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

Abstract

The invention discloses application of a reagent for detecting an anti-GLUL autoantibody in diagnosis of nervous system autoimmune diseases, and belongs to the technical field of preparation of protein diagnostic reagents. By comparing the serum of a patient with the symptoms of the nervous system autoimmune disease with the serum of a healthy person, the following results are obtained: compared with the serum of the healthy person, the anti-GLUL autoantibody exists in the serum of the patient with the symptoms of the nervous system autoimmune disease, and a large number of experiments verify that the anti-GLUL autoantibody exists in the serum of the patient with the symptoms of the nervous system autoimmune disease. It is clear that the anti-GLUL autoantibody can be used as a marker for diagnosing the autoimmune diseases of the nervous system, and especially can be used as an anti-nerve cell antibody for auxiliary diagnosis of the autoimmune diseases of the nervous system. By detecting the anti-GLUL autoantibody, diagnosis of autoimmune diseases of a nervous system can be realized, and especially auxiliary diagnosis of autoimmune encephalitis can be realized.

Description

Technical Field

[0001] This invention belongs to the field of protein diagnostic reagent preparation technology, specifically relating to the application of a reagent for detecting anti-GLUL autoantibodies in the diagnosis of autoimmune diseases of the nervous system. Background Technology

[0002] Neurological autoimmune diseases constitute a significant class of conditions in neurology, affecting the central nervous system, peripheral nervous system, and neuromuscular junctions. Their pathological mechanisms primarily involve autoimmune factors and antibodies attacking the nervous system. Neurological autoimmune diseases are relatively rare, exhibiting the complexity of immune disorders and the high lethality and disabling potential of neurological diseases, thus attracting considerable attention from clinicians and researchers. While progress has been made in identifying autoantibody-mediated immune mechanisms in a large proportion of patients with neurological disorders, specific "neurological" antibodies remain undetectable in some patient samples, and a number of cases with unknown etiologies remain undiagnosed even after extensive evaluation of infectious causes. A better understanding of the immune mechanisms in seronegative patients could open new treatment options for affected individuals. Therefore, the identification of neoantigens that bind to autoantibodies is necessary to improve diagnostic and therapeutic outcomes.

[0003] Glutamate-ammonia ligase (GLUL) is a key neurometabolistic enzyme mainly distributed in astrocytes of the central nervous system. Its core function is to catalyze the combination of glutamate and ammonia to produce glutamine. GLUL plays an irreplaceable role in maintaining glutamate homeostasis in the central nervous system, clearing neurotoxic ammonia, regulating neurotransmitter metabolism, and protecting nerve cells from excitotoxic damage. Recent studies have found that abnormal expression or dysfunction of GLUL is closely related to various neurological diseases, such as brain injury, stroke, and Alzheimer's disease.

[0004] Autoantibodies are antibodies produced by the body against its own tissues or cellular components, playing a crucial role in the development and progression of various diseases, especially autoimmune diseases, tumors, and neurodegenerative diseases. Accurate detection and in-depth research of autoantibodies are irreplaceable for early disease diagnosis, disease monitoring, treatment planning, and elucidation of pathogenesis. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide the application of reagents for detecting anti-GLUL autoantibodies in the diagnosis of neurological autoimmune diseases, to specifically diagnose neurological autoimmune diseases, to improve the accuracy of the diagnosis of neurological autoimmune diseases, and especially to realize the auxiliary diagnosis of neurological autoimmune diseases.

[0006] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses the application of reagents for detecting anti-GLUL autoantibodies in the preparation of products for diagnosing autoimmune diseases of the nervous system.

[0007] Preferably, in this invention, the reagent for detecting anti-GLUL autoantibodies preferably includes one or more of GLUL protein, cells expressing GLUL protein, vectors expressing GLUL protein, and tissues containing GLUL protein, and more preferably GLUL protein.

[0008] More preferably, the GLUL protein of the present invention refers to an immunogenic polypeptide capable of binding to GLUL autoantibodies. The polypeptide is preferably a polymer of ≥2 amino acids, more preferably a polymer of 2-70 amino acids, and even more preferably a polymer of 2, 3, 4, 5, 6, 7, 8, 10, 12, 20, 30, 40, 50, 60, or 70 amino acids. The polypeptide of the present invention preferably contains one or more epitopes derived from the GLUL protein.

[0009] More preferably, the polypeptide of the present invention further includes a fusion protein incorporating GLUL and other amino acids, wherein the amino acids are preferably linked to the N-terminus or C-terminus and have the function of promoting the purification, immobilization, precipitation or identification of the polypeptide or protein. The amino acids can constitute tags known in the art, such as His tags, thioredoxins, maltose-binding proteins, glutathione S-transferases, flag tags, myc tags or strep tags.

[0010] In this invention, there is no particular limitation on the source of the polypeptide; recombinant polypeptides and / or purified polypeptides may be used. The GLUL protein described in this invention is preferably an immobilized protein, and more preferably immobilized on a solid support.

[0011] Preferably, the amino acid sequence of the GLUL protein includes any one of a) to c): a) The amino acid sequence shown in SEQ ID NO.1; b) The amino acid sequence shown in SEQ ID NO.1, after modification or mutation, can recognize the amino acid sequence of anti-GLUL autoantibody; c) has an amino acid sequence identity of ≥70% and <100% with a) or b), and is capable of recognizing the amino acid sequence of anti-GLUL autoantibody.

[0012] More preferably, in b), the amino acid sequence shown in SEQ ID NO.1, after being modified or mutated, can recognize the amino acid sequence of the anti-GLUL autoantibody as shown in SEQ ID NO.3.

[0013] More preferably, in this invention, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1 preferably includes any one of I) to III): I) the nucleotide sequence shown in SEQ ID NO.2; II) a nucleotide sequence that, after mutation of the nucleotide sequence shown in SEQ ID NO.2, can encode an amino acid sequence that recognizes anti-GLUL autoantibody; III) a nucleotide sequence that has ≥70% and <100% identity with the nucleotide sequence in I) or II) and can encode an amino acid sequence that recognizes anti-GLUL autoantibody.

[0014] The sequence represented by SEQ ID NO.1 is as follows: MTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPEWNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRACLYAG VKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNWNGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIRTCLLNETGDEPFQYKN.

[0015] The sequence shown in SEQ ID NO.2 is as follows:

[0016] The sequence shown in SEQ ID NO.3 is as follows: MTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPEWNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLPQGPYYCGVGADRAYGRDIVEAHYRACLYAGVKIAGTNAEVMPAQWEFQIGP CEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNWNGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIRTCLLNETGDEPFQYKN.

[0017] Preferably, in this invention, the cells preferably include bacterial cells or eukaryotic cells; the bacterial cells preferably include Escherichia coli cells; the eukaryotic cells preferably include immortalized human cells, insect cells, or yeast. In specific implementations of this invention, selectable cells may include HEK293 cells, HeLa cells, CHO, Pichia pastoris, Saccharomyces cerevisiae, sf9, BL21, or Rosetta, etc.

[0018] Preferably, the vectors described in this invention include vectors from the pTriEx vector family, pcDNA3 family, pET series, or pBac series. The tissues described in this invention are preferably brain tissue, and more preferably brain tissue from humans, rats, primates, mice, goats, horses, sheep, or cattle.

[0019] This invention involves a serological survey of patients with symptoms of neurological autoimmune diseases. Rat brain tissue sections were incubated with patient serum and healthy human serum. Signal amplification using fluorescent secondary antibodies revealed the presence of autoantibodies in patient serum compared to healthy human serum. A biodetector chip was used to screen for autoantibodies in patient serum that recognize the GLUL antigen. The authenticity of the target antigen was verified by co-staining commercially available antibodies with patient serum on neurons, astrocytes, and GLUL-overexpressing cells, as well as by serum neutralization experiments. Further collection of serum from patients with or without neurological diseases and healthy individuals was used as control samples for screening using CBA to verify the specificity of the target antigen.

[0020] In this invention, the symptoms of the neurological autoimmune disease preferably include one or more of the following: mental and behavioral abnormalities, cognitive impairment, recent memory loss, epileptic seizures, speech disorders, motor disorders, involuntary movements, decreased level of consciousness and coma, and autonomic dysfunction. Preferably, the neurological autoimmune disease of this invention is autoimmune encephalitis; the patient with symptoms of the neurological autoimmune disease is preferably a patient to whom a physician highly suspects autoimmune encephalitis, and whose serum sample tests for autoimmune encephalitis-related autoantibodies (NMDAR, AMPA1, AMPA2, LGI1, CASPR2, GABABR, DPPX, IgLON5, GlyRα1, GABAARα1, GABAARβ3, GABAARγ2) are all negative.

[0021] In this invention, the diagnosis is preferably an auxiliary diagnosis. Anti-GLUL autoantibodies are closely related to autoimmune diseases of the nervous system and can distinguish between autoimmune diseases of the nervous system and other autoimmune diseases, especially autoimmune encephalitis and other autoimmune diseases. In the field of autoimmune encephalitis detection, the diagnostic criteria for autoimmune encephalitis generally include four aspects: clinical manifestations, auxiliary examinations, confirmatory tests, and exclusion of other causes. When a patient's clinical manifestations and auxiliary examinations are consistent with autoimmune encephalitis, and other causes are excluded, a diagnosis of possible autoimmune encephalitis is made. When a patient's clinical manifestations and auxiliary examinations are consistent with autoimmune encephalitis, and other causes are excluded, and the detection of autoimmune encephalitis-related markers (anti-neuronal cell antibodies) is positive, a diagnosis of confirmed autoimmune encephalitis is made. This invention uses anti-GLUL autoantibodies as markers for the diagnosis of autoimmune encephalitis. Anti-GLUL autoantibodies are closely related to autoimmune diseases of the nervous system, especially autoimmune encephalitis, and can be used for the diagnosis of autoimmune encephalitis, thus belonging to the auxiliary diagnosis of autoimmune encephalitis. This invention uses anti-GLUL autoantibodies as biomarkers. By detecting anti-GLUL autoantibodies, doctors can combine the results with the patient's clinical manifestations, auxiliary examinations, and disease biomarker test results to determine whether the patient has the neurological autoimmune-related disease described in this invention or to rule out the possibility of having another neurological autoimmune disease. This helps doctors select a more promising treatment plan or drug for the patient.

[0022] The present invention also provides a kit for diagnosing autoimmune diseases of the nervous system, including reagents for detecting anti-GLUL autoantibodies.

[0023] The details of the reagent for detecting anti-GLUL autoantibodies described in this invention have been described above and will not be repeated here. As one embodiment, the kit described in this invention can be a kit based on enzyme-linked immunosorbent assay (ELISA) or a kit based on protein immunoblotting. Preferably, the kit described in this invention further includes one or more of a labeled antibody, a reaction buffer, and a sample diluent. This invention does not impose strict requirements on the specific composition of the labeled antibody, buffer, and sample diluent; conventional selections are acceptable.

[0024] This invention establishes a kit for diagnosing autoimmune diseases of the nervous system, using reagents for detecting anti-GLUL autoantibodies as the main component. This kit can qualitatively or quantitatively analyze anti-GLUL autoantibodies, thereby diagnosing autoimmune diseases of the nervous system, and is particularly useful as an auxiliary diagnostic tool. When using this kit for detection, there are no strict requirements on the detection method used; any method well-known to this invention can be used, such as CBA, TBA, ELISA, immunogold assay, Western blotting, immunospot assay, membrane strip assay, chemiluminescence, radioimmunoassay, liquid chromatography-array assay, lateral chromatography, or flow cytometry.

[0025] Compared with the prior art, the present invention has the following beneficial effects: This invention, by comparing the serum of patients with symptoms of neurological autoimmune diseases with that of healthy individuals, yielded the following results: Anti-GLUL autoantibodies were present in the serum of patients with symptoms of neurological autoimmune diseases, compared to healthy individuals. Extensive experimental verification confirmed that anti-GLUL autoantibodies can serve as a biomarker for diagnosing neurological autoimmune diseases, particularly as an auxiliary diagnostic marker for these diseases. Detection of anti-GLUL autoantibodies enables the diagnosis of neurological autoimmune diseases, especially as an auxiliary diagnostic tool for autoimmune encephalitis.

[0026] This invention also provides a kit for diagnosing autoimmune diseases of the nervous system, including reagents for detecting anti-GLUL autoantibodies. This invention is the first to establish a kit for diagnosing autoimmune diseases of the nervous system using reagents for detecting anti-GLUL autoantibodies as the main component. Using this kit, anti-GLUL autoantibodies can be qualitatively or quantitatively analyzed; the operation is simple, enabling the diagnosis of autoimmune diseases of the nervous system, especially as an auxiliary diagnostic tool for autoimmune encephalitis. Attached Figure Description

[0027] Figure 1 The image shows the CBA results of five patients (H36, H64, H151, H211, H227) that produced positive signals when reacting with the same antigen GLUL on the biodetection chip. Figure 2The staining results of serum from 5 patients (H36, H64, H151, H211, H227) and healthy individuals on primary neurons are shown. Figure 3 The staining results show the co-localization of patient serum and neuronal marker MAP2 antibody on primary neurons. Figure 4 The staining results of serum from 5 patients (H36, H64, H151, H211, H227) and healthy individuals on primary astrocytes are shown. Figure 5 The staining results of co-localization of the GFAP antibody, a specific marker for astrocytes, in primary astrocytes; Figure 6 The staining results of GLUL antibody on rat primary astrocytes to verify the signal detected by patient serum; Figure 7 The staining results of GLUL antibody on GLUL-overexpressing cell slides to verify the signal detected in patient serum; Figure 8 Images of GLUL antibody and patient WB results; Figure 9 The images show the staining results of the serum neutralization assay on rat primary neuronal cell slides, rat primary astrocyte slides, GLUL-overexpressing cell slides, and rat brain tissue sections, validating the signals detected in the patient's serum. In the images, A represents patient serum + GLUL neutralizing protein; B represents patient serum + control neutralizing protein. Figure 10 The graph shows the results of the neutralization experiment of patient serum on WB. Figure 11 The staining results are shown in the serum of some patients who are positive for anti-GLUL antibodies, the serum of disease controls, and the serum of healthy individuals. Figure 12 This image shows the staining results of a portion of serum on a cell slide overexpressing the recombinant KCTD3 deletion mutant. Detailed Implementation

[0028] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0029] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0030] The technical terms involved in this invention are defined as follows: The term "diagnosis" described in this invention refers to the process of making a clear determination of a patient's health status and disease-related attributes. It covers situations including: determining whether an individual has a certain disease; identifying the specific type and subtype of the disease suffered by an individual who has already contracted the disease; determining the severity, stage, or grade of the disease; tracing the cause or trigger of the disease; differentiating multiple suspected diseases with overlapping symptoms; further confirming the disease after screening asymptomatic individuals for abnormalities; adjusting the original diagnosis according to changes in the condition during disease treatment or follow-up; or a combination of the above.

[0031] The term "assisted diagnosis" as described in this invention refers to the process of providing objective evidence, supplementary information, or decision-making reference for "diagnosis" through specific methods, technologies, or test results (including but not limited to laboratory testing reagents, imaging techniques, pathological staining techniques, molecular detection methods, etc.). It cannot independently complete a full diagnosis and needs to be combined with core clinical information such as patient medical history and physical examination. The scope includes: in the disease screening stage, initially identifying high-risk individuals who may have the disease; in the disease diagnosis stage, providing key evidence to support "whether the patient has the disease" and "what disease the patient has"; in the disease differential diagnosis stage, excluding or narrowing the range of suspected diseases; in the disease classification and staging stage, assisting in clarifying the specific attributes of the disease; in the disease follow-up or disease monitoring stage, assisting in assessing disease activity, treatment response, or recurrence; in the prognostic assessment stage, assisting in predicting disease outcome or recovery; or a combination of the above. The core objective of "assisted diagnosis" is to reduce diagnostic uncertainty, improve diagnostic accuracy, shorten the diagnostic cycle, and provide reliable support for diagnostic decisions.

[0032] The term "identity" percentage described in this invention refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when two sequences are optimally aligned. The amino acid sequence identity percentage alignment can be performed using various methods within the art, such as software well-known in the field, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA.

[0033] To further illustrate the present invention, the application of the anti-GLUL autoantibody provided by the present invention in the preparation of products for diagnosing autoimmune diseases of the nervous system is described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments: The patient serum described in this invention was donated by the hospital with the consent of the individual. The serum from healthy individuals came from the serum of healthy individuals undergoing physical examinations donated by the hospital's physical examination center with the consent of the individual undergoing the physical examinations.

[0035] Example 1: Confirmation and Identification of Novel Autoantibodies Step 1: Sample Collection Collected data from January 1, 2019 to December 31, 2022, for 12 tests related to autoimmune encephalitis (NMDAR, AMPA1, AMP A2, LGI1, CASPR2, GABA). B R, DPPX, IgLON5, GlyR, GABA A Rα1, GABA A Rγ2, GABA A Serum samples from patients who tested negative for Rβ3 and all 12 autoantibody tests were further screened, with the following inclusion criteria: (1) Symptoms of an autoimmune disease of the nervous system, including one or more of the following: abnormal mental behavior, cognitive impairment, recent memory loss, seizures, speech disorders, motor disorders, involuntary movements, decreased level of consciousness and coma, and autonomic dysfunction; (2) At least one inflammatory condition, such as cerebrospinal fluid leukocytosis (>5×10⁻⁶). 6 / L), elevated oligoclonal bands or IgG index in cerebrospinal fluid, MRI showing encephalitis (high signal on brain MRI limited to one or both medial temporal lobes, or involving gray matter, white matter, or both in multifocal areas), brain biopsy showing lymphocytic infiltration; (3) Detection of autoantibodies (NMDAR, AMPA1, AMPA2, LGI1, CASPR2, GABA) associated with autoimmune encephalitis using cell-based immunofluorescence assay (CBA). B R, DPPX, IgLON5, GlyR, GABA A Rα1, GABA A Rγ2, GABA A All Rβ3 were negative; (4) Age ≥ 14 years; (5) Exclude infectious diseases (including viral encephalitis, neurosyphilis, and nervous system infections caused by bacteria, fungi and parasites), metabolic and toxic encephalopathy (including hepatic encephalopathy and pulmonary encephalopathy, toxic encephalopathy caused by antibiotics, chemotherapy drugs or immunosuppressants, radiation encephalopathy), hereditary diseases (mitochondrial encephalopathy, methylmalonic acidemia, adrenoleukodystrophy), and neurodegenerative diseases (Lewy body dementia, frontotemporal dementia, multiple system atrophy and hereditary cerebellar degeneration).

[0036] Patients who did not meet the inclusion criteria were excluded, resulting in data from 269 patients, randomly assigned to numbers H1-H269. Serum samples were then randomly collected from 20 healthy individuals, assigned to numbers J1-J20.

[0037] Step 2: Expanding the antibody profile detection The CBA method was used to detect more reported neurological-related autoantibodies in samples from the aforementioned 269 patients. These included autoantibodies related to paraneoplastic syndromes (Hu, Ri, YO, Ma1, Ma2, CV2 / CRMP5, Amphiphysin, PKCγ). Detection of Zic4, SOX1, Tr(DNER), Titin, Recoverin, GAD65; detection of central nervous system demyelination-related autoantibodies (AQP4, MOG, MBP, GFAP, AQP1, Flotillin1 / 2); and detection of 60 other reported nervous system-related autoantibodies (mGluR5, D2R, Neurexin-3α, KCTD16, GAD67, KCNA4(Kv1.4), KLHL11, AK5, TRIB2, GluR3, Gephyrin, CaVα2δ(CACNA2D1), TPO, TGM2, TGM6, MUNC18-1, mGluR1, GABAARAP, Drebrin, AGO, NAE, PDE). 10A, ADAM22, ADAM23, ROCK2, mGluR3, mGluR4, CACNB1, VAMP2, CRMP2, CACNA1A, Homer3, ATP1A3, ARHGAP26, ITPR1, septin-5, NCDN, GRID2, AP3B2, mGluR2, GRIK2(GluR6 / GluK2), Rab6A, Rab6B, CA8(CARPVIII), PDE10A, PLP1, NF155, NF186, CNTN1, CNTN2, CASPR1, Gliomedin, Agrin, SYN1, AChR, LRP4, MuSK, MAG, neurobeachin, VGLUT2).

[0038] The target antigens of the aforementioned 80 neurological-related autoantibodies were identified. Cell slides overexpressing these target antigens were prepared, and these cell slides were assembled into a biodetection chip. Immunofluorescence was used to detect whether patient serum could react with the biodetection chip, thus exploring whether the patient serum contained specific autoantibodies for this series of genes. The specific steps are as follows: (1) Construction of recombinant vectors: The gene sequences encoding the above 80 proteins were found on NCBI and sent to the sequencing company. The gene sequences encoding the above 80 proteins were ligated into pCDNA3.1 to obtain 80 recombinant vectors. After the constructed recombinant vectors were correctly sequenced, they were extracted for later use. (2) Transfection of target genes: 81 culture dishes were used with 293T cells with 6cm×6cm climbing slices at the bottom. When the cell density reached 60%~80%, the recombinant vectors of 80 corresponding genes and the empty vector pCDNA3.1 were transfected into 293T cells using PEI transfection reagent and labeled. (3) Cell fixation: Cells grown for 24-48 hours after transfection were washed twice with PBS, fixed with acetone for 5 minutes, washed twice with PBS, and dried. The cell covers were cut into 2.5 mm × 2.5 mm pieces, and 81 types of 2.5 mm × 2.5 mm cell covers were attached to a glass slide to prepare a biodetection chip for screening target antigens. (4) Immunofluorescence staining: The serum of 269 patients and 20 healthy individuals were diluted with PBST at a volume ratio of 1:10 and incubated on the biodetector chip. The chips were incubated at room temperature for 1 hour and washed with PBST 3 times for 5 minutes each time. FITC-labeled goat anti-human IgG secondary antibody diluted 1:200 was used and incubated at room temperature for 30 minutes. The chips were washed with PBST 3 times for 5 minutes each time. The results were observed under a fluorescence microscope and photographed.

[0039] Some CBA test results are shown in the image below. Figure 1 As shown in Table 1, the CBA screening antibody positive sample numbers and corresponding positive indicators are as follows: 13 CBA positive and 256 CBA negative in the serum of 269 patients. The positive indicators detected include AQP4, Ma2, GFAP, Titin, LRP4, Amphiphysin, MOG, GRID2 and Hu.

[0040] Table 1. CBA Screening Antibody Positive Sample Numbers and Corresponding Positive Indicators

[0041] After excluding 13 previously reported patients with positive neurological autoantibodies, a total of 256 patient data were obtained.

[0042] Step 3: Detect the fluorescence signal of patient serum in rat brain tissue using immunofluorescence assay. Serum samples from these 256 patients were analyzed using tissue-based immunofluorescence (TBA) assay, as follows: (1) Preparation of frozen sections of rat brain tissue: Adult rats were anesthetized, and after the rats' limbs were stiffened, the abdominal cavity was opened to expose the apex of the heart. PBS was perfused from the left apex of the heart to facilitate systemic circulation. Then the brain tissue was removed and fixed with methanol for 10-30 min. The sample was transferred into a 30 wt.% sucrose solution for dehydration and placed at 4°C until the tissue block settled to the bottom. A small amount of embedding agent OCT was added to the specimen stage and placed in the freezing stage of a -20°C cryostat for sectioning to obtain frozen sections of rat brain tissue.

[0043] (2) Serum incubation: The serum of 256 patients and 20 healthy individuals were diluted with PBST at a volume ratio of 1:10 and incubated on frozen sections of rat brain tissue. The mixture was incubated at room temperature for 1 hour, washed three times with PBST for 5 minutes each time, and then diluted with FITC-labeled goat anti-human IgG secondary antibody. The mixture was incubated at room temperature for 30 minutes, washed three times with PBST for 5 minutes each time, and the results were observed and photographed under a fluorescence microscope.

[0044] Of the 256 patients' serum samples, 111 showed positive signals in the cerebellum and thalamic white matter on frozen sections of rat brain tissue, while no positive signals were found in the serum samples of 145 patients and 20 healthy individuals. The patient sample numbers and corresponding TBA test results are shown in Table 2.

[0045] Table 2 TBA test results

[0046] It can be seen that the serum of 111 patients showed a significantly stronger positive signal than that of healthy human serum on frozen sections of rat brain tissue, indicating that the serum of 111 patients contained antibodies that bound to antigens in the cerebellum and thalamic white matter of rat brain tissue. However, according to the results of CBA in step 2 of Example 1, the serum of 111 patients may contain new autoantibodies that can recognize neurons, which are different from those previously reported.

[0047] Step 4: Screening of target antigens We identified 100 proteins with relatively high expression levels in the human brain, cerebellum, and hypothalamus from the human protein atlas (https: / / www.proteinatlas.org / ). We then prepared a biodetector chip and used immunofluorescence to detect whether patient serum could generate an immune response after incubation with the biodetector chip. This study aimed to explore whether the serum of the 111 previously reported patients who were negative for neurological-related autoantibodies (CBA) but positive for TBA contained novel autoantibodies that could recognize neurons, unlike those previously reported. The specific steps are as follows: (1) Preparation of biodetection chips: The gene sequences encoding each protein were found on NCBI and sent to a sequencing company to synthesize the genes into pCDNA3.1. The constructed recombinant vectors were sequenced and then extracted for later use. The above recombinant vectors were transfected into 293T cells grown on 6cm×6cm cell slides using PEI transfection reagent. After 24-48 hours of transfection, the cells were washed, fixed and dried. The 6cm×6cm cell slides were cut into 2.5mm×2.5mm pieces and these cell slides were used to prepare biodetection chips for later use.

[0048] (2) Immunofluorescence staining: The serum of 111 patients and 20 healthy individuals were diluted with PBST at a volume ratio of 1:10 and incubated onto the prepared biodetector chip. The chips were incubated at room temperature for 1 h and washed with PBST 3 times for 5 min each time. FITC-labeled goat anti-human IgG secondary antibody diluted 1:200 was used and incubated at room temperature for 30 min. The chips were washed with PBST 3 times for 5 min each time. The results were observed under a fluorescence microscope and photographed.

[0049] The results are as follows Figure 1 As shown, the serum of 5 patients (H36, H64, H151, H211, and H227) showed a significantly stronger colorimetric reaction with the same antigen on the biodetection chip than that of healthy serum, producing positive signals. Additionally, the serum of 13 patients showed significantly stronger colorimetric reactions with 11 different antigens than those of healthy serum, producing positive signals. The serum of the remaining 93 patients and 20 healthy serum did not produce signals with any of the antigens on the biodetection chip. Upon investigation, this target antigen was identified as GLUL (glutamate-ammonia ligase, accession number: NM_001033044.4), with the amino acid sequence shown in SEQ ID NO.1 and the nucleotide sequence encoding this protein shown in SEQ ID NO.2.

[0050] Step 5: Serological survey of patient samples Serum samples from these five patients were used as biological samples for serological investigation, and the discovery process of anti-GLUL autoantibodies is described in detail. Information on the five patients included in this invention is shown in Table 3.

[0051] Table 3 Clinical information of patients H36, H64, H151, H211, and H227

[0052] Step 6: Screen for fluorescence signals in primary neurons from the patient's serum. (1) Isolation of primary rat neurons Rats were anesthetized with 10% chloral hydrate at a concentration of 3 mL / kg body weight. After anesthesia, the rats were disinfected by immersion in 75% alcohol. The rat brain tissue was then removed from a biosafety cabinet and placed in pre-cooled DMEM containing 1% BSA. The rat brain tissue was then cut into a paste. 10 mL of papain was added, and the mixture was digested at 37°C for 30 minutes. After digestion, the cells were transferred to new centrifuge tubes, and 10 mL of DMEM containing 1% BSA was added to resuspend the cells. The tubes were then centrifuged at 400g and 4°C for 5 minutes, and the supernatant was discarded. Another 5 mL of DMEM containing 1% BSA was added to resuspend the cells, and the tubes were centrifuged again at 200g and 4°C for 5 minutes, and the supernatant was discarded again. Finally, 20 mL of DMEM was added to resuspend the cells, and the cells were cultured at an appropriate density in cell culture dishes containing smears to obtain primary neurons from rat brain tissue.

[0053] (2) Fixation of cell spread sheets Rat primary neurons were fixed with 4% paraformaldehyde (FPA) for 10 minutes, then washed twice with PBS solution, terminated with 1.25M glycine for 10 minutes, and washed twice with PBS to obtain rat primary neuron cell slices for later use.

[0054] (3) Serum incubation Serum from 5 patients (H36, H64, H151, H211, H227) and 5 healthy individuals (J1, J2, J3, J4, J5) were diluted 1:10 using PBST and incubated separately in rat primary neuronal cell slides at room temperature for 1 hour. The cells were then washed three times with PBST for 5 minutes each time. Next, the cells were incubated with FITC-labeled goat anti-human IgG secondary antibody diluted 1:200 at room temperature for 30 minutes, followed by three washes with PBST for 5 minutes each time. DAPI was then used to stain the cell nuclei. Cells were stained at room temperature for 10 minutes, washed three times with PBST for 5 minutes each time, and serum-incubated cell smears were obtained. The results were observed and photographed under a fluorescence microscope. The results showed that the serum of five patients (H36, H64, H151, H211, and H227) showed positive signals on primary neurons, while the serum of healthy individuals did not show positive signals. This demonstrates that antibodies binding to primary neurons were present in the serum of the five patients (H36, H64, H151, H211, and H227). Some staining results are shown below. Figure 2 As shown.

[0055] (4) Antibody colocalization The neuronal cell-specific scaffold marker antibody MAP2 was diluted 1:500 using PBST and incubated with cell smears infused with serum from the five patients (H36, H64, H151, H211, and H227) for co-staining. The cells were incubated overnight at 4°C, washed three times with PBST for 5 minutes each time. The same neuronal cell smears were then incubated with Alexa Fluor 594-labeled goat anti-mouse IgG secondary antibody diluted 1:200 at room temperature for 30 minutes, followed by three washes with PBST for 5 minutes each time. The resulting co-stained cell smears were observed and photographed under a fluorescence microscope. The results showed that the staining signal from the serum of the five patients (H36, H64, H151, H211, and H227) overlapped with the signal of the MAP2 antibody on primary neurons, indicating that the antigen recognized by the antibody in the serum of the five patients (H36, H64, H151, H211, and H227) was expressed on the neurons. Some staining results are shown below. Figure 3 As shown.

[0056] Step 7: Screen for fluorescence signals in primary astrocytes from patient serum. (1) Isolation and culture of primary rat astrocytes Referring to step 6(1) of Example 1, after discarding the supernatant, 20 mL of DMEM medium containing 10% fetal bovine serum was added to the centrifuge tube to resuspend the cells. The cells were then cultured at an appropriate density in a cell culture dish containing a scab to obtain primary rat astrocytes. After fixation according to step 6(2) of Example 1, a scab of primary rat astrocytes was obtained.

[0057] (2) Serum incubation Referring to step (3) of Example 1, serum from 5 patients (H36, H64, H151, H211, H227) and 5 healthy individuals (J1, J2, J3, J4, J5) was incubated on rat primary astrocyte smears. The results were observed and photographed under a fluorescence microscope. The results showed that the serum from the 5 patients (H36, H64, H151, H211, H227) showed positive signals on the primary astrocytes, while the serum from the healthy individuals did not show positive signals. This proved that the serum from the 5 patients (H36, H64, H151, H211, H227) contained antibodies that bound to the primary astrocytes. Some staining results are shown below. Figure 4 As shown.

[0058] (3) Antibody colocalization Astrocyte-specific marker GFAP (glial fibrillary acidic protein) antibody was diluted 1:500 using PBST and incubated on cell smears incubated with the above serum for antibody co-staining. The cells were incubated overnight at 4°C, washed three times with PBST for 5 minutes each time. Goat anti-rabbit IgG secondary antibody, diluted 1:200 with Alexa Fluor 594, was then incubated on the above astrocyte smears at room temperature for 30 minutes, washed three times with PBST for 5 minutes each time, to obtain antibody-co-stained cell smears. The cells were observed and photographed under a fluorescence microscope. The results showed that the staining signal from the serum of five patients (H36, H64, H151, H211, and H227) overlapped with the signal of the GFAP antibody on primary astrocytes, indicating that the antigen recognized by the antibody in the serum of these five patients (H36, H64, H151, H211, and H227) was highly expressed on astrocytes. Some staining results are shown below. Figure 5 As shown.

[0059] Example 2: Validation of GLUL antibodies in patient serum Step 1: Verify GLUL signal detected in neurons using commercial antibodies. Preparation of rat primary astrocyte smears: Following step 7 of Example 1, rat primary astrocyte smears were obtained. Serum from five patients (H36, H64, H151, H211, H227) was incubated and co-stained with a commercially available GLUL antibody. The smears were observed and photographed under a fluorescence microscope. Results showed that the staining signals from the serum of the five patients (H36, H64, H151, H211, H227) on rat primary astrocytes overlapped with the staining signals from the commercially available GLUL antibody. This indicates that the antibody in the serum of the five patients (H36, H64, H151, H211, H227) specifically recognizes the GLUL protein on rat primary astrocytes. Some staining results are shown below. Figure 6 As shown.

[0060] Step 2: Verify the GLUL signal detected by serum on overexpression cell smears using commercial antibodies. Following the procedure in step 2 of Example 1, GLUL-overexpressing cell smears and sera from 5 patients (H36, H64, H151, H211, H227) were prepared and co-stained with a commercially available GLUL antibody. The samples were observed and photographed under a fluorescence microscope. Results showed that the commercially available antibody showed a positive signal on the GLUL-overexpressing cell smears, indicating successful overexpression of the GLUL protein on 293T cells. The staining signals from the sera of the 5 patients (H36, H64, H151, H211, H227) overlapped with those from the commercially available GLUL antibody on the GLUL-overexpressing cell smears, indicating that the antibody in the sera of the 5 patients (H36, H64, H151, H211, H227) specifically recognized the GLUL protein on the GLUL-overexpressing cell smears. Some results are shown below. Figure 7 As shown.

[0061] Step 3: Verify the GLUL signal detected by serum on Western blotting using commercial antibodies. (1) Preparation of protein samples: When the density of cultured 293T cells reaches 30%~40%, the recombinant vector in step 4 of Example 1 is transfected using PEI transfection reagent. GLUL pCDNA3.1 and empty pCDNA3.1 were transfected into 293T cells and labeled. After 24-48 hours, one dish of 293T cells overexpressing GLUL was collected, the supernatant was discarded, and the cells were scraped into a 1.5mL centrifuge tube using a cell scraper. The cells were centrifuged at 800rpm at room temperature to remove the supernatant, and 200μL of PBS was added. The cells were then sonicated (10% power, 3s disruption, 6s pause, 1 minute total) to obtain the overexpressed GLUL protein. One dish of cells transfected with empty pCDNA3.1 was used to prepare the control protein, using the same conditions and methods as the GLUL overexpression protein preparation. (2) Sample loading and transfer: 20 μg of overexpressed GLUL protein and 20 μg of empty pCDNA3.1 protein were loaded into two groups. After electrophoresis, wet transfer was performed at 300 mA for 90 min. 5% skim milk powder was used for blocking at room temperature for 1 h. (3) Antibody incubation and color development: GLUL antibody was diluted 1:2000 with TBST, and 5 patients (H36, H64, H151, H211, H227) were diluted 1:200 as primary antibodies, and incubated overnight at 4°C. The next day, the cells were washed 3 times with TBST for 5 min each time. HRP-labeled goat anti-rabbit / human secondary antibody was added and incubated at room temperature for 1 h. The cells were washed 3 times with TBST for 5 min each time. Chemiluminescence solution was added for color development and photographs were taken. The results showed that both GLUL antibody and the immunoblot of patients' serum overexpressing GLUL protein had specific bands (42KD), while there was no signal compared with the control protein. This indicates that there are autoantibodies that react with GLUL protein in the serum of patient H36. Some results are shown below. Figure 8 As shown.

[0062] Step 4: Serum neutralization test to verify the signal detected in the patient's serum (1) Preparation of neutralizing protein: Refer to step 4 of Example 2 to prepare overexpressed GLUL protein as neutralizing protein and pCDNA3.1 protein as control protein.

[0063] (2) Serum neutralization test to verify the signal detected in patient serum on primary neuronal cell slices. Following step 5 of Example 1, primary neuronal cell smears were prepared. Three portions of commercially available GLUL antibody diluted 1:200 were prepared using PBST, and two portions of patient H36 serum diluted 1:10 were prepared using PBST, 100 μL of each portion. 20 μL of GLUL neutralizing protein and 20 μL of control protein were added to each portion, and the cells were incubated at room temperature for 30 minutes. Two portions of patient H64 serum, two portions of patient H151 serum, two portions of patient H211 serum, and two portions of patient H227 serum were prepared using the same method and incubated with the prepared primary neuronal cell smears at room temperature for 1 hour. The cells were washed three times with PBST for 5 minutes each time. FITC-labeled goat anti-rabbit IgG secondary antibody diluted 1:200 was added, and the cells were incubated at room temperature for 30 minutes. The cells were washed three times with PBST for 5 minutes each time. The cells were observed and photographed under a fluorescence microscope.

[0064] (3) Serum neutralization test: The signal detected in the patient's serum was verified on primary astrocyte smears. Primary astrocyte smears were prepared according to step 5 of Example 1. Referring to part (2) of step 5 of Example 2, the neutralizing protein group and control protein group of serum from 5 patients (H36, H64, H151, H211, H227) were incubated on the primary astrocyte smears, observed under a fluorescence microscope, and photographed.

[0065] (4) Serum neutralization assay: The signal detected in the patient's serum was verified on a smear of GLUL-overexpressing cells. Following the procedure in step 2 of Example 1, GLUL-overexpressing cell smears were prepared. Following the procedure in step 5 (2) of Example 2, the neutralizing protein group and control protein group of serum from 5 patients (H36, H64, H151, H211, H227) were incubated into GLUL-overexpressing cell smears, observed under a fluorescence microscope, and photographed.

[0066] (5) Serum neutralization test to verify the signal detected in patient serum on rat brain tissue sections. Following the procedure in Example 1, frozen sections of rat brain tissue were prepared. Following the procedure in step 5(2) of Example 2, the neutralizing protein group and control protein group of serum from 5 patients (H36, H64, H151, H211, H227) were incubated onto the rat brain tissue sections, observed under a fluorescence microscope, and photographed.

[0067] The results showed that the commercially available GLUL antibody signal and the serum signals from five patients (H36, H64, H151, H211, and H227) were blocked by GLUL neutralizing protein on primary neuronal cell slices, primary astrocyte slices, GLUL-overexpressing cell slices, and rat brain tissue sections. However, the control protein did not block the signal appearing on primary neuronal cell slices. This indicates that the signals appearing in the serum of the five patients (H36, H64, H151, H211, and H227) on the aforementioned primary neuronal cell slices, primary astrocyte slices, GLUL-overexpressing cell slices, and rat brain tissue sections are signals specifically recognizing the GLUL antigen. Some results are shown below. Figure 9 As shown.

[0068] (5) Serum neutralization test to verify the signal detected in patient serum on WB. Following the procedure in step 4 of Example 2, the overexpressed GLUL protein was subjected to SDS-PAGE gel electrophoresis and transferred to a membrane. Two 1:200 diluted serum samples from patient H64 were prepared using TBST, 2 mL each. 400 μL of GLUL neutralizing protein and 400 μL of control protein were added to each sample, respectively, and incubated at room temperature for 30 minutes. Two more serum samples from patients H64, H151, H211, and H227 were prepared using the same method. The neutralizing protein group and the control protein group were incubated onto membranes, and chemiluminescent buffer was added for color development and imaging. The results showed that a specific band (42 KD) was observed in the immunoblot of the overexpressed GLUL protein from patient H64 serum, and the band in the neutralizing protein group was significantly weaker than that in the control protein group. This indicates that the band in the immunoblot of the overexpressed GLUL protein from patient H64 serum specifically recognizes GLUL. Some results are shown below. Figure 10 As shown.

[0069] Example 3: Verification of the clinical specificity and detection rate of anti-GLUL autoantibodies using cell-based immunofluorescence assay Step 1: Sample Collection To verify the clinical specificity and detection rate of anti-GLUL antibodies, serum samples submitted between January 1, 2023 and June 31, 2025 were collected. Inclusion criteria were: (1) age ≥ 14 years; (2) discharge diagnosis information. Patients who did not meet the inclusion criteria were excluded, resulting in data from 902 patients.

[0070] Patients were classified and numbered based on their discharge diagnosis. Among them, 413 patients had suspected or confirmed autoimmune encephalitis, including 112 confirmed cases and 301 possible cases. 341 patients had other confirmed neuroimmune diseases, including paraneoplastic syndromes, myasthenia gravis, multiple sclerosis, neuromyelitis optica spectrum disorders, Guillain-Barré syndrome, and immune-related peripheral neuropathy. 148 patients had non-neurological autoimmune diseases, including infectious diseases (viral encephalitis, meningitis, and neurological infections caused by bacteria, fungi, and parasites), metabolic and toxic encephalopathy (including hepatic encephalopathy and pulmonary encephalopathy, toxic encephalopathy caused by antibiotics, chemotherapy drugs, or immunosuppressants, and radiation encephalopathy), tumors (gliomatosis, primary central nervous system lymphoma, and multiple metastatic cancers), and patients without neurological symptoms. Serum samples were randomly collected from 129 healthy individuals and numbered J21-J150. Detailed patient classifications and numbers are shown in Table 4.

[0071] Table 4. Patient Classification and Numbering in the Validation Set

[0072] Step 2: Detection of anti-GLUL autoantibodies in CBA validation set samples (1) Following the procedure in step 2 of Example 1, GLUL-overexpressing cell slides and empty pCDNA3.1 control cell slides were prepared. GLUL-overexpressing and empty pCDNA3.1 control cell slides were used for detection to screen for GLUL autoantibody-positive serum samples. A total of 12 GLUL autoantibody-positive samples were screened. Some results are shown below. Figure 11 As shown.

[0073] (2) The results showed that 12 anti-GLUL antibody positive cases were detected in 902 serum samples; among them, 8 anti-GLUL antibody positive cases were detected in serum samples of suspected autoimmune encephalitis patients, with a detection rate of 2.66%; among them, 3 anti-GLUL antibody positive cases were detected in serum samples of confirmed autoimmune encephalitis patients, with a detection rate of 2.68%. These 3 patients were positive for anti-CASPR2 antibody, anti-NMDAR antibody, and anti-MGLUR1 antibody, respectively; 1 positive case was detected in the samples of 341 patients with other neurological immune-related diseases other than autoimmune encephalitis. This patient was positive for anti-GFAP antibody, with a detection rate of 0.29%; in addition, no anti-GLUL antibody was detected in the samples of 148 patients with other non-neurological immune-related diseases and 129 healthy controls, with a detection rate of 0.00%.

[0074] (3) The five patients who were positive for anti-GLUL antibodies in the previous clinical collection (numbered H36, H64, H3, H211, H227) and the 12 newly added patients who were positive for anti-GLUL antibodies (numbered PAE5, PAE19, PAE37, PAE103, PAE106, PAE121, PAE174, PAE289, AE27, AE31, AE80, MS25) were integrated, totaling 17 patients. All patients were verified by CBA to be positive for the target antibody, and cases with other neurological infections, tumors and metabolic diseases were excluded. Clinical symptoms were classified into 9 categories (psychiatric abnormalities, cognitive impairment, recent memory loss, epileptic seizures, speech disorders, motor disorders, involuntary movements, decreased level of consciousness and coma, and autonomic dysfunction). To avoid double counting, if a patient presented with both "incoherent speech and hallucinations", the patient was only classified as "psychiatric abnormalities". The symptom distribution data of 17 patients who were positive for anti-GLUL antibodies were statistically analyzed, and the results are shown in Table 5.

[0075] Table 517 Symptom Distribution of Anti-GLUL Antibody Positive Patients

[0076] According to the results in Table 5, recent memory loss, motor disorders, and abnormal mental behavior are the most frequent symptoms associated with positive anti-GLUL antibodies.

[0077] Step 3: Sensitivity and specificity analysis of anti-GLUL antibodies in the auxiliary diagnosis of autoimmune encephalitis: (1) First, based on the detection results of step 2, the sensitivity and specificity of anti-GLUL antibody detected by CBA method in the auxiliary diagnosis of autoimmune diseases of the nervous system, especially autoimmune encephalitis, were systematically evaluated. The correspondence between the detection results and clinical diagnosis is shown in Table 6.

[0078] Table 6. Correspondence between test results and clinical diagnosis

[0079] Based on Table 6, the sensitivity, specificity, positive predictive value, negative predictive value, and 95% confidence interval (95% CI) of anti-GLUL antibodies for the auxiliary diagnosis of autoimmune encephalitis were calculated, and the results are as follows: Sensitivity (positive concordance rate) = 2.66%, 95% CI: 1.38%~4.70%; Specificity (negative concordance rate) = 99.84%, 95% CI: 99.15%~99.99%; Positive predictive value (PPV) = 91.67%, 95% CI: 61.52%–99.79%; Negative predictive value (NPV) = 60.55%, 95% CI: 57.43%–67.59%; Accuracy (overall compliance rate) = 61.00%, 95% CI: 57.90%~63.90%.

[0080] Based on the above verification results, this invention provides a novel antigen that binds to autoantibodies for the diagnosis of autoimmune diseases of the nervous system, especially autoimmune encephalitis; anti-GLUL autoantibodies can be detected in patients with autoimmune diseases of the nervous system with neurological symptoms, indicating that the antibody has an auxiliary role in the diagnosis of autoimmune diseases of the nervous system, especially in the diagnosis of autoimmune encephalitis.

[0081] Example 7: Detection of GLUL autoantibodies in patient serum using GLUL mutants This embodiment selects a person. GLUL One mutant of the gene, which lacks amino acids 114-156 of the GLUL protein, has the following sequence: RHTCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPG.

[0082] A mutant lacking amino acids 114-156 of the GLUL protein (amino acid sequence shown in SEQ ID NO. 3) was constructed according to the steps described in Example 2. Cell slides overexpressing the recombinant GLUL deletion mutant were prepared, and serum samples from 17 patients (numbered H36, H64, H151, H211, H227, PAE5, PAE19, PAE37, PAE103, PAE106, PAE121, PAE174, PAE289, AE27, AE31, AE80, MS25) who were positive for anti-GLUL antibodies, as well as serum samples from 5 healthy individuals (numbered J21, J22, J23, J24, J25), were tested. The results showed that the serum from all 17 patients who were positive for anti-GLUL antibodies produced a positive signal on the cell slides overexpressing the recombinant GLUL deletion mutant, while no positive signal was produced in the 5 healthy individuals. Therefore, the GLUL deletion mutant can still recognize anti-GLUL antibodies in patient serum. Some results are shown below. Figure 12 As shown.

[0083] As can be seen from the above embodiments, anti-GLUL autoantibodies can be used as biomarkers for diagnosing autoimmune diseases of the nervous system, and the detection of anti-GLUL autoantibodies can achieve auxiliary diagnosis of autoimmune diseases of the nervous system.

[0084] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

[0085] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. Application of reagents for detecting anti-GLUL autoantibodies in the preparation of products for diagnosing autoimmune diseases of the nervous system.

2. The application as described in claim 1, characterized in that, The reagent for detecting anti-GLUL autoantibodies includes one or more of the following: GLUL protein, cells expressing GLUL protein, vectors expressing GLUL protein, and tissues containing GLUL protein.

3. The application as described in claim 1, characterized in that, The amino acid sequence of the GLUL protein includes any one of a) to c): a) The amino acid sequence shown in SEQ ID NO.1; b) The amino acid sequence shown in SEQ ID NO.1, after modification or mutation, can recognize the amino acid sequence of anti-GLUL autoantibody; c) has an amino acid sequence identity of ≥70% and <100% with a) or b), and is capable of recognizing the amino acid sequence of anti-GLUL autoantibody.

4. The application as described in claim 3, characterized in that, In b), the amino acid sequence shown in SEQ ID NO.1, after being modified or mutated, can recognize the amino acid sequence of the anti-GLUL autoantibody as shown in SEQ ID NO.

3.

5. The application as described in claim 3, characterized in that, The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1 preferably includes any one of (I) to (III): Ⅰ) The nucleotide sequence shown in SEQ ID NO.2; Ⅱ) A nucleotide sequence that, after mutation of the nucleotide sequence shown in SEQ ID NO.2, is able to encode an amino acid sequence that recognizes anti-GLUL autoantibodies; III) A nucleotide sequence that has ≥70% and <100% identity with the nucleotide sequence in I) or II) and is capable of encoding an amino acid sequence that recognizes an anti-GLUL autoantibody.

6. The application as described in claim 1, characterized in that, The cells expressing the GLUL protein include bacterial cells or eukaryotic cells; preferably, they include HEK293 cells, HeLa cells, CHO, Pichia pastoris, Saccharomyces cerevisiae, sf9, BL21, or Rosetta. The vectors expressing GLUL protein include the pTriEx vector family, pcDNA3 family, pET series vectors, or pBac series vectors. The tissue containing GLUL protein is brain tissue; preferably, it includes brain tissue from humans, rats, primates, mice, goats, horses, sheep, or cattle.

7. The application as described in claim 1, characterized in that, The aforementioned autoimmune disease of the nervous system is autoimmune encephalitis; Symptoms of the aforementioned autoimmune neurological disorders include one or more of the following: mental and behavioral abnormalities, cognitive impairment, recent memory loss, seizures, speech disorders, motor disorders, involuntary movements, decreased level of consciousness and coma, and autonomic dysfunction.

8. A reagent kit for diagnosing autoimmune diseases of the nervous system, characterized in that, The reagent includes a method for detecting anti-GLUL autoantibodies, which includes one or more of the following: GLUL protein, cells expressing GLUL protein, vectors expressing GLUL protein, and tissues containing GLUL protein.

9. The kit for diagnosing autoimmune diseases of the nervous system according to claim 8, characterized in that, The amino acid sequence of the GLUL protein includes any one of a) to c): a) The amino acid sequence shown in SEQ ID NO.1; b) The amino acid sequence shown in SEQ ID NO.1, after modification or mutation, can recognize the amino acid sequence of anti-GLUL autoantibody; c) The amino acid sequence identity with a) or b) is ≥70% and <100%, and the amino acid sequence is capable of recognizing anti-GLUL autoantibodies; Preferably, in b), the amino acid sequence shown in SEQ ID NO.1 is modified or mutated to recognize the amino acid sequence of the anti-GLUL autoantibody as shown in SEQ ID NO.

3.

10. The kit for diagnosing autoimmune diseases of the nervous system according to claim 8, characterized in that, The kit is a kit that uses enzyme-linked immunosorbent assay (ELISA) for detection, or a kit that uses protein immunoblotting for detection. When using the kit described above for detection, the following methods may be selected: CBA, TBA, ELISA, immunogold assay, immunoblotting, immunospot assay, membrane strip assay, chemiluminescence assay, radioimmunoassay, liquid chromatography-array assay, lateral chromatography, or flow cytometry.

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