Diagnosis and kit for autoimmune nervous system diseases

Abstract

The invention relates to diagnosis and a kit for autoimmune nervous system diseases. The diagnosis and the kit comprise application of a reagent for detecting an anti-EEF1D autoantibody to preparation of a preparation for diagnosing the autoimmune nervous system diseases in a subject.

Description

Technical Field

[0001] The present invention relates to the field of disease diagnosis, and in particular to a diagnosis and kit for autoimmune nervous system diseases. Background Art

[0002] The diagnosis of neurological diseases is a difficult problem in biomedical science because neurological diseases can be caused by various causes, such as infection, genetics, drug abuse, malnutrition, injury, mental illness, immune deficiency and cancer.

[0003] Because the clinical manifestations of neurological diseases are usually disease-specific, it is often difficult to confirm the diagnosis through physical examinations and the medical history provided by the patient. Early diagnosis of neurological diseases is very important. Although there are currently no curable drugs for many neurological diseases, such as Alzheimer's disease and Parkinson's disease, early diagnosis and early intervention can allow patients to obtain drugs that can slow the progression of the disease in the early stages of the disease, thereby delaying the development and deterioration of the disease as much as possible.

[0004] For autoimmune nervous system diseases, the pathogenic mechanism of nervous system autoantibodies is complex, and the clinical manifestations caused are different. With the advancement of science and technology, people's understanding of nervous system autoantibodies has continued to deepen, and more and more nervous system autoantibodies have been discovered. Due to the complexity of nervous system diseases, they are rarely associated with unique clinical manifestations, so it is difficult to make an accurate diagnosis simply by observing or examining the patient. Specific autoantibodies in patients are often closely related to clinical manifestations. Some antibodies have obvious pathogenicity, and some antibodies are involved in the occurrence and development of the disease, providing a basis for the diagnosis of the disease and assisting in typing and prognosis judgment. For example, the detection of some autoantibodies can help distinguish different types of neurological diseases with similar clinical manifestations (differential diagnosis). The detection of some autoantibodies can directly exclude the neurological diseases corresponding to this autoantibody (exclusion diagnosis). Therefore, the detection of nervous system autoantibodies is of great significance for the study of the mechanism, diagnosis and treatment of the disease, and prognosis judgment. Summary of the invention

[0005] The present invention unexpectedly found in the clinic that in autoimmune cerebellar ataxia combined with Sjögren's syndrome, patients have a positive response to immunosuppressive therapy, but the autoantibody screening did not show that their serum contained known autoantibodies. This indicates that a new type of autoantibody related to autoimmune neurological diseases exists in the patient's body. Through further experimental research, the inventor found this autoantibody related to autoimmune neurological diseases, which can bind to EEF1D protein. At the same time, the inventor also found that the administration of artificially synthesized EEF1D protein can competitively inhibit the binding of the autoantibody to the autoantigen in the body. Thus, the present invention is completed.

[0006] Specifically, the present invention provides a new autoantibody related to autoimmune neurological diseases and its target antigen, EEF1D, and develops a detection kit, a pharmaceutical composition and a medical device for detecting the autoantibody.

[0007] In a first aspect, the present invention provides a use of a reagent for detecting anti-EEF1D autoantibodies in the preparation of a preparation for diagnosing autoimmune neurological diseases in a subject.

[0008] In one embodiment, the formulation is for contact with a sample from a subject.

[0009] In a preferred embodiment, the agent is selected from EEF1D polypeptide or its homologue or a polypeptide comprising one or more epitopes derived from EEF1D polypeptide, and the preparation is used to contact the sample from the subject with EEF1D polypeptide or its homologue or a polypeptide comprising one or more epitopes derived from EEF1D polypeptide, and detect the binding of the anti-EEF1D autoantibody to the EEF1D polypeptide or its homologue or a polypeptide comprising one or more epitopes derived from EEF1D polypeptide.

[0010] In a preferred embodiment, the EEF1D polypeptide or a homologue thereof comprises a sequence according to SEQ ID NOs: 10-19 and SEQ ID NOs: 42-64 or a sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity thereto.

[0011] In a preferred embodiment, the sample is a body fluid containing autoantibodies selected from the group consisting of whole blood, serum, cerebrospinal fluid and saliva.

[0012] In one embodiment, the EEF1D polypeptide or homologue comprises a lysate derived from a recombinant cell expressing the EEF1D protein.

[0013] In a preferred embodiment, a sample from a subject is contacted with a fragment of an EEF1D polypeptide or a homologue according to claim 3, or a homologue of said fragment, said fragment having at least 20, 30, 40, 50, 60, 70, 80, 90 or 100 consecutive amino acids of a sequence according to SEQ ID NOs: 10-19 and SEQ ID NOs: 42-64, said homologue of said fragment having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity thereto, and binding of anti-EEF1D autoantibodies from the sample to the fragment of the EEF1D polypeptide or homologue is detected.

[0014] In a preferred embodiment, the EEF1D polypeptide or homologue comprises a protein having a binding function to an EEF1D autoantibody after modification of the amino acid sequence shown in SEQ ID NOs: 10-19 and SEQ ID NOs: 42-64, or a splice variant having a binding function to an EEF1D autoantibody obtained after splicing of the amino acid sequence shown in SEQ ID NOs: 10-19 and SEQ ID NOs: 42-64;

[0015] In a preferred embodiment, the modification includes phosphorylation modification, glycosylation modification, acetylation modification or ubiquitination modification.

[0016] In a preferred embodiment, the EEF1D polypeptide or homologue or fragment thereof is linked to a reporter molecule or a solid phase.

[0017] In one embodiment, the autoimmune neurological disease is selected from neuromyelitis optica, anti-NMDA receptor encephalitis, and cerebellar ataxia.

[0018] In a preferred embodiment, the subject has one or more symptoms selected from the following or the autoimmune disease is associated with one or more symptoms selected from the following: decreased vision and spinal cord function, primary dysfunction, hypoventilation, mild hemiparesis, loss of consciousness, cerebellar oculomotor disorder, dysarthria, ataxia, weakness, incoordination, unsteady gait, eye movement difficulties, dysphagia, hypotonia, inflammatory changes in cerebrospinal fluid, and dysmetria.

[0019] In a second aspect, the present invention provides the use of the above-mentioned EEF1D polypeptide or homologue or a polypeptide comprising one or more epitopes derived from the EEF1D polypeptide or a fragment thereof in the preparation of a medicament for diagnosing an autoimmune neurological disease in a subject, wherein a sample from the subject is contacted with the EEF1D polypeptide or homologue or fragment according to claim 3, and the binding of anti-EEF1D autoantibodies from the sample to the EEF1D polypeptide or homologue or fragment is detected.

[0020] In one embodiment, the method for detecting the binding of anti-EEF1D autoantibodies to EEF1D polypeptides or homologues or fragments comprises at least one of CBA, TBA, ELISA, immunogold method, immunoblotting, immunofluorescence, immunospot, membrane strip method, chemiluminescence, radioimmunoassay, liquid phase chip method, lateral flow and flow cytometry.

[0021] In a preferred embodiment, the method for detecting the binding of anti-EEF1D autoantibodies to EEF1D polypeptides or homologues or fragments thereof comprises x.

[0022] In a preferred embodiment, the method for detecting the binding of anti-EEF1D autoantibodies to EEF1D polypeptides or homologues or fragments thereof comprises x.

[0023] The third aspect of the present invention provides a detection kit for detecting autoantibodies, which comprises the above-mentioned EEF1D polypeptide or homologue or a polypeptide or fragment thereof derived from one or more epitopes of the EEF1D polypeptide.

[0024] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned EEF1D polypeptide or homologue or a polypeptide or fragment thereof derived from one or more epitopes of the EEF1D polypeptide.

[0025] In a fifth aspect of the present invention, a medical device is provided, which is coated with the EEF1D polypeptide or homologue according to claim 3 or a polypeptide or fragment thereof comprising one or more epitopes derived from the EEF1D polypeptide; and / or coated with the pharmaceutical composition according to claim 15.

[0026] In a preferred embodiment, the medical device is selected from the group consisting of: a slide, preferably for microscopy, a biochip, a microtiter plate, a test strip, a membrane; preferably a line blot, a chromatography column, and magnetic beads.

[0027] In a sixth aspect, the present invention provides the use of the above-mentioned EEF1D polypeptide or homologue or a polypeptide comprising one or more epitopes derived from the EEF1D polypeptide or a fragment thereof in the preparation of a pharmaceutical composition or a medical device for treating an autoimmune neurological disease in a subject, wherein the treatment comprises the following steps:

[0028] a. taking blood or plasma from a subject,

[0029] b. contacting the blood or plasma with the above-mentioned pharmaceutical composition or the above-mentioned medical device to remove disease-associated antibodies, and

[0030] c. re-administering the blood or plasma to the subject.

[0031] In one embodiment, the autoimmune neurological disease is selected from neuromyelitis optica, anti-NMDA receptor encephalitis, and cerebellar ataxia.

[0032] In a preferred embodiment, the subject has one or more symptoms selected from the following or the autoimmune disease is associated with one or more symptoms selected from the following: decreased vision and spinal cord function, primary dysfunction, hypoventilation, mild hemiparesis, loss of consciousness, cerebellar oculomotor disorder, dysarthria, ataxia, weakness, incoordination, unsteady gait, eye movement difficulties, dysphagia, hypotonia, inflammatory changes in cerebrospinal fluid, and dysmetria.

[0033] Compared with the prior art, the advantages of the present invention are:

[0034] 1. The present invention is the first to discover autoantibodies associated with autoimmune neurological diseases in humans, and to determine that their corresponding antigen is EEF1D.

[0035] 2. The present invention verifies the correspondence between the autoantibodies related to autoimmune neurological diseases and EEF1D through experimental means. The two can combine to form an antigen-antibody complex, and based on this, the diagnosis of autoimmune neurological diseases in subjects can be achieved.

[0036] 3. The present invention also unexpectedly discovered that providing exogenous EEF1D can competitively inhibit the binding of the autoantibodies to the autoantigens in the body, thereby providing a separation and treatment direction for autoantibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : shows an indirect immunofluorescence assay using frozen sections of rat cerebellum tissue;

[0038] Figure 2 : Shows that patient serum autoantibodies specifically bind to EEF1D. DETAILED DESCRIPTION

[0039] Although the present invention can be implemented in many different forms, what is disclosed here is its specific illustrative embodiment that verifies the principle of the present invention.It should be emphasized that the present invention is not limited to the specific embodiment illustrated.In addition, any section heading used herein is only for organizational purposes and is not to be interpreted as limiting the subject matter described.

[0040] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, terms in the singular should include plural forms, and terms in the plural should include singular forms. More specifically, as used in this specification and the appended claims, unless the context otherwise clearly indicates, the singular forms "a", "an" and "the" include plural indicators. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the ranges provided in the specification and the appended claims include endpoints and all values ​​between the endpoints.

[0041] Generally, terms relating to cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein and their techniques are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, e.g., Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Abbas et al., Cellular and Molecular Immunology, 6th ed., WB Saunders Company (2010); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly used in the art. Additionally, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0042] definition

[0043] In order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0044] As used herein, the term "diagnosis" refers to any type of procedure intended to obtain information that is helpful in assessing whether a patient has suffered from or is likely to suffer from, or is more likely to suffer from, a certain disease or condition than the average or comparison subjects (who preferably have similar symptoms), in the past, at the time of diagnosis, or in the future; to discover how a disease is progressing or is likely to progress in the future; or to evaluate a patient's responsiveness to a certain treatment (e.g., administration of an immunosuppressive drug). In other words, the term "diagnosis" includes not only diagnosis, but also the process of predicting and / or monitoring a disease or condition.

[0045] In many cases, simple detection (in other words, whether there is detectable level of antibody in the assay sample) is sufficient for diagnosis. If autoantibodies can be detected, this will be helpful information for the clinician's diagnosis, and indicates the possibility of increased patients suffering from the disease. In a preferred embodiment, if one or more methods, preferably immunoprecipitation detection autoantibodies can be used to be selected from the group comprising immunoprecipitation, indirect immunofluorescence, ELISA or linear dot blot, then the autoantibodies are considered to be detectable. Experimental details are described in the experimental section of the application, or as described in the textbook or practical manual available on the priority date of the application. In a preferred embodiment, the relative concentration of the antibody in serum can be determined compared to the level that can be found in average healthy subjects. Although in many cases it may be sufficient to determine whether an autoantibody is present or detectable in a sample, the method implemented in order to obtain helpful information for diagnosis may include determining whether the concentration is at least 0.1 times, preferably 0.2, 0.5, 1, 2, 5, 10, 20, 25, 50, 100, 200, 500, 1000, 10000 or 100000 times higher than the concentration found in an average healthy subject. In a preferred embodiment, one or more methods selected from the group comprising semiquantitative immunoprecipitation, semiquantitative indirect immunofluorescence, ELISA or semiquantitative linear dot blot, preferably ELISA, are used to determine the relative concentration of the autoantibody. The experimental details are described in the experimental section of the application, or as described in a textbook or practical manual available on the priority date of the application.

[0046] Those skilled in the art will appreciate that clinicians typically do not conclude whether a patient suffers from or may suffer from a disease, condition or illness based solely on a single diagnostic parameter, but need to consider other aspects, such as the presence of other autoantibodies, markers, blood parameters, clinical assessment of patient symptoms, or the results of medical imaging or other non-invasive methods (e.g., polysomnography) to reach a conclusive diagnosis. The value of a diagnostic agent or method may also be that it may exclude a disease, thereby allowing indirect diagnosis of another disease. In a preferred embodiment, the meaning of any symptom or disease mentioned throughout the application is consistent with that understood by those skilled in the art at the time of the filing date of the application or the preferred earliest priority date, as demonstrated by textbooks and scientific publications.

[0047] Thus, the term "diagnosis" preferably does not imply that the diagnostic method or reagent according to the invention will be definitive and sufficient for completing a diagnosis based on a single test (let alone a parameter), but may refer to a contribution to a so-called "differential diagnosis", i.e. a systematic diagnostic procedure that takes into account the likelihood of a range of possible conditions based on a range of diagnostic parameters. Thus, the method, polypeptide or use of the invention, which is optionally used to determine whether a patient suffers from a disease, may comprise: obtaining a sample from a patient, preferably a human patient; determining the presence of autoantibodies that bind to EEF1D in said sample, wherein said determination is performed by contacting the sample with a polypeptide of the invention and detecting whether binding occurs between said polypeptide and said autoantibodies, preferably by using a labeled secondary antibody, wherein said autoantibodies, if present in the sample, bind to said polypeptide; and diagnosing the patient as suffering from or more likely to suffer from said neurological disease if the autoantibodies are determined to be present in the sample.

[0048] As used herein, the term "diagnosis" may also refer to methods or agents used to distinguish between two or more conditions associated with similar or identical symptoms.

[0049] As used herein, the term "diagnosis" may also refer to a method or reagent for selecting the most promising treatment regimen for a patient. In other words, the method or reagent may involve selecting a treatment regimen for a subject. For example, detection of autoantibodies may indicate that immunosuppressive therapy will be selected, which may include administering one or more immunosuppressive drugs to the patient.

[0050] Autoantibodies and Antigens

[0051] As used herein, "EEF1D protein" refers to an EEF1D polypeptide or a homolog or a polypeptide derived from one or more epitopes of an EEF1D polypeptide or a fragment thereof.

[0052] As used herein, the term "antibody" may include complete antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or single chains thereof. Antibodies are generally produced by the immune system after foreign proteins or other substances (especially pathogenic bacteria) enter the body, and are used for immune response to eliminate foreign harmful substances. Autoantibodies refer to antibodies against one's own tissues, organs, cells and cell components. The growth, development and survival of the human body are maintained by a complete autoimmune tolerance mechanism, and the normal immune response has a protective defense effect, that is, it does not react to one's own tissues and components. Once the integrity of self-tolerance is destroyed, the body regards its own tissues and components as "foreign matter", and an autoimmune reaction occurs, producing autoantibodies.

[0053] As used herein, "autoantibodies" and "EEF1D antibodies" are interchangeable and refer to autoantibodies that bind to EEF1D protein in a sample from a subject. Antibodies usually bind to their cognate antigens with high affinity and specificity. -7 -10 -11 M or lower. Any KD greater than about 10 -6 A KD of M is generally considered to indicate nonspecific binding.

[0054] As used herein, an antibody that "specifically binds" an antigen refers to an antibody that binds the antigen and substantially identical antigens with high affinity, meaning an antibody with a binding affinity of 10 -7 M or less, preferably 10 -8 M or less, even more preferably 5 x 10 -9 M or less, and most preferably 10 -8 M to 10 -10 An antigen is "substantially identical" to a given antigen if it exhibits a high degree of sequence identity to the given antigen, for example, if it exhibits at least 80%, at least 90%, preferably at least 95%, more preferably at least 97%, or even more preferably at least 99% sequence identity to the given antigen sequence.

[0055] In a specific embodiment of the present invention, the antigen includes an artificially synthesized polypeptide and / or a lysate of a recombinant cell expressing the EEF1D protein.

[0056] As used herein, the term "recombinant" refers to a polypeptide produced at any stage of the production process by using genetic engineering methods, for example by fusing a nucleic acid encoding the polypeptide with a strong promoter for overexpression in a cell or tissue or by modifying the sequence of the polypeptide itself. Those skilled in the art are familiar with methods for modifying nucleic acids and encoded polypeptides. The polypeptides (including any variants) used to implement the teachings of the present invention are preferably designed so that they contain at least one epitope that is recognized by an autoantibody that binds to the EEF1D protein and / or specifically binds to an autoantibody that binds to the EEF1D protein. Any epitope is more preferably an epitope that is only recognized by such an autoantibody compared to antibodies other than autoantibodies to the EEF1D protein. In one embodiment, such an epitope comprises 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more, preferably at least 9 but no more than 16 consecutive amino acids from the EEF1D protein.

[0057] EEF1D gene

[0058] As used herein, "EEF1D" refers to the eEF1Bδ subunit of the translation elongation factor 1 complex encoded by the EEF1D gene, which is involved in regulating translation elongation in eukaryotes. The mammalian translation elongation factor 1 complex consists of two parts (Sourd et al., 2006), eEF1A (corresponding to EF-Tu in bacteria) and eEF1B (corresponding to EF-Ts in bacteria). eEF1B plays the role of guanine nucleotide exchange factor (GEF) in the complex (Janssen et al., 1988; Pittman et al., 2006). eEF1B includes a structural subunit (eEF1Bγ) and two catalytic subunits (eEF1Bα, eEF1Bδ). eEF1Bδ originates from the repeat of eEF1Bα and has a unique leucine zipper structure (Amons et al., 1994; Minella et al., 1996; Guerrucci et al., 1999), suggesting that it may interact with other proteins.

[0059] The present invention provides a new autoantibody related to autoimmune neurological diseases and its target antigen, EEF1D, and develops a detection kit and a therapeutic drug for the target antigen.

[0060] Detection Kits

[0061] The present invention provides a detection kit for detecting autoantibodies, which comprises the EEF1D polypeptide or homologue or a polypeptide derived from one or more epitopes of the EEF1D polypeptide or a fragment thereof.

[0062] In a specific embodiment of the present invention, the detection kit comprises a solid phase carrier, an antigen coated on the solid phase carrier and other detection reagents, wherein the antigen is selected from the EEF1D polypeptide or homologue or a polypeptide or fragment thereof derived from one or more epitopes of the EEF1D polypeptide. In a specific embodiment of the present invention, the reagent comprises an artificially synthesized polypeptide and / or a lysate of a recombinant cell expressing the EEF1D protein. The autoantibodies detected by the kit are IgG, IgA and / or other types of antibodies.

[0063] In one embodiment, the antigen coated on the solid phase carrier binds to the autoantibody in the sample to be tested to obtain an antigen-antibody complex adsorbed on the solid phase carrier; then the detection reagent is used to perform qualitative or quantitative analysis on the obtained antigen-antibody complex. The detection reagent is a detection reagent for visible light colorimetry, chemiluminescence or fluorescence luminescence, and the detection reagent includes a secondary antibody with a marker, a blocking buffer and / or a washing buffer. In one embodiment, the kit also includes a control substance that does not contain the autoantibody; preferably, the control substance comes from the body fluid of a healthy individual, which is selected from whole blood, serum, cerebrospinal fluid and saliva.

[0064] The polypeptide of the present invention can be a polypeptide coated on a solid phase carrier. In a preferred embodiment, the term "coated on a solid phase carrier" as used herein refers to a molecule that is bound to a solid carrier that is insoluble in an aqueous solution, more preferably by covalent bonds, electrostatic interactions, encapsulation or encapsulation (e.g., by denaturing a spherical polypeptide in a gel), or by hydrophobic interactions, most preferably by one or more covalent bonds. Various suitable carriers, such as paper, polystyrene, metal, silicon or glass surfaces, microfluidic channels, membranes, magnetic beads, column chromatography media, biochips, polyacrylamide gels, etc. The immobilized molecules can be separated from the aqueous solution together with the insoluble carrier, for example, by filtration, centrifugation or dialysis. The immobilized molecules can be immobilized in a reversible or irreversible manner.

[0065] In one embodiment of the present invention, the detection kit comprises the medical device according to the present invention as described below.

[0066] diagnosis

[0067] The present invention provides a use of a reagent for detecting anti-EEF1D autoantibodies for diagnosing autoimmune neurological diseases in a subject.

[0068] The present invention also provides use of a reagent for detecting anti-EEF1D autoantibodies in preparing a preparation for diagnosing autoimmune neurological diseases in a subject.

[0069] Such methods include identifying individuals with autoimmune neurological diseases for treatment or monitoring the progress of autoimmune neurological diseases, including contacting a patient or a sample obtained from a patient (in vivo or in vitro) with the RAB6A polypeptide or homologue described herein or a polypeptide or fragment thereof comprising one or more epitopes derived from the RAB6A polypeptide, and detecting the presence or absence or binding level of antibodies bound to a bound or free target molecule in the sample. In some embodiments, the polypeptide will comprise a detectable label or reporter molecule as described herein.

[0070] Samples can be analyzed by a variety of assays, in a specific embodiment, for example, by radioimmunoassays, enzyme immunoassays (e.g., ELISA), competitive binding assays, fluorescent immunoassays, immunoblotting assays, Western blot analysis, and flow cytometry assays. Compatible in vivo diagnostics or diagnostic assays can include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computerized tomography (e.g., CAT scans), positron emission tomography (e.g., PET scans), radiography, ultrasound, etc., known to those skilled in the art.

[0071] Pharmaceutical composition

[0072] The present invention provides a pharmaceutical composition comprising the EEF1D polypeptide or homologue or a polypeptide derived from one or more epitopes of the EEF1D polypeptide or a fragment thereof.

[0073] In another specific embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and the above-mentioned reagent that specifically binds to the autoantibody, and optionally an immunosuppressant or anti-inflammatory agent. The pharmaceutical composition is used to treat autoimmune neurological diseases, preferably autoimmune neurological diseases associated with autoantibodies that specifically bind to the EEF1D protein.

[0074] In one embodiment, the pharmaceutical composition is used to remove autoantibodies; specifically, an excess of the pharmaceutical composition is contacted with the autoantibodies to form a complex through binding and the complex is separated.

[0075] In another specific embodiment, the pharmaceutical composition is used to competitively bind to the autoantibody to block the binding of the autoantibody to the autoantigen in the body; preferably, to block the binding of the autoantibody to the autoantigen in the nervous system.

[0076] The pharmaceutical compositions provided herein comprise an effective amount of EEF1D protein, and optionally, an immunosuppressant or anti-inflammatory agent dissolved or dispersed in a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other adverse reactions when appropriately administered to an animal (e.g., a human). "Pharmaceutically acceptable carriers" as used herein include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, drugs, drug stabilizers, gelling agents, adhesives, excipients, disintegrants, lubricants, sweeteners, fragrances, dyes, similar substances and combinations thereof, as known to those of ordinary skill in the art. Unless any conventional carrier is incompatible with the active ingredient, its use in a therapeutic composition or pharmaceutical composition is contemplated. The pharmaceutical composition may comprise different types of carriers, depending on whether it is administered in solid, liquid or aerosol form, and whether it needs to be sterile for administration routes such as injection. The pharmaceutical compositions provided herein can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, superficially, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intracapsularly, transmucosally, intrapericardially, intraumbilically, intraocularly, orally, superficially, topically, inhaled (e.g., aerosol inhalation), injected, infused, continuously infused, directly localized perfusion bathing target cells, by catheter, by lavage, with cream, with lipid compositions (e.g., liposomes), or by other methods known to those of ordinary skill in the art or any combination thereof. The actual dose of the composition administered to the patient can be determined by physical and physiological factors, such as body weight, severity of illness, type of disease treated, previous or simultaneous therapeutic intervention, idiopathic disease of the patient, and route of administration. In any event, the medical personnel responsible for administration will determine the concentration of active ingredient in the composition and the appropriate dose for individual subjects.

[0077] The EEF1D protein in the pharmaceutical composition of the present invention can be used in combination with an anti-inflammatory agent. The anti-inflammatory agent defined herein refers to an agent known or beneficial for treating or preventing inflammation in a subject. Corticosteroids are the main type of anti-inflammatory agents. Corticosteroids can be short-term, medium-term or long-term, and can be delivered in a variety of ways. A non-limiting list of cortices expected in embodiments of the present invention includes oral corticosteroids, such as cortisone, hydrocortisone, prednisone and dexamethasone. Another main type of anti-inflammatory agent is a non-steroidal anti-inflammatory agent. Non-steroidal anti-inflammatory agents include a class of drugs used to treat inflammation and pain. The exact mode of action of this class of drugs is unknown. Examples of members of this class of agents include, but are not limited to, ibuprofen, ketoprofen, flurbiprofen, nabumetone, piroxicam, naproxen, diclofenac, indomethacin, sulindac, tolmetin, etodolac, flufenamic acid, diflunisal, oxaprozin, rofecoxib and celecoxib. Those of ordinary skill in the art will be familiar with these agents. This class includes derivatives of salicylates and salicylates, such as acetylsalicylic acid, sodium salicylate, choline salicylate, choline magnesium salicylate and diflunisal. Other anti-inflammatory agents include anti-rheumatic agents, such as gold salts (e.g., gold sodium thiomalate, thioglucose and auranofin), anti-rheumatic agents (e.g., chloroquine, hydroxychloroquine and penicillamine), antihistamines (e.g., diphenhydramine, chlorpheniramine, clemastine, hydroxyzine and triprolidine) and immunosuppressants (e.g., methotrexate, nitrogen mustard, cyclophosphamide, chlorambucil, cyclosporin and azathioprine). Other immunosuppressants contemplated by embodiments of the present invention are tacrolimus and everolimus. Tacrolimus suppresses the production of interleukin-2 associated with T cell activation, suppresses the differentiation and proliferation of cytotoxic T cells. Today, it is recognized worldwide as the cornerstone of immunosuppressant therapy. Those of ordinary skill in the art will be familiar with these agents and other members of this class of agents, as well as their mechanisms of action and the indications for their use.

[0078] Medical Devices

[0079] The present invention provides a medical device coated with the EEF1D polypeptide or homologue or a polypeptide comprising one or more epitopes derived from the EEF1D polypeptide or a fragment thereof; and / or coated with the pharmaceutical composition.

[0080] In a specific embodiment, the medical device is selected from the group consisting of: a slide, preferably for microscopy, a biochip, a microtiter plate, a test strip, a membrane, preferably a line blot, a chromatography column, and magnetic beads.

[0081] In one embodiment, the medical device is a diagnostic device for diagnosing a disease, preferably an autoimmune neurological disease in a subject.

[0082] In one embodiment, the medical device is used to reduce the concentration of the autoantibody in a sample.

[0083] In a specific embodiment, the antigen coated on the solid phase carrier binds to the autoantibody in the sample to obtain an antigen-antibody complex adsorbed on the solid phase carrier and a separated sample, and the concentration of the autoantibody in the separated sample is reduced.

[0084] In a specific embodiment, the medical device can separate the antigen-antibody complex adsorbed on the solid phase carrier from the free sample, thereby reducing the concentration of the autoantibody in the separated sample. The medical device can perform separation by centrifugation, gravity or dialysis.

[0085] Sequence Listing Overview

[0086] This application is accompanied by a sequence listing containing a number of nucleic acid and amino acid sequences. Table A below provides an overview of the sequences contained.

[0087] mRNA nucleic acid sequences encoded by human EEF1D gene (NCBI database accession numbers: NM_001130053, NM_001130055, NM_001130056, NM_001195203, NM_001289950, NM_001317743, NM_001330646, NM_001960, NM_032378) and proteins Sequence (NCBI database accession number: NP_001123525, NP_001123527, NP_001123528, NP_001182132, NP_001276879, NP_001304672, NP_001317575, NP_001951, NP_115754; Uniprot database accession number: P29692).

[0088] The mRNA nucleic acid sequence encoded by the rat Eef1d gene (NCBI database accession number: XM_008765558.2, XM_008765562, XM_039078816, XM_017594766, XM_039078819, XM_008765552, XM_017594768, XM_008765561, XM_039078815, XM_0087655 57,XM_017594765,XM_039078814,XM_008765551,XM_017594769, XM_039078817,XM_017594767,XM_039078812,XM_039078810, PCR products (NCBI database accession numbers: XP_008763780, XP_008763784, XP_038934744, XP_017450255, XP_038934747, XP_008763774, XP_017450257, XP_008763783, XP_038934743, XP_008763779, XP_017450254, XP_0 38934742, XP_008763773, XP_017450258, XP_008763781, XP_038934746, XP_038934745, XP_017450256, XP_038934740,.XP_038934738, XP_038934741, XP_038934739; Uniprot database accession number: Q68FR9).

[0089] Table A

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] Example

[0130] The invention generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the invention. These examples are not intended to represent that the experiments below are all or the only experiments performed.

[0131] Example 1: Patient Selection

[0132] Patients were recruited from the hospital and clinically evaluated by experienced neurologists.

[0133] The patient had dizziness and unsteady gait for 3 months, accompanied by short-term tinnitus, which was relieved by bed rest. After 3-4 days, he developed unsteady gait and lower limb weakness, and could not coordinate his steps when going up and down stairs, which was more obvious in the left leg, accompanied by hip pain. He also had frequent lower limb twitching during sleep, about a dozen times a night.

[0134] Example 2: Method

[0135] 2.1 Indirect immunofluorescence assay

[0136] The immunofluorescence results of patient serum and cerebrospinal fluid and rat cerebellum tissue showed positive results. TBA detection of patient serum was performed using rat cerebellum tissue as a matrix piece. First, each matrix piece was incubated with 30 μL of PBS-diluted samples at 4°C for 3 hours, washed with PBS-Tween and immersed in PBS-Tween for 5 minutes. Then goat anti-human IgG labeled with fluorescein isothiocyanate (FITC) was incubated at room temperature for 30 minutes. The slides were washed again with PBS-Tween rinse solution and then immersed in PBS-Tween for 5 minutes. Glycerol was used to seal the slides (approximately 10 μL per field of view) and examined by fluorescence microscopy. The experiment set up positive and negative controls, and the untransfected cells and control samples were directly compared. Based on the fluorescence intensity of the transfected cells, the samples were divided into positive or negative. The endpoint titer refers to the last dilution that shows visible fluorescence.

[0137] 2.2 Tissue immunoprecipitation and identification of antigens

[0138] Rat cerebellar tissue sections and patient serum (1:100 dilution) tissue were incubated at 4°C for 3 hours and washed 3 times in PBS containing 0.2% Tween (w / v). The immune complexes were lysed at 4°C for 1 hour with a cell lysis buffer containing protease inhibitors, and then the mixture was blown evenly and centrifuged at 16000G for 15 minutes at 4°C. The supernatant was incubated with Protein G Dynabeads at 4°C overnight to capture the immune complexes. The beads were then washed 3 times with PBS and eluted with a sample eluent at 100°C for 10 minutes, followed by mass spectrometry analysis.

[0139] 2.3 Mass spectrometry analysis

[0140] Take 20ul of the sample eluate for 12% gel electrophoresis, stain with Coomassie Brilliant Blue and decolorize, cut out the target band, extract the protein with a gel protein extraction kit, and then resuspend in lysis buffer for mass spectrometry analysis.

[0141] 2.4 Immunoblotting

[0142] After SDS-PAGE electrophoresis, the gel proteins were transferred to polyvinylidene fluoride (PVDF) membranes by slot blotting. The membranes were blocked with 5% skim milk powder for 1 hour and incubated with patient serum or anti-EEF1D antibody at 4°C overnight. Subsequently, three washing steps were performed using universal blotting buffer and incubated with HRP-labeled anti-human IgG and anti-rabbit IgG for 60 minutes, respectively. After three washing steps, the membranes were developed using an enhanced chemiluminescence detection kit.

[0143] 2.5 Recombinant expression of eEF1D protein in HEK293 cells

[0144] To prepare the cell matrix for expressing the recombinant protein, HEK293 were cultured on sterile coverslips and transfected with the recombinant plasmid pCMV6-EEF1D to express the EEF1D protein for 48 hours. The coverslips were washed with PBS, fixed with 4% paraformaldehyde for 20 minutes, permeabilized in PBS containing 0.3% Triton X-100 for 10 minutes, washed three times with cold PBS, and then blocked with PBS containing 5% calf serum for 1 hour.

[0145] Example 3: Results

[0146] 3.1 Characterization of patient autoantibodies

[0147] We found that the serum of a patient with autoimmune cerebellar ataxia binds to an unknown antigen in rat cerebellum tissue. Indirect immunofluorescence assay was performed on frozen sections of brain tissue.

[0148] The results are as follows Figure 1As shown, the patient's serum IgG can stain the Purkinje cell layer, molecular layer and granular layer of the rat cerebellum, while there is no fluorescent staining in the healthy control.

[0149] 3.2 Identification of EEF1D as an autoantibody target antigen

[0150] After mass spectrometry analysis of immunoprecipitates of patient sera, healthy control sera and rat cerebellum, the protein EEF1D was specifically detected in the patient group, and the specific binding of autoantibodies in patient sera to EEF1D was subsequently experimentally verified.

[0151] 1) In the western blot experiment, the patient's serum can be used as the primary antibody to specifically bind to the EEF1D recombinant protein, and the blot band produced by the experiment is consistent with the position of the anti-EEF1D antibody binding band. The healthy control serum has no binding to the EEF1D recombinant antigen (such as Figure 2 a).

[0152] 2) After the patient's serum was pre-adsorbed with EEF1D, the positive signal disappeared in the immunofluorescence experiment of rat cerebellum tissue (e.g. Figure 2 b).

[0153] 3) Patient serum and cerebrospinal fluid showed specific positive fluorescence signals in immunofluorescence experiments with recombinant EEF1D-transfected cells, while healthy control serum did not have this feature (e.g. Figure 2 c).

[0154] It should be understood that although the present invention has been described exemplarily according to its preferred embodiments, it should not be limited to the above-described embodiments, and for those skilled in the art, the present invention can have various changes and variations. The selection and application of specific reagents can be adjusted and changed accordingly according to specific needs. Therefore, for those skilled in the art, without departing from the concept and principle of the present invention, several simple replacements can also be made, which should all be included in protection scope of the present invention.

Claims

1. Use of a reagent for detecting anti-EEF1D autoantibodies in the preparation of a preparation for diagnosing autoimmune neurological diseases in a subject.

2. The use according to claim 1, wherein the preparation is used for contact with a sample from a subject.

3. The use according to claim 2, wherein the agent is selected from the group consisting of an EEF1D polypeptide or a homologue thereof or a polypeptide comprising one or more epitopes derived from an EEF1D polypeptide, and the preparation is used to contact the sample from the subject with an EEF1D polypeptide or a homologue thereof or a polypeptide comprising one or more epitopes derived from an EEF1D polypeptide, and to detect binding of the anti-EEF1D autoantibody to the EEF1D polypeptide or a homologue thereof or a polypeptide comprising one or more epitopes derived from an EEF1D polypeptide.

4. Use according to claim 3, wherein the EEF1D polypeptide or a homologue thereof comprises a sequence according to SEQ ID NOs: 10-19 and SEQ ID NOs: 42-64 or a sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity thereto.

5. The use according to claim 2, wherein the sample is a body fluid containing autoantibodies, which is selected from whole blood, serum, cerebrospinal fluid and saliva.

6. The use according to claim 3, wherein the EEF1D polypeptide or homologue comprises a lysate derived from a recombinant cell expressing the EEF1D protein.

7. The use according to claim 3, wherein a sample from a subject is contacted with a fragment of an EEF1D polypeptide or a homologue according to claim 3, or a homologue of said fragment, said fragment having at least 20, 30, 40, 50, 60, 70, 80, 90 or 100 consecutive amino acids of a sequence according to SEQ ID NOs: 10-19 and SEQ ID NOs: 42-64, said homologue of said fragment having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity thereto, and binding of anti-EEF1D autoantibodies from the sample to the fragment of the EEF1D polypeptide or homologue is detected.

8. The use according to claim 3, wherein the EEF1D polypeptide or homologue comprises a protein having a binding function to an EEF1D autoantibody after modification of the amino acid sequence shown in SEQ ID NOs: 10-19 and SEQ ID NOs: 42-64, or a splice variant having a binding function to an EEF1D autoantibody obtained after splicing of the amino acid sequence shown in SEQ ID NOs: 10-19 and SEQ ID NOs: 42-64; The modification includes phosphorylation modification, glycosylation modification, acetylation modification or ubiquitination modification.

9. The use according to claim 3, wherein the EEF1D polypeptide or homologue or fragment thereof is linked to a reporter molecule or a solid phase.

10. The use according to claim 1, wherein the autoimmune neurological disease is selected from neuromyelitis optica, anti-NMDA receptor encephalitis and cerebellar ataxia.

11. The method of claim 10, wherein the subject has one or more symptoms selected from the group consisting of: decreased vision and spinal cord function, primary dysfunction, hypoventilation, hemiparesis, loss of consciousness, cerebellar oculomotor disorder, dysarthria, ataxia, weakness, incoordination, gait instability, eye movement difficulties, dysphagia, hypotonia, inflammatory changes in cerebrospinal fluid, and dysmetria.

12. Use of the EEF1D polypeptide or homologue according to claim 3 or a polypeptide comprising one or more epitopes derived from the EEF1D polypeptide or a fragment thereof in the preparation of a medicament for diagnosing an autoimmune neurological disease in a subject, wherein a sample from the subject is contacted with the EEF1D polypeptide or homologue or fragment according to claim 3, and binding of anti-EEF1D autoantibodies from the sample to the EEF1D polypeptide or homologue or fragment is detected.

13. The use according to claim 12, wherein the method for detecting the binding of anti-EEF1D autoantibodies to EEF1D polypeptides or homologues or fragments comprises: At least one of CBA, TBA, ELISA, immunogold method, immunoblotting, immunofluorescence, immunospot, membrane strip method, chemiluminescence, radioimmunoassay, liquid phase chip method, lateral flow and flow cytometry; more preferably immunofluorescence; most preferably CBA.

14. A test kit for detecting autoantibodies, comprising the EEF1D polypeptide or homologue according to claim 3, or a polypeptide derived from one or more epitopes of the EEF1D polypeptide or a fragment thereof.

15. A pharmaceutical composition comprising the EEF1D polypeptide or homologue according to claim 3 or a polypeptide derived from one or more epitopes of the EEF1D polypeptide or a fragment thereof.

16. A medical device coated with the EEF1D polypeptide or homologue according to claim 3 or a polypeptide comprising one or more epitopes derived from the EEF1D polypeptide or a fragment thereof; and / or coated with the pharmaceutical composition according to claim 15.

17. The medical device according to claim 16, selected from the group consisting of: a slide, preferably for microscopy, a biochip, a microtiter plate, a test strip, a membrane; preferably a line blot, a chromatography column, and magnetic beads.

18. Use of the EEF1D polypeptide or homologue according to claim 3 or a polypeptide comprising one or more epitopes derived from the EEF1D polypeptide or a fragment thereof in the preparation of a pharmaceutical composition or a medical device for treating an autoimmune neurological disease in a subject, wherein the treatment comprises the following steps: a. taking blood or plasma from a subject, b. contacting the blood or plasma with the pharmaceutical composition according to claim 15 or the medical device according to claim 16 or 17 to remove disease-associated antibodies, and c. re-administering the blood or plasma to the subject.

19. The use according to claim 18, wherein the autoimmune neurological disease is selected from neuromyelitis optica, anti-NMDA receptor encephalitis and cerebellar ataxia.

20. The use according to claim 19, wherein the subject has one or more symptoms selected from the following or the autoimmune disease is associated with one or more symptoms selected from the following: decreased vision and spinal cord function, primary dysfunction, hypoventilation, mild hemiparesis, loss of consciousness, cerebellar oculomotor disorder, dysarthria, ataxia, weakness, incoordination, unsteady gait, eye movement difficulties, dysphagia, hypotonia, inflammatory changes in cerebrospinal fluid, dysmetria.

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