, Gephyrin, ATP1A3, CACNA2d1, SEZ6L2, GRIK2, GluR3, KCTD16, TGM2, TGM6, KCNA4, TRIB2, ROCK2, SYN1, RGS8, GAD65 / 67, Hu (ANNA-1, HuB / C / D), Ri (ANNA-2, Nova1 / 2), Yo, CV2, Ma1, Ma2, Amphiphysin, PKCγ, Zic4, SOX1, and Tr, as described in the following steps:

[0071] (1) Construction of recombinant vectors: The gene sequences encoding the above 56 proteins were ligated to pCDNA3.1 by molecular cloning using PCR or artificial synthesis methods to obtain 56 recombinant vectors. The constructed recombinant vectors were sequenced and then collected for future use.

[0072] (2) Target gene transfection: 6 cm × 6 cm 293T cell slides were laid on the bottom of culture dishes and cultured in a cell culture incubator at 37°C and 5% CO2 using 10% FBS-DMEM high glucose medium. A total of 57 dishes were used. When the cell density reached 30% to 40%, 56 recombinant vectors of corresponding genes and empty pCDNA3.1 were transfected into 293T cells using PEI transfection reagent (manufacturer: thermo, catalog number: BMS1003) and labeled.

[0073] (3) Fixation of cell slides: cells grown for 48 h after transfection were washed twice with PBS, fixed with acetone for 5 min, washed twice with PBS, and dried at 45°C for 30 min. The slides were cut into 2.5 mm × 2.5 mm pieces, and 57 2.5 mm × 2.5 mm cell slides were attached to a glass slide to prepare a bioassay chip for screening target antigens for later use.

[0074] (4) Immunofluorescence staining: The sera of 4 patients and 5 healthy subjects were diluted with PBST at a volume ratio of 1:10 and then incubated on the bioassay chip. The cells were incubated at room temperature for 1 hour and washed three times with PBST for 5 minutes each. FITC-labeled goat anti-human IgG antibody was diluted 1:200 and incubated at room temperature for 30 minutes. The cells were washed three times with PBST for 5 minutes each. Observation under a fluorescence microscope revealed that the sera of the 4 patients did not produce a color reaction with the 56 bioassay chips with a signal significantly stronger than that of healthy subjects, indicating that the sera of the 4 patients did not contain autoantibodies that bound to the 56 known autoimmune brain target antigens.

[0075] Example 2

[0076] Indirect immunofluorescence assay to screen patient serum signals

[0077] 1. Isolation of Primary Rat Neuronal Cells and Preparation of Slides

[0078] Pregnant Sprague-Dawley mice (gestational age 1 to 16 days) were anesthetized with 10% chloral hydrate and disinfected by immersion in 75% (v / v) alcohol for 3 minutes. The fetuses were removed from the biosafety cabinet and rinsed twice with pre-chilled Hank's solution (Thermo, catalog number 11575032). The brains were dissected in pre-chilled Hank's solution and immersed in pre-chilled Dulbecco's Modified Eagle's Methionine (DMEM) cell culture medium. The DMEM was discarded, and the tissue fragments were minced into a minced mixture using ophthalmic scissors. Papain was added and digested at 37°C for 30 minutes. After digestion, the cells were transferred to a fresh centrifuge tube, resuspended in DMEM cell culture medium, centrifuged at 400g for 5 minutes at 4°C, the supernatant discarded, and the cells resuspended in 20 mL of Neurobasal medium (GIBCO, catalog number 21103-049) in a fresh centrifuge tube. In subsequent experiments, neurons were isolated from the resuspended cells using Opti Prep medium (Sigma, Catalog No. 92339-11-2) and cultured in a cell culture dish containing cell slides. Primary rat cell slides were fixed with 2% paraformaldehyde (PFA) for 10 minutes, washed twice with Hank's solution, terminated with 1.25 M glycine for 10 minutes, and washed twice with Hank's solution to obtain primary neuronal cell slides.

[0079] 2. Immunofluorescence Staining of Primary Neuronal Cells

[0080] The serum of 4 patients and 3 healthy subjects was diluted with PBS buffer at a volume ratio of 1:10, and incubated on the primary neuronal cell slides of rat brain tissue. The slides were incubated at room temperature for 30 minutes, and washed twice with Hank's solution, each time for 5 minutes. Then, 1:200 diluted FITC-labeled goat anti-human IgG antibody was added, and the slides were incubated at room temperature for 30 minutes. The slides were washed twice with Hank's solution, each time for 5 minutes. The slides were stained at room temperature for 10 minutes, and washed twice with Hank's solution, each time for 5 minutes. The slides were observed under a microscope. Figure 1 As shown. Figure 1 It can be seen that the sera of four patients showed positive signals on primary cell neurons of rat brain tissue, while the sera of three healthy people did not show positive signals on primary cell neurons of rat brain tissue, suggesting that antibodies that recognize primary neuronal cell antigens may exist in the sera of these four patients.

[0081] 3. Antibody Co-staining

[0082] The slides incubated with the serum of patients 1 to 3 in step 2 were co-stained with the neuron-specific marker MAP2 antibody (manufacturer: Wuhan Sanying, catalog number: 17490-1-AP). The MAP2 antibody was diluted 1:200 and washed twice with Hank's solution for 5 minutes each. The slides were incubated with AlexaFluor 594-labeled goat anti-rabbit IgG antibody (manufacturer: Jackson) at room temperature for 30 minutes and washed twice with Hank's solution for 5 minutes each. The results were observed under a microscope. Figure 2 As shown. Figure 2 It can be seen that the signals stained by the sera of patients 1 to 3 overlap with the signals of MAP2 antibody (MAP2 is a protein used as a mature neuronal marker) on primary neuronal cells, indicating that the antigens recognized by the antibodies in the sera of patients 1 to 3 are present on neuronal cells.

[0083] Example 3

[0084] Immunoprecipitation of primary tissue / cell lysates and validation and identification of target antigens

[0085] 1. Immunoprecipitation experiments with primary tissue / cell lysates

[0086] (1) Seven 50mg portions of rat brain tissue were minced, transferred to a mortar and ground into a fine powder by adding liquid nitrogen. 500μL of RIPA lysis buffer (water as solvent, containing 150mM NaCl, 1mM EDTA, 100mM Tris-HCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% TritonX-100, 5% glycerol, pH 7.5) was added to each sample group. Protease inhibitors were added at a final concentration of 1× and sonicated (10% power, supersonication for 3 seconds and rest for 6 seconds) for 1 minute. After sonication, the samples were lysed on ice for 30 minutes, shaken every 5 minutes, and centrifuged at 15000rpm for 30 minutes. The supernatants were collected for later use. 1μL of serum from each of the four patients and three controls was added to the supernatants of the seven rat brain tissue extracts. The samples were incubated at 4°C overnight to obtain seven serum-reacted brain tissue extracts.

[0087] (2) Take 7 dishes of primary cultured rat neuronal cells, discard the supernatant, wash twice with PBS buffer, fix with 2 wt.% paraformaldehyde for 10 minutes, and wash three times with 1× Hank's solution; dilute the serum of 4 patients and 3 controls with 1× Hank's solution at a volume ratio of 1:200, filter with 0.22 μm filter membrane, add to the fixed primary cultured neuronal cells, and incubate at 4°C overnight; place the incubated cells on ice, discard the supernatant, wash twice with PBS buffer, add 500 μL of RIPA lysis buffer, collect the cells, add protease inhibitor cocktail (manufacturer: Roche, catalog number: 04693132001) with a final concentration of 1×, lyse for 30 minutes, shake at intervals, centrifuge at 15,000 rpm for 30 minutes, take the supernatant, and obtain 7 portions of neuronal extract after reaction with serum.

[0088] 2. The seven serum-reacted brain tissue extracts and seven serum-reacted neuronal extracts prepared in step 1 were added to the treated Protein A / G immunoprecipitation magnetic beads, respectively, and incubated with rotation at 4°C overnight; the incubated magnetic beads were washed four times with RIPA lysis buffer, eluted with 100 μL 1× loading buffer, and the eluate was collected; 5× SDS-PAGE loading buffer was first added to the eluate, followed by DTT at a final concentration of 0.01 M, heated at 95°C for 5 minutes, and then iodoacetamide at a final concentration of 2 wt.% was added, and the mixture was allowed to stand at room temperature for 15 minutes to obtain the immunoprecipitated complexes of the four patient serum samples and three control serum samples; the prepared immune complexes were subjected to SDS-PAGE electrophoresis and stained using a silver staining kit (manufacturer: thermo). The results are as follows: Figure 3 As shown. Figure 3It can be seen that in rat brain tissue / primary neuronal proteins, a protein of approximately 100KDa to 130KDa was detected in the immunoprecipitates captured using sera from four patients ( Figure 3 The protein was not present in the control group prepared from 3 control sera.

[0089] 3. After step 2 SDS-PAGE electrophoresis, wet transfer was performed at 300 mA for 120 min. 5% skim milk powder was then used to block the membrane at room temperature for 1 h. The serum of 4 patients and 3 controls was diluted with TBST at a volume ratio of 1:200 and incubated at room temperature for 2 h. The membrane was washed 3 times with TBST for 5 min each. HRP-labeled goat anti-human IgG antibody (manufacturer: Jackson, catalog number 109-035-098) was added and incubated at room temperature for 1 h. The membrane was washed 3 times with TBST for 5 min each. Chemiluminescent solution was added for color development. The results are shown in Figure 2. Figure 4 As shown. Figure 4 It can be seen that in the immunoprecipitates captured in rat brain tissue / primary neuronal proteins using the sera of four patients, there are bands that react with the autoantibodies in the sera of four patients, and the target protein band is between 100KDa and 130KDa ( Figure 4 In the immunoprecipitates captured by the three control sera in rat brain tissue / primary neuronal proteins, no reactive bands were found between 100 kDa and 130 kDa, indicating that the sera of the four patients contained autoantibodies that reacted with neuronal proteins.

[0090] 4. Mass spectrometry detection and confirmation of target bands: gel cutting and recovery of bands at 100KDa to 130KDa ( Figure 3 The sample was sent to Novogene for mass spectrometry analysis. Proteins with a prot.score>100 (the higher the score, the higher the confidence of the detected corresponding protein) were screened. Combined with the property analysis of these proteins, preliminary verification and, after excluding another suspicious protein, the NCAM2 protein (accession number: NM_004540.5) was finally identified as the possible target protein.

[0091] Example 4

[0092] Commercial antibody validation target antigen

[0093] 1. Verify the target antigen in the IP eluted sample using a commercial NCAM2 antibody

[0094] The immunoprecipitates captured in primary neuronal cells using the serum of patient 1 and control 1 prepared in Example 3 were subjected to SDS-PAGE electrophoresis; after electrophoresis, wet transfer was performed at 300 mA for 120 min; 5% skim milk powder was then used for blocking at room temperature for 1 h; the commercial antibody NCAM2 (manufacturer: proteintech, item number: 13850-1-AP) was diluted with TBST at a volume ratio of 1:1000, and the serum of patient 1 and control 1 were diluted at a volume ratio of 1:200, respectively, and incubated at room temperature for 2 h; washed 3 times with TBST for 5 min each; HRP-labeled goat anti-rabbit / goat anti-human IgG antibody (manufacturer: Jackson) was added and incubated at room temperature for 1 h; washed 3 times with TBST for 5 min each; chemiluminescent solution was added for color development. The results are shown in FIG. Figure 5 As shown. Figure 5 It can be seen that a band reactive with the commercial NCAM2 antibody exists in the immunoprecipitate captured by the serum of Patient 1 in primary neuronal cells, and this band also exists in the WB results of the complex between the serum of Patient 1 and the immunoprecipitate, but does not exist in the WB results with the serum of Control 1, indicating that the protein detected in the immunoprecipitate captured using the serum of Patient 1 is NCAM2 protein.

[0095] 2. Use commercial NCAM2 antibodies to verify the target antigen in overexpressed cell slides

[0096] (1) Construction of recombinant vector: The NCAM2 gene (SEQ ID NO: 3) was linked to pCDNA3.1 by molecular cloning using PCR or artificial synthesis, with the insertion site being Nhe I / Not I, to obtain the recombinant vector pCDNA3.1-NCAM2. The constructed recombinant vector was sequenced correctly and then used in bulk.

[0097] (2) 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 correctly sequenced recombinant vector pCDNA3.1-NCAM2 was transfected into the 293T cells using PEI transfection reagent (manufacturer: thermo, catalog number: BMS1003). After transfection, the cells were grown for 48 hours and washed twice with PBS buffer, fixed with acetone for 30 minutes, discarded the acetone, and dried at 42°C for 30 minutes. The slides were cut into 2.5 mm × 2.5 mm pieces and pasted for later use. Empty slides containing pCDNA3.1 were prepared using the same transfection and fixation methods.

[0098] (3) Immunofluorescence staining: The serum of patients 1 to 4 was diluted at a ratio of 1:10 and incubated on the cell slides overexpressing NCAM2 protein, incubated at room temperature for 1 hour, and washed 3 times with PBST buffer, each time for 5 minutes; FITC-labeled goat anti-human IgG antibody was diluted at 1:200, incubated at room temperature for 30 minutes, and washed 3 times with PBST, each time for 5 minutes; commercial NCAM2 antibody was diluted at 1:1000, incubated on the cell slides overexpressing NCAM2 incubated with the serum of patients 1 to 4, incubated at room temperature for 1 hour, and washed 3 times with PBST, each time for 5 minutes; AlexaFluor 594-labeled goat anti-rabbit IgG antibody was diluted at 1:200 and observed under a fluorescence microscope. The results are shown in the figure. Figure 6 It can be seen that the staining signals of the sera of patients 1 to 4 on the cell slides overexpressing NCAM2 overlap with the staining signals of the commercial NCAM2 antibody, indicating that the antibodies in the sera of patients 1 to 4 specifically recognize the NCAM2 protein on the cell slides overexpressing NCAM2.

[0099] Example 5

[0100] Serum neutralization assay validates the signal detected in patient serum

[0101] 1. Preparation of Neutralizing Protein

[0102] Referring to the method in step 2 of Example 4, the correctly sequenced recombinant vector pCDNA3.1-NCAM2 and the empty pCDNA3.1 were transfected into 293T cells, respectively, and grown for 48 h after transfection. One dish of 293T cells overexpressing NCAM2 was taken, the supernatant was discarded, and the cells were scraped into a 1.5 mL centrifuge tube with a cell scraper. The cells were centrifuged at 800 rpm at room temperature to remove the supernatant, and 200 μL of PBS was added. The cells were ultrasonically disrupted for 1 min (10% power, disruption for 3 seconds, pause for 6 seconds) to prepare the NCAM2 neutralizing protein. A control protein was prepared from one dish of cells transfected with the empty pCDNA3.1, using the same preparation conditions and method as the NCAM2 neutralizing protein.

[0103] 2. Validation of Neutralizing Protein

[0104] The protein samples prepared in step 1 were subjected to electrophoresis, membrane transfer, and blocking steps. The commercial NCAM2 antibody was used, and the serum of patient 1 and control 1 were incubated on the transferred PVDF membrane, respectively, and incubated at 4°C overnight. The next day, the membrane was washed with TBST three times, each time for 5 minutes. HRP-labeled goat anti-rabbit / goat anti-human IgG antibody (manufacturer: Jackson) was added and incubated at room temperature for 1 hour. The membrane was washed with TBST three times, each time for 5 minutes. ECL chemiluminescence solution was added for color development and photography. The results are shown in Figure 2. Figure 7 As shown. Figure 7It can be seen that the commercial NCAM2 antibody and patient 1 serum have specific signals on the immunoblot of overexpressed NCAM2 protein, while the control 1 serum has no obvious band signal here, indicating that both the commercial NCAM2 antibody and patient 1 serum can specifically recognize the signals of NCAM2 antigen in overexpressed cells, and the neutralizing protein was successfully prepared.

[0105] 3. Serum neutralization experiments verify the signals detected by patient serum in primary neuronal cells

[0106] Referring to Example 2, primary neuronal cell fixed slides were prepared. Three portions of patient 1 serum diluted 1:200 were prepared using Hank's solution, 100 μL each, and 20 μL PBST, 20 μL NCAM2 neutralizing protein, and 200 μL control neutralizing protein were added. Three portions of patient 2-4 serum were prepared in the same manner and incubated with primary neuronal cell slides, incubated at room temperature for 1 hour, washed twice with Hank's solution, each for 5 minutes; then diluted FITC-labeled goat anti-human IgG antibody was added, incubated at room temperature for 30 minutes, washed twice with Hank's solution, each for 5 minutes; and observed under a microscope. The results are as follows. Figure 8 As shown. Figure 8 It can be seen that on the fixed slides of primary neuronal cells, the signals of the sera of patients 1 to 4 were blocked by the NCAM2 neutralizing protein, while the control protein did not block the signals of the sera of patients 1 to 4 on the fixed slides of primary neuronal cells, indicating that the signals of the sera of patients 1 to 4 on the fixed slides of primary neuronal cells are signals that specifically recognize the NCAM2 antigen.

[0107] 4. Serum neutralization experiment verifies the signal detected by patient serum on overexpression cell slides

[0108] Referring to Example 4, NCAM2-overexpressing cell slides were prepared. Three 1:200 diluted serum samples from patient 1 were prepared using PBST, 100 μL each, and 20 μL PBST, 20 μL NCAM2 neutralizing protein, and 200 μL control neutralizing protein were added. Three serum samples from patients 2 to 4 were prepared in the same manner and incubated with NCAM2-overexpressing cell slides, incubated at room temperature for 1 hour, and washed three times with PBST for 5 minutes each. Diluted FITC-labeled goat anti-human IgG antibody was then added, incubated at room temperature for 30 minutes, and washed five times with PBST for 5 minutes each. The results were observed under a microscope. Figure 9 As shown. Figure 9It can be seen that on the NCAM2-overexpressing cell slides, the signals of the sera of patients 1 to 4 were blocked by the NCAM2 neutralizing protein, while the control protein did not block the signals of the sera of patients 1 to 4 on the NCAM2-overexpressing cell slides, indicating that the signals of the sera of patients 1 to 4 on the NCAM2-overexpressing cell slides were signals that specifically recognized the NCAM2 antigen.

[0109] 5. Serum neutralization experiment verifies the signal detected by patient serum on immunoblot

[0110] The mouse brain tissue protein and primary neuronal cell protein prepared in Example 3 were used for gel electrophoresis, transfer and blocking steps. Three 1:200 dilutions of patient 1 serum were prepared using PBST, 1000 μL each, 200 μL PBST, 20 μL NCAM2 neutralizing protein and 200 μL control neutralizing protein were added, and the membranes were incubated on the transferred PVDF membranes and incubated at 4°C overnight. The next day, the membranes were washed three times with TBST for 5 minutes each. HRP-labeled goat anti-human IgG antibody (manufacturer: Jackson) was added and incubated at room temperature for 1 hour. The membranes were washed three times with TBST for 5 minutes each. ECL chemiluminescent solution was added for color development and photography. The results are shown in Figure 3. Figure 10 As shown. Figure 10 It can be seen that in the lanes of mouse brain tissue proteins and primary neuronal cells, the serum signal of patient 1 was blocked by the NCAM2 neutralizing protein, while the control protein did not block the signal of patient 1 serum binding to mouse brain tissue proteins and primary neuronal cell proteins, indicating that the signal of patient 1 binding to mouse brain tissue proteins and primary neuronal cell proteins is a signal that specifically recognizes the NCAM2 antigen.

[0111] Example 6

[0112] Validation of clinical specificity and sensitivity of anti-NCAM2 autoantibodies by immunofluorescence assay

[0113] 1. Summary of the detection of anti-NCAM2 antibodies in various diseases

[0114] In order to screen out more cases related to anti-NCAM2 antibodies and verify the clinical specificity and sensitivity of anti-NCAM2 antibodies, 820 samples were collected in this example (Table 2). The primary neuronal cell slides prepared in Example 2 were used to perform the test according to the method of step 2 of Example 2 to screen patient samples with staining patterns similar to those of patients 1 to 4. The NCAM2-overexpressing cell slides prepared in Example 4 were used to perform immunofluorescence staining according to the method of step (3) of Example 4 to screen NCAM2 autoantibody-positive samples. The test results were statistically analyzed. The immunofluorescence staining results are shown in FIG. Figure 11As shown; among them, patient 5 serum is a sample from a patient with possible autoimmune encephalitis, patient 6 cerebrospinal fluid is a sample from a patient with possible autoimmune encephalitis, and patient 7 serum is a positive sample from a patient with confirmed autoimmune encephalitis; disease control 1 serum is a sample from a patient with clinical diagnosis of myasthenia gravis; disease control 2 serum is a sample from a patient with clinical diagnosis of central demyelination.

[0115] Table 1 Sample information

[0116]

[0117]

[0118] According to Table 1 and Figure 11 It can be seen that among the 820 samples, 4 cases were detected with positive anti-NCAM2 antibodies; among them, 3 cases were detected with positive anti-NCAM2 antibodies among the 98 samples of patients with possible autoimmune encephalitis, and the CBA staining results of 2 patients (patients 5 and 6) were shown in Figure 2. Figure 11The detection rate was 3.06%. Among 143 confirmed autoimmune encephalitis patient samples, one anti-NCAM2 antibody-positive serum (patient 7 sample) was detected, with a detection rate of 0.70%. This patient with confirmed autoimmune encephalitis was anti-Neurexin-3α antibody-positive and showed symptoms such as epileptic seizures, impaired consciousness, and central hypoventilation. The positive rate of anti-NCAM2 antibodies in 241 patients clinically diagnosed with autoimmune encephalitis / possible autoimmune encephalitis was 1.66%. In addition to autoimmune encephalitis, In 147 samples from patients with other neurological autoimmune diseases besides encephalitis (including 64 cases of central nervous system demyelinating diseases, 48 ​​cases of myasthenia gravis, and 35 cases of peripheral neuropathy), no anti-NCAM2 antibody was detected, with a detection rate of 0.00%. In 105 samples from patients with other non-neurological autoimmune diseases (including 47 cases of viral encephalitis, 32 cases of central nervous system tumors, and 26 cases of mitochondrial encephalopathy) and 327 healthy controls as controls, no anti-NCAM2 antibody was detected, with a detection rate of 0.00%. Therefore, anti-NCAM2 autoantibodies can be detected in patients with autoimmune encephalitis / possible autoimmune encephalitis suffering from the symptoms described in the present invention, especially in patients with possible autoimmune encephalitis who were negative for 56 antibodies. Anti-NCAM2 autoantibodies were not detected in other patients or healthy controls, indicating that these autoantibodies have an auxiliary role in the diagnosis of autoimmune encephalitis, especially for the above-mentioned patients with possible autoimmune encephalitis. Since 3 of the samples from these patients with possible autoimmune encephalitis were positive for anti-NCAM2 antibodies, clinicians classified the 3 patients with positive anti-NCAM2 antibodies, screened from 98 patients with possible autoimmune encephalitis in this example, as confirmed patients with autoimmune encephalitis. Therefore, anti-NCAM2 antibodies can be used as a biomarker for the diagnosis of patients with autoimmune encephalitis.

[0119] 2. Analysis of the sensitivity and specificity of anti-NCAM2 antibodies in the auxiliary diagnosis of autoimmune encephalitis

[0120] Based on the test results in Table 1 , the sensitivity and specificity of anti-NCAM2 antibodies detected by immunofluorescence assay in the auxiliary diagnosis of autoimmune diseases of the nervous system, especially autoimmune encephalitis, were analyzed. The results are shown in Table 2 .

[0121] Table 2 Detection results of anti-NCAM2 antibodies

[0122]

[0123]

[0124] Sensitivity = 4 / (4 + 237) × 100% = 1.66%;

[0125] Specificity = 579 / (579+0) x 100% = 100%.

[0126] Based on the above, it can be seen that the specificity of anti-NCAM2 antibodies in assisting the diagnosis of autoimmune diseases of the nervous system, especially autoimmune encephalitis, is 100% and the sensitivity is 1.66%. The 2022 edition of the expert consensus on the detection of antibodies related to central nervous system autoimmune diseases reported the positive rates of multiple antibodies, including the positive rates of anti-LGI1 antibodies at 12.8% to 22.0%, anti-GABA antibodies at 1.6% to 2.8%, and anti-GABA antibodies at 1.6% to 2.8%. B The positive rate of receptor antibodies is 1.3%, and the positive rate of anti-AMPA receptor antibodies is 0.6% (Neuroimmunology Group of the Neurology Branch of the Chinese Medical Association. Expert Consensus on the Detection of Antibodies Related to Autoimmune Diseases of the Central Nervous System 2022 [J]. Chinese Journal of Neurology, 2023, 56 (3): 257-268). The anti-NCAM2 antibody of the present invention has a similar positive rate to some autoimmune encephalitis markers known in the art when used for auxiliary diagnosis of autoimmune encephalitis. According to the Expert Consensus on the Diagnosis and Treatment of Autoimmune Encephalitis in China (2022 Edition), combined with the patient's clinical manifestations, auxiliary examinations, and anti-NCAM2 antibody detection results, after excluding other causes, it can be judged whether the patient suffers from autoimmune encephalitis. Therefore, the present invention provides a new antigen that binds to autoantibodies for the diagnosis of patients with autoimmune encephalitis. Anti-NCAM2 autoantibodies can be detected in patients with neurological symptoms, especially in patients with autoimmune encephalitis, indicating that the autoantibody has an auxiliary effect on the diagnosis of autoimmune diseases of the nervous system, especially autoimmune encephalitis.

[0127] Example 7

[0128] Detection of anti-NCAM2 autoantibodies in patient sera using NCAM2 mutants

[0129] A mutant of the human NCAM2 gene, lacking amino acids 651 to 738 of the NCAM2 protein (SEQ ID NO: 3), was selected. Vector construction, cell slides overexpressing the NCAM2 deletion mutant were prepared according to the procedures described in Example 4, and the sera of patients 1 to 4 were tested. The results demonstrated that the NCAM2 deletion mutant could still recognize anti-NCAM2 antibodies in the patient sera.

[0130] Based on the above, it can be seen that anti-NCAM2 autoantibodies can be detected in patients with neurological autoimmune diseases with neurological symptoms, and have an auxiliary role in the diagnosis of neurological autoimmune diseases, especially autoimmune encephalitis.

[0131] Although the above embodiment provides a detailed description of the present invention, 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 scope of protection of the present invention.

Claims

1. Use of a reagent for detecting anti-NCAM2 autoantibodies in the preparation of a product for detecting and / or diagnosing autoimmune diseases of the nervous system.

2. The use according to claim 1, characterized in that The products include kits.

3. The use according to claim 1, characterized in that The reagent for detecting anti-NCAM2 autoantibodies includes one or more of NCAM2 protein, lysate containing NCAM2 protein, and biological materials expressing NCAM2 protein; the biological materials include one or more of recombinant vectors, cells, and tissues; The lysate includes cell lysate and / or tissue lysate.

4. The use according to claim 3, characterized in that The amino acid sequence of the NCAM2 protein includes any one of a) to c): a) the amino acid sequence shown in SEQ ID NO: 1; b) an amino acid sequence that is ≥70% and <100% identical to the amino acid sequence of SEQ ID NO: 1 and that can recognize an NCAM2 autoantibody; c) The amino acid sequence in a) or b) is modified or mutated to be able to recognize the amino acid sequence of the NCAM2 autoantibody.

5. The use according to claim 4, characterized in that After the amino acid sequence in a) or b) is modified or mutated, the amino acid sequence that can recognize the NCAM2 autoantibody is as shown in SEQ ID NO:

3.

6. The use according to claim 4, characterized in that The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 includes any one of I) to III): Ⅰ) the nucleotide sequence shown in SEQ ID NO: 2; II) having an identity of ≥70% and <100% with the nucleotide sequence of SEQ ID NO: 2, and capable of expressing an amino acid sequence that recognizes an NCAM2 autoantibody; III) After the nucleotide sequence in I) or II) is mutated, the amino acid sequence of the autoantibody that recognizes NCAM2 can be expressed.

7. The use according to any one of claims 1 to 6, characterized in that 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 level of consciousness, coma and autonomic dysfunction.

8. The use according to any one of claims 1 to 6, characterized in that: The autoimmune disease of the nervous system is autoimmune encephalitis.

9. A kit for detecting and / or diagnosing autoimmune diseases of the nervous system, characterized in that: The invention comprises a labeled antibody and a reagent for detecting anti-NCAM2 autoantibodies.

10. The kit according to claim 9, characterized in that The reagent for detecting anti-NCAM2 autoantibodies includes one or more of NCAM2 protein, lysate containing NCAM2 protein, and biological materials expressing NCAM2 protein; the biological materials include one or more of recombinant vectors, cells, and tissues; The lysate includes cell lysate and / or tissue lysate.

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