Application of reagent for detecting anti-GALNTL6 autoantibody in diagnosis of nervous system autoimmune diseases

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a reagent for detecting an anti-GALNTL6 autoantibody in diagnosis of nervous system autoimmune diseases. The invention provides application of a reagent for detecting an anti-GALNTL6 autoantibody in preparation of a product for detecting and / or diagnosing autoimmune diseases of a nervous system. The invention provides a novel antibody for diagnosing the autoimmune diseases of the nervous system, the antibody is an anti-GALNTL6 autoantibody, the anti-GALNTL6 autoantibody is determined as a marker for detecting and / or diagnosing the autoimmune diseases of the nervous system for the first time, GALNTL6 is used as a detection antigen, and the GALNTL6 is used as a marker for detecting and / or diagnosing the autoimmune diseases of the nervous system for detecting and / or diagnosing the autoimmune diseases of the nervous system. By detecting the expression of the anti-GALNTL6 autoantibody, the nervous system autoimmune diseases can be detected, biomarkers for identifying the nervous system autoimmune diseases are enriched, and the accuracy of detection and / or diagnosis of the nervous system autoimmune diseases is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the use of a reagent for detecting anti-GALNTL6 autoantibodies in diagnosing autoimmune diseases of the nervous system. Background Art

[0002] Autoimmune diseases of the nervous system are autoimmune diseases whose primary pathogenic mechanism is the attack of the nervous system by autoimmune cells and molecules. They share the complex nature of immune diseases and the high mortality and disabling nature of neurological diseases. In the immune response, pathogenic antibodies that act on autoantigens of the nervous system are collectively referred to as nervous system autoantibodies. Nervous system autoantibodies are important molecules mediating neurological immune diseases. Based on the distribution of their target antigens, they are divided into peripheral nervous system antibodies and central nervous system antibodies. Based on the localization of the target antigen within nerve cells, they can be further divided into antibodies against intracellular neuronal antigens and antibodies against neuronal surface antigens. Antibodies against intracellular antigens (such as Hu, Yo, Ri, and CV2) are often associated with paraneoplastic syndromes of the nervous system. The discovery of an increasing number of autoantibodies to the nervous system has effectively improved the clinical diagnosis and treatment of these diseases. However, due to the wide variety of autoantibodies and their diverse mechanisms of action, the clinical presentations of patients are complex. Therefore, the exploration and discovery of autoantibodies for autoimmune diseases of the nervous system is of great significance for the auxiliary diagnosis and treatment of the diseases.

[0003] Paraneoplastic neurological syndromes (PNS) are a series of neurological syndromes that occur in patients with certain tumors or potential tumors, causing acute or subacute damage to the nervous system due to remote effects in the absence of metastasis. These syndromes can affect any part of the nervous system, ranging from central nervous system involvement to cerebellar degeneration, encephalomyelitis, and limbic encephalitis; peripheral nervous system involvement to polyneuropathy and mononeuritis complex; and neuromuscular junction involvement to myasthenia gravis, Lambert-Eaton myasthenic syndrome, neuromyotonia, and polymyositis. Paraneoplastic antibodies are key mediators of these syndromes. Tumor cells in certain tissues, such as small cell lung cancer, ovarian cancer, thymoma, and lymphoma, can express neural antigens, inducing the production of autoantibodies, leading to neural damage. Currently recognized classic paraneoplastic antibodies include those against Hu, Yo, Ri, CV2, Ma, and Amphiphysin. These antibodies are closely related to the clinical manifestations and prognosis of paraneoplastic nervous system syndromes. In the diagnostic criteria for paraneoplastic nervous system syndromes, autoantibodies are also considered as one of the important diagnostic bases.

[0004] In 2010, Peng et al. discovered a new member of the human ppGalNAc-T family, ppGalNAc-T20. Using PCR in a human small cell lung cancer cell line, they cloned GALNTL6, named GalNAcT20, encoding a 601-amino acid type II membrane protein primarily expressed in the gastrointestinal tract, testis, prostate, brain, and muscle. Currently, there are no reports of anti-GALNTL6 autoantibodies in patients with autoimmune neurological diseases, suggesting that GALNTL6 may serve as a novel target for these diseases. Summary of the Invention

[0005] In order to provide a new target that can be used for diagnosing and / or detecting autoimmune diseases of the nervous system, the present invention provides a reagent for detecting anti-GALNTL6 autoantibodies for use in diagnosing autoimmune diseases of the nervous system, specifically including the following technical solutions:

[0006] Use of a reagent for detecting anti-GALNTL6 autoantibodies in the preparation of a product for detecting and / or diagnosing autoimmune diseases of the nervous system.

[0007] Preferably, the reagent comprises a polypeptide and / or a biological material expressing the polypeptide; the biological material comprises one or more of cells, vectors and tissues;

[0008] The polypeptide is an immunogenic polypeptide that binds to anti-GALNTL6 autoantibodies.

[0009] Preferably, the polypeptide comprises the GALNTL6 protein.

[0010] Preferably, the amino acid sequence of the GALNTL6 protein includes any one of a) to c):

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

[0012] b) the amino acid sequence in SEQ ID NO: 1, after modification or mutation, can recognize the amino acid sequence of anti-GALNTL6 autoantibodies;

[0013] 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-GALNTL6 autoantibody.

[0014] Preferably, the amino acid sequence capable of recognizing anti-GALNTL6 autoantibodies in b) includes the amino acid sequence shown in SEQ ID NO: 3.

[0015] Preferably, the cells include neuronal cells, HEK293 cells, Hela cells or CHO cells;

[0016] The vector includes pTriEx series vectors, pCDNA3 series vectors, pET series vectors or pBac series vectors;

[0017] The tissue includes mammalian brain tissue.

[0018] Preferably, the autoimmune disease of the nervous system comprises a paraneoplastic syndrome.

[0019] The present invention also provides a kit for detecting and / or diagnosing autoimmune diseases of the nervous system, comprising a reagent for detecting anti-GALNTL6 autoantibodies and a labeled antibody.

[0020] Preferably, the reagent for detecting anti-GALNTL6 autoantibodies comprises a polypeptide and / or a biological material expressing the polypeptide; the biological material comprises one or more of cells, vectors and tissues;

[0021] The polypeptide is an immunogenic polypeptide that binds to anti-GALNTL6 autoantibodies.

[0022] Preferably, the polypeptide comprises a GALNTL6 protein, and the amino acid sequence of the GALNTL6 protein comprises any one of a) to c):

[0023] a) the amino acid sequence shown in SEQ ID NO: 1;

[0024] b) the amino acid sequence in SEQ ID NO: 1, after modification or mutation, can recognize the amino acid sequence of anti-GALNTL6 autoantibodies;

[0025] 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-GALNTL6 autoantibody.

[0026] The beneficial effects of the present invention are:

[0027] The present invention provides the use of a reagent for detecting anti-GALNTL6 autoantibodies in the preparation of a product for detecting and / or diagnosing autoimmune diseases of the nervous system. This invention identifies, for the first time, anti-GALNTL6 autoantibodies as markers for detecting and / or diagnosing nervous system-related diseases. Using GALNTL6 as a detection antigen, detecting the expression of anti-GALNTL6 autoantibodies can detect nervous system-related diseases, enriching the biomarkers for identifying nervous system-related diseases and improving the accuracy of detecting and / or diagnosing nervous system-related diseases.

[0028] The present invention also for the first time uses a reagent for detecting anti-GALNTL6 autoantibodies as the main component to establish a kit for detecting nervous system-related diseases. The kit can qualitatively or quantitatively analyze anti-GALNTL6 autoantibodies, and can help distinguish whether patients with symptoms of nervous system diseases have nervous system autoimmune diseases. Doctors can combine the patient's clinical symptoms, physiological and biochemical test indicators, disease marker test results and other comprehensive conditions to determine whether the patient has a nervous system autoimmune-related disease or rule out the possibility of having another nervous system autoimmune disease. This helps doctors choose a more promising treatment plan or therapeutic drug for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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.

[0030] Figure 1 The staining results of the serum of patients 1 to 5 and healthy subjects on rat primary neuronal cells in Example 1 of the present invention are shown in FIG. 1 ; wherein the scale bar is 200 μm;

[0031] Figure 2 The co-localization staining results of the sera of patients 1 to 5 and the neuronal marker β-TUBB3 antibody on rat primary neuronal cells in Example 1 of the present invention are shown; wherein, the scale bar is 200 μm;

[0032] Figure 3 The staining results of the serum of patients 1 to 5 and healthy subjects on the GALNTL6-overexpressing cell slides in Example 4 of the present invention are shown in FIG. 1 , wherein the scale bar is 200 μm.

[0033] Figure 4 This is the staining result of co-localization of GALNTL6 autoantibody and neuronal marker β-TUBB3 on rat primary neuronal cells in Example 4 of the present invention;

[0034] Figure 5 This is the WB result of GALNTL6 autoantibody verification of overexpressed GALNTL6 protein in Example 5 of the present invention;

[0035] Figure 6 The results of the neutralization protein verification of the GALNTL6 autoantibody in Example 5 of the present invention on a cell slide overexpressing GALNTL6 are shown; wherein, the scale bar is 200 μm;

[0036] Figure 7 The WB results of the GALNTL6 autoantibody on mouse brain tissue protein and monkey brain tissue protein in Example 5 of the present invention are shown;

[0037] Figure 8The staining results of co-localization of sera from patients 1 to 5 and GALNTL6 autoantibodies on GALNTL6-overexpressing cell slides in Example 6 of the present invention are shown; the scale bar is 200 μm;

[0038] Figure 9 The staining results of the serum neutralization experiment in Example 7 of the present invention on rat primary neuronal cell slides verifying the signals detected by the sera of patients 1 to 5; wherein, the scale bar is 200 μm;

[0039] Figure 10 The staining results of the serum neutralization experiment in Example 7 of the present invention are to verify the signals detected by the sera of patients 1 to 5 on the cell slides overexpressing GALNTL6; wherein, the scale bar is 200 μm;

[0040] Figure 11 These are the staining results of some anti-GALNTL6 autoantibody-positive patient sera, disease control sera, and healthy human sera in Example 8 of the present invention. DETAILED DESCRIPTION

[0041] The present invention provides use of a reagent for detecting anti-GALNTL6 autoantibodies in preparing a product for detecting and / or diagnosing autoimmune diseases of the nervous system.

[0042] In the present invention, as an embodiment, the symptoms of the autoimmune disease of the nervous system preferably include one or more of sleep disorder, dizziness, unsteady gait, weakness in the limbs, ataxia, nystagmus, slow reaction, cognitive impairment, coughing when drinking water, dysarthria, dysphagia, mental abnormalities, epilepsy, confusion, slurred speech, dementia and tumors, and more preferably sleep disorder, dizziness, unsteady gait, weakness in the limbs, ataxia, nystagmus, slow reaction, cognitive impairment, coughing when drinking water, dysarthria, dysphagia, mental abnormalities, epilepsy, confusion, slurred speech, dementia and tumors. The autoimmune disease of the nervous system of the present invention is preferably a paraneoplastic syndrome.

[0043] In one embodiment, the product includes a kit. In one embodiment, the kit is based on an immune response assay; in another embodiment, the immune response includes, but is not limited to, cell-based immunofluorescence assay (CBA), tissue-based immunofluorescence assay (TBA), enzyme-linked immunosorbent assay (ELISA), immunocolloidal gold assay, immunoblot, immunospot, membrane strip assay, chemiluminescence, radioimmunoassay, liquid phase chip assay, lateral flow assay, or flow cytometry.

[0044] As one embodiment, the reagent of the present invention includes a polypeptide and / or a biological material expressing the polypeptide; the biological material includes one or more of cells, vectors and tissues; the polypeptide is an immunogenic polypeptide that binds to an anti-GALNTL6 autoantibody.

[0045] In one embodiment, the polypeptide comprises a GALNTL6 protein. In one embodiment, the amino acid sequence of the GALNTL6 protein comprises any one of a) to c): a) the amino acid sequence set forth in SEQ ID NO: 1; b) an amino acid sequence of SEQ ID NO: 1 that, after modification or mutation, can recognize an anti-GALNTL6 autoantibody; c) an amino acid sequence that has ≥70% and <100% identity with the amino acid sequence in a) or b) and can recognize an anti-GALNTL6 autoantibody. In one embodiment, the amino acid sequence set forth in b) of the present invention that can recognize an anti-GALNTL6 autoantibody is the amino acid sequence set forth in SEQ ID NO: 3, which is a sequence lacking amino acids 201-210 of the GALNTL6 gene sequence. As an optional embodiment, the amino acid sequence in c) of the present invention has an identity of ≥80% and <100% with the amino acid sequence of the GALNTL6 protein; as an optional embodiment, the amino acid sequence in c) of the present invention has an identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 94%, 95%, 96%, 97%, 98% or 99% with the amino acid sequence of the GALNTL6 protein.

[0046] SEQ ID NO:1: MKRKQKRFLQMTLLFTVALIFLPNVGLWSLYKDKHLVK SAEPGEQQTFPLGLGDGQFYSWTDGLRRKDWHDYESIQKEAMRSGKGEHGKPYPLTEEDHDDSAYRENGFNIFVSNNIALERSLPDIRHANCKHKMYLERLPNTSIIIPFHNEGWTSLLRTIHSIINRTPGSLIAEIILVDDFSEREHLKDKLEEYMARFSKVRIVRTKKREGLIRTRLLGASMARGEVLTFLDSHCEVNVNWLPPLLNQIALNHKTIVCPMIDVIDHNHFGYEAQAGDAMRGAFDWEMYYKRIPIPPELQRADPSDPFESPVMAGGLFAVDRKWFWELGGYDPGLEIWGGEQYEISFKVWMCGGEMFDVPCSRVGHIYRKYVPYKVPSGTSLARNLKRVAETWMDEFAEYIYQRRPEYRHLSTGDISAQKELRKQLKCKDFKWFMAAVAWDVPKYYPPVEPPPAAWGEIRNVAANLCVDSKHGATGTELRLDICVKDGSERTWSHEQLFTFGWREDIRPGEPLHTRKFCFDAISHNSPVTLYDCHGMKGNQLWGYRKDRTLFHPVSNSCMDCNPAEKKIFMARCDPLSETQQWIFEHINMTVLEKFNHHANS。

[0047] As an implementation manner, the present invention also provides the nucleotide sequence of the GALNTL6 protein, as shown in SEQ ID NO:2.

[0048]

[0049] SEQ ID NO:3: MKRKQKRFLQMTLLFTVALIFLPNVGLWSLYKDKHLVK.

[0050] In one embodiment, the GALNTL6 protein described herein is expressed by transferring a recombinant vector containing the GALNTL6 gene into a corresponding expression system. The type of expression vector is selected based on the expression system, which can be a prokaryotic expression system, a yeast expression system, a baculovirus expression system, or a mammalian cell expression system. In one embodiment, the GALNTL6 protein can be obtained by purification using affinity chromatography, molecular sieve chromatography, ion exchange chromatography, or hydrophobic chromatography.

[0051] In one embodiment, the cells include neuronal cells, HEK293 cells, Hela cells or CHO cells.

[0052] As an embodiment, the vector includes a pTriEx series, a pCDNA3 series, a pET series or a pBac series vector.

[0053] In one embodiment, the tissue comprises mammalian brain tissue; in one embodiment, the mammalian brain tissue comprises human, rat, primate, rat, goat, horse, sheep, or cow brain tissue.

[0054] In one embodiment, the autoimmune disease of the nervous system includes a paraneoplastic syndrome. In one embodiment, the symptoms of the paraneoplastic syndrome include one or more of sleep disorders, dizziness, unsteady gait, limb weakness, ataxia, nystagmus, slow reaction, cognitive impairment, coughing when drinking water, dysarthria, dysphagia, mental abnormalities, epilepsy, confusion, slurred speech, dementia, and tumors.

[0055] The present invention identifies, for the first time, anti-GALNTL6 autoantibodies as markers for detecting and / or diagnosing autoimmune diseases of the nervous system. By detecting anti-GALNTL6 autoantibodies bound to the GALNTL6 protein in a sample, the present invention can diagnose autoimmune diseases of the nervous system, and can particularly aid in the diagnosis and treatment of autoimmune diseases of the nervous system. These anti-GALNTL6 autoantibodies bound to the GALNTL6 protein can be detected in samples from patients with symptoms of autoimmune diseases of the nervous system, but not in samples from healthy individuals.

[0056] The present invention also provides a kit for detecting and / or diagnosing autoimmune diseases of the nervous system, characterized in that it comprises a reagent for detecting anti-GALNTL6 autoantibodies and a labeled antibody.

[0057] In one embodiment, the reagent of the present invention comprises a polypeptide and / or a biological material expressing the polypeptide; the biological material comprises one or more of cells, vectors, and tissues; and the polypeptide is an immunogenic polypeptide that binds to an anti-GALNTL6 autoantibody. In one embodiment, the reagent of the present invention is as described above and will not be further described here. The types of kits of the present invention are also as described above and will not be further described here.

[0058] In one embodiment, the polypeptide comprises a GALNTL6 protein. In one embodiment, the amino acid sequence of the GALNTL6 protein comprises any one of a) to c): a) the amino acid sequence set forth in SEQ ID NO: 1; b) an amino acid sequence in SEQ ID NO: 1 that, after modification or mutation, can recognize an anti-GALNTL6 autoantibody; c) an amino acid sequence that has ≥70% and <100% identity with the amino acid sequence in a) or b) and can recognize an anti-GALNTL6 autoantibody.

[0059] The kit of the present invention can be used for the detection of anti-GALNTL6 autoantibodies. By detecting anti-GALNTL6 autoantibodies, a basis can be provided for the diagnosis and treatment of autoimmune diseases of the nervous system. It can also be used to distinguish autoimmune diseases, especially autoimmune diseases of the nervous system from non-autoimmune diseases. In addition, the patient's disease severity and treatment effect can be monitored by detecting changes in the titer of the autoantibodies.

[0060] The present invention also provides a method for detecting anti-GALNTL6 autoantibodies, which is performed by using the kit as described above.

[0061] As an embodiment, the method for detecting anti-GALNTL6 autoantibodies described in the present invention is not particularly limited. The detection methods that can be used include enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay, immunoblotting, immunoprecipitation, immunomagnetic bead assay, protein microarray, or luminescence detection. As an embodiment, the indirect immunofluorescence assay includes TBA and CBA. As an embodiment, in the method for detecting anti-GALNTL6 autoantibodies, the device used to observe the results can be a fluorescence microscope, a microplate reader, a chemiluminescence analyzer, a flow cytometer, or a slide viewer.

[0062] In one embodiment, the solid phase carrier used in the method for detecting anti-GALNTL6 autoantibodies can be a polyethylene plate, a membrane (nylon membrane, nitrocellulose membrane, PVDF membrane), a glass slide, magnetic beads, a chromatography filler, a microfluidic channel, a polyacrylamide gel, etc. In one embodiment, the labeled antibody used in the method for detecting anti-GALNTL6 autoantibodies can be a horseradish peroxidase-labeled antibody, an alkaline phosphatase-labeled antibody, a biotin-labeled antibody, a FITC-labeled antibody, an AlexaFluor dye, etc.

[0063] In one embodiment, the sample used in the method for detecting anti-GALNTL6 autoantibodies includes an in vitro fluid sample and / or an in vitro tissue sample containing the antibody. In one embodiment, the in vitro fluid sample includes cerebrospinal fluid, blood, lymph, or interstitial fluid. In one embodiment, the in vitro tissue sample includes neural tissue.

[0064] The invention also discloses the process of discovering antibodies, determining antigens and verifying signals.

[0065] The process of discovering antibodies described in the present invention is to use the serum of patients with paraneoplastic neurological syndrome who are negative for 14 paraneoplastic neurological syndrome antibodies donated by the hospital, screen the genes expressed in human brain tissue, detect the reactivity of the patient serum with recombinant cells overexpressing the genes, and screen the positive patient serum; compare the signals of the patient's positive serum and healthy human serum on the recombinant cells overexpressing the genes, and find that the signal exists in the patient's serum but not in the healthy human serum; then conduct co-localization and neutralization experiments on the antibody signals of the corresponding proteins to verify the positive signal; the 14 paraneoplastic neurological syndrome antibodies are 14 paraneoplastic neurological syndrome-specific antibodies, including anti-Hu, Yo, Ri, Ma1, Ma2, CV2, Amphiphysin, Tr, Zic4, PKCγ, Recoverin, Titin, SOX1, and GAD65 antibodies.

[0066] The antigen determination process described herein is to compare the signals of patient serum and healthy human serum on primary cells. The presence of a positive signal in the patient's serum but not in the healthy human serum indicates the presence of antibodies in the patient's serum that are not present in the healthy human serum, and that these antibodies can recognize antigens localized in neuronal cells. The primary cells include primary cells from mammalian brain tissue, such as monkey brain and mouse brain, preferably rat brain.

[0067] The process of verifying the signal of the present invention includes verifying the signal of the patient serum on primary cells, brain tissue, and overexpressing cells. The primary cells include primary cells of mammalian brain tissue such as monkey brain and mouse brain. As an optional embodiment, the tissue includes mammalian brain tissue such as human, rat, primate, rat, goat, horse, sheep or cattle. As an optional embodiment, the overexpressing cells include HEK293, Hela, CHO cells that overexpress GALNTL6 protein. As an optional embodiment, the method of verifying the signal includes TBA, CBA, WB, etc.

[0068] The patient sera in the examples of the present invention were all donated by the hospital and have been approved by the patients themselves. The serum of healthy people was donated by the hospital physical examination center and has been approved by the patients themselves. The serum of 5 patients with confirmed paraneoplastic syndrome of the nervous system who were negative for 14 antibodies of PNS was included as biological samples for serological investigation, and the discovery process of anti-GANTL6 autoantibodies was described in detail. The inclusion conditions are as follows: (1) Cases with clinical symptoms or auxiliary examinations consistent with paraneoplastic syndrome of the nervous system; (2) Using cell-based immunofluorescence assay (CBA) to detect 14 antibodies of paraneoplastic syndrome of the nervous system (Hu, Yo, Ri, Ma2, Ma1, CV2, Amphiphysin, Tr, Zic4, PKCγ, Recoverin, Titin, SOX1, GAD65), the results were negative; (3) Using tissue-based immunofluorescence assay (TBA) to detect immune reaction.

[0069] The information of the five patients with symptoms of autoimmune diseases of the nervous system who underwent serological investigation in the present invention is as follows:

[0070] Patient 1: Male, 69 years old; in the past 3 months, he suffered from memory loss, epileptic seizures, abnormal behavior, mood swings, anxiety, and weight loss of 5kg; brain MRI showed abnormal signals in the medial temporal lobes of both sides; small cell lung cancer; TBA showed signals in the molecular layer of the cerebellum and in the hippocampus; diagnosed with paraneoplastic syndrome of the nervous system.

[0071] Patient 2: Male, 52 years old; unstable gait, with the onset of walking deviation without obvious cause 4 weeks ago, requiring the support of a wall when walking, which gradually worsened to the point where he could not stand independently, with dizziness, nausea, and slurred speech; brain MRI showed no abnormalities; TBA showed signals in the molecular layer of the cerebellum and in the hippocampus; diagnosed with paraneoplastic syndrome of the nervous system.

[0072] Patient 3: Female, 40 years old; numbness of both lower limbs, instability when holding objects, swaying from side to side when standing, accompanied by diplopia, vertigo, ataxia, and significantly elevated tumor marker CA125; ovarian cancer; TBA showed signals in the cerebellar molecular layer and hippocampus; diagnosed with paraneoplastic syndrome of the nervous system.

[0073] Patient 4: Male, 49 years old; confusion, slurred speech, convulsive epileptic seizures in the limbs, lower limb weakness, mild cerebrospinal fluid leukocytosis; right testicular seminoma; TBA showed signals in the molecular layer of the cerebellum and in the hippocampus; diagnosed with paraneoplastic syndrome of the nervous system.

[0074] Patient 5: Female, 42 years old; coughing when drinking water, hoarseness, limb muscle twitching, hallucinations, slurred speech, examination revealed a nodule in the right upper lobe of the lung; lung adenocarcinoma; TBA showed signals in the molecular layer of the cerebellum and in the hippocampus; diagnosed with paraneoplastic syndrome of the nervous system.

[0075] To exclude the possibility that the five patients mentioned above had other autoimmune diseases of the nervous system, antibodies related to other nervous system immune diseases were detected, including autoimmune encephalitis-related autoantibodies (NMDAR, AMPA1, AMPA2, LGI1, CASPR2, GABABR) and central nervous system demyelination-related autoantibodies (AQP4, MOG, MBP, GFAP, AQP1, Flotillin1 / 2), all of which were negative.

[0076] To further illustrate the present invention, the following detailed description of the use of the reagents provided herein for detecting anti-GALNTL6 autoantibodies in the diagnosis of autoimmune diseases of the nervous system is provided in conjunction with the accompanying drawings and examples. However, these descriptions are not to be construed as limiting the scope of the present invention. Unless otherwise specified, the reagents, instruments, and methods used in the present invention are all conventionally selected in the art.

[0077] Example 1 Screening of fluorescence signals of patient serum on primary neuronal cells

[0078] 1. Isolation of primary rat neurons

[0079] Rats were anesthetized with 10% chloral hydrate at a standard concentration of 3 mL / kg body weight. After anesthesia, the rats were immersed in 75% alcohol and disinfected for 3 minutes. The rat brain tissue was then removed from a biosafety cabinet and placed in pre-chilled DMEM containing 1% BSA. The brain tissue blocks were minced using ophthalmic scissors. 10 mL of papain was added, and the mixture was digested at 37°C for 30 minutes. After digestion, the cells were transferred to a new centrifuge tube and resuspended in 10 mL of DMEM containing 1% BSA. The cells were then centrifuged at 400g and 4°C for 5 minutes, and the supernatant discarded. The cells were again resuspended in 5 mL of DMEM containing 1% BSA and centrifuged at 200g and 4°C for 5 minutes. The supernatant was again discarded. The cells were then resuspended in 20 mL of DMEM containing 10% fetal bovine serum and plated at an appropriate density on a cell culture dish containing a slide to obtain primary neuronal cell slides from rat brain tissue.

[0080] 2. Fixation of cell slides

[0081] Rat brain tissue cell slides were fixed with 4% paraformaldehyde (FPA) for 10 minutes, then washed twice with PBS solution, terminated with 1.25M glycine for 10 minutes, and then washed twice with PBS to obtain rat primary neuronal cell slides for use.

[0082] 3. Serum incubation

[0083] The sera of patients 1 to 5 and 10 healthy subjects were diluted with PBST at a ratio of 1:10 and incubated in rat primary neuronal cell slides respectively. The cells were incubated at room temperature for 1 hour and then washed three times with PBST for 5 minutes each. FITC-labeled goat anti-human IgG secondary antibody (manufacturer: Jackson) was then used at a dilution of 1:200 and incubated at room temperature for 30 minutes. The cells were then washed three times with PBST for 5 minutes each to obtain serum-incubated cell slides.

[0084] The serum-incubated cell slides were observed under a fluorescence microscope and photographed. Figure 1 As shown, the sera of patients 1 to 5 showed positive signals on primary neuronal cells, while the sera of healthy people did not show positive signals, proving that there were antibodies in the sera of patients 1 to 5 that bound to primary neuronal cells.

[0085] 4. Antibody co-staining

[0086] Use PBST to dilute the neuronal cell-specific skeleton marker marker β-TUBB3 antibody at a ratio of 1:1000 (manufacturer: Wuhan Sanying; catalog number: 66375-1-Ig) and incubate the cell slides incubated with the serum of patients 1 to 5 in step 3 for antibody co-staining. Incubate overnight at 4°C and wash three times with PBST for 5 minutes each. Use a 1:200 dilution of AlexaFluor 594-labeled goat anti-mouse IgG secondary antibody to incubate the above neuronal cell slides, incubate at room temperature for 30 minutes, and wash three times with PBST for 5 minutes each to obtain antibody co-stained cell slides.

[0087] The antibody-costained cell slides were observed and photographed under a fluorescence microscope. Figure 2 As shown, the signals stained by the sera of patients 1 to 5 overlap with the signals of the β-TUBB3 antibody on primary neuronal cells, indicating that the antigens recognized by the antibodies in the sera of patients 1 to 5 exist on neuronal cells.

[0088] Example 2 Serum expanded antibody spectrum screening

[0089] 1. Target screening of known nervous system-related autoantibodies

[0090] Previously, patient sera were screened for more reported autoantibodies related to the nervous system. The target antigens of reported autoantibodies related to the nervous system were identified, cell slides overexpressing the target antigens were prepared, and multiple cell slides overexpressing the target antigens were assembled into bioassay chip materials. Immunofluorescence staining of patient samples was performed. The specific operation method is as follows:

[0091] 60 reported target antigens of nervous system autoantibodies were found, including DPPX, IgLON5, GlyR, GABAARα1, GABAARγ2, GABAARβ3, mGluR5, D2R, Neurexin-3α, GAD67, KCNA4 (Kv1.4), KLHL11, AK5, TRIB2, GLuR3, Gephyrin, CaVα2δ (CACNA2D1), TGM2, TGM6, MUNC18-1, mGLuR1, GABAARAP, Drebrin, AGO, NAE, PDE10A, ADAM22, ROC K2, mGluR3, mGluR4, CACNB1, VAMP2, CRMP2, CACNA1A, Homer3, ATP1A3, ARHGAP26, ITPR1, septin-5, NCDN, GRID2, AP3B2, mGluR2, GRIK2(Gl uR6 / GluK2), Rab6A, Rab6B, CA8(CARPVIII), PDE10A, PLP1, NF155, NF186, CNTN1, CNTN2, CASPR1, Gliomedin, Agrin, AChR, LRP4, MuSK and MAG.

[0092] The gene sequences encoding the above 60 proteins were searched on NCBI and sent to a sequencing company for gene synthesis. Recombinant vectors encoding the corresponding genes were obtained. The recombinant vectors were then transfected into 293T cells to obtain recombinant cells. Bioassay chip materials that overexpressed the target antigens were prepared. Immunofluorescence was used to detect whether the patient's serum could produce an immune reaction after incubation with the bioassay chip to explore whether the patient's serum contained specific autoantibodies to the series of genes. The specific steps are as follows:

[0093] (1) Construction of recombinant vectors: The gene sequences encoding the above 60 proteins were ligated to the pCDNA3.1 vector by molecular cloning or synthetic methods to obtain 60 recombinant vectors. The constructed recombinant vectors were sequenced and then collected for future use.

[0094] (2) Target gene transfection: 61 293T cells were cultured in a 10% FBS-DMEM high-glucose medium at 37°C in a cell culture incubator with 5% CO2 and 6 cm × 6 cm slides on the bottom of the culture dishes. When the cell density reached 30% to 40%, PEI transfection reagent (manufacturer: thermo, catalog number: BMS1003) was used to transfect the 60 recombinant vectors of the corresponding genes and the empty pCDNA3.1 into the 293T cells and label them.

[0095] (3) Fixation of cell slides: Cell slides 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 61 cell slides of 2.5 mm × 2.5 mm pieces were attached to a glass slide to prepare a bioassay chip for screening target antigens.

[0096] (4) Immunofluorescence staining: The sera of patients 1 to 5 and 3 healthy subjects were diluted 1:10 with PBST and incubated on the biodetection chip. The cells were incubated at room temperature for 1 hour and washed 3 times with PBST, each time for 5 minutes. FITC-labeled goat anti-human IgG secondary antibody was diluted 1:200 and incubated at room temperature for 30 minutes. The cells were washed 3 times with PBST, each time for 5 minutes, to obtain the biodetection chip incubated with serum.

[0097] The bioassay chips incubated with serum were observed under a fluorescence microscope, and it was found that the sera of patients 1 to 5 and the 60 bioassay chips did not show a color reaction with a signal significantly stronger than that of healthy human serum. However, according to the primary neuronal cell staining results in Example 1, the sera of patients 1 to 5 had obvious staining results in primary neuronal cells. Therefore, the sera of patients 1 to 5 may contain new autoantibodies that can recognize neuronal cells, which are different from those previously reported.

[0098] Example 3 Immunofluorescence screening of autoantibodies in patient serum

[0099] The human protein atlas (https: / / www.proteinatlas.org / ) was used to search for 100 proteins with relatively high expression levels in human brain tissue. A bioassay chip was prepared according to the preparation method of Example 2. Immunofluorescence was used to test whether the patient's serum could produce an immune reaction after incubation with the bioassay chip. This was done to explore whether the patient's serum contained new autoantibodies that could recognize neuronal cells, different from those previously reported. The specific steps were as follows:

[0100] (1) Preparation of biological detection chips: Search the gene sequences encoding each protein from NCBI and send them to a sequencing company to synthesize the genes into pCDNA3.1. Transform the synthesized recombinant vector into the cloning bacteria TOP10 for amplification, and then extract the plasmid for use. The above recombinant vectors are transfected into 293T cells grown on 6cm×6cm slides using PEI transfection reagent. After 48 hours of transfection, the 6cm×6cm cell slides are washed, fixed and dried, and then cut into 2.5mm×2.5mm sizes for use. After these cell slides are prepared, they are pasted on glass slides to prepare biological detection chips for sample detection.

[0101] (2) Immunofluorescence staining: Patient serum and healthy human serum were diluted 1:10 with PBST and incubated on the bioassay chip prepared in the above step (1), incubated at room temperature for 1 hour, and washed 3 times with PBST, each time for 5 minutes; FITC-labeled goat anti-human IgG secondary antibody was diluted 1:200, incubated at room temperature for 30 minutes, and washed 3 times with PBST, each time for 5 minutes, to obtain the bioassay chip incubated with serum.

[0102] The bioassay chip incubated with serum was placed under a fluorescence microscope to observe the results and take pictures. Figure 3 As shown, it can be seen that the sera of patients 1 to 5 reacted with one antigen on the biological detection chip to produce obvious fluorescent signals, while the sera of healthy people did not produce any signals with all the antigens on the biological detection chip.

[0103] After verification, the target antigen was found to be GALNTL6 (polypeptide N-acetylgalactosaminyltransferase like 6, accession number: NM_001034845), the amino acid sequence of which is shown in SEQ ID NO: 1, and the nucleotide sequence encoding this protein is shown in SEQ ID NO: 2.

[0104] Example 4: Verification of the expression of target antigens by commercial antibodies in rat primary neuronal cells

[0105] 1. Live cell staining: 6 dishes of rat primary neuronal cells were prepared according to the method described in Example 1, the supernatant was discarded, and the cells were washed twice with PBS. A commercial anti-GALNTL6 autoantibody was diluted 1:100 with PBS and incubated with the prepared neuronal cells. The cells were incubated at room temperature for 1 hour and washed three times with PBS for 5 minutes each. The cells were incubated with a 1:200 dilution of AlexaFluor 594-labeled goat anti-rabbit IgG secondary antibody at room temperature for 30 minutes and washed three times with PBS for 5 minutes each. The nuclei of rat primary neuronal cells were stained with DAPI at room temperature for 10 minutes and washed three times with PBS for 5 minutes each to obtain neuronal cells stained with anti-GALNTL6 autoantibodies.

[0106] 2. β-TUBB3 antibody co-staining: Use 4% paraformaldehyde to fix the neuronal cells stained with anti-GALNTL6 autoantibodies at room temperature for 10 minutes, wash 3 times with PBST, each time for 5 minutes; use PBST to dilute β-TUBB3 antibody 1:100, incubate on the neuronal cell slides, incubate overnight at 4°C, wash 3 times with PBST, each time for 5 minutes; incubate 1:200 diluted FITC-labeled goat anti-mouse IgG secondary antibody, incubate on the above neuronal cell slides, incubate at room temperature for 30 minutes, wash 3 times with PBST, each time for 5 minutes to obtain β-TUBB3 antibody co-stained neuronal cell slides.

[0107] The neuronal cell slides co-stained with β-TUBB3 antibody were observed under a fluorescence microscope and photographed. Figure 4 As shown, it can be seen that the commercial anti-GALNTL6 autoantibody has a signal on primary cells, and the stained signal overlaps with the signal of the β-TUBB3 antibody on primary neuronal cells, indicating that the two antibodies are co-localized. Therefore, it is shown that this signal is located on the rat brain neuronal cells and the GALNTL6 protein is expressed on primary neuronal cells.

[0108] Example 5 WB verification of GALNTL6 expression on mouse brain tissue protein and monkey brain tissue protein

[0109] 1. Preparation of WB membrane

[0110] The brain tissue was removed according to the operation as shown in Example 1, and the rat brain tissue was minced. The minced rat brain tissue was transferred to a mortar and added with liquid nitrogen for freezing and grinding until it was fine powder. 500 μl of lysis buffer (150 mM NaCl, 1 mM EDTA, 100 mM Tris-HCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, pH 7.5) was added, and a protease inhibitor was added at a final concentration of 1×. The cells were lysed for 30 minutes, shaken intermittently, and centrifuged at 15,000 rpm for 30 minutes. The supernatant was used as a Western Blot sample for SDS-PAGE electrophoresis. After electrophoresis, the membrane was transferred using a wet method (transfer conditions: 200 mA, 80 minutes); and 5% skim milk powder was blocked at room temperature for 1 hour to obtain rat brain tissue protein.

[0111] Mouse brain tissue protein and monkey brain tissue protein were prepared according to the above method and set aside.

[0112] 2. Preparation and validation of neutralizing protein

[0113] The pCDNA3.1-GALNTL6 plasmid was transfected into 293T cells using the method described in Example 2. One dish (10 cm dish) of 293T3 cells overexpressing GALNTL6 was collected, the supernatant 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 (disruption conditions: 10% power, disruption for 3 seconds, pause for 6 seconds, and total ultrasonication for 1 minute) to serve as the GALNTL6 neutralizing protein.

[0114] According to the above method, cells transfected with empty pCDNA3.1 plasmid were used to prepare control neutralizing protein for later use.

[0115] (1) WB verification of neutralizing protein

[0116] GALNTL6 neutralizing protein and empty pCDNA3.1 control protein were subjected to SDS-PAGE gel electrophoresis experiments, with the sample amount of 40 μg. After the electrophoresis, the membrane was transferred using the wet transfer condition of 300 mA for 90 minutes; 5% skim milk powder was blocked at room temperature for 1 hour; anti-GALNTL6 autoantibody was diluted 1:1000 in TBST and incubated at 4°C overnight; the next day, TBST was used to wash three times, each time for 5 minutes; HRP-labeled goat anti-rabbit secondary antibody (manufacturer: Jackson) was added and incubated at room temperature for 1 hour; TBST was used to wash three times, each time for 5 minutes; chemiluminescence solution was added to develop the color and take pictures. The results are shown as follows Figure 5 shown.

[0117] Depend on Figure 5It can be seen that the anti-GALNTL6 autoantibody has a specific signal on the immunoblot of the overexpressed GALNTL6 protein, but no signal with the control protein, indicating that the neutralizing protein was successfully prepared.

[0118] (2) Neutralization experiments of anti-GALNTL6 autoantibodies to verify the role of neutralizing proteins

[0119] Reference Example 2 Preparation method for preparing GALNTL6 overexpressing cell slides, using PBST to prepare a total of 3 portions of commercial anti-GALNTL6 autoantibodies diluted at 1:100, each 100 μL, 20 μL PBST, 20 μL GALNTL6 neutralizing protein and 20 μL control protein were added, and incubated at room temperature for 30 min. The prepared GALNTL6 overexpressing cell slides were incubated with them, incubated at room temperature for 1 h, and washed 3 times with PBST for 5 min each; incubated with 1:200 diluted AlexaFluor 594-labeled goat anti-rabbit IgG secondary antibody, incubated at room temperature for 30 min, and washed 3 times with PBST for 5 min each to obtain cell slides with GALNTL6 neutralizing protein and control protein.

[0120] The cell slides containing the GALNTL6 neutralizing protein and the control protein were placed under a fluorescence microscope for observation and photography. Figure 6 As shown in the figure, on the GALNTL6-overexpressing cell slides, the commercial anti-GALNTL6 autoantibody signal was blocked by the GALNTL6 neutralizing protein, but not by the control protein, indicating that the signal specifically recognizes the GALNTL6 antigen and the neutralizing protein was successfully prepared.

[0121] 3. Sample incubation

[0122] Prepare three commercial anti-GALNTL6 autoantibodies at a dilution of 1:1000 using TBST. 100 μL of each was added to 20 μL of PBST, 20 μL of GALNTL6 neutralizing protein, and 20 μL of control protein, respectively, and incubated at room temperature for 30 minutes. Let it stand at room temperature for 10 minutes to bind. Then, incubate the three antibodies onto the membrane prepared in step 1 above. After incubation at room temperature for 2 hours, wash three times with TBST for 5 minutes each. Then, add HRP-labeled secondary antibodies, incubate at room temperature for 1 hour, and wash three times with TBST for 5 minutes each. Add chemiluminescent solution for color development and take pictures. The results are shown in Figure 2. Figure 7 shown.

[0123] Depend on Figure 7It can be seen that anti-GALNTL6 autoantibodies have bands that react with rat brain tissue, mouse brain tissue and monkey brain tissue. GALNTL6 neutralizing protein significantly weakened the signals of anti-GALNTL6 autoantibodies appearing on the above three tissue proteins, that is, the positive signals were blocked by GALNTL6 neutralizing protein, while the control protein did not block the positive signals appearing on the above three proteins, further proving that antigens that react with anti-GALNTL6 autoantibodies exist in rat brain tissue, mouse brain tissue and monkey brain tissue.

[0124] Example 6: Verification of signals detected by patient serum using commercial antibodies on cell slides overexpressing GALNTL6

[0125] 1. Preparation of cell slides

[0126] Refer to the method described in Example 2 to prepare cell slides overexpressing GALNTL6 and empty pCDNA3.1 for use;

[0127] 2. Serum incubation

[0128] The sera of patients 1 to 5 were diluted at a ratio of 1:10 using PBST and then incubated on cell slides overexpressing GALNTL6. 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 secondary antibody diluted 1:200 was added and incubated at room temperature for 30 minutes. The cells were washed three times with PBST for 5 minutes each to obtain cell slides incubated with the sera of patients 1 to 5.

[0129] 3. Antibody co-staining

[0130] Commercial anti-GALNTL6 autoantibodies were diluted 1:100 in PBST and incubated in the cell slides incubated with the sera of patients 1 to 5. The cells were incubated at room temperature for 30 minutes, washed three 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 incubated at room temperature for 30 minutes. The cells were washed twice with PBST, each time for 5 minutes. The cell nuclei were stained with DAPI at room temperature for 10 minutes, and washed three times with PBST, each time for 5 minutes. The cells were observed and photographed under a fluorescence microscope. The results are shown in the figure below. Figure 8 shown.

[0131] Depend on Figure 8 It can be seen that the commercial antibody has a positive signal on the cell slides overexpressing GALNTL6, and the GALNTL6 protein is successfully overexpressed on 293T cells; the staining signals of patients 1 to 5 sera and commercial anti-GALNTL6 autoantibodies on the cell slides overexpressing GALNTL6 overlap, indicating that the antibodies in patients 1 to 5 sera specifically recognize the GALNTL6 protein on the cell slides overexpressing GALNTL6.

[0132] Example 7 Serum Neutralization Experiment Verification of Signals Detected in Patient Serum

[0133] 1. Serum neutralization experiments were performed on rat primary neuronal cells to verify the signals detected by patient serum

[0134] Reference Example 1 Preparation method Rat primary neuronal cell slides were prepared; 3 1:10 diluted patient 1 serums were prepared using PBST, 100 μL each, and 20 μL PBST, 20 μL GALNTL6 neutralizing protein and 20 μL control neutralizing protein were added respectively and incubated at room temperature for 30 min. According to the same method, 3 patient 2 serums, 3 patient 3 serums, 3 patient 4 serums, and 3 patient 5 serums were prepared and incubated with them on the prepared rat primary neuronal cell slides, incubated at room temperature for 1 h, and washed 3 times with PBST for 5 min each; 1:200 diluted FITC-labeled goat anti-human IgG secondary antibody was added, incubated at room temperature for 30 min, and washed 3 times with PBST for 5 min each; the above cell slides were observed under a fluorescence microscope and photographed. The results are as follows Figure 9 shown.

[0135] Depend on Figure 9 It can be seen that on the rat primary neuronal cell slides, the serum signals of patients 1 to 5 were blocked by the GALNTL6 neutralizing protein, while the control protein did not block the signals appearing on the rat primary neuronal cell slides, indicating that the signals of patients 1 to 5 sera appearing on the rat brain tissue sections are signals that specifically recognize the GALNTL6 antigen.

[0136] 2. Serum neutralization experiment verifies the signal detected by patient serum on cell slides overexpressing GALNTL6

[0137] Reference Example 2 Preparation method for preparing GALNTL6 overexpressing cell slides, using PBST to prepare a total of 3 1:10 diluted patient 1 serum, each 100 μL, were added 20 μL PBST, 20 μL GALNTL6 neutralizing protein and 20 μL control neutralizing protein, respectively, and incubated at room temperature for 30 min. According to the same method, 3 patient 2 serum, 3 patient 3 serum, 3 patient 4 serum, and 3 patient 5 serum were prepared, and the prepared GALNTL6 overexpressing cell slides were incubated therewith, incubated at room temperature for 1 h, washed 3 times with PBST, each for 5 min; 1:200 diluted FITC-labeled goat anti-human IgG secondary antibody was added, incubated at room temperature for 30 min, washed 3 times with PBST, each for 5 min, and the above cell slides were placed under a fluorescence microscope for observation and photography. The results are as follows Figure 10 shown.

[0138] Depend on Figure 10It can be seen that on the GALNTL6-overexpressing cell slides, the serum signals of patients 1 to 5 were blocked by the GALNTL6 neutralizing protein, while the control protein did not block the signals appearing on the GALNTL6-overexpressing cell slides, indicating that the signals appearing on the GALNTL6-overexpressing cell slides from the serum of patients 1 to 5 were signals that specifically recognized the GALNTL6 antigen.

[0139] Example 8: Verification of the clinical specificity and detection rate of anti-GALNTL6 autoantibodies by cell-based immunofluorescence assay

[0140] In order to verify the clinical specificity and detection rate of anti-GALNTL6 autoantibodies, a total of 377 serum samples were collected in the present invention. Cell slides overexpressing GALNTL6 and control cell slides with empty pCDNA3.1 were prepared according to Example 2. According to the method of step 3 of Example 6, cell slides overexpressing GALNTL6 were used for detection to screen serum samples positive for anti-GALNTL6 autoantibodies. A total of 10 anti-GALNTL6 autoantibody-positive samples were screened. The immunofluorescence detection results are shown in Table 1, and some staining results are shown in Table 1. Figure 11 shown.

[0141] Table 1 Immunofluorescence detection results

[0142]

[0143] According to Table 1 and Figure 11It can be seen that in 377 serum samples, 9 cases of anti-GALNTL6 autoantibodies were detected, with a detection rate of 2.4%. Among them, 3 cases of anti-GALNTL6 autoantibodies were detected in PNS patients diagnosed with 14 PNS antibodies, including 1 anti-Hu antibody and 2 anti-Yo antibody, and the remaining 52 cases were all anti-GALNTL6 autoantibody negative; in addition, 6 cases of anti-GALNTL6 autoantibody positive were detected in serum samples of PNS patients diagnosed with 14 PNS antibodies, and the remaining 43 cases were all anti-GALNTL6 autoantibody negative; 123 patients with other diseases (including autoimmune encephalitis, central nervous system demyelinating disease, myasthenia, Guillain-Barré syndrome, Alzheimer's, viral encephalitis, tuberculous meningitis, motor neuron disease, multiple sclerosis, craniosynostosis, myasthenia gravis ... No positive anti-GALNTL6 autoantibodies were detected in 150 samples of samples from 150 healthy subjects; therefore, anti-GALNTL6 autoantibodies can be detected in confirmed PNS patients with the symptoms described in the present invention, especially in PNS patients diagnosed with negative results in the 14 PNS antibodies, and are not detected in other patients and healthy subjects, indicating that this autoantibody has an auxiliary role in the diagnosis of paraneoplastic syndromes of the nervous system. In particular, for PNS patients diagnosed with negative results in the 14 PNS antibodies, anti-GALNTL6 antibodies can be used as a biomarker for the diagnosis of PNS. Therefore, the present invention provides a new antigen that binds to autoantibodies for the diagnosis of PNS. Anti-GALNTL6 autoantibodies can be detected in patients with neurological symptoms, indicating that this autoantibody has an auxiliary role in the diagnosis of autoimmune diseases of the nervous system.

[0144] Example 9 GALNTL6 mutant detection of anti-GALNTL6 autoantibodies in patient serum

[0145] In this example, a mutant of the human GALNTL6 gene was selected, i.e., a mutant lacking the gene encoding amino acids 201-210 of the GALNTL6 gene sequence. Vector construction, preparation of cell slides overexpressing the deleted GALNTL6 gene, and testing of patient serum were performed according to the steps of Example 1. The results showed that the lack of the gene encoding amino acids 201-210 did not affect the detection of antibodies in patient serum.

[0146] The amino acid sequence of the mutant of the gene lacking the 201st to 210th amino acids encoding the GALNTL6 gene sequence is shown in SEQ ID NO: 3.

[0147] In summary, the present invention provides a new antigen to be detected that binds to autoantibodies for the diagnosis of autoimmune diseases of the nervous system, especially autoimmune encephalitis; anti-GALNTL6 autoantibodies can be detected in patients with autoimmune diseases of the nervous system with neurological symptoms, indicating that the antibody has an auxiliary effect on the diagnosis of autoimmune diseases of the nervous system, especially autoimmune encephalitis.

[0148] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments like 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-GALNTL6 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 reagent includes a polypeptide and / or a biological material expressing the polypeptide; the biological material includes one or more of a cell, a carrier and a tissue; The polypeptide is an immunogenic polypeptide that binds to anti-GALNTL6 autoantibodies.

3. The use according to claim 2, characterized in that The polypeptide includes the GALNTL6 protein.

4. The use according to claim 3, characterized in that The amino acid sequence of the GALNTL6 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-GALNTL6 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-GALNTL6 autoantibody.

5. The use according to claim 4, characterized in that The amino acid sequence capable of recognizing anti-GALNTL6 autoantibodies in b) includes the amino acid sequence shown in SEQ ID NO:

3.

6. The use according to claim 2, characterized in that The cells include neuronal cells, HEK293 cells, Hela cells or CHO cells; The vector includes pTriEx series vectors, pCDNA3 series vectors, pET series vectors or pBac series vectors; The tissue includes mammalian brain tissue.

7. The use according to claim 1, wherein The autoimmune diseases of the nervous system include paraneoplastic syndromes.

8. A kit for detecting and / or diagnosing autoimmune diseases of the nervous system, characterized in that: Includes reagents and labeled antibodies for detecting anti-GALNTL6 autoantibodies.

9. The kit according to claim 8, wherein The reagent for detecting anti-GALNTL6 autoantibodies includes a polypeptide and / or a biological material expressing the polypeptide; the biological material includes one or more of cells, vectors, and tissues; The polypeptide is an immunogenic polypeptide that binds to anti-GALNTL6 autoantibodies.

10. The kit according to claim 9, wherein The polypeptide includes a GALNTL6 protein, and the amino acid sequence of the GALNTL6 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-GALNTL6 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-GALNTL6 autoantibody.

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