Use of reagents for detecting anti-tnfaip3 autoantibodies

Abstract

This application provides a reagent for detecting anti-TNFAIP3 autoantibodies in the preparation of products for detecting and / or assisting in the diagnosis of neurological autoimmune diseases. The reagent includes: (1) TNFAIP3 protein; (2) biomaterial expressing the TNFAIP3 protein; (3) an antigenic peptide capable of binding to the anti-TNFAIP3 autoantibody; and (4) a solid-phase carrier coupled with at least one antigen from (1) to (3). The amino acid sequence of the TNFAIP3 protein is the amino acid sequence shown in SEQ ID NO:1, or the amino acid sequence shown in SEQ ID NO:1 modified or mutated to recognize the amino acid sequence of the TNFAIP3 autoantibody. The application of the reagent of this application can detect and / or assist in the diagnosis of neurological autoimmune diseases, improving the accuracy of neurological autoimmune disease diagnosis, thereby identifying the pathogenic factors of neurological autoimmune diseases such as AE and GBS, solving diagnostic and treatment dilemmas, and having significant implications for clinical diagnosis and treatment challenges.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, and more specifically, to the use of a reagent for detecting anti-TNFAIP3 autoantibodies in the preparation of products for detecting and / or assisting in the diagnosis of autoimmune diseases of the nervous system. Background Technology

[0002] Neurological autoimmune diseases are caused by the immune system's abnormal attack on the nervous system, affecting both the central and peripheral nervous systems and leading to dysfunction. Currently, the diagnosis of neurological autoimmune diseases relies on a comprehensive system of "clinical manifestations + imaging examinations + laboratory tests + differential diagnosis," with autoantibody detection being the core basis for diagnosis and classification.

[0003] Since the discovery of anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis in 2007, a series of antibodies against the surface of neurons have been subsequently identified. However, some patients suspected of having immune-mediated encephalitis still lack detectable antibodies, posing challenges to diagnosis and treatment. Diagnostic uncertainty may delay immunotherapy, thereby affecting patient prognosis. In 2016, GRAUS et al. published international consensus criteria for diagnosing AEs based on clinical characteristics, aiming to provide a basis for initiating immunomodulatory therapy for AEs as early as possible before antibody test results are available. Simultaneously, this consensus proposed diagnostic criteria for antibody-negative autoimmune encephalitis (AbNAE). Currently, AbNAE has become the major subtype of AEs, and some studies even suggest that the incidence of AbNAE may be similar to that of antibody-positive AEs.

[0004] Guillain-Barré syndrome (GBS) is a common autoimmune disease of the peripheral nervous system. Its core pathogenesis involves infection-induced molecular mimicry, where pathogen antigens are homologous to peripheral nerve components, inducing the production of autoantibodies that attack nerves, leading to conduction disorders. However, in the acute phase of GBS, 20%–40% of patients do not have detectable known related autoantibodies, posing a significant challenge to diagnosis and treatment.

[0005] In summary, the development and progression of neurological autoimmune diseases such as acute exacerbations (AE) and generalized systemic neuropathy (GBS) are closely related to autoantibodies. However, a large number of patients present negative results for known autoantibodies, suggesting the presence of undiscovered novel autoantibodies. These novel autoantibodies may be key pathogenic factors in these patients, and their absence leads to diagnostic and treatment challenges. Therefore, identifying relevant novel autoantibodies and improving the pathogenic antibody profile are of great significance for solving clinical diagnostic and treatment problems and are core issues that urgently need to be addressed in this field. Summary of the Invention

[0006] One objective of this application is to provide a reagent for detecting anti-TNFAIP3 autoantibodies for use in the preparation of products for detecting and / or assisting in the diagnosis of autoimmune diseases of the nervous system, which can at least solve the problem of the lack of detection reagents for autoimmune diseases of the nervous system in the prior art.

[0007] To achieve the above objectives, this application provides the following technical solutions.

[0008] According to an embodiment of this application, a reagent for detecting anti-TNFAIP3 autoantibodies is provided for use in the preparation of products for detecting and / or assisting in the diagnosis of autoimmune diseases of the nervous system. The reagent comprises: (1) TNFAIP3 protein; (2) biomaterial expressing the TNFAIP3 protein; (3) an antigenic peptide capable of binding to the anti-TNFAIP3 autoantibody; and (4) a solid-phase carrier coupled with at least one antigen from (1) to (3). The amino acid sequence of the TNFAIP3 protein is the amino acid sequence shown in SEQ ID NO:1 or the amino acid sequence shown in SEQ ID NO:1 modified or mutated to recognize the amino acid sequence of the TNFAIP3 autoantibody.

[0009] Optionally, the TNFAIP3 protein is an immunogenic polypeptide capable of binding to the TNFAIP3 autoantibody, and the polypeptide is a polymer having two or more amino acids.

[0010] Optionally, the biomaterial includes any one or more of recombinant vectors, cells, and tissues.

[0011] Optionally, the recombinant vector includes any one or more of the pTriEx series vectors, pCDNA3 series vectors, pET series vectors, or pBac series vectors.

[0012] Optionally, the cells include bacterial cells or eukaryotic cells, the bacterial cells including Escherichia coli cells, and the eukaryotic cells including at least one of immortalized human cells, insect cells, or yeast.

[0013] Optionally, the tissue includes mammalian brain tissue, wherein the mammal is selected from any one or more of humans, rats, primates, mice, goats, horses, sheep, or cattle.

[0014] Optionally, the solid-phase support includes any one or more of the following: chip, enzyme-labeled plate, membrane carrier, microsphere carrier, and microplate.

[0015] Optionally, the amino acid sequence of the TNFAIP3 protein has at least 70% identity with the amino acid sequence shown in SEQ ID NO:1.

[0016] Optionally, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1 includes any one of I) to III): I) the nucleotide sequence shown in SEQ ID NO:2; II) an amino acid sequence that is ≥70% identical to the nucleotide sequence shown in SEQ ID NO:2 and can express an amino acid sequence that recognizes TNFAIP3 autoantibody; III) a nucleotide sequence that, after mutation in I) or II), can express an amino acid sequence that recognizes TNFAIP3 autoantibody.

[0017] Optionally, the neurological autoimmune diseases include autoimmune encephalitis and Guillain-Barré syndrome.

[0018] Optionally, the product is a reagent kit.

[0019] Optionally, the kit may further include labeled antibodies, including FITC-labeled antibodies and Alexa Fluor dye-labeled antibodies, horseradish peroxidase-labeled antibodies, and alkaline phosphatase-labeled antibodies.

[0020] The application of the reagent for detecting anti-TNFAIP3 autoantibodies according to the embodiments of this application enables qualitative or quantitative analysis of anti-TNFAIP3 autoantibodies. The reagent is easy to use and can detect and / or assist in the diagnosis of neurological autoimmune diseases, improving the accuracy of detection and / or auxiliary diagnosis of neurological autoimmune diseases, especially autoimmune encephalitis and Guillain-Barré syndrome. This helps identify the pathogenic factors of neurological autoimmune diseases such as AE and GBS, solving diagnostic and treatment dilemmas and having significant implications for clinical diagnosis and treatment.

[0021] Other features and advantages of this application will become clear from the following detailed description of exemplary embodiments with reference to the accompanying drawings. Attached Figure Description

[0022] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the present application and, together with their description, serve to explain the principles of the present application.

[0023] Figure 1 The results of immunofluorescence staining of cerebrospinal fluid at the Purkinje location in the cerebellum of 4 patients in Example 2 of this application; Figure 2 The results of silver staining of immunoprecipitates of cerebrospinal fluid in 24 cases in Example 3 of this application include 4 cases in the disease study group, 10 cases in the disease control group, and 10 healthy subjects as controls. Figure 3 The mass spectrometry results of the cerebrospinal fluid immunoprecipitate from patient 1 in Example 4 of this application; Figure 4The Western blot results of immunoprecipitates of cerebrospinal fluid from patients 1 and 2 were used to verify the commercial TNFAIP3 antibody in Example 4 of this application. Figure 5 The Western blot results of the commercially available TNFAIP3 antibody in Example 5 of this application were used to verify the overexpression of TNFAIP3 protein. Lane 1 is the marker, lane 2 is the control protein, and lane 3 is the overexpression of TNFAIP3 protein. Figure 6 The results of Western blotting in Example 5 of this application to verify the anti-TNFAIP3 antibody in the cerebrospinal fluid of patients 1-4 are shown. Lane 1 is the marker, lane 2 is the empty pcDNA3.4 control protein, and lane 3 is the overexpressed TNFAIP3 protein. Figure 7 This is the result of immunofluorescence staining of cerebrospinal fluid from patients 1-3 at the Purkinje position in the cerebellum, as verified by antibody co-staining in Example 6 of this application; Figure 8 This is to verify the immunofluorescence staining results of the cerebrospinal fluid of patients 1-3 at the Purkinje location in the cerebellum in the neutralization experiment of Example 6 of this application; Figure 9 This is the result of immunofluorescence staining of cerebrospinal fluid from patients 1-4 on TNFAIP3-overexpressing smears, as verified by antibody co-staining in Example 7 of this application. Figure 10 This is to verify the immunofluorescence staining results of cerebrospinal fluid from patients 1-4 on TNFAIP3-overexpressing smears in the neutralization experiment of Example 7 of this application; Figure 11 The results of immunofluorescence staining of cerebrospinal fluid from 5 patients, as detected by cell-based immunofluorescence in Example 8 of this application; Figure 12 The results of Western blotting of cerebrospinal fluid from 11 patients in Example 9 of this application are shown. Detailed Implementation

[0024] The embodiments of this application will now be described in detail. Examples of these embodiments are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0025] The terms "first" and "second" in the specification and claims of this application may explicitly or implicitly include one or more of the features. In the description of this application, unless otherwise stated, "multiple" means two or more. Furthermore, "and / or" in the specification and claims indicates at least one of the connected objects, and the character " / " generally indicates that the preceding and following objects are in an "or" relationship.

[0026] The following section defines and explains the terms used in this application.

[0027] The term "diagnosis" refers to the process of making a clear determination of a patient's health status and disease-related attributes. It covers situations such as: determining whether an individual has a certain disease; identifying the specific type and subtype of the disease in an individual who is already ill; determining the severity, stage, or grade of the disease; tracing the cause or trigger of the disease; differentiating between multiple suspected diseases with overlapping symptoms; further confirming the disease after screening asymptomatic individuals for abnormalities; adjusting the original diagnosis based on changes in the condition during disease treatment or follow-up; or a combination of the above.

[0028] The term "auxiliary diagnosis" 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 test reagents, imaging techniques, pathological staining techniques, molecular detection methods, etc.). It cannot independently complete a full diagnosis and must be combined with core clinical information such as the patient's medical history and physical examination. This 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 the determination of "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 "auxiliary diagnosis" is to reduce diagnostic uncertainty, improve diagnostic accuracy, shorten the diagnostic cycle, and provide reliable support for diagnostic decisions.

[0029] The term "identity percentage" refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when two sequences are optimally aligned. Amino acid sequence identity percentage alignment can be performed using various methods within the relevant technical field, such as software well-known in the art, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA.

[0030] The TNFAIP3 (tumor necrosis factor-α-inducible protein 3) gene is located on chromosome 6q23. Transcription and translation produce the TNFAIP3 protein (also known as zinc finger protein A20), a 790-amino acid protein with a relative molecular mass of 90 kDa. TNFAIP3 is an important negative regulatory molecule in the immune system, widely involved in immune regulation. It limits inflammatory responses and maintains immune homeostasis by inhibiting the nuclear factor κB signaling pathway. Mutations in the TNFAIP3 gene can lead to TNFAIP3 protein dysfunction, thereby disrupting the normal regulatory mechanisms of the immune system. This may be associated with the occurrence and development of various autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, and ulcerative colitis.

[0031] This application, through comparative analysis of the cerebrospinal fluid (CSF) of patients with symptoms of neurological autoimmune diseases and healthy individuals, discovered that, compared to the CSF of healthy individuals, the CSF of these patients specifically contained anti-TNFAIP3 autoantibodies. Anti-TNFAIP3 autoantibodies are a type of anti-neuronal cell antibody and can serve as an effective marker for diagnosing neurological autoimmune diseases. They can differentiate between neurological autoimmune diseases and other autoimmune diseases, especially autoimmune encephalitis, Guillain-Barré syndrome, and other autoimmune diseases.

[0032] Based on this, this application proposes the use of a reagent capable of detecting anti-TNFAIP3 autoantibodies in the preparation of products for detecting and / or assisting in the diagnosis of autoimmune diseases of the nervous system.

[0033] The reagents for detecting anti-TNFAIP3 autoantibodies in the embodiments of this application will be described in detail below.

[0034] The reagent for detecting anti-TNFAIP3 autoantibodies according to embodiments of this application includes: (1) TNFAIP3 protein; (2) Biomaterials expressing the TNFAIP3 protein; (3) An antigenic peptide capable of binding to the anti-TNFAIP3 autoantibody; (4) A solid-phase carrier coupled with at least one of the antigens (1) to (3); The amino acid sequence of the TNFAIP3 protein is the amino acid sequence shown in SEQ ID NO:1 or the amino acid sequence shown in SEQ ID NO:1 after modification or mutation, which can recognize the amino acid sequence of the TNFAIP3 autoantibody.

[0035] The reagent for detecting anti-TNFAIP3 autoantibodies according to the embodiments of this application enables qualitative or quantitative analysis of anti-TNFAIP3 autoantibodies. It is easy to operate and can detect and / or assist in the diagnosis of neurological autoimmune diseases, improve the accuracy of neurological autoimmune diseases, thereby finding the pathogenic key of neurological autoimmune diseases such as AE and GBS, solving the diagnostic and treatment dilemma, and is of great significance to clinical diagnosis and treatment problems.

[0036] Optionally, according to one embodiment of this application, the TNFAIP3 protein is an immunogenic polypeptide capable of binding to the TNFAIP3 autoantibody, and the polypeptide is a polymer having two or more amino acids.

[0037] In other words, the TNFAIP3 protein according to this application is an immunogenic polypeptide capable of binding to TNFAIP3 autoantibodies. The polypeptide is preferably a polymer of ≥2 amino acids, more preferably a polymer of 2 to 70 amino acids, specifically a polymer of 2, 3, 4, 5, 6, 7, 8, 10, 12, 20, 30, 40, 50, 60, or 70 amino acids. Optionally, the polypeptide contains one or more epitopes derived from the protein TNFAIP3.

[0038] In some embodiments, the TNFAIP3 protein may be a fusion protein of TNFAIP3 protein and other amino acids. The other 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. These other 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. The purification includes affinity chromatography, molecular sieve chromatography, ion exchange chromatography, or hydrophobic chromatography.

[0039] Optionally, in some specific embodiments of this application, the biological material expressing the TNFAIP3 protein includes any one or more of recombinant vectors, cells, and tissues.

[0040] The recombinant vector may include any one or more of the pTriEx series vectors, pCDNA3 series vectors, pET series vectors, or pBac series vectors.

[0041] The cells may include bacterial cells or eukaryotic cells, wherein the bacterial cells include Escherichia coli cells, and the eukaryotic cells include at least one of immortalized human cells, insect cells, or yeast. In some embodiments, the cells may be neuronal cells, HEK293 cells, HeLa cells, CHO cells, Pichia pastoris, Saccharomyces cerevisiae, sf9, BL21, or Rosetta, etc.

[0042] The tissue includes mammalian brain tissue, wherein the mammal is selected from any one or more of the following: human, rat, primate, mouse, goat, horse, sheep or cow.

[0043] Optionally, according to some embodiments of this application, the solid-phase carrier includes any one or more of the following: chip, enzyme-labeled plate, membrane carrier, microsphere carrier, and microplate.

[0044] In some optional embodiments of this application, the amino acid sequence of the TNFAIP3 protein has at least 70% identity with the amino acid sequence shown in SEQ ID NO:1. The at least 70% includes: 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99% or more, or 100%.

[0045] Optionally, in some specific embodiments of this application, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1 includes any one of (I) to (III): I) The nucleotide sequence shown in SEQ ID NO:2; II) The nucleotide sequence identity with SEQ ID NO:2 is ≥70%, and the amino acid sequence that can express the autoantibody that recognizes TNFAIP3 is expressed; After mutation of the nucleotide sequence in (III), (I), or (II), an amino acid sequence that recognizes TNFAIP3 autoantibodies can be expressed.

[0046] The sequence information of SEQ ID NO:1 and SEQ ID NO:2 involved in this document is as follows: SEQ ID NO.1: MAEQVLPQALYLSNMRKAVKIRERTPEDIFKPTNGIIHHFKTMHRYTLEMFRTCQFCPQFREIIHKALIDRNIQATLESQKKLNWCREVRKLVALKTNGDGNCLMHATSQYMWGVQDTDLVLRKALFSTLKETDTRNFKFRWQLESLKSQEFVETGLCYDTRNWNDEWDNLIKMASTDTPMARSGLQYNSLEEIHIFVLCNILRRPIIVISDKMLRSLESGSNFAPLKVGGIYLPLHWPAQECYRYPIVLGYDSHHFVPLVTLKDSGPEIRAVPLVNRDRGRFEDLKVHFLTDPENEMKEKLLKEYLMVIEIPVQGWDHGTTHLINAAKLDEANLPKEINLVDDYFELVQHEYKKWQENSEQGRREGHAQNPMEPSVPQLSLMDVKCETPNCPFFMSVNTQPLCHECSERRQKNQNKLPKLNSKPGPEGLPGMALGASRGEAYEPLAWNPEESTGGPHSAPPTAPSPFLFSETTAMKCRSPGCPFTLNVQHNGFCERCHNARQLHASHAPDHTRHLDPGKCQACLQDVTRTFNGICSTCFKRTTAEASSSLSTSLPPSCHQRSKSDPSRLVRSPSPHSCHRAGNDAPAGCLSQAARTPGDRTGTSKCRKAGCVYFGTPENKGFCTLCFIEYRENKHFAAASGKVSPTASRFQNTIPCLGRECGTLGSTMFEGYCQKCFIEAQNQRFHEAKRTEEQLRSSQRRDVPRTTQSTSRPKCARASCKNILACRSEELCMECQHPNQRMGPGAHRGEPAPEDPPKQRCRAPACDHFGNAKCNGYCNECFQFKQMYG SEQ ID NO.2: Based on the reagents for detecting anti-TNFAIP3 autoantibodies described above, this application proposes the use of the reagents for detecting anti-TNFAIP3 autoantibodies according to any of the above embodiments in the preparation of products for detecting and / or assisting in the diagnosis of autoimmune diseases of the nervous system.

[0047] In other words, this application also provides an application of a reagent for detecting anti-TNFAIP3 autoantibodies, particularly in the preparation of products for detecting and / or assisting in the diagnosis of autoimmune diseases of the nervous system, wherein the symptoms of said autoimmune diseases of the nervous system include one or more of the following: acute symmetrical limb weakness, muscle weakness, mild sensory abnormalities in the distal extremities, fever, headache, mental and behavioral abnormalities, altered consciousness and seizures, sensory and motor disorders, and cranial nerve dysfunction.

[0048] Optionally, the neurological autoimmune diseases include autoimmune encephalitis and Guillain-Barré syndrome.

[0049] Based on this, this application also proposes a kit for detecting and / or assisting in the diagnosis of autoimmune diseases of the nervous system, including a reagent for detecting anti-TNFAIP3 autoantibodies according to any one of the above embodiments.

[0050] Optionally, the kit may also include labeled antibodies, including FITC-labeled antibodies and Alexa Fluor dye-labeled antibodies, horseradish peroxidase-labeled antibodies, and alkaline phosphatase-labeled antibodies.

[0051] In other words, this application also provides a product for diagnosing or assisting in the diagnosis of autoimmune diseases of the nervous system, comprising: the reagent for detecting anti-TNFAIP3 autoantibodies as described in any of the foregoing embodiments.

[0052] In some embodiments, the product may also include markers or a reagent kit.

[0053] Optionally, the kit can be a kit for detection based on enzyme-linked immunosorbent assay (ELISA) or a kit for detection based on protein immunoblotting.

[0054] In some embodiments, the kit may further include one or more of labeled antibodies, reaction buffers, and sample diluents. This application does not impose strict requirements on the specific composition of the labeled antibodies, buffers, and sample diluents; conventional selections are acceptable.

[0055] This application establishes a kit for diagnosing autoimmune diseases of the nervous system, using reagents for detecting anti-TNFAIP3 autoantibodies as the main component. This kit can qualitatively or quantitatively analyze anti-TNFAIP3 autoantibodies, thereby diagnosing autoimmune diseases of the nervous system, and has a particularly auxiliary diagnostic role. When using the kit for detection, this application does not have strict requirements on the detection method used; any method well-known to this application 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.

[0056] Furthermore, embodiments of this application also provide biological materials selected from any of the following: (A) A nucleic acid molecule encoding the TNFAIP3 protein described in any of the foregoing embodiments; (B) A carrier containing the nucleic acid molecule; (C) Cells containing the carrier.

[0057] In some embodiments, the nucleotide sequence encoding the amino acid sequence of the TNFAIP3 protein is the nucleotide sequence shown in SEQ ID NO.2.

[0058] Optionally, the nucleotide sequence of the TNFAIP3 protein has at least 70% identity with the sequence shown in SEQ ID NO:2. The at least 70% includes: 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99% or more, or 100%.

[0059] In some embodiments, the carrier and cells are the same as those described in any of the foregoing embodiments, and will not be repeated here.

[0060] Therefore, this application provides the application of reagents for detecting anti-TNFAIP3 autoantibodies in the preparation of detection and / or auxiliary diagnosis of autoimmune diseases of the nervous system, especially autoimmune encephalitis and Guillain-Barré syndrome, and the corresponding detection kits.

[0061] This application establishes a kit for detecting and / or assisting in the diagnosis of neurological diseases, using reagents for detecting anti-TNFAIP3 autoantibodies as the main component. The kit is capable of qualitative or quantitative analysis of anti-TNFAIP3 autoantibodies, is easy to operate, and can detect and / or assist in the diagnosis of neurological autoimmune diseases, thereby improving the accuracy of detection and / or auxiliary diagnosis of neurological autoimmune diseases, especially autoimmune encephalitis and Guillain-Barré syndrome.

[0062] In summary, this application uses anti-TNFAIP3 autoantibodies as biomarkers for the diagnosis of autoimmune encephalitis and Guillain-Barré syndrome. Anti-TNFAIP3 autoantibodies are closely related to autoimmune diseases of the nervous system, especially autoimmune encephalitis and Guillain-Barré syndrome, and can be used for their diagnosis. This application uses anti-TNFAIP3 autoantibodies as biomarkers. By detecting these autoantibodies and combining the results with the patient's clinical manifestations, auxiliary examinations, and disease biomarker detection results, this application can determine whether the patient has the autoimmune diseases of the nervous system described in this application or rule out the possibility of another autoimmune disease of the nervous system, helping to select a more appropriate treatment plan or drug for the patient.

[0063] To further illustrate the technical solution of this application, the following description, in conjunction with the accompanying drawings and specific embodiments, details the new biomarkers provided by this application for auxiliary diagnosis of autoimmune diseases of the nervous system, but these should not be construed as limiting the scope of protection of this application.

[0064] It should be noted that the cerebrospinal fluid samples studied in the following embodiments of this application were all from the People's Hospital of Xinjiang Uygur Autonomous Region. The discovery process of anti-TNFAIP3 autoantibodies was described in detail by including cerebrospinal fluid samples from 37 patients with neurological diseases, 31 patients with other diseases, and 10 healthy subjects. Information on the patients and healthy subjects included in this study is shown in Table 1. Table 1. Information on patients and healthy participants included in this study. The inclusion criteria for patients with neurological disorders in Example 1 of this application are as follows: (1) Symptoms of an autoimmune disease of the nervous system, including one or more of the following: acute symmetrical limb weakness, muscle weakness, mild sensory abnormalities in the distal extremities, fever, headache, mental and behavioral abnormalities, altered consciousness and seizures, sensory and motor disorders, and cranial nerve dysfunction. (2) The autoantibodies against the following antigens were detected by cell-based immunofluorescence assay (CBA): NMDAR, AMPA1, AMPA2, LGI1, CASPR2, GABABR, DPPX, IgLON5, GlyR, GABAARα1, GABAARγ2, GABAARβ3, GM1, GM2, GM3, GM4, GD1a, GD1b, GD2, GD3, GT1a, GT1b, GQ1b and sulfates. All results were negative. (3) Age ≥ 14 years; (4) Exclude hereditary diseases (mitochondrial encephalopathy, methylmalonic acidemia, adrenoleukodystrophy), neurodegenerative diseases (Lewy body dementia, frontotemporal dementia, multiple system atrophy and hereditary cerebellar degeneration), etc.

[0065] Example 1: Screening of the nervous system autoantibody profile Using a biodetector chip, samples from 37 disease study group participants, 31 disease control group participants, and 10 healthy subjects were screened for an expanded antibody profile of neurological autoantibodies. The reported neurological autoantibody profile included 52 antigens (different from the antibodies described in the patient inclusion criteria above), namely SNAP91, CACNA1A, MX1, SYN1, INA, Drebrin (DBN1), ARHGAP10, NF186, AK5, vimentin, TNFAIP3, ARHGAP17, p0, TRIM9, TRIM67, PDE10A, CA2, SLC3a2, and TNFAI. P3, IDE, Ncam1, TNFAIP3, ARHGAP26, NF140, ANNA3(DACH1), DNM1, mGluR3, NBCe1, MUNC18-1, mGluR5, MAP1b (PCA-2), KLHL11, SEZ6L2, NAE , TRIM46, CHRM1, SAMD14, Neurexin-3α, Homer3, ATP1A3, Septin5, GlyT2, TGM2(TG2), KCNA4, ADAM23, VAMP2, DAGLA, gAchR, GRP78, plexin D1, LGI4, Neurabin-1.

[0066] The specific steps are as follows: (1) Construction of recombinant vectors: The gene sequences encoding the above 52 proteins were ligated into pcDNA3.4 by PCR or artificial synthesis to obtain 52 recombinant vectors. After the constructed recombinant vectors were correctly sequenced, they were extracted for later use. (2) Transfection of target genes: 6cm×6cm 293T cell smears were laid at the bottom of the culture dish and cultured in a 37℃, 5% CO2 cell culture incubator using 10% FBS-DMEM high glucose culture base. A total of 53 dishes were used. When the cell density reached 30% to 40%, the recombinant vectors of 52 corresponding genes and the empty vector pcDNA3.4 were transfected into 293T cells using PEI transfection reagent and labeled. (3) Cell fixation: Cells that have grown for 48 hours after transfection were washed twice with PBS, fixed with acetone for 5 minutes, washed twice with PBS, dried at 45°C for 30 minutes, cut the cell covers to 2.5 mm × 2.5 mm, and attached 53 types of 2.5 mm × 2.5 mm cell covers to a glass slide to prepare a biodetection chip for screening target antigens for later use; (4) Immunofluorescence staining: Cerebrospinal fluid samples from 37 disease study group, 31 disease control group and 10 healthy subjects were incubated onto a biodetector chip using PBST and incubated at room temperature for 1 h. The samples were washed three times with PBST for 5 min each time. FITC-labeled goat anti-human IgG antibody diluted 1:200 was used and incubated at room temperature for 30 min. The samples were washed three times with PBST for 5 min each time. The samples were then observed under a fluorescence microscope.

[0067] The results showed that in the disease study group (n=37), cerebrospinal fluid samples from 17 patients produced significant fluorescence signals with one or more target antigens on the 52 biodetection chips, while no significant antigen-antibody fluorescence reaction was observed in the remaining 20 samples. In the disease control group (n=31), cerebrospinal fluid samples from 12 patients showed significant fluorescence signals with ≥1 target antigen on the chip, while no positive fluorescence reaction was observed in 19 samples. In the healthy subject group (n=10), none of the cerebrospinal fluid samples produced significant fluorescence signals with the target antigens of the 52 biodetection chips.

[0068] To investigate the presence of potential autoantibodies in the cerebrospinal fluid of patients with antibody-negative diseases, this application was designed as follows: Cerebrospinal fluid samples from 20 antibody-negative patients, identified through systematic screening, were collected as the disease study group. Simultaneously, 19 antibody-negative patient samples were used as the disease control group, and 10 healthy subjects were included as the healthy control group. All samples were initially screened using frozen sections of rat brain tissue via tissue immunofluorescence assay (TBA) to identify samples with characteristic fluorescent signals from the disease study group. The selected positive samples will subsequently undergo target antigen identification and verification using the methods detailed in Examples 3 to 7.

[0069] Example 2: Screening of cerebrospinal fluid signals in patients using indirect immunofluorescence based on tissue. 1. Preparation of frozen sections of rat brain tissue: Adult rats were anesthetized, and after the limbs of the rats 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, brain tissue was removed and fixed with methanol for 10–30 min. The sample was transferred to 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 the specimen was placed in the freezing stage of a -20°C cryostat. When the tissue turned slightly white, a thin layer of OCT was applied to the surface of the specimen, and the specimen was frozen for another 20 min to obtain frozen sections of rat brain tissue.

[0070] 2. Sample incubation: Cerebrospinal fluid samples from 20 patients in the disease study group, 19 patients in the disease control group, and 10 healthy subjects were incubated onto frozen sections of rat brain tissue. The samples were incubated overnight at 4°C, washed three times with PBST for 5 minutes each time, and then incubated with diluted FITC-labeled anti-human secondary antibody at room temperature for 30 minutes. The samples were washed three times with PBST for 5 minutes each time, stained with DAPI, and observed under a fluorescence microscope.

[0071] Results: In cerebrospinal fluid (CSF) samples from 20 patients in the disease study group, characteristic similar fluorescence patterns were observed in the Purkinje region of the cerebellum in 4 samples from frozen sections of rat brain tissue. In cerebrospinal fluid samples from 19 patients in the disease control group, punctate fluorescence signals were observed in the molecular layer and Purkinje cell region of rat brain tissue in 1 sample, while the remaining 18 samples did not show specific binding reactions with rat brain tissue sections. No positive fluorescence signals were observed in CSF samples from 10 healthy subjects. These results suggest that the CSF of the 4 patients in the disease study group may contain unknown autoantibodies that can target and bind to neuronal proteins. Typical staining results of the 4 samples from the disease study group showing characteristic fluorescence patterns in the Purkinje region of the cerebellum, as well as some samples from the disease control group (n=19) and healthy subjects (n=10), are shown below. Figure 1 As shown.

[0072] Among them, the clinical characteristics of the four patients with characteristic similar fluorescence patterns observed in Example 2, namely Patient 1, Patient 2, Patient 3, and Patient 4, are as follows: Patient 1: DSC-1, 58 years old, male, discharged diagnosis Guillain-Barré syndrome, chief complaint of numbness in the extremities and double vision for 9 days. Physical examination revealed incomplete motor function in both eyes, diminished tendon reflexes in the extremities, slightly diminished bilateral pharyngeal reflexes, muscle strength grade IV+ in both upper extremities, and a suspected positive result for the eye-closed standing test. Ancillary examinations: Cranial MRI showed an ischemic lesion near the frontal horn of the left ventricle; electromyography / NCV indicated damage to multiple nerve axons or myelin sheaths, decreased amplitude of bilateral peroneal nerve H-reflexes, and suspected neurogenic damage to multiple muscle groups; cerebrospinal fluid routine examination showed elevated protein levels, cytology showed predominantly lymphocytes and monocytes, intracranial pressure was normal, and viral testing showed positive results for herpes simplex virus, cytomegalovirus, and Epstein-Barr virus capsid antigen IgG.

[0073] Patient 2: DSC-2, 46 years old, male, diagnosed with acute Guillain-Barré syndrome, chief complaint of sudden onset of dizziness, unsteady gait, and blurred vision for 5 days. Physical examination revealed vertical gaze palsy in both eyes and limited eye movement in multiple directions. Head MRI showed multiple ischemic and infarct lesions in the subcortical areas of the bilateral frontal lobes and the centrum semiovale. Cerebrospinal fluid test showed positive for cytomegalovirus IgG.

[0074] Patient 3: DSC-3, 47 years old, female, diagnosed with viral encephalitis and ANCA-associated vasculitis, chief complaint of fever with urinary frequency and urgency for 1 week and somnolence for 1 day. Physical examination revealed bilateral superficial sensory loss, grade VI muscle strength in both lower extremities, and neck stiffness of 3 fingerbreadths. Enhanced MRI of the head showed suspicious enhancement of the meninges, mainly in the bilateral cerebellum, and intracranial infection could not be ruled out. Cerebrospinal fluid examination showed a lymphocyte percentage as high as 79.5%, positive cytomegalovirus IgG, incomplete electromyography, and missing routine cerebrospinal fluid data.

[0075] Patient 4: DSC-4, 28 years old, male, discharged with Guillain-Barré syndrome and fecal occult blood. Neurological examination revealed proximal muscle weakness of V in both upper limbs. - Grade III peripheral nerve injury, grade III distal nerve injury, grade III proximal nerve injury and grade I distal nerve injury in both lower limbs; normal muscle tone; no tendon reflexes elicited in both lower limbs. Head MRI (DWI+MRA) showed no significant abnormalities; electromyography / nerve conduction velocity indicated damage to the axons and myelin sheaths of peripheral nerve motor fibers, with normal F waves and decreased H-reflex amplitude; electromyography showed neurogenic damage. Cerebrospinal fluid examination showed elevated protein (1.02 g / L), positive cytomegalovirus IgG, and no other abnormalities in routine and biochemical indicators.

[0076] Example 3: Using immunoprecipitation (IP) to capture antigens recognized by autoantibodies in patient cerebrospinal fluid samples. This embodiment uses cerebrospinal fluid (CSF) samples from 4 disease study group patients (DSC-1 to DSC-4) who showed similar fluorescence patterns in the Purkinje region of the cerebellum in Example 2, 19 disease control CSF samples (DCC-1 to DCC-19), and 10 healthy control CSF samples (HCC-1 to HCC-10) for IP experiments. The specific operating steps are as follows: Tissue lysis: Rat brain tissue was minced, transferred to a mortar, and ground into a fine powder using liquid nitrogen. The powder was then aliquoted into 50 portions, each approximately 50 mg. Each sample was added to 500 μL of RIPA lysis buffer, followed by a 1× protease inhibitor concentration. The mixture was sonicated (10% power, 3 sec on, 6 sec off) for 1 min. After sonication, the tissue was lysed on ice for 30 min, with shaking every 5 min. The mixture was then centrifuged at 15000 rpm for 30 min, and the supernatant was collected for later use. Serum incubation: 50 μL of cerebrospinal fluid from 4 patients in the disease study group, 19 disease controls, and 10 healthy subjects were added to the supernatant of 33 extracted mouse brain tissue extracts and incubated overnight at 4°C to obtain 33 mixed supernatants of brain tissue after reaction with cerebrospinal fluid.

[0077] Magnetic bead binding: Take 50 μL of magnetic beads for each reaction, wash twice with magnetic bead binding / washing buffer (50 mM Tris, 150 mM NaCl, 0.1% Triton X-100) for 5 min each time, add 1% BSA to block for 2 h; discard the blocking BSA, add the mixed supernatant collected in step (2) (i.e. antigen-antibody complex), and incubate overnight at 4°C by rotation; Elution: Discard the supernatant and wash three times with magnetic bead binding / washing buffer, 5 min each time; first add 5×SDS-PAGE loading buffer to the eluent, then add DTT to a final concentration of 0.01M, heat at 95℃ for 5 min, then add iodoacetamide to a final concentration of 2wt.%, and let stand at room temperature for 15 min to obtain the IP elution sample of 33 cerebrospinal fluid samples; SDS-PAGE: Prepare 8-15% gradient gels using a gradient gel generation system, and load 20 μL of each of the IP eluted samples obtained above. Silver staining: Silver staining was performed using the THERMO Pierce™ Silver Stain Kit. The target band was observed. The silver staining results are as follows: Figure 2 As shown.

[0078] according to Figure 2 The silver staining results shown indicate that, after IP and silver staining analysis, samples from the four patients in the disease study group (numbered DSC-1, DSC-2, DSC-3, and DSC-4, respectively) all showed suspected specific protein bands in the relative molecular mass range of 70-100 kDa. Figure 2 As indicated by the red arrow in the middle, one patient (numbered DCC-1) in the 19 disease control groups also showed a suspected specific protein band in the same location in their cerebrospinal fluid sample. No obvious specific protein band was found in this location in the remaining 18 disease control group samples and 10 healthy subject samples (only the silver staining results of the 10 disease control group samples are shown here). These results suggest that the target protein identified in the samples from these four disease study group patients and the sample from the one disease control group patient (DCC-1) may be the same protein.

[0079] Example 4: Mass Spectrometry Results Analysis and Validation Using a clean blade, bands with relative molecular masses between 70 and 100 kDa were cut from the IP gels of patient DSC-1 and healthy subject HCC-1 samples from Example 3. These bands were then subjected to mass spectrometry analysis by Beijing Novogene. The results are as follows: Figure 3 As shown. Based on the molecular weight (corresponding to the silver staining band) and the protein quantification value of the corresponding sample (excluding keratin), TNFAIP3 protein was selected as the target antigen for verification (TNFAIP3, tumor necrosis factor α-induced protein, expressed in the cytoplasm and nucleus, moderately expressed in neuronal cells, with a molecular weight of approximately 90KD). The amino acid sequence of this protein is shown in SEQ ID NO:1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:2.

[0080] 2. Validation of mass spectrometry results Referring to Example 3, the IP elution samples from patients DSC-1 and DSC-2, and healthy subjects HCC-1 and HCC-2, were subjected to SDS-PAGE electrophoresis using a wet transfer method at 300 mA for 120 min. The membrane was then blocked with 5% skim milk at room temperature for 1 h. A commercially available TNFAIP3 antibody (manufacturer: Santa, catalog number: sc-166692) was diluted 1:200 using TBST and incubated overnight at 4°C. The membrane was washed three times with TBST for 5 min each time. HRP-labeled anti-mouse IgG antibody was added and incubated at room temperature for 1 h. The membrane was washed three times with TBST for 5 min each time. Chemiluminescent buffer was added, and the results were observed. The results are as follows: Figure 4 As shown.

[0081] according to Figure 4 The results show that in the immunoprecipitates (IP elution samples) captured from rat brain tissue lysates of DSC-1 and DSC-2 patients, bands reacting with commercially available TNFAIP3 antibody were present, with the target protein band ranging from 70 kDa to 100 kDa. Figure 4 (middle arrow position), while the immunosuppressants (IP elution samples) captured from rat brain tissue lysates of healthy subjects HCC-1 and HCC-2 did not show bands reacting with commercially available TNFAIP3 antibodies, demonstrating the presence of autoantibodies reacting with TNFAIP3 protein in the cerebrospinal fluid of patients DSC-1 and DSC-2.

[0082] Example 5: Western blotting confirms the presence of anti-TNFAIP3 antibodies in patient cerebrospinal fluid samples. 1. Construction of recombinant vectors Based on the cDNA sequence of human TNFAIP3 protein (GenBank accession number NM_006290.4), a coding gene with a 6×His tag at its C-terminus was synthesized by Shanghai Sangon Biotech Co., Ltd., and cloned into the pcDNA3.4 plasmid vector to obtain the recombinant vector pCDNA3.4- TNFAIP3-6His After the constructed recombinant vector is correctly sequenced, it is extracted and ready for use.

[0083] 2. Cell transfection HEK293T cells were cultured in 10% FBS-DMEM high-glucose medium. Once the cells reached confluence, they were passaged at a ratio of 1:5 to 1:6 and cultured overnight at 37°C in a 5% CO2 incubator. When the cell density reached 30%–40%, pcDNA3.4- TNFAIP3-6His The recombinant vector was transferred into the cells and cultured for another 48 hours.

[0084] 3. Take one dish of 293T cells overexpressing TNFAIP3, discard the supernatant, scrape the cells into a 1.5 mL centrifuge tube using a cell scraper, centrifuge at 800 rpm at room temperature to remove the supernatant, add 200 μL PBS, and lyse using RIPA lysis buffer. Centrifuge again to obtain the supernatant overexpressing TNFAIP3 protein. Take one dish of cells transfected with empty pcDNA3.4 to prepare the control protein, using the same conditions and methods as for the preparation of the overexpressing TNFAIP3 protein.

[0085] 4. Protein Validation Overexpressing TNFAIP3 protein and control protein were subjected to SDS-PAGE gel electrophoresis. After electrophoresis, wet transfer was performed. The membrane was blocked with 5% skim milk powder at room temperature for 1 hour. Commercial TNFAIP3 antibody and commercial 6×His antibody (manufacturer: abcam, catalog number: ab18184) were diluted 1:200 using TBST and incubated overnight at 4°C. The next day, the membrane was washed three times with TBST for 5 minutes each time. HRP-labeled anti-mouse secondary antibody was added and incubated at room temperature for 1 hour. The membrane was washed three times with TBST for 5 minutes each time. Chemiluminescence solution was added for development and photographing. The photographic results are shown below. Figure 5 As shown.

[0086] Depend on Figure 5 The results show that both the commercial TNFAIP3 antibody and the 6×His antibody showed specific signals at 90KD in the lanes overexpressing TNFAIP3 protein, while no signal was observed at the same position in the control protein lane, indicating that the preparation of TNFAIP3 protein overexpression was successful.

[0087] 5. Western blotting confirmed the presence of anti-TNFAIP3 antibodies in the patient's cerebrospinal fluid samples. Referring to step 4, SDS-PAGE was performed using overexpressed TNFAIP3 protein and control protein. After electrophoresis, the membrane was transferred and blocked. Cerebrospinal fluid samples from patients DSC-1 to DSC-4 in the disease study group, patients DSC-1 in the disease study group, and HCC-1 to HCC-4 from four healthy subjects were diluted 1:5 using TBST as primary antibodies and incubated overnight at 4°C. Then, HRP-labeled anti-human secondary antibody was added for incubation, and finally, chemiluminescent solution was added for color development. The color development results are shown below. Figure 6 As shown.

[0088] observe Figure 6 The colorimetric results show that in the cerebrospinal fluid samples of the four patients (DSC-1 to DSC-4) in the disease study group, clear and specific bands appeared near the relative molecular mass of 90 kDa on the electrophoretic lanes overexpressing TNFAIP3 protein. Furthermore, the molecular weight position of these bands completely coincided with the target band position obtained from previous immunoprecipitation (IP) combined with silver staining (e.g., ...). Figure 2 (As shown in the figure). This result clearly indicates that anti-TNFAIP3 autoantibodies are present in the cerebrospinal fluid of these 4 patients. After testing the cerebrospinal fluid samples of the 4 healthy subjects under the same Western blot conditions, no specific band appeared at the 90kDa position of the lane overexpressing TNFAIP protein. Combined with the previous silver staining experiment showing no band in the healthy subjects, it can be confirmed that the cerebrospinal fluid of the healthy subjects does not contain anti-TNFAIP3 autoantibodies. For the 1 disease control sample (DCC-1), although there was a suspected band at the 90kDa position in the silver staining gel image of its previous IP elution sample, no specific band appeared at the corresponding position of the lane overexpressing TNFAIP3 protein in this Western blot validation. This suggests that the band shown in the previous silver staining was not a product of the specific binding of TNFAIP3 protein and the sample antibody. Therefore, it can be determined that the cerebrospinal fluid of the disease control group DCC-1 does not contain anti-TNFAIP3 autoantibodies.

[0089] Example 6: Verification of staining signal in patient's cerebellar cerebrospinal fluid at Purkinje location. 1. Co-localization of staining between the patient's cerebrospinal fluid and commercially available TNFAIP3 antibody. Using frozen sections of rat brain tissue from Example 1, following the steps outlined in Example 1, cerebrospinal fluid from DSC-1 to DSC-3 patients in the disease study group was added and incubated overnight at 4°C. The tissue was washed three times with PBST, and FITC-labeled anti-human IgG secondary antibody was added for signal amplification. Then, diluted commercial TNFAIP3 antibody was added for co-staining, and Alexa Fluor 594-labeled anti-human IgG fluorescent secondary antibody was used for signal amplification. After washing, the results were observed under a microscope. The results are as follows: Figure 7 As shown.

[0090] from Figure 7The staining results show that the cerebrospinal fluid signals of DSC-1 to DSC-3 patients in the disease study group at the Purkinje position of the cerebellum can colocalize with the signals of commercially available TNFAIP3 antibody, indicating that there are autoantibodies in the cerebrospinal fluid of these three patients that can recognize the cerebellar Purkinje target antigen TNFAIP3.

[0091] 2. Neutralization experiments confirmed the presence of anti-TNFAIP3 antibodies in the patient's cerebrospinal fluid samples. The overexpressed TNFAIP3 protein and control protein prepared in Example 5 were used as the neutralizing and control proteins in the neutralization experiment, respectively. Two 100 μL portions of cerebrospinal fluid from DSC-1 to DSC-3 patients in the disease study group were taken from each portion. 20 μL of the neutralizing and control proteins were added to each portion, and the mixtures were incubated on a shaker at room temperature for 1 hour. After pre-neutralizing the cerebrospinal fluid from DSC-1 to DSC-3 patients, the mixtures were transferred to frozen sections of rat brain tissue and incubated at room temperature for 1 hour. After washing three times with PBST, FITC-labeled fluorescent secondary antibody was added, and the mixtures were incubated at room temperature for 30 minutes. After washing three times with PBST, the results were observed under a fluorescence microscope. The results are as follows: Figure 8 As shown.

[0092] from Figure 8 The results show that after neutralizing the cerebrospinal fluid of patients DSC-1 to DSC-3 with overexpressed TNFAIP3 protein, the immune response in the Purkinje region of the cerebellum in frozen sections of rat brain tissue was significantly weakened, indicating that the cerebrospinal fluid of these three patients DSC-1 to DSC-3 contained autoantibodies that recognize TNFAIP3 protein.

[0093] Example 7: Verification of staining signal of patient cerebrospinal fluid on TNFAIP3 overexpressing smears 1. Preparation of TNFAIP3 overexpression slices The recombinant vector pcDNA3.4- prepared using the examples TNFAIP3-6His Transfection was performed. 48 hours after transfection, cells overexpressing TNFAIP3 protein were obtained. The culture medium was discarded, and the cells were washed twice with PBS buffer. Acetone was added for fixation for 30 minutes, the acetone was discarded, and the cells were dried at 42°C for 30 minutes. The cell slides were cut to 2.5 mm × 2.5 mm size and pasted together for later use, thus obtaining cell slides overexpressing TNFAIP3 protein. Control cell slides were transfected with empty pcDNA3.4, and their preparation method was the same as that for the TNFAIP3 protein overexpressing cell slides.

[0094] 2. Co-localization of staining between the patient's cerebrospinal fluid and commercially available TNFAIP3 antibody. CBA staining: Cell smears overexpressing TNFAIP3 protein were incubated overnight at 4°C using undiluted cerebrospinal fluid from patients' DSC-1 to DSC-4. After washing three times with PBST, FITC-labeled fluorescent secondary antibody was added, and the cells were incubated at room temperature for 30 minutes. After washing three times with PBST, diluted commercial TNFAIP3 antibody was added for co-staining. Signal amplification was achieved using Alexa Fluor 594-labeled anti-human IgG fluorescent secondary antibody. The results were observed under a microscope after washing. The results are as follows: Figure 9 As shown.

[0095] from Figure 9 The staining results shown indicate that the TNFAIP3 overexpression signal in the cerebrospinal fluid of patients DSC-1 to DSC-4 can colocalize with the signal of commercial TNFAIP3 antibody, indicating that autoantibodies that can recognize the target antigen TNFAIP3 protein are present in the cerebrospinal fluid of patients DSC-1 to DSC-4.

[0096] 3. Neutralization experiments confirmed the presence of anti-TNFAIP3 antibodies in the patient's cerebrospinal fluid samples. The overexpressing TNFAIP3 protein and control protein prepared in Example 5 were used as the neutralizing and control proteins in the neutralization experiment, respectively. Two portions of 100 μL of cerebrospinal fluid from patients DSC-1 to DSC-3 were taken from each portion, with 20 μL of neutralizing and control protein added to each. The mixtures were incubated on a shaker at room temperature for 1 hour. This pre-neutralized cerebrospinal fluid from patients DSC-1 to DSC-3 was then transferred to TNFAIP3-overexpressing cell slides for CBA staining. The staining results were observed as follows: Figure 10 As shown.

[0097] according to Figure 10 The results show that after neutralizing the cerebrospinal fluid of patients DSC-1 to DSC-4 with TNFAIP3 overexpression protein, the immune response on the cell smears of patients with TNFAIP3 overexpression protein was significantly weakened, indicating that the cerebrospinal fluid of patients DSC-1 to DSC-4 all contained autoantibodies that recognize TNFAIP3 protein.

[0098] To verify the diagnostic efficacy of anti-TNFAIP3 autoantibodies, Examples 8-9 of this application collected cerebrospinal fluid samples submitted by the People's Hospital of Xinjiang Uygur Autonomous Region from January 2024 to April 2025. A total of 271 patients with neurological diseases and 10 healthy subjects were included as research samples. Recombinant cells overexpressing TNFAIP3 protein were used for indirect immunofluorescence and Western blotting to screen for cases related to anti-TNFAIP3 antibodies and to verify the association between anti-TNFAIP3 antibodies and specific diseases.

[0099] Example 8: Detection of anti-TNFAIP3 antibody using cell-based immunofluorescence assay 1. Construction of recombinant vectors Based on the cDNA sequence of human TNFAIP3 protein (GenBank accession number NM_006290.4), a coding gene with a 6×His tag at its C-terminus was synthesized by Shanghai Sangon Biotech Co., Ltd., and cloned into the pcDNA3.4 plasmid vector to obtain the recombinant vector pCDNA3.4. -TNFAIP3-6His After the constructed recombinant vector is correctly sequenced, it is extracted and ready for use.

[0100] 2. Cell transfection HEK 293T cells were cultured in 10% FBS-DMEM high-glucose medium. Once the cells reached confluence, they were passaged at a ratio of 1:5 to 1:6 and cultured overnight at 37°C in a 5% CO2 incubator. When the cell density reached 30%–40%, pcDNA3.4- TNFAIP3-6His The recombinant vector was transferred into the cells and cultured for another 48 hours.

[0101] 3. Preparation of TNFAIP3 overexpression slices 48 h after transfection, cells overexpressing TNFAIP3 protein were obtained. The culture medium was discarded, and the cells were washed twice with PBS buffer. Acetone was added for fixation for 30 min, the acetone was discarded, and the cells were dried at 42℃ for 30 min. The cell slides were cut to 2.5 mm × 2.5 mm size and pasted together for later use, thus obtaining cell slides overexpressing TNFAIP3 protein. Control cell slides were transfected with empty pcDNA3.4, and their preparation method was the same as that for the TNFAIP3 overexpressing cell slides.

[0102] 4. Immunofluorescence assay for anti-TNFAIP3 antibodies: Cerebrospinal fluid from 271 patients with neurological diseases and 10 healthy subjects were used to incubate TNFAIP3-overexpressing cell slides. The slides were incubated overnight at 4°C, washed three times with PBST, and then incubated with FITC-labeled fluorescent secondary antibody at room temperature for 30 minutes. After washing three times with PBST, the results were observed under a microscope. The results are shown below. Figure 11 As shown.

[0103] from Figure 11 The staining results show that, among the cerebrospinal fluid (CSF) samples from 271 patients with neurological diseases and 10 healthy subjects, five patients (numbered 1-5) exhibited characteristic fluorescent signals on the TNFAIP3 overexpression slides, indicating the presence of autoantibodies that recognize the target antigen TNFAIP3 protein in their CSF. The remaining patients and the 10 healthy subjects did not show characteristic fluorescent signals on the TNFAIP3 overexpression slides.

[0104] Example 9: Western blotting method for detecting anti-TNFAIP3 antibody 1. Following steps 1 and 2 of Example 8, 293T cells overexpressing TNFAIP3 and cells with empty pcDNA3.4 were cultured for 48 hours.

[0105] 2. Take one dish of 293T cells overexpressing TNFAIP3, discard the supernatant, scrape the cells into a 1.5 mL centrifuge tube using a cell scraper, centrifuge at 800 rpm at room temperature to remove the supernatant, add 200 μL PBS, and lyse using RIPA lysis buffer. Centrifuge again to obtain the supernatant overexpressing TNFAIP3 protein. Take one dish of cells transfected with empty pcDNA3.4 to prepare the control protein, using the same conditions and methods as for the preparation of the overexpressing TNFAIP3 protein.

[0106] 3. The overexpressing TNFAIP3 protein and the control protein were subjected to SDS-PAGE gel electrophoresis. After electrophoresis, wet transfer was performed. The membrane was blocked with 5% skim milk powder at room temperature for 1 hour. Cerebrospinal fluid from 271 patients with neurological diseases and 10 healthy subjects were diluted 1:5 with PBST and incubated overnight at 4°C. The next day, the membrane was washed three times with TBST for 5 minutes each time. HRP-labeled anti-mouse secondary antibody was added and incubated at room temperature for 1 hour. The membrane was washed three times with TBST for 5 minutes each time. Chemiluminescent solution was added for color development and photographing. The photographic results are shown below. Figure 12 As shown.

[0107] from Figure 12 The Western blotting results shown indicate that among the cerebrospinal fluid (CSF) of 271 patients with neurological disorders and 10 healthy subjects, 11 patients (numbered 3, 6–15) exhibited a specific band at 90 kDa in the lane overexpressing TNFAIP3 protein, indicating the presence of autoantibodies that recognize the target antigen TNFAIP3 protein in their CSF. No specific band was observed at 90 kDa in the CSF of the remaining patients with neurological disorders and the 10 healthy subjects.

[0108] Example 10: Clinical association study between anti-TNFAIP3 autoantibodies and patients with neurological diseases Using recombinant cells overexpressing TNFAIP3 protein, indirect immunofluorescence assay and Western blotting were employed to detect anti-TNFAIP3 autoantibodies in the cerebrospinal fluid of 271 patients with neurological diseases. Fifteen samples were identified as positive for anti-TNFAIP3 autoantibodies, as shown in Table 2. Analysis of the detection of anti-TNFAIP3 autoantibodies in samples from 271 patients and 10 healthy subjects yielded statistical results regarding the sensitivity and specificity of anti-TNFAIP3 autoantibodies in neurological autoimmune diseases, as shown in Tables 3 and 4. These results indicate that TNFAIP3 can serve as one of the relevant autoantibody recognition antigens for neurological autoimmune diseases, and detection reagents targeting anti-TNFAIP3 autoantibodies can aid in the auxiliary diagnosis of such diseases.

[0109] Table 2 Summary information of 15 patients with positive anti-TNFAIP3 antibody Table 3. Summary of anti-TNFAIP3 antibody detection results in 271 patients. Table 4. Analysis of various indicators in the anti-TNFAIP3 antibody detection results. The sensitivity and specificity analysis results of the anti-TNFAIP3 autoantibody are as follows: Sensitivity (true positive rate) = TP / (TP + FN) = 11 / 132 ≈ 8.33% Specificity (true negative rate) = TN / (TN + FP) = 135 / 139 ≈ 97.12% Youden's index = Sensitivity + Specificity - 1 ≈ 0.0833 + 0.9712 - 1 = 0.0545 Positive likelihood ratio (LR+) = Sensitivity / (1 - Specificity) ≈ 0.0833 / (1 - 0.9712) ≈ 2.90 Negative likelihood ratio (LR-) = (1 - sensitivity) / specificity ≈ (1 - 0.0833) / 0.9712 ≈ 0.944 Positive predictive value (PPV) = TP / (TP + FP) = 11 / 15 ≈ 73.33% Negative predictive value (NPV) = TN / (TN + FN) = 35 / 256 ≈ 52.73% The above results indicate that anti-TNFAIP3 autoantibodies were mainly detected in patients with autoimmune encephalitis or GBS, but not in healthy subjects, suggesting that this autoantibody has auxiliary diagnostic value for autoimmune encephalitis and GBS and can serve as a potential diagnostic biomarker for these diseases.

[0110] According to the "Expert Consensus on Antibody Detection for Autoimmune Diseases of the Central Nervous System 2022," in autoimmune encephalitis, besides the high positive rate of anti-NMDAR antibody (approximately 54%–80%), the positive rates of other antibodies are 12.8%–22.0% for anti-LGI1 antibody, 1.3% for anti-GABAB receptor antibody, and 0.6% for anti-AMPA receptor antibody. This shows that in AE encephalitis, apart from the high positive rate of anti-NMDAR antibody, the positive rates of other antibodies are not high. In Guillain-Barré syndrome, besides the high positive rates of anti-GM1 antibody (AMAN (IgG, detectable in 30% of patients)), MMN (IgM, detectable in 46% of patients), CIDP (IgM, detectable in 3%), and anti-GQ1b antibody (detectable in 95%–99% of MSF), the detection rates of other antiganglioside antibodies are low.

[0111] The anti-TNFAIP3 antibody described in this application exhibits a similar positive rate to some known biomarkers for autoimmune encephalitis and Guillain-Barré syndrome when used as an adjunct in the diagnosis of these conditions. Detection of TNFAIP3 antibody, combined with the patient's clinical presentation and auxiliary examinations, after ruling out other causes, can aid in the diagnosis of autoimmune encephalitis and Guillain-Barré syndrome. The detectability of anti-TNFAIP3 autoantibody in patients with neurological symptoms, particularly those with autoimmune encephalitis and Guillain-Barré syndrome, indicates that this autoantibody plays an auxiliary role in the diagnosis of neurological diseases, especially autoimmune encephalitis and Guillain-Barré syndrome.

[0112] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments without departing from the scope and spirit of this application. The scope of this application is defined by the appended claims.

Claims

1. The application of a reagent for detecting anti-TNFAIP3 autoantibodies in the preparation of products for detecting and / or assisting in the diagnosis of autoimmune diseases of the nervous system, characterized in that, The reagents include: (1) TNFAIP3 protein; (2) Biomaterials expressing the TNFAIP3 protein; (3) An antigenic peptide capable of binding to the anti-TNFAIP3 autoantibody; (4) A solid-phase carrier coupled with at least one of the antigens (1) to (3); The amino acid sequence of the TNFAIP3 protein is the amino acid sequence shown in SEQ ID NO:1 or the amino acid sequence shown in SEQ ID NO:1 after modification or mutation, which can recognize the amino acid sequence of the TNFAIP3 autoantibody.

2. The application according to claim 1, characterized in that, The TNFAIP3 protein is an immunogenic polypeptide that can bind to the TNFAIP3 autoantibody, and the polypeptide is a polymer having two or more amino acids.

3. The application according to claim 1, characterized in that, The biomaterials include any one or more of recombinant vectors, cells, and tissues.

4. The application according to claim 3, characterized in that, The recombinant vector includes any one or more of the pTriEx series vectors, pCDNA3 series vectors, pET series vectors, or pBac series vectors.

5. The application according to claim 3, characterized in that, The cells include bacterial cells or eukaryotic cells, the bacterial cells including Escherichia coli cells, and the eukaryotic cells including at least one of immortalized human cells, insect cells, or yeast.

6. The application according to claim 3, characterized in that, The tissue includes mammalian brain tissue, wherein the mammal is selected from any one or more of the following: human, rat, primate, mouse, goat, horse, sheep or cow.

7. The application according to claim 1, characterized in that, The solid-phase carrier includes any one or more of the following: chip, enzyme-labeled plate, membrane carrier, microsphere carrier, and microplate.

8. The application according to claim 1, characterized in that, The amino acid sequence of the TNFAIP3 protein has at least 70% identity with the amino acid sequence shown in SEQ ID NO:

1.

9. The application according to claim 1, characterized in that, The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1 includes any one of (I) to (III): I) The nucleotide sequence shown in SEQ ID NO:2; II) The nucleotide sequence identity with SEQ ID NO:2 is ≥70%, and the amino acid sequence that can express the autoantibody that recognizes TNFAIP3 is expressed; After mutation of the nucleotide sequence in (III), (I), or (II), an amino acid sequence that recognizes TNFAIP3 autoantibodies can be expressed.

10. The application according to claim 1, characterized in that, The aforementioned autoimmune diseases of the nervous system include autoimmune encephalitis and Guillain-Barré syndrome.

11. The application according to claim 1, characterized in that, The product in question is a reagent kit.

12. The application according to claim 11, characterized in that, The kit also includes labeled antibodies, including FITC-labeled antibodies, Alexa Fluor dye-labeled antibodies, horseradish peroxidase-labeled antibodies, and alkaline phosphatase-labeled antibodies.

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