Methods and reagents for detecting autoantibodies

Abstract

The present invention relates to a method comprising the steps of detecting an antibody in a sample that specifically binds to NECAB1; the antibody specifically binding to NECAB1; a carrier, such as a diagnostically useful carrier having a solid phase having a fixed polypeptide comprising an antigen or at least one epitope thereof or a variant of the antigen or an epitope thereof, wherein the antigen is NECAB1; and the use of the antibody for diagnosing autoimmune diseases or cancer.

Description

Technical Field

[0001] The present invention relates to a method comprising the steps of detecting an antibody in a sample that specifically binds to NECAB1; the antibody specifically binding to NECAB1; a carrier, preferably a diagnostically useful carrier, the carrier having a solid phase having a fixed polypeptide comprising NECAB1 or at least one epitope thereof or a variant of NECAB1 or an epitope thereof; and the use of the antibody for diagnosing autoimmune diseases of the nervous system or cancer. Background Technology

[0002] Neurological autoimmune diseases are rare but potentially treatable. They are characterized by neurological symptoms and an autoimmune response, typically manifested by autoantibodies that specifically bind to brain structures, such as polypeptides that play a crucial role in neurotransmission. They can affect any region of the nervous system, including the central, peripheral, and autonomic nervous systems. While systemic involvement is often multifocal, as in encephalomyelitis, it can also affect a single system, such as in cerebellar degeneration.

[0003] Neurological autoimmune diseases can be associated with cancer. If this is the case, it is called PNS (paraneoplastic syndrome). Neurological symptoms may precede or follow a cancer diagnosis, but in some cases, the primary cancer may not be found even at autopsy. Most PNS reflect a neurological-specific autoimmune attack triggered by tumor neuroantigens released into peripheral lymphoid tissue from a primary or recurrent tumor that was never suspected. Typically, cerebrospinal fluid (CSF) studies in these patients show increased lymphocytes, elevated protein levels, increased IgG synthesis, and oligoclonal bands, supporting the immunopathology. Antibodies targeting accessible membrane targets can directly cause the disease, as seen with acetylcholine receptor antibodies in myasthenia gravis and P / Q voltage-gated calcium channels in Lambert-Eaton syndrome. Often, cancer is asymptomatic when it presents with neurological syndromes. Some paraneoplastic antibodies are cancer-specific, while others are not.

[0004] Autoimmune encephalitis is another major autoimmune neurological disorder, particularly anti-NMDA receptor autoimmune encephalitis, which affects approximately 1.5 per million people annually. The nature of the symptoms (such as delusions, psychosis, and violent behavior) is initially often psychotic, but as the disease progresses, this can be accompanied by seizures, cognitive impairment, memory deficits, and speech problems. In later stages, patients may require intensive care if they develop medical emergency symptoms, including cerebellar ataxia, autonomic dysfunction, and catatonia. Autoantibodies against the NR1 subunit of the NMDA receptor have been identified as the cause (Dalmau et al., Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies, Lancet Neurology, Vol. 7, No. 12, 2008).

[0005] Early diagnosis and treatment of autoimmune diseases of the nervous system are crucial because any delay can lead to rapidly progressing and irreversible neurological damage. Conversely, early and appropriate treatment, often through some form of immunosuppressive therapy, can reverse some of the damage. In some cases, complete recovery is possible. For example, 80% of patients with NMDA receptor encephalitis have good outcomes with treatment, but long-term mental health problems or relapses are possible.

[0006] However, diagnosing autoimmune diseases of the nervous system is often difficult. One reason is the lack of a specific clinical pattern and specific imaging and laboratory abnormalities. A combination of clinical and laboratory evaluations must be used to achieve early diagnosis. Treating any underlying cancer (if present) is important in the treatment of neurological disorders.

[0007] Neurological autoantibodies are helpful in diagnosis. Their presence, alone or in combination with other indicators, helps establish the autoimmune nature of the disease and helps clinicians differentiate new neurological symptoms from those caused by infection, treatment-related complications such as toxic neurological drug abuse, and mental illness. Furthermore, they help detect disease relapse in patients who are already seropositive.

[0008] However, many patients suspected of having neurological autoimmune diseases (30% to 40% of PNS cases) will not have any antibodies that can be detected by current technology, at least because many diagnostically relevant antibodies remain unknown. There is a risk that these patients may go undiagnosed or even be misdiagnosed. In the past, some of them have been admitted to psychiatric wards, where they could have led relatively normal lives with appropriate immunosuppressive therapy. Summary of the Invention

[0009] This invention is based on an unexpected discovery by the inventors: antibodies against NECAB1 are present and detectable in samples from patients with neurological autoimmune diseases and / or cancers, but are undetectable in samples from healthy subjects. Therefore, these antibodies can be used to aid in the diagnosis of diseases.

[0010] Therefore, the problem upon which this invention is based is to provide reagents and methods for diagnosing autoimmune diseases of the nervous system and / or cancers, wherein the diseases are preferably associated with the presence of antibodies that specifically bind to NECAB1.

[0011] Another problem upon which this invention is based is to distinguish between autoimmune diseases caused by antibodies that specifically bind to NECAB1 and diseases caused by other causes (especially infections).

[0012] Another problem upon which this invention is based is to provide assays with improved diagnostic reliability, particularly in terms of specificity and / or sensitivity, for the diagnosis of autoimmune diseases of the nervous system or cancer, optionally in combination with prior art assays.

[0013] The problem on which this invention is based is addressed by the subject matter of the appended independent and dependent claims.

[0014] In a first aspect, the problem on which the present invention is based is solved by a method comprising the step of detecting an antibody in a sample that specifically binds to an antigen, preferably a mammalian autoantibody, wherein the antigen is NECAB1, preferably as shown in SEQ ID NO1 or SEQ ID NO21.

[0015] In a second aspect, the problem upon which the present invention is based is solved by a method for isolating antibodies that specifically bind to an antigen, preferably mammalian autoantibodies, wherein the antigen is NECAB1, preferably as shown in SEQ ID NO1 or SEQ ID NO21, the method comprising the following steps:

[0016] a) Immobilizing a polypeptide containing an antigen or at least one epitope thereof, or a variant of said antigen or epitope thereof, onto a carrier.

[0017] b) Under conditions compatible with the formation of the complex, a sample containing an antibody is contacted with the polypeptide, wherein the antibody binds to the polypeptide.

[0018] c) Separate the complex formed in step a) from the sample.

[0019] d) Optionally, the antibody is separated from the polypeptide, and

[0020] e) Optionally detect the antibody or complex.

[0021] In a third aspect, the problem upon which this invention is based is solved by a method comprising the following steps:

[0022] a) Immobilizing a polypeptide comprising an antigen or at least one epitope thereof, or a variant thereof, on a carrier, wherein the antigen is NECAB1, preferably as shown in SEQ ID NO1 or SEQ ID NO21.

[0023] b) Contact the carrier with a liquid containing an antibody that specifically binds to the antigen, preferably a mammalian autoantibody, wherein the candidate drug is present in the liquid and / or the liquid does not contain a sample from the subject to be diagnosed, preferably wherein the mammalian autoantibody is at a known concentration.

[0024] c) Contact the carrier with a tool for detecting the immobilized antibody, and

[0025] d) Detection of presence, preferably in a quantitative manner.

[0026] In a fourth aspect, the problem is addressed by an antibody that specifically binds to the antigen, preferably a mammalian autoantibody, preferably in a liquid comprising one or more, more preferably all, of the group consisting of artificial buffers, preservatives and artificial anticoagulants, wherein the antigen is NECAB1, preferably as shown in SEQ ID NO1 or SEQ ID NO21.

[0027] In a fifth aspect, the problem is addressed by: a carrier, preferably a diagnostically useful carrier, the carrier having a solid phase having a fixed polypeptide comprising antigen I or at least one epitope thereof or a variant of antigen I or an epitope thereof, and a) a negative control and / or b) at least one additional antigen II, preferably a diagnostically useful antigen II, or at least one epitope thereof or a variant of antigen II or an epitope thereof, wherein the polypeptide is spatially separated from the negative control or the additional antigen II or an epitope thereof or a variant of antigen II or an epitope thereof on the carrier; or a first carrier, preferably a first diagnostically useful carrier having a solid phase having a fixed polypeptide comprising antigen I or at least one epitope thereof or a variant of antigen I or an epitope thereof, and a second carrier, preferably a second diagnostically useful carrier comprising a solid phase having a fixed a) negative control and / or b) at least one additional polypeptide comprising at least one fixed antigen II or at least one epitope thereof or a variant of antigen II or an epitope thereof, wherein antigen I is NECAB1, preferably as shown in SEQ ID NO1 or SEQ ID NO21.

[0028] In a preferred embodiment, at least one additional antigen II is at least one antigen from the group consisting of, preferably all, the following: NMDAR, LgI1, AMPA1, AMPA2, CASPR2, GABA B, GABA A, DPPX, IGLON5, Hu, Yo, CV2 / CRMP5, Ri, Ma2, dual-carrier protein, recovery protein, RGS8, DAGLA, STX1B, AK5, AP3B2, raft protein 1+2, GRM1, GRM2, GRM5, GLURD2, ITPR1, KCNA2, NCDN, septum 3+5+6+7+11, and Sez6L2.

[0029] In a sixth aspect, the problem is addressed by the following for identifying patients at risk of developing and / or for the diagnosis of neurological autoimmune diseases or cancers: a) antibodies that specifically bind to antigens, preferably mammalian autoantibodies and / or recombinant antibodies; or b) a combination of a polypeptide comprising an antigen or at least one epitope thereof or a variant thereof, with a tool for detecting or capturing IgG antibodies; or c) a carrier comprising a polypeptide containing an antigen or at least one epitope thereof or a variant thereof, wherein the antigen is NECAB1, preferably as shown in SEQ ID NO1 or SEQ ID NO21.

[0030] In a seventh aspect, the problem is solved by a kit comprising a polypeptide containing an antigen or at least one epitope thereof or a variant of said antigen or epitope thereof, and a carrier, preferably a diagnostically useful carrier, more preferably a carrier according to the invention, wherein said antigen is NECAB1, preferably as shown in SEQ ID NO1 or SEQ ID NO21, wherein

[0031] a) The polypeptide is immobilized on a carrier, preferably a diagnostically useful carrier, more preferably a carrier according to the invention, and the kit further includes a tool for detecting immobilized antibodies that specifically bind to the antigen, preferably a secondary antibody that specifically binds to human immunoglobulins, or

[0032] b) The polypeptide and the carrier are configured to immobilize the polypeptide on the surface of the carrier, preferably via an affinity tag and a ligand binding to the affinity tag, and the kit further includes a tool for detecting the immobilized antibody, preferably a secondary antibody that specifically binds to human immunoglobulins, or

[0033] c) The carrier is immobilized with a tool for capturing antibodies, preferably a secondary antibody that specifically binds to human immunoglobulins, and the kit further includes a tool for detecting the captured antibodies, preferably containing a detectable labeled peptide, or

[0034] d) The carrier and the tool for capturing antibodies, preferably a secondary antibody that specifically binds to human immunoglobulins, are configured to immobilize the tool for capturing antibodies on the surface of the carrier, preferably via an affinity tag and a ligand binding to the affinity tag, and the kit further includes a tool for detecting the immobilized antibodies, preferably containing a detectable labeled peptide.

[0035] And preferably from one or more, more preferably all, of the following: recombinant antibodies that specifically bind to the antigen, isolated antibodies (preferably mammalian autoantibodies) that specifically bind to the antigen, chemical solutions that react with detectable labels, positive controls, negative controls, leak-proof containers for incubating samples with carriers or reagents, wash buffers, and calibrators (preferably a set of calibrators).

[0036] In a preferred embodiment, the kit includes a secondary antibody and / or a polypeptide containing NECAB1 or an epitope thereof or a variant of NECAB1 or an epitope thereof, wherein optionally the secondary antibody and / or the polypeptide is labeled.

[0037] In an eighth aspect, the problem is addressed by the use of a polypeptide comprising an antigen or at least one epitope thereof or a variant of the antigen or an epitope thereof, or an antibody, preferably an autoantibody, or a recombinant antibody that specifically binds to the antigen, for the preparation of a kit or medical device, preferably a diagnostic device, preferably for the diagnosis or auxiliary diagnosis of a disease, preferably associated with the presence of a mammalian antibody, preferably an autoantibody, that specifically binds to NECAB1, more preferably an autoimmune disease of the nervous system and / or cancer, wherein the antigen is NECAB1, preferably as shown in SEQ ID NO1 or SEQ ID NO21.

[0038] In a ninth aspect, the problem is solved by using an antibody that specifically binds to the antigen, preferably a mammalian autoantibody, or a recombinant antibody that specifically binds to the antigen as a positive control for detecting an antibody, preferably a mammalian autoantibody, in a sample, wherein the antibody is preferably recognized by a secondary antibody against a human immunoglobulin, preferably a human immunoglobulin of the IgG class, wherein the antigen is NECAB1, preferably as shown in SEQ ID NO1 or SEQ ID NO21.

[0039] In a tenth aspect, the problem is solved by an in vitro method for removing antibodies that specifically bind to an antigen from blood, preferably patient serum, preferably mammalian autoantibodies, wherein the antigen is NECAB1, preferably as shown in SEQ ID NO1 or SEQ ID NO21.

[0040] In an eleventh aspect, the problem is addressed by a method for diagnosing or assisting in the diagnosis of a disease, preferably associated with the presence of mammalian antibodies, preferably autoantibodies, that specifically bind to NECAB1, such as autoimmune diseases associated with antibodies, preferably mammalian autoantibodies, that specifically bind to NECAB1 (preferably as shown in SEQ ID NO1 or SEQ ID NO21). In one embodiment, the method for diagnosing the disease is an in vivo method. In one embodiment, the method for diagnosing the disease is an ex vivo method. In one embodiment, the method for diagnosing the disease includes using a polypeptide comprising an antigen or at least one epitope thereof or a variant of said antigen or epitope thereof, wherein said antigen is NECAB1, preferably as shown in SEQ ID NO1 or SEQ ID NO21. In one embodiment, the disease is an autoimmune disease, preferably a neurological autoimmune disease, such as those described herein, and / or cancer, such as those described herein. In one embodiment, the method for diagnosing the disease includes the step of detecting antibodies, preferably autoantibodies, that specifically bind to an antigen from blood, preferably patient serum, wherein said antigen is NECAB1, preferably as shown in SEQ ID NO1 or SEQ ID NO21.

[0041] In a twelfth aspect, the problem is addressed by a kit comprising a polypeptide containing NECAB1 or an epitope thereof or a variant of NECAB1 or an epitope thereof, and tools for detecting and / or capturing antibodies. In one embodiment, the polypeptide may contain a label. In one embodiment, the kit comprises a secondary antibody that specifically binds to mammalian, preferably human, immunoglobulin. In one embodiment, the secondary antibody may contain a label.

[0042] In a preferred embodiment, the disease associated with the presence of mammalian antibodies, preferably autoantibodies, that specifically bind to NECAB1, preferably autoimmune diseases, is a neurological autoimmune disease associated with the presence of antibodies, preferably mammalian autoantibodies, that specifically bind to NECAB1, preferably selected from the group consisting of: paraneoplastic neurological syndromes (PNS), dementia, psychotic symptoms, auditory hallucinations, seizures, encephalitis (such as limbic encephalitis, brainstem encephalitis, or autoimmune encephalitis), peripheral polyneuropathy, Guillain-Barré syndrome (GBS) symptoms, quadriplegia, cerebellar / brainstem syndrome, hearing loss, symmetrical macular degeneration, cerebellar-pontine lesions extending to the spinal cord, personality changes, cognitive impairment and emotional disorders, gait impairment, gait and standing ataxia, multiple cerebral ischemia, and / or cancer (such as carcinoma, brain metastases, breast cancer, lung cancer (such as lung carcinoma) and melanoma). In another preferred embodiment, the autoimmune disease is a neurological autoimmune disease associated with the presence of an antibody that specifically binds to NECAB1, preferably selected from the group consisting of: paraneoplastic neurological syndromes (PNS), personality changes, cognitive impairment and mood disorders, gait impairment, encephalopathy, encephalomyelitis, myelitis, cerebellar inflammation, neuropathy, dementia, encephalitis (such as limbic encephalitis, brainstem encephalitis or autoimmune encephalitis), neuronal neuropathy, cerebellar / brainstem syndrome and / or cancer (such as breast cancer and lung cancer (such as lung cancer)).

[0043] In a preferred embodiment, the polypeptide is a recombinant, isolated, and / or purified polypeptide.

[0044] In a preferred embodiment, the antigen to which the antibody binds is NECAB1.

[0045] In a preferred embodiment, the antibody is a mammalian, preferably human, antibody and / or the sample is a mammalian, preferably human, sample, said sample comprising a representative group of antibodies, preferably selected from the group comprising whole blood, plasma, serum, cerebrospinal fluid, and saliva. In a preferred embodiment, the antibody is an autoantibody. In a preferred embodiment, the antibody specifically binds to an antigen. In a preferred embodiment, the antibody comprises a human Fc region. In a preferred embodiment, the antibody (preferably an autoantibody) comprises one or more sequences selected from the group comprising SEQ ID NO24, SEQ ID NO25, SEQ ID NO26, SEQ ID NO27, SEQ ID NO28, SEQ ID NO29, SEQ ID NO30, SEQ ID NO31, SEQ ID NO32, SEQ ID NO33, SEQ ID NO34, SEQ ID NO35, SEQ ID NO36, SEQ ID NO37, and SEQ ID NO38, preferably selected from the group comprising SEQ ID NO28, SEQ ID NO29, SEQ ID NO30, and SEQ ID NO31. In one embodiment, the antibody is isolated. In one embodiment, the antibody is in a blood sample such as whole blood, said blood sample optionally being diluted.

[0046] In a preferred embodiment, an antibody or complex is detected using a detection method selected from the group consisting of: immunodiffusion, immunoelectrophoresis, light scattering immunoassay, agglutination, labeled immunoassay such as labeled immunoassay from the group consisting of radiolabeled immunoassay, enzyme immunoassay, more preferably ELISA, chemiluminescent immunoassay, preferably electrochemiluminescent immunoassay, and immunofluorescence, preferably indirect immunofluorescence.

[0047] In a preferred embodiment, the carrier is selected from the group consisting of: glass slides, preferably glass slides for microscopy, biochips, microtiter plates, side-flow devices, test strips, membranes, preferably line blots, chromatographic columns, and beads, preferably magnetic beads or fluorescent beads.

[0048] This invention is based on an unexpected discovery by the inventors: antibodies against NECAB1 (preferably as shown in SEQ ID NO1 or SEQ ID NO21) are present and detectable in samples from patients with neurological autoimmune diseases and / or cancer, but are undetectable in samples from healthy subjects. Therefore, these antibodies can be used to aid in the diagnosis of diseases such as autoimmune diseases, such as the neurological autoimmune diseases described herein, and / or to differentiate neurological autoimmune diseases from neurological diseases with different etiologies but similar symptoms, such as infectious neurological diseases or aseptic non-autoimmune neurological diseases. For example, this can help differentiate autoimmune encephalitis (including limbic system encephalitis and brainstem encephalitis) from infectious and / or aseptic non-autoimmune encephalitis or meningitis, including encephalitis of infectious etiology (such as viral encephalitis, bacterial encephalitis, tick-borne encephalitis, herpes encephalitis, CMV encephalitis, toxoplasmosis encephalitis), meningitis of infectious etiology (such as viral meningitis, bacterial meningitis), and aseptic meningitis and encephalitis that are not associated with autoantibodies (especially autoantibodies that specifically bind to NECAB1).

[0049] SEQ ID NO1 represents the sequence of NECAB1 (N-terminal EF-chiral calcium-binding protein 1). This protein has the uniprot database reference number UniProtKB-Q8N987 (NECA1_HUMAN) and exhibits calcium-binding activity. It participates in various biological processes, such as blastocyst hatching and regulation of amyloid precursor protein biosynthesis. An isotype of this protein is known (uniprot reference number Q8N987-2, SEQ ID NO22). Therefore, in one embodiment, NECAB1 comprises SEQ ID NO1 or a variant thereof. In one embodiment, NECAB1 comprises one or more sequences selected from the group consisting of SEQ ID NO18, SEQ ID NO19, SEQ ID NO20, SEQ ID NO21, SEQ ID NO22, and SEQ ID NO23 or variants thereof. In a preferred embodiment, NECAB1 is selected from the group consisting of SEQ ID NO1, SEQ ID NO18, SEQ ID NO19, SEQ ID NO20, SEQ ID NO21, SEQ ID NO22 and SEQ ID NO23 or variations thereof, and more preferably from the group consisting of SEQ ID NO1, SEQ ID NO18, SEQ ID NO19 and SEQ ID NO20.

[0050] In a preferred embodiment, the antigen used in the method of the present invention comprises NECAB1 and / or variants thereof. Variants of NECAB1 may be selected from the group comprising SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5, SEQ ID NO6, SEQ ID NO7, SEQ ID NO8, SEQ ID NO9, SEQ ID NO10, SEQ ID NO18, SEQ ID NO19, SEQ ID NO20, SEQ ID NO21, SEQ ID NO22, and SEQ ID NO23 or variants thereof, preferably the group comprising SEQ ID NO18, SEQ ID NO19, SEQ ID NO20, SEQ ID NO21, SEQ ID NO22, and SEQ ID NO23 or variants thereof, even more preferably the group comprising SEQ ID NO18, SEQ ID NO19, SEQ ID NO20, and SEQ ID NO22, and even more preferably the group comprising SEQ ID NO18, SEQ ID NO19, and SEQ ID NO20 or variants thereof. In other preferred embodiments, the antibody (preferably an autoantibody) that specifically binds to NECAB1 is associated with autoimmune diseases of the nervous system and / or cancer, said diseases preferably selected from the group consisting of: personality changes, cognitive impairment and mood disorders, breast cancer, lung cancer (such as lung cancer), PNS, encephalitis (such as limbic encephalitis, brainstem encephalitis or autoimmune encephalitis), neuropathy, neuronal neuropathy, cerebellitis, myelitis and / or cerebellar syndrome.

[0051] In a preferred embodiment, the antigen of the present invention is derived from a mammal selected from the group consisting of: humans, non-human primates, rodents, cattle, horses, dogs, cats, bears, donkeys, sheep, goats, camels, and dromedary camels, more preferably humans. In another preferred embodiment, the antigen of the present invention comprises at least one polypeptide, said polypeptide comprising at least one protein selected from the group consisting of SEQ ID NO1, SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5, SEQ ID NO6, SEQ ID NO7, SEQ ID NO8, SEQ ID NO9, SEQ ID NO10, SEQ ID NO18, SEQ ID NO19, SEQ ID NO20, SEQ ID NO21, SEQ ID NO22, and SEQ ID NO23 or variants thereof, preferably selected from SEQ ID NO18, SEQ ID NO19, SEQ ID NO20, and SEQ ID NO23. The sequence of NO22 or its variants, and optionally having at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, The length of 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 380, 400, 420, 440, 450, 460, 480, 500, 520, 540, 550, 560, 580, 600, 620, 640, 650, 660, 680, 700, 720, 740, 750, 760, 780, 800, 820, 840, 850, 860, 880, 900, 920, 940, 950, 960, 980, 1000, 1050, 1100, 1150, 1200, 1250 or more amino acids.

[0052] In a preferred embodiment, the method according to the invention includes the step of providing a carrier according to the invention. A polypeptide containing an antigen (such as NECAB1) or at least one epitope thereof or a variant of said antigen or its epitope may be immobilized on a solid surface of the carrier, or a tool may be configured to immobilize a carrier containing said antigen onto the carrier, which may be done in a later stage but before sample removal and washing of the carrier. The carrier may then be contacted with a sample suspected of containing antibodies, under conditions allowing any antibody to bind to NECAB1. The sample may then be removed and the carrier may be washed to remove any remaining sample. The tool for detecting antibodies, carrying a detectable label (such as a fluorescent dye), may then be contacted with the carrier, under conditions allowing any bound antibody to form a complex with the tool. The carrier may be washed again. Finally, the presence of antibodies is detected by checking whether the tool (such as a secondary antibody) can be detected. Preferably, an ELISA or immunofluorescence assay is used, which uses mammalian cells or tissues expressing polypeptides containing the antigen of the invention or variants thereof. In the case of immunofluorescence, a unique pattern determined by the expression pattern of the antigen in the cells or tissue indicates the presence of antibodies, as in the examples (…). Figure 1 As shown in the diagram. In the case of ELISA or another semi-quantitative or quantitative detection method, a value higher than the cutoff value as known in the art indicates the presence of antibody.

[0053] Competitive assays, capture-bridge assays, immunometric assays, class-specific secondary antibodies on a solid phase, and direct or indirect class capture assays can also be used. The principles of each of these forms are detailed in *The Immunoassay Handbook*, 3rd edition, edited by David Wild, Elsevier, 2005. In short, in the competitive form, the antibody to be detected can compete with a recombinant antibody for binding sites (such as epitopes) on an antigen (which is NECAB1 or a variant thereof). Alternatively, a sample containing the antibody can be pre-incubated with an antigen in one reaction, subsequently exposed to an immobilized antigen or an antigen configured for immobilization, and can be exposed to the antigen in another reaction without pre-incubation to show specific binding. In a capture-bridge assay, two antigen molecules bind to two antigen-binding sites on the antibody to be detected. One of the antigen molecules is labeled, and the other is immobilized or configured for immobilization, preferably via an affinity tag and a ligand that specifically binds to said affinity tag. In an immunometric assay, the antibody to be detected binds to an antigen that has been immobilized before or after binding. Antibodies are detected using a tool for detecting antibodies (such as a labeled secondary antibody). In a direct class capture assay, the antibody to be detected is fixed using a tool for capturing antibodies and detected using a labeled antigen. In an indirect class capture assay, the antibody to be detected is fixed using a tool for capturing antibodies. It is detected using at least one (preferably two) antigen molecules that bind to the antibody to be detected and a tool for detecting antibodies (such as a labeled secondary antibody). In any of the aforementioned assays, one or more polypeptides containing at least one epitope of the antigen or a variant of the antigen or its epitope may be used instead of the antigen.

[0054] In a preferred embodiment, a carrier and a tool for capture and / or a tool for detecting antibodies are provided, preferably wherein the carrier and the tool for capture and / or the tool for detection are configured to immobilize the tool onto a solid surface of the carrier. A particularly preferred method of configuration or immobilization is to modify the carrier and / or the tool such that it contains an affinity tag and a ligand for the affinity tag. Alternatively, the carrier or tool may contain reactive chemical groups, such as thiol groups, amino groups, epoxy groups, ester groups, and anhydride groups. In a preferred embodiment, the term "immobilized" as used herein refers to the binding of molecules to a solid carrier insoluble in aqueous solutions, more preferably by covalent or non-covalent bonding, electrostatic interactions, encapsulation, nonspecific adsorption, printing, or embedding, for example by denaturing globular peptides in a gel, or by hydrophobic interactions, most preferably by one or more covalent bonds. Various suitable supports, such as paper, polystyrene, metal, silicon or glass surfaces, microfluidic channels, membranes, beads (such as magnetic beads), column chromatography media, biochips, polyacrylamide gels, etc., have been described in the literature, for example in Kim, D. and Herr, AE (2013), Protein immobilization techniques for microfluidic assays, Biomicrofluidics 7(4), 041501. In this way, immobilized molecules can be separated from aqueous solutions directly along with the insoluble support, for example by centrifugation or decantation. The immobilized molecules can be immobilized in a reversible or irreversible manner. For example, immobilization is reversible if the molecules interact with the support through ionic interactions (which can be masked by adding a high concentration of salt), or if the molecules are bound by cleavable covalent bonds (such as disulfide bonds that can be cleaved by adding a thiol-containing reagent). In contrast, immobilization is irreversible if the molecule is attached to the carrier via covalent bonds that cannot be broken in aqueous solution (e.g., bonds formed by the reaction of epoxy and amino groups, often used to couple lysine side chains to affinity columns). Proteins can be immobilized indirectly, for example by immobilizing antibodies or other entities with affinity for the molecule, subsequently forming a complex to achieve the effect of immobilizing a molecule-antibody complex. Various methods of molecule immobilization are described in the literature, for example in Kim, D. and Herr, AE (2013), Protein immobilization techniques for microfluidic assays, Biomicrofluidics 7(4), 041501. Furthermore, various reagents and kits for immobilization reactions are commercially available, for example from Pierce Biotechnology.

[0055] According to the present invention, cells can be provided comprising an expression vector containing a nucleotide sequence encoding a polypeptide under the control of a promoter (optionally a strong promoter and / or an inducible promoter), the polypeptide containing an antigen or at least one epitope thereof or a variant of the antigen or an epitope thereof, wherein the antigen is NECAB1 or a variant thereof. The vector may encode an N-terminal or C-terminal affinity tag fused to the polypeptide, preferably via a linker sequence. Cells can be cultured under conditions that allow expression of the antigens of the present invention. Any expressed antigen can then be purified, preferably using an affinity tag. However, in some embodiments, unpurified antigens may also be used. This is then immobilized onto a carrier according to the present invention. In a preferred embodiment, the cells, nucleic acids, or vectors can be used to prepare a kit for diagnosing autoimmune diseases of the nervous system.

[0056] According to the invention, a medical or diagnostic device (such as the carrier of the invention, preferably a diagnostically useful carrier) can be prepared by expressing a recombinant polypeptide in cells (such as eukaryotic or prokaryotic cells) comprising NECAB1 or at least one epitope thereof or a variant of NECAB1 or an epitope thereof, which contains an affinity tag and optionally has an artificial linker that may include a protease cleavage site. The polypeptide is contacted with a ligand that specifically binds to the affinity tag, the ligand being immobilized on a solid phase. The solid phase is washed to remove non-specifically bound material from the cells, and the expressed variant is eluted from the solid phase, preferably by adding an excess of the immobilized ligand. The variant can then be immobilized on the device. Optionally, the affinity tag can be removed prior to immobilization by contacting the variant with a protease (preferably a protease that recognizes a protease cleavage site). Affinity tags can be selected from the following tag groups: His, immobilized nickel, glutathione, chitin, 18A, ACP, Aldehyd, Avi, BCCP, calmodulin, chitin-binding protein, E-tag, ELK16, FLAG, flash, polyglutamic acid, polyaspartic acid, GST, GFP, HA, Isope, maltose-binding protein, myc, nus, NE, ProtA, ProtC, Tho1d4, S-tag, SnoopTag, SpyTag, SofTag, streptavidin, Strep-tag II, T7 epitope tag, TAP, TC, thioredoxin, Ty, V5, VSV, biotin, Xpress tag, and recombinant antibodies that bind to ligands targeting affinity tags. Useful proteases include, but are not limited to, TEV, thrombin, Faktor Xa, or intestinal peptidase. Suitable linkers are part of a vector, such as the pET vector series (Novagen).

[0057] In a preferred embodiment, the detection of the presence or absence of at least one antibody other than the antibody specifically binding to the antigen of the present invention is preferably from the group consisting of: autoantibodies specifically binding to NMDAR, autoantibodies specifically binding to LgI1, autoantibodies specifically binding to AMPA1, autoantibodies specifically binding to AMPA2, autoantibodies specifically binding to CASPR2, autoantibodies specifically binding to GABA B, and autoantibodies specifically binding to GABA B. Autoantibodies specifically binding to A, DPPX, IGLON5, Hu, Yo, CRMP5, Ri, Ma2, dual-linked protein, recovery protein, RGS8, DAGLA, NSF, STX1B, DNM1, VAMP2, Anna-3, and Zic-4. The study includes autoantibodies specifically binding to SOX1 (US7314721), PCA2, Tr, glutamate decarboxylase, AK5, AP3B2, raft protein 1 / 2, GRM1, GRM2, GRM5, GLURD2, ITPR1, KCNA2, NCDN, septal proteins 3+5+6+7+11, and Sez6L2. In a preferred embodiment, the detection of any of these autoantibodies aids in the diagnosis of autoimmune diseases (such as neurological autoimmune diseases) or suggests such a diagnosis. In a preferred embodiment, two or more autoantibodies are detected in the same sample, preferably substantially simultaneously. The carrier according to the invention can be configured to detect two or more autoantibodies in the same sample, preferably substantially simultaneously.

[0058] In a preferred embodiment, the presence or absence of two or more antibodies is detected and distinguished. In other words, the signal indicating the presence of an antibody indicates which of the two antibodies is present. In a preferred embodiment, the presence or absence of two or more antibodies is detected in a spatially separated reaction, more preferably in different reaction mixtures in separated containers. In another preferred embodiment, the signal indicating the presence of one of the two antibodies can be distinguished from the signal indicating the presence of the other antibody. This can be achieved by using different detectable labels, more preferably distinguishable fluorophores. For example, a green-emitting fluorophore and another red-emitting fluorophore can be used.

[0059] In a preferred embodiment, the presence or absence of two or more antibodies is detected, but not distinguished. In a preferred embodiment, their presence or absence is detected in a one-pot reaction, preferably in two or more reactions within the same reaction vessel without spatial separation, and without signal differentiation. In other words, the signal indicates the presence of at least one of the two antibodies, but not which one. In a preferred embodiment, two or more antigens may be present in the mixture.

[0060] In a preferred embodiment, the sample comprises a group of representative antibodies, more preferably IgG, IgA, and IgM antibodies, and most preferably IgG antibodies. It is preferably selected from the group consisting of whole blood, plasma, serum, cerebrospinal fluid (CSF), and saliva. The sample can be a liquid sample or a dried blood spot prepared using a sample (preferably whole blood, plasma, serum, or capillary blood, preferably capillary blood).

[0061] In a preferred embodiment, the sample is derived from an organism possessing a brain and producing antibodies, preferably from the group comprising mammals and birds, more preferably from mammals comprising humans, non-human primates, rodents (such as rats or mice), cattle, horses, dogs, cats, bears, donkeys, sheep, goats, camels, and dromedary camels, with humans being the most preferred. In another preferred embodiment, the sample is derived from birds, more preferably from the group comprising chickens, parrots, and falcons. The animals may have been extensively trained, for example, for assisting people in need, for riding, or for hunting.

[0062] The antibody to be detected, or the tool used to detect or capture the antibody, specifically binds to its interacting partner, which is an antigen in the case of an antibody, or an antibody in the case of a tool used to detect an antibody, preferably an antibody that specifically binds to NECAB1 or a variant thereof. In a preferred embodiment, the term "specific binding" as used herein means a binding reaction that is strong and characterized by a dissociation constant of 1 x 10⁻⁶. -5 M, more preferably 1 x 10 -7 M, more preferably 1 x 10-8 M, more preferably 1 x 10 -9 M, more preferably 1 x 10 -10 M, more preferably 1 x 10 -11 M, more preferably 1 x 10 -12 The binding reaction of M (as determined by surface plasmon resonance at 25°C in pH 7 PBS buffer using a Biacore device).

[0063] In a preferred embodiment, the term "autoantibody," as used herein, refers to an antibody that specifically binds to a structure (preferably an antigen, more preferably at least one epitope of the antigen) from an organism that produces the antibody. The organism is preferably a patient suspected of or actually suffering from a disease, preferably a mammalian patient, and more preferably a human patient. Such an autoantibody has a constant region, like other antibodies of the same class from the same organism. Particularly preferably, the autoantibody is a mammalian autoantibody, even more preferably a human autoantibody, and even more preferably a human autoantibody of the IgG, IgM, or IgA (preferably IgG) class. Variable domains are capable of specifically binding to the antigen. Constant domains specifically bind to molecules such as secondary antibodies that recognize the constant domain of the immunoglobulin class (preferably IgG antibodies) of the antibody. It has sequence elements shared with other antibodies (preferably IgG antibodies from the same organism).

[0064] According to the present invention, antibodies that specifically bind to NECAB1 or its variants are provided, isolated, or used. Those skilled in the art are familiar with the isolation or purification of antibodies. Comprehensive descriptions are available in the prior art, for example in GE Healthcare's Affinity Chromatography Vol. 1 Antibody, www.gelifesciences.com, April 2016, and Thermo Scientific Pierce Antibody Production and Purification Technical Handbook, Version 2, www.thermoscientific.com. For example, specific purification steps may involve affinity chromatography using peptides comprising the antigen of the present invention or variants thereof, said peptides being immobilized to beads by coupling with primary amines and / or protein G.

[0065] In a preferred embodiment, the subject or patient, as used herein, is a mammal, preferably a human subject or mammal, preferably a human patient. In a preferred embodiment, the patient is a subject who has or is suspected of having a disease associated with the presence of mammalian antibodies that specifically bind to NECAB1.

[0066] The teachings of this invention can be carried out not only with polypeptides (particularly polypeptides containing a natural sequence of a polypeptide called NECAB1 or a variant thereof) or nucleic acids having an exact sequence explicitly stated (e.g. by function, name, sequence or accession number) or implicitly mentioned in this application, but also with variants of such polypeptides or nucleic acids.

[0067] In a preferred embodiment, the term "variant" as used herein may refer to at least one fragment of the full-length sequence mentioned, and more specifically, to one or more amino acid or nucleic acid sequences that are truncated at one or both ends of the full-length sequence. Such fragments contain or encode peptides having an original sequence or a variant thereof of at least 6, 7, 8, 10, 12, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 380, 400, 450, 500, 600, 620, 640, or 660 consecutive amino acids. The total length of the variant can be at least 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 380, 400, 450, 500, 520, 540, 560, 580, 600, 610, 620, 630, 640, 650, 660, or 670 or more amino acids.

[0068] The term "variant" refers not only to at least one fragment, but also to a polypeptide or fragment thereof comprising an amino acid sequence having at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% (preferably at least 80% or 99%) identity with the reference amino acid sequence or fragment thereof, preferably wherein amino acids other than those essential for biological activity (e.g., the ability to bind to antigens and antibodies, or the folding or structure of the polypeptide) are deleted or substituted, and / or one or more such essential amino acids are substituted in a conserved manner, and / or amino acids are added such that the biological activity of the polypeptide is preserved. In other words, in a preferred embodiment, the variant retains the biological activity (of the full-length protein from which it originates). Existing techniques include various methods that can be used to compare two given nucleic acid or amino acid sequences and calculate the degree of identity, see, for example, Arthur Lesk (2008), Introduction to Bioinformatics, Oxford University Press, 2008, 3rd edition. In a preferred embodiment, ClustalW software is used with default settings (Larkin, MA, Blackshields, G., Brown, NP, Chenna, R., McGettigan, PA, McWilliam, H., Valentin, F., Wallace, IM, Wilm, A., Lopez, R., Thompson, JD, Gibson, TJ, Higgins, DG (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948).

[0069] In a preferred embodiment, the polypeptide and its variants may include tags, such as affinity tags, wherein the tags are preferably selected from the group consisting of: His tag, GST tag, E tag, FLAG tag, HA tag, myc tag, V5 tag, S tag, SnoopTag, SpyTag, SofTag, Strep tag, Strep tag II, T7 epitope tag, biotin tag, ALFA tag, AviTag tag, C- tag, calmodulin tag, iCap tag, polyglutamic acid tag, polyarginine tag, NE tag, Rho1D4 tag, SBP tag, Softag1, Softag3, Spot tag, T7 tag, TC tag, Ty tag, VSV tag, and Xpress tag.

[0070] In a preferred embodiment, the polypeptide and its variants may also contain chemical modifications, such as isotopic labeling or covalent modifications, including glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, methylation, hydroxylation, ubiquitination, etc. Methods for modifying polypeptides are familiar to those skilled in the art. Any modification is designed such that it does not eliminate the biological activity of the antigen, epitope, or variant of the present invention.

[0071] Furthermore, variants can also be generated by fusing peptides, fragments, or variants thereof with the N-terminus and / or C-terminus of other known peptides or variants thereof or artificial sequences (such as adapters), and contain an active moiety or domain, preferably having at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity when compared with the active moiety of a reference sequence, wherein the term "active moiety" as used herein refers to an amino acid sequence that is shorter than the full-length amino acid sequence, or, in the case of a nucleic acid sequence, encoding an amino acid sequence shorter than the full-length amino acid sequence, and / or a variant of a native sequence that retains at least some biological activity. Preferably, the active moiety is the active moiety of NECAB1. Peptides may contain additional sequences, preferably artificial sequences, such as adapters or binding epitopes. Any fusion sequence is selected such that the ability of the antigen or variant of the present invention to specifically bind to the antibody to be detected, or the diagnostic reliability (particularly sensitivity and / or specificity), is not significantly altered, let alone eliminated.

[0072] In a preferred embodiment, the “variant” of the nucleic acid includes a nucleic acid whose complementary strand preferably hybridizes with a reference or wild-type nucleic acid under stringent conditions. The stringency of the hybridization reaction can be readily determined by those skilled in the art and is typically determined by empirical calculations of probe length, washing temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to re-anneal with the present complementary strand at an environment below its melting temperature: the higher the degree of homology desired between the probe and the hybridizable sequence, the higher the relative temperature that can be used. Therefore, higher relative temperatures tend to make the reaction conditions more stringent, while lower temperatures are less stringent. For further details and explanations regarding the stringency of the hybridization reaction, see Ausubel, FM (1995), Current Protocols in Molecular Biology. John Wiley & Sons, Inc. In addition, those skilled in the art can follow the instructions given in the manual Boehringer Mannheim GmbH (1993) The DIG System Users Guide for Filter Hybridization, Boehringer Mannheim GmbH, Mannheim, Germany, and Liebl, W., Ehrmann, M., Ludwig, W. and Schleifer, KH (1991) International Journal of Systematic Bacteriology 41: 255-260 on how to determine DNA sequences by means of hybridization. In a preferred embodiment, any hybridization is performed under strict conditions, i.e., hybridization will only occur if the probe and target sequence have 70% or higher identity. Probes with low identity to the target sequence can hybridize, but such hybrids are unstable and will be removed in a washing step under stringent conditions, such as reducing the salt concentration to 2 × SSC, or optionally and subsequently to 0.5 × SSC, while the temperature, in an increasing preferred order, is approximately 50°C-68°C, approximately 52°C-68°C, approximately 54°C-68°C, approximately 56°C-68°C, approximately 58°C-68°C, approximately 60°C-68°C, approximately 62°C-68°C, approximately 64°C-68°C, and approximately 66°C-68°C. In a particularly preferred embodiment, the temperature is approximately 64°C-68°C or approximately 66°C-68°C. The salt concentration can be adjusted to 0.2 × SSC or even 0.1 × SSC.Nucleic acid sequences with at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a reference or wild-type sequence can be isolated. In a preferred embodiment, a variant of the nucleic acid sequence, as used herein, refers to any nucleic acid sequence encoding the same amino acid sequence as the reference nucleic acid sequence (preferably NECAB1 or a variant thereof), conforming to the degeneracy of the genetic code.

[0073] The polypeptide (including any variants) used for teaching this invention is preferably designed to contain at least one epitope that is recognized and / or specifically bound to an antibody that binds to the antigen of this invention (i.e., NECAB1 or a variant thereof). This epitope may exhibit the strongest binding to an antibody bound to the corresponding natural antigen compared to observed binding to other antibodies. In one embodiment, such a polypeptide comprises 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 113, 125 or more (preferably at least 9 but not more than 16) consecutive amino acid segments from the antigen of this invention. Those skilled in the art are familiar with guidelines for designing peptides with sufficient immunogenicity, such as those described in Jackson, DC, Fitzmaurice, CJ, Brown, LE, Zeng, W. (1999), Preparation and properties of totally synthetic immunogenes, Vaccine, Vol. 18, No. 3–4, Sept. 1999, pp. 355–361; and Black, M., Trent, A., Tirrell, M. and Olive, C. (2010), Advances in the design and delivery of peptide subunit vaccines with a focus on Toll-like receptor agonists, Expert Rev Vaccines, Feb. 2010; 9(2): 157–173. In short, peptides are expected to meet as many of the following requirements as possible: (a) they have a high degree of hydrophilicity, (b) they contain one or more residues selected from the group consisting of aspartic acid, proline, tyrosine and phenylalanine, (c) for higher specificity they have little or no homology with other known peptides or polypeptides, (d) they need to have sufficient solubility, and (e) they do not contain glycosylation or phosphorylation sites unless required for a specific reason.Alternatively, bioinformatics methods can be followed, such as those described in Moreau, V., Fleury, C., Piquer, D., Nguyen, C., Novali, N.,Villard, S., Laune, D., Granier, C. and Molina, F. (2008), PEPOP: Computational design of immunogenic peptides, BMC Bioinformatics 2008, 9:71.

[0074] When used according to the invention, the polypeptides of the invention can be provided in any kind of conformation. For example, the polypeptide can be substantially unfolded, partially unfolded, or fully folded. In a preferred embodiment, the polypeptide is folded, meaning that the epitope is essential for binding to the antibody of the invention, or that the protein as a whole or a variant thereof takes the folding that a natural protein takes in its natural environment. Those skilled in the art are familiar with methods suitable for determining whether a polypeptide is folded and, if so, what structure it has, such as limited proteolysis, NMR spectroscopy, CD spectroscopy, or X-ray crystallography (see, for example, Banaszak LJ (2008), Foundations of Structural Biology, Academics Press, or Teng Q. (2013), Structural Biology: Practical Applications, Springer), with CD spectroscopy being preferred.

[0075] The polypeptides of the present invention can be fusion proteins comprising an amino acid sequence other than that derived from the antigen of the present invention, particularly a C-terminal or N-terminal tag, preferably an N-terminal tag, which, in a preferred embodiment, as used herein, is an additional sequence motif or polypeptide having a function (the function having some biological or physical function) and can be used, for example, for the purification, immobilization, precipitation, or identification of the polypeptides of the present invention. In a more preferred embodiment, the tag is a sequence or domain capable of specifically binding to a ligand, for example selected from tags comprising His tags, thioredoxins, maltose-binding proteins, glutathione S-transferases, and fluorescent tags (e.g., from the group comprising green fluorescent protein).

[0076] In a preferred embodiment, the term "sensitivity" refers to the number of samples correctly identified as positive relative to the total number of samples examined. In a preferred embodiment, the term "specificity" refers to the number of samples correctly identified as negative relative to the total number of samples examined.

[0077] The variant possesses biological activity. In a preferred embodiment, such biological activity is the ability to specifically bind to an antibody that binds to a corresponding antigen (such as NECAB1), such that the antibody is found in patients with an autoimmune disease associated with the presence of such an antibody in a sample, the disease preferably being selected from the group comprising: PNS, dementia, psychotic symptoms, auditory hallucinations, seizures, encephalitis (such as limbic encephalitis, brainstem encephalitis, or autoimmune encephalitis), peripheral polyneuropathy, neuronal neuropathy (such as sensorimotor neuron neuropathy), Guillain-Barré syndrome (GBS) symptoms, encephalopathy, cerebrospinal fluid disorders. Myelitis, cerebellar encephalitis, encephalomyelitis, spinal cord encephalitis, quadriplegia, cerebellar / brainstem syndrome, hearing loss, symmetrical macular degeneration, cerebellar syndrome, cerebellar-pontine lesions extending into the spinal cord, personality changes, cognitive impairment and emotional disorders, gait disturbances, gait and standing ataxia, multiple cerebral ischemia, and cancers (such as carcinoma, brain metastases, lung cancers (such as lung cancer), breast cancer, and melanoma), even more preferably selected from the group comprising PNS, personality changes, cognitive impairment and emotional disorders, gait disturbances, encephalitis (such as limbic encephalitis, brainstem encephalitis, or autoimmune encephalitis), breast cancer, and lung cancers (such as lung cancer). In a preferred embodiment, the dementia is rapidly progressive dementia. In other preferred embodiments, the encephalitis is limbic encephalitis, brainstem encephalitis, or autoimmune encephalitis. In yet another preferred embodiment, the cancer is breast cancer or lung cancer, such as SCLC or NSCLC. For example, the biological activity of a peptide variant can be examined by determining whether it specifically binds to an antibody from a sample from such a patient (which contains an antibody that specifically binds to a wild-type antigen), preferably as determined by indirect immunofluorescence as described in the experimental section of this application. In a preferred embodiment, those skilled in the art will consider, when designing the variant, that domains exposed to the cytoplasm or extracellular space may contain epitopes of the antibody to be detected. In even more preferred embodiments, the variant may be a peptide comprising at least one sequence listed in SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5, SEQ ID NO6, SEQ ID NO7, SEQ ID NO8, SEQ ID NO9, SEQ ID NO10, SEQ ID NO18, SEQ ID NO19 and / or SEQ ID NO20.

[0078] According to the present invention, cells are provided that overexpress a polypeptide containing an antigen or at least one epitope thereof or a variant of said antigen or epitope thereof, wherein said antigen is NECAB1, said cells are preferably combined with at least one additional cell that overexpresses another antigen or a variant thereof contained in the polypeptide from the group consisting of: Hu, Yo, Ri, CV2, PNMA1, PNMA2, DNER / Tr, ARHGAP26, ITPR1 (EP14003703.7), ATP1A3, NBC1 (EP14003958.7), Neurochrondrin (EP15001186), CARPVIII, Zic4, SOX1 (US7314721), Ma, MAG, MPO, MBP, GAD65, dual-carrier protein, recovery protein, GABA A receptor, GABA B receptor, glycine receptor, pontocin, IgLON5, DPPX, aquaporin-4, MOG, NMDA receptor, AMPA receptor, GRM1, GRM5, LGI1, VGCC, mGluR1, CASPR2, ATP1A3 (also known as the α3 subunit of human neuronal Na(+) / K(+) ATPase) (EP14171561.5) and raft protein 1 / 2 (EP3101424).

[0079] In a preferred embodiment, as used herein, the term "overexpression" means that cells have been transfected with nucleic acids containing a nucleic acid sequence encoding a polypeptide under the control of a promoter, the polypeptide containing the antigen of the present invention or other antigens or variants thereof. Thus, transfected cells express at least 10%, 20%, 30%, 50%, 100%, 200%, or 500% more of the polypeptide that is specifically bound to the detection antibody compared to what would normally be expressed by cells of the same type (as determined by quantitative Western blotting). The promoter may be an inducible promoter, which allows expression to be induced by the addition of an inducer. Those skilled in the art are familiar with protocols and vectors for transient overexpression of polypeptides in eukaryotic cells, such as the pTriEx system from Novagen, and with protocols and vectors for stable transfection of eukaryotic cells, such as the pcDNA™4 / TO vector system from Invitrogen.

[0080] In a preferred embodiment, fixed mammalian cells can be used. In a preferred embodiment, the term "fixed" cells, as used herein, refers to cells treated with a reactive compound such that the cells no longer possess metabolic activity but still exhibit their epitopes for immunostaining with antibodies and subsequent detection (e.g., by fluorescence). More preferably, the reactive compound is selected from the group consisting of acetone, formalin, methanol, and ethanol, or mixtures thereof, preferably all of them. Those skilled in the art are familiar with methods that can be used to prepare fixed cells.

[0081] According to the present invention, cells can be mounted on a support for microimmunofluorescence analysis. Such a support can be a glass slide. Cells on the slide can be covered with a mounting buffer. The mounting medium is a liquid that helps maintain a near-physiological pH to preserve the molecular structure of any diagnostically relevant molecules and their epitopes, is compatible with the emission of fluorescence signals, and prevents premature loss of fluorescence due to bleaching of fluorophores. It can be selected from the group consisting of water, glycerol, natural oils, or plastics or mixtures thereof, preferably water and glycerol. Various compositions are described in the prior art, for example in "Mountants and Antifades" published by Wright Cell Imaging Facility, Toronto Western Research Institute University Health Network (https: / / de.scribd.com / document / 47879592 / Mountants-Antifades), Krenek et al. (1989) J. Immunol. Meth 117, 91-97, and Nairn et al. (1969) Clin. Exp. Immunol. 4, 697-705.

[0082] A coverslip can be placed on top of the composition containing the sample and mounting medium. Slides with coverslips (FB 112d-1005-1 or ZZ 3000-0112) are available from EUROIMMUN Medizinische Labordiagnostika, AG. However, any carrier compatible with fluorescence-modulated microscopy can be used. The carrier can contain simulated transfected cells that have been transfected with a carrier identical to cells overexpressing an antigen (e.g., NECAB1 or a variant thereof) or at least one epitope thereof, or a variant thereof, but without nucleic acid encoding the latter. Such simulated transfected cells can serve as a negative control. The carrier is configured for analysis using immunofluorescence microscopy.

[0083] In a preferred embodiment, the carrier may include regions containing cells according to the invention. Additionally, the carrier may include other regions. These regions are preferably surrounded by a hydrophobic surface. Each of these regions may contain cells overexpressing another antigen or a variant thereof. Regions may contain slices of primate cerebellum, rat cerebellum, or rat hippocampus and thalamus. In a preferred embodiment, the cells are eukaryotic cells overexpressing a polypeptide, such as cells selected from the group comprising HEK, HeLa, CHO, and Jurkat cells and their derivatives. In a preferred embodiment, the cells are recombinant cells overexpressing a polypeptide, preferably under the control of a heterologous strong promoter.

[0084] In another preferred embodiment, the carrier, such as a diagnostically useful carrier, is a bead. Various beads for a variety of applications are commercially available and are primarily based on carbohydrates, such as agarose gel or agarose, or plastics. They may contain active or activatable chemical groups, such as carboxyl, toluenesulfonyl, or ester groups, which can be used to immobilize tools for specifically capturing antibodies. Preferably, the beads are beads with an average diameter of 0.1 µm to 10 µm, 0.5 µm to 8 µm, 0.75 µm to 7 µm, or 1 µm to 6 µm. Preferably, the beads are provided in the form of an aqueous suspension with a bead content of 10% to 90%, preferably 20% to 80%, more preferably 30% to 70%, and more preferably 40% to 60% (w / w). Those skilled in the art are familiar with such beads (Diamindis, EP, Chriopoulus, TK, Immunoassays, 1996, AcademicPress), which are commercially available, such as Bio-Plex COOH beads MC10026-01 or 171-506011 from Bio-Rad.

[0085] In another preferred embodiment, the carrier is a microtiter plate containing at least eight wells suitable for ELISA. At least one well is coated with a tool for direct or indirect specific capture of antibodies, preferably containing peptides of SEQ ID NO1, SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5, SEQ ID NO6, SEQ ID NO7, SEQ ID NO8, SEQ ID NO9 and / or SEQ ID NO10 or variants thereof. A calibration curve for semi-quantitative analysis can be established using at least three, preferably four, more preferably five calibrators at defined concentrations. When performing the method of the present invention, calibrators typically covering a concentration range encompassing the calibration curve can be processed and developed in parallel with the sample. Secondary antibodies containing labels capable of detecting markers such as enzyme activity, for example, labels with horseradish peroxidase or alkaline phosphatase activity, or enzymes capable of chemiluminescence, can be provided.

[0086] In another preferred embodiment, the carrier is a microarray. In a preferred embodiment, as used herein, the term "microarray" refers to a chip spotted with a variety of spatially separated antigens, preferably at least 5, more preferably 10, 20, 30, 40, 50, 80, or 100. Preferably, each antigen is a peptide comprising or consisting of 5 to 25, preferably 7 to 15 consecutive amino acids, spanning a fragment of a mammalian protein. At least one antigen is a polypeptide comprising NECAB1 or an epitope thereof or a variant of NECAB1 or an epitope thereof. Preferably, two or more antigens are polypeptides comprising NECAB1 or an epitope thereof or a variant of NECAB1 or an epitope thereof, such as SEQ ID NO1, SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5, SEQ ID NO6, SEQ ID NO7, SEQ ID NO8, SEQ ID NO9, SEQ ID NO10, SEQ ID NO18, SEQ ID NO19, and / or SEQ ID NO20 or a variant thereof. The detection can be performed using a secondary antibody containing a label (preferably a fluorescent label) that specifically binds to human immunoglobulins (preferably human IgG). Preferably, at least one additional antigen or a variant thereof is spotted.

[0087] In a preferred embodiment, an ELISA is used to determine the presence of antibodies binding to NECAB1 or its variants in a sample from the subject. In another preferred embodiment, immunofluorescence, preferably indirect immunofluorescence, is used to determine the presence of antibodies binding to NECAB1 or its variants in a sample from the subject.

[0088] According to the present invention, a tool for detecting antibodies is used, which is a molecule that specifically binds to and allows detection of the antibody, and typically contains a detectable label. In a preferred embodiment, the tool for detecting antibodies is a secondary antibody, or a polypeptide containing an antigen or at least one epitope thereof or a variant of the antigen or an epitope thereof, wherein the antigen is NECAB1 or a variant thereof. In a preferred embodiment, the label, as used herein, is a detectable label. In a preferred embodiment, the detectable label can be used to distinguish a group of molecules from other groups of molecules using a biophysical detection method. It is preferably selected from the group containing fluorescent, radioactive, chemiluminescent labels, heavy metal labels such as gold labels, nanoparticles, beads, or labels with enzyme activity, preferably labels that catalyze colorimetric reactions. In a preferred embodiment, the fluorescent label is selected from the group containing Alexa dye, FITC, TRITC, and green fluorescent protein (GFP). Iodine-125 can be used as a radioactive label. In a preferred embodiment, the enzyme activity label is selected from the group containing horseradish peroxidase, glucose oxidase, β-galactosidase, alkaline phosphatase, and luciferase. In a preferred embodiment, the chemiluminescent label is selected from the group consisting of luminol or its derivatives, acrid esters, and luciferase. Those skilled in the art are able to select suitable labels and ligate them to proteins, nucleic acids, and other molecules (Hassanzadeh L, Chen S, Veedu RN. Radiolabeling of Nucleic Acid Aptamers for Highly Sensitive Disease-Specific Molecular Imaging). Pharmaceuticals (Basel) .2018;11(4):106. Published on October 15, 2018. doi:10.3390 / ph11040106, Greg T. Hermanson's Bioconjugate Techniques, 3rd Edition(2013), Obermaier C, Griebel A, Westermeier R. Principles of protein labeling techniques. Methods Mol Biol. 2015; 1295:153-65), and a wide range of labeled molecules are commercially available. According to the invention, a tool for capturing antibodies (such as IgG antibodies) is a molecule that specifically binds to and is capable of immobilizing the antibody to be captured and / or immobilized, as it is immobilized itself or configured for immobilization, preferably via an affinity tag. Preferably, the tool for capturing antibodies is a secondary antibody or a polypeptide containing NECAB1 or its epitope or a variant of NECAB1 or its epitope. The tool for detecting antibodies (such as immobilized antibodies) can be a ligand that specifically binds to mammalian immunoglobulins, preferably human immunoglobulins, more preferably human IgG, and / or mammalian, preferably human NECAB1 or a variant thereof, and / or can be selected from the group consisting of: secondary antibodies, polypeptides containing NECAB1 or at least one epitope of NECAB1 or a variant thereof, specifically binding ligands, protein G or a variant thereof, protein A or a variant thereof, or aptamers or antibodies that specifically bind to said antibody. Preferably, the tool for detecting antibodies is a secondary antibody or a polypeptide containing NECAB1 or an epitope of NECAB1 or a variant thereof. For example, the tool for capturing antibodies that specifically bind to NECAB1 can be a secondary antibody, and the tool for detecting said antibody can be a polypeptide containing NECAB1 or an epitope of NECAB1 or a variant thereof, which is optionally labeled, preferably with a detectable label. As another example, the tool for capturing an antibody that specifically binds to NECAB1 may be a polypeptide containing NECAB1 or its epitope or a variant of NECAB1 or its epitope, and the tool for detecting the antibody may be a secondary antibody, which is optionally labeled, preferably with a detectable label.

[0089] According to the present invention, the term "secondary antibody" should be understood in its broadest sense as any kind of "binding moiety," preferably a binding protein, capable of specifically binding to immunoglobulins, preferably mammalian, more preferably human immunoglobulins, and even more preferably to IgA, IgG, and / or IgM antibodies or fragments thereof of a selected species (preferably human) such as constant domains of a specific Ig class. In one embodiment, the secondary antibody specifically binds to one or more sequences, or specifically binds to an antibody comprising one or more sequences selected from the group consisting of SEQ ID NO24, SEQ ID NO25, SEQ ID NO26, SEQ ID NO27, SEQ ID NO28, SEQ ID NO29, SEQ ID NO30, SEQ ID NO31, SEQ ID NO32, SEQ ID NO33, SEQ ID NO34, SEQ ID NO35, SEQ ID NO36, SEQ ID NO37, and SEQ ID NO38, preferably specifically binding to one or more sequences selected from the group consisting of SEQ ID NO28, SEQ ID NO29, SEQ ID NO30, and SEQ ID NO31. Non-limiting examples of conjugates include: antibodies (e.g., antibodies derived from the immunology or genetics of any species such as humans, chickens, camels, llamas, lampreys, sharks, goats, rodents, cattle, dogs, rabbits, etc.), antibody fragments, their domains or portions, such as Fab, Fab', F(ab')2, scFab, Fv, scFv, VH, VHH, VL, VLR, etc., biantibodies, monoclonal antibodies (mAb), polyclonal antibodies (pAb), mAbdAb, phage display-derived conjugates, affinities, heteroconjugated antibodies, bispecific antibodies, evibody, lipid transport proteins, anticalin, affinities, avime. r, macrobody, heat shock proteins (such as GroEL and GroES), transbody, DARPin, aptamers, C-type lectin domains (such as tetralectin); human γ-lens proteins and human ubiquitin-derived conjugates (such as affilin), PDZ domain-derived conjugates; scorpion venom and / or Kunitz-type domain conjugates, fibronectin-derived conjugates (e.g., adnectin), receptors, ligands, lectins, streptavidin, biotin, including their derivatives and / or combinations, such as bispecific / multispecific forms formed from two or more of these conjugate molecules.Various antibody-derived and alternative (i.e. non-antibody) binding protein scaffolds are known in the art, including their generation methods (e.g., Chiu ML et al., Antibodies (Basel), (2019) 8(4):55; Simeon R. & Chen Z., Protein Cell. (2018) 9(1):3-14; and Chapter 7 – Non-Antibody Scaffolds from Handbook of Therapeutic Antibodies (2007), Stefan Dübel (ed.), US 7,166,697; Rothe C and Skerra A., BioDrugs. (2018)32(3):233-243; Gebauer M and Skerra A, Curr Opin Biotechnol. (2019) 60:230-241; Feldwisch, J and Tolmachev, V. (2012) Methods Mol. Biol.). 899:103-126; Wikman M et al., Protein Eng Des Sel. (2004) 17(5):455-62; Silverman J et al. (2005), NatBiotechnol 23:1556–1561; Plückthun A., Annu Rev Pharmacol Toxicol. (2015) 55:489-511; Hosse RJ et al. (2006) Protein Sci 15:14-27; Hackel BJ et al. (2008) J MolBiol 381:1238–1252 (reviewed in these journals). In a preferred embodiment, the secondary antibody is an antibody that binds to all antibodies from antibody or immunoglobulin classes, preferably human antibody classes, preferably IgA and / or IgG and / or IgM antibodies, preferably IgG. Secondary antibodies can recognize constant domains of the target Ig class, or one or more epitopes in sequences or 3D structures common to antibodies of the target Ig class. Secondary antibodies are typically derived from mammals other than humans or from birds, preferably from chickens, rabbits, mice, rats, horses, pigs, donkeys, goats, cattle, camels, llamas, or non-human primates. Secondary antibodies can be monoclonal antibodies (preferably recombinant antibodies) or polyclonal antibodies. A wide range of these are commercially available. In a preferred embodiment, the secondary antibody specifically binds to human IgG, more preferably to constant regions of human IgG.

[0090] In a preferred embodiment, the ligand for the affinity tag, as used herein, is an artificial entity that specifically binds to the affinity tag, typically a chemically synthesized modified or recombinant protein or peptide linked to the target molecule. The ligands targeting the affinity tag depend on the type of affinity tag chosen and can be selected from the following groups: His, immobilized nickel, glutathione, chitin, 18A, ACP, Aldehyd, Avi, BCCP, calmodulin, chitin-binding protein, E-tag, ELK16, FLAG, flash, polyglutamic acid, polyaspartic acid, GST, GFP, HA, Isope, maltose-binding protein, myc, nus, NE, ProtA, ProtC, Tho1d4, S-tag, SnoopTag, SpyTag, SofTag, streptavidin, Strep-tag II, T7 epitope tag, TAP, TC, thioredoxin, Ty, V5, VSV, biotin, Xpress tag, and recombinant antibodies that bind to ligands targeting the affinity tag.

[0091] According to the present invention, an antibody specifically binding to NECAB1 is provided, preferably in a solution comprising one or more, more preferably all, of a group consisting of artificial buffers, preservatives, and artificial anticoagulants. An artificial buffer is a buffer that is synthetic and / or may not be present in the patient or at least at a concentration much lower than that used. The buffer may be selected from the group consisting of Tris, phosphates, Tricine, acetates, MOPS, MES, carbonates, citrates, and HEPES. In a preferred embodiment, the term "preservative," as used herein, refers to a substance that inhibits microbial growth and / or chemical degradation in a liquid solution, and may preferably be selected from the group consisting of azides, lactic acid, nitrates, nitrites, antibiotics, protease inhibitors, and ethanol.

[0092] Various methods or uses according to the invention can be performed using samples from subjects as described herein. These methods or uses can also be characterized as “in vitro” methods or “in vitro” uses.

[0093] In a preferred embodiment, the term "chemical solution reacting with a detectable label" refers to a compound in a liquid that emits a detectable signal when exposed to the detectable label. The solution may contain a chromogenic substrate of the label with enzymatic activity. For example, if the label is peroxidase, 3,3',5,5'-tetramethylbenzidine / H₂O₂ may be used. The solution may contain small inorganic or organic compounds capable of reacting with the chemiluminescent label. In the case of acridinium esters, a mixture of H₂O₂ and sodium hydroxide is commonly used as the chemical solution. Various other chemical solutions and detectable labels are known in the art (Weeks, I., Beheshti, I., McCapra, F., Campbell, AK, Woodhead, JS (1983) Acridinium esters as high specific activity labels in immunoassay). Clin Chem 29: 1474-1479), Thermo Scientific Pierce Antibody Production and PurificationTechnical Handbook, Version 2, www.thermoscientific.com).

[0094] In a preferred embodiment, the term “diagnosis” as used herein should be used in its broadest possible sense and can refer to any kind of procedure aimed at obtaining information that helps assess whether a patient (known or anonymized from a cohort) has had, is likely to have, or is more likely to have than average or control subjects (preferably with similar symptoms) a certain disease or disorder in the past, at diagnosis, or in the future, to discover how the disease progresses or is likely to progress in the future, or to evaluate the overall responsiveness of one or more patients to a particular treatment (e.g., administration of an immunosuppressive drug), or to determine whether a sample is from such a patient. Such information can be used for clinical diagnosis, but can also be obtained by laboratory and / or research laboratories for research purposes in general, such as to determine the proportion of subjects with the disease in a patient cohort or population. In other words, the term “diagnosis” includes not only diagnosis but also the prediction and / or monitoring of the progression of a disease or disorder, including monitoring the response of one or more patients to the administration of a drug or candidate drug, for example, to determine its efficacy. While for clinical diagnostic applications, results may be assigned to a specific patient and may be communicated to the physician or medical institution treating said patient, this is not necessarily the case for other applications, such as in a diagnosis for research purposes, where assigning results to samples from anonymized patients may be sufficient. Therefore, in some embodiments, the person to be diagnosed (i.e., the “subject” or “patient”) is an anonymous blood donor whose blood may be donated or used to obtain antibodies that are therapeutically or diagnostically useful. The term “diagnosis” also refers to a negative diagnosis, where no antibody specifically binding to NECAB1 or its variants is found in a sample from the patient. In these embodiments, the absence of an antibody specifically binding to NECAB1 or its variants indicates that the patient may have a disease other than one associated with the presence of said antibody (as described herein). Therefore, in one embodiment, “diagnosis” also includes a situation where the absence of an antibody specifically binding to NECAB1 or its variants helps rule out certain diseases (such as those associated with the presence of such antibodies, as described herein), which may lead to, aid in, or support an indirect diagnosis of another disease. In another preferred embodiment, the detection of an antibody specifically binding to NECAB1 or its variants is considered to signify a definitive diagnosis of an autoimmune disease of the nervous system because of the presence of the antibody.

[0095] Therefore, in a preferred embodiment, the terms “diagnosis,” “diagnosing,” or “diagnostic” cover the diagnosis, prognosis, therapeutic diagnosis, and monitoring of autoimmune diseases (preferably neurological autoimmune diseases, such as those described herein) and / or cancers (such as those described herein). As used herein, the term “therapeutic diagnosis” refers to identifying (e.g., by diagnostic methods) a patient who may benefit from a particular therapy, and optionally subsequently treating said patient.

[0096] In a preferred embodiment, the methods and products according to the invention can be used for interaction studies, including determining whether a candidate drug or other compound may interfere with the binding of an antibody that specifically binds to NECAB1 or a variant thereof, or may affect any downstream process or the strength of its binding to its target. In a preferred embodiment, they can be used to monitor immune responses, more preferably, after administration, for example, to a mammal (which may be a mammal other than a human, such as a laboratory animal) of an immunogenic composition comprising a polypeptide containing NECAB1 or a variant thereof or an immunogenic variant thereof, to monitor the presence and / or titer of antibodies against the antigens of the invention.

[0097] In a preferred embodiment, the methods and products may be used to provide reagents, such as antibodies against NECAB1 or variants thereof, which may serve as positive controls or calibrators for diagnostic tests or for the development and / or validation of diagnostic tests. In a preferred embodiment, the term "validation," as used herein, refers to procedures for establishing an assay in a specific setting based on previously known principles and confirming that it produces useful results. For example, while this application discloses the usefulness of antibodies against the antigens of the present invention as biomarkers for diagnosis, clinical or research laboratories may need to confirm the diagnostic value of the results before routinely using the test on their patients or a new group of patients (e.g., a group of animals that may not have been previously known to have a disease or condition).

[0098] In another preferred embodiment, the method and product according to the invention can be used to determine the concentration of an antibody that specifically binds to NECAB1 or a variant thereof. In a more preferred embodiment, the antibody is an autoantibody derived from a patient suffering from a neurological autoimmune disease. In another preferred embodiment, the antibody is a recombinant antibody that binds to NECAB1 or a variant thereof, but is recognized by a secondary antibody that specifically binds to human immunoglobulins (preferably human IgG antibodies, preferably IgG1, IgG2, IgG3, and IgG4 isotypes). In a more preferred embodiment, such a concentration needs to be determined for research purposes, for the preparation or monitoring of the quality of reagents, animal models, or devices (which may or may not be used for the diagnosis of neurological autoimmune diseases).

[0099] In many cases, detecting antibodies alone (in other words, determining the presence of detectable levels of antibodies in a sample) is sufficient for diagnosis. In a more preferred embodiment, this may involve determining whether the concentration is at least 10%, preferably 20%, 50%, 100%, 200%, 500%, 1000%, 2000%, 2500%, 5000%, 10000%, 20000%, 50000%, 100000%, 100000%, or 10000000%. If antibodies are detectable, this information will be helpful for clinicians in making a diagnosis. It can indicate an increased likelihood that a patient has a disease.

[0100] Those skilled in the art will understand that clinicians typically do not draw conclusions about whether a patient has or may have a disease, condition, or disorder based solely on a single diagnostic parameter. Instead, they consider other factors, such as the presence of other antibodies, biomarkers, blood parameters, clinical assessment of the patient's symptoms, or the results of medical imaging or other non-invasive methods (such as polysomnography), to arrive at a definitive diagnosis. See Baenkler HW (2012), General aspects of autoimmune diagnostics, in Renz, H., Autoimmune diagnostics, 2012, deGruyter, p. 3. The value of diagnostic agents or methods can also lie in ruling out the possibility of one disease, thereby allowing for an indirect diagnosis of another. In a preferred embodiment, any symptom or disease mentioned throughout this application is understood by those skilled in the art as of the filing date or preferably the earliest priority date of this application, as demonstrated by textbooks and scientific publications. In a preferred embodiment, the method, use, or product is not used alone to arrive at a definitive final diagnosis.

[0101] In a preferred embodiment, any information or data demonstrating the presence or absence of antibodies may be communicated orally (preferably by telephone), in writing (preferably by fax or letter), or electronically (by fax or via the Internet, such as as email or text message) to the patient or the physician treating the patient.

[0102] The teachings of this invention can also be used in methods for the prevention or treatment of diseases, preferably after a diagnosis according to the invention, the method comprising steps a) reducing the concentration of antibodies in the blood of a subject that bind to NECAB1 or a variant thereof and / or b) administering one or more immunosuppressive drug substances, preferably selected from the group consisting of: rituximab, prednisone, methylprednisolone, cyclophosphamide, mycophenolate mofetil, intravenous immunoglobulin, tacrolimus, cyclosporine, methotrexate and azathioprine.

[0103] According to the present invention, the presence of antibodies can be determined qualitatively or quantitatively. In a preferred embodiment, the term "quantitative detection" as used herein means not only detecting the presence of antibodies but also obtaining results containing information about the absolute or relative amount of antibodies in the sample. In a more preferred embodiment, values ​​representing absolute concentrations are obtained. In another more preferred embodiment, values ​​representing relative concentrations or changes in concentration are obtained. In another preferred embodiment (also referred to as a "semi-quantitative" method), the antibody concentration is placed in one of several groups, most preferably, a concentration window that means it is not actually present, a concentration window that means a boundary result is obtained, and a concentration window that means the presence of antibodies. Further distinctions can be made between categories such as "weak positive" or "strong positive" signals.

[0104] In a preferred embodiment, the term "autoantibody" as used herein refers to an antibody that specifically binds to an endogenous molecule of an animal, preferably a mammal, more preferably a human, that produces the autoantibody, wherein the level of such an antibody is more preferably elevated compared to the average healthy subject. Thus, an autoantibody is an endogenous molecule produced in the body of a subject. The subject is preferably a patient suspected of or actually suffering from a disease, preferably a mammal, more preferably a human patient. Such an autoantibody has a constant region, like other antibodies of the same class from the same organism. Particularly preferably, the autoantibody is a mammalian autoantibody, even more preferably a human autoantibody, even more preferably a human autoantibody of the IgG, IgM, or IgA (preferably IgG) class. Its variable domain is capable of specifically binding to the antigen described herein. In one embodiment, the constant domain specifically binds to molecules such as secondary antibodies that recognize the constant domain of human immunoglobulins (preferably IgG class antibodies). Therefore, the autoantibody may have a sequence of the constant region of the antibody derived from the animal (preferably human) from which it was produced, but the variable region is capable of specifically binding to the endogenous molecules of said animal, more particularly to NECAB1 or its variants, and even more preferably to SEQ ID NO1, SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5, SEQ ID NO6, SEQ ID NO7, SEQ ID NO8, SEQ ID NO9, SEQ ID NO10, SEQ ID NO18, SEQ ID NO19 and / or SEQ ID NO20. In a preferred embodiment, the autoantibody is isolated and / or purified from a sample (preferably tissue, serum, plasma, blood or CSF) from an animal (preferably human). The autoantibody can be isolated as a mixture of polyclonal, natural antibodies from an animal or patient. It is not a synthetic, monoclonal or recombinant antibody. Recombinant antibodies that specifically bind to NECAB1 or its variants can be generated using standard methods. The autoantibody or recombinant antibody can be used as a positive control and for detection assays, such as sandwich assays or competitive assays.

[0105] As used herein, the term "isolated" means that a molecule referred to as isolated contains or substantially contains at least one of the components found in nature. For example, a molecule (such as an antibody) may contain or substantially contain some or all of the components found in its natural environment. In the case of an antibody, such components may include, for example, red blood cells, white blood cells, platelets, plasma, proteins, nucleic acids, salts, lipids, and nutrients.

[0106] The method according to the present invention is preferably an in vitro method.

[0107] According to the present invention, the polypeptide (preferably a polypeptide comprising an antigen or at least one epitope thereof or a variant of said antigen or epitope thereof, wherein said antigen is NECAB1 or a variant thereof) may be a recombinant protein. In a preferred embodiment, the term “recombinant” as used herein refers to a polypeptide produced at any stage of the production process using genetic engineering methods, such as by fusing a nucleic acid encoding the polypeptide with a strong promoter for overexpression in cells or tissues, or by engineering the sequence of the polypeptide itself. Those skilled in the art are familiar with methods for engineering nucleic acids and encoded polypeptides (e.g., described in Sambrook, J., Fritsch, EF and Maniatis, T. (1989), Molecular Cloning, CSH or Brown TA (1986), Gene Cloning – an introduction, Chapman & Hall), as well as methods for producing and purifying natural or recombinant polypeptides (e.g., GE Healthcare Life Sciences’ manuals “Strategies for Protein Purification”, “Antibody Purification”, and in Burgess, RR, Deutscher, MP (2009): Guide to Protein Purification). In another preferred embodiment, the polypeptide (such as a polypeptide comprising an antigen or at least one epitope thereof or a variant of said antigen or epitope thereof, wherein said antigen is NECAB1) or antibody provided or used according to the invention is an isolated polypeptide, wherein the term "isolated" means that the polypeptide has been enriched compared to its state when it was produced using biotechnological or synthetic methods, and preferably is pure, i.e., as determined by SDS-polyacrylamide gel electrophoresis followed by Coomassie blue staining and visual inspection, in the corresponding liquid, at least 60%, 70%, 80%, 90%, 95%, or 99% of the polypeptide consists of said polypeptide. Preferably, any polypeptide on the carrier used as a tool for capturing antibodies is pure.

[0108] Patients with antibodies against NECAB1 or its variants can have a variety of cancers, including breast cancer, lung cancer (such as NSCLC), brain metastases, and melanoma. In a preferred embodiment, the term "cancer," as used herein, refers to a group of diseases involving the abnormal growth of cells with the potential to invade or spread to other parts of the body.

[0109] In a preferred embodiment, the terms "breast cancer" or "breast cancer," as used interchangeably herein, are defined as cancer originating in the breast gland, with cells exhibiting a malignant form and structure, the ability to grow uncontrollably, and the potential or ability to invade or metastasize. In a preferred embodiment, the breast cancer has spread to other organs, such as lymph nodes. In another preferred embodiment, the breast cancer is invasive and may be metastatic.

[0110] In a preferred embodiment, the term "non-small cell lung cancer (NSCLC)" as used herein includes epidermoid carcinoma cells, adenocarcinoma cells, and large undifferentiated carcinoma cells. The most common symptoms associated with NSCLC are cough (including hemoptysis), weight loss, shortness of breath, and chest pain.

[0111] In a preferred embodiment, the term "brain metastasis," as used herein, refers to a tumor that originates outside the central nervous system (CNS, such as the brain and spinal cord) and spreads to it via the bloodstream or invades directly from adjacent tissues. Metastatic tumors are the most common tumors affecting the brain and spinal cord.

[0112] In a preferred embodiment, the term "melanoma" as used herein refers to a malignant tumor of melanocytes. Melanocytes are cells that produce the dark pigment melanin, which is responsible for skin color. They are primarily found in the skin, but also in other parts of the body, including the intestines and eyes. Melanoma can occur in any part of the body that contains melanocytes.

[0113] Additional background and definitions relating to cancer and its diagnosis or differential diagnosis (including antibody testing) may be derived from neurology textbooks available at the earliest priority date or filing date of this disclosure, such as Kaye, Textbook of Medical Oncology, 3rd edition, Taylor & Francis; Shøenfeld, Meroni and Gershwin, Autoantibodies, 3rd edition, Elsevier, including in particular Part 11 of Chapter 76; and Darnell and Posner, Paraneoplastic Syndromes, Oxford University Press, 2011.

[0114] Patients with antibodies against NECAB1 or its variants have a variety of autoimmune diseases, such as neurological autoimmune diseases including PNS, dementia, psychotic symptoms, auditory hallucinations, seizures, encephalitis, encephalopathy, encephalomyelitis, myelitis, cerebellar encephalitis, neuropathy, dementia, peripheral polyneuropathy, Guillain-Barré syndrome (GBS) symptoms, quadriplegia, cerebellar / brainstem syndrome, hearing loss, symmetrical macular degeneration, cerebellar-pontine lesions extending into the spinal cord, personality changes, cognitive impairment and mood disorders, gait and standing ataxia, multiple cerebral ischemia, cancer, brain metastases and melanoma, preferably rapidly progressive dementia, limbic encephalitis, brainstem encephalitis, autoimmune encephalitis, breast cancer and lung cancer, such as lung cancer (such as NSCLC).

[0115] In a preferred embodiment, as used herein, the term "ataxia" refers to a lack of voluntary muscle motor coordination, which may include gait abnormalities, speech changes, and eye movement abnormalities. Ataxia is a clinical manifestation indicating dysfunction of the nervous system components that coordinate movement, such as the cerebellum.

[0116] In a preferred embodiment, as used herein, the term "polyneuropathy" refers to a disease (peripheral neuropathy) affecting substantially the same area of ​​the peripheral nerves on both sides of the body, characterized by weakness, numbness, and burning pain.

[0117] In a preferred embodiment, the term "encephalitis" as used herein refers to inflammation of the brain, with symptoms including decreased or altered consciousness, personality changes, psychotic delusions, rigidity, headache, fever, confusion, neck stiffness, and vomiting. Complications may include seizures, hallucinations, speech difficulties, memory problems, and hearing problems. The illness can be a result of infection or an autoimmune disease; therefore, antibody detection according to the present invention can be used to differentiate between these two types of encephalitis.

[0118] In the preferred embodiment, the term “dementia,” as used herein, broadly refers to any disorder, disease, or syndrome characterized by an abnormally high and progressive loss of brain function. While symptoms of dementia can vary considerably, hallmarks of dementia include impairment in several core mental functions, including memory, communication and language, the ability to focus and pay attention, reasoning and judgment, and visual perception. People with dementia may have problems with short-term memory, keeping a handbag or wallet safe, paying bills, planning and preparing meals, remembering appointments, or leaving home. Many cases of dementia are progressive, meaning that symptoms begin slowly and gradually worsen. Dementia can be determined using standard clinical procedures, and the degree of dementia is defined by a score in the Mini-Mental State Examination (MMSE), as detailed in Folstein MF, Folstein SE, and McHugh PR, J Psychiatry Res., 12:189-198 (1975). Examples of dementia or dementia-related neurological disorders include, but are not limited to, Alzheimer's disease (AD), progressive supranuclear palsy (PSP), Huntington's disease (HD), mixed dementia, Parkinson's disease, diffuse Lewy body dementia, vascular dementia, frontotemporal dementia, semantic dementia, and Lewy body dementia. In a more preferred embodiment, the dementia is rapidly progressive dementia.

[0119] In a preferred embodiment, the term "ischemia" as used herein refers to a condition resulting from a reduction or lack of blood flow and oxygen to a part of the body, such as the brain, heart, or other tissues. Ischemic injury generally refers to damage to distal or otherwise affected tissues caused by loss of blood flow and oxygen. Ischemic injury is usually a result of a lack of oxygen and fluid, but can also include inflammatory cascades. For example, ischemia and ischemic injury can occur due to heart, lung, or brain injury, organ transplantation or surgery, or disease or disorder (such as sickle cell anemia or sickle cell disease). In a more preferred embodiment, the ischemia is multiple cerebral ischemia.

[0120] In a preferred embodiment, the term "paraneoplastic neurological syndrome (PNS)," as used herein, refers to a neurological syndrome associated with the presence of cancer that is associated with a tumor presenting antigens typically unique to the nervous system. As a result, antibodies that specifically bind to these neuroantigens are generated, which can then impair the nervous system. Manifestations of PNS include, but are not limited to, encephalitis (often associated with seizures), psychotic manifestations (such as hallucinations, anxiety, and depression), cerebellar symptoms (such as ataxia, nystagmus, and dysarthria), oculoclonus-myoclonus, and sensory neuron neuropathy; and Lambert-Eaton myasthenia gravis. It should be noted that PNS-associated tumors are typically small, slow-growing, and may not be detectable when neurological symptoms appear. PNS can be associated with one or more antibodies that can specifically bind to antigens from the group comprising: NMDAR, LgI1, AMPA1, AMPA2, CASPR2, GABA B, GABA A, DPPX, IGLON5, Hu, Yo, CRMP5, Ri, Ma2, dual-carrier protein, recovery protein, RGS8, DAGLA, NSF, STX1B, DNM1 and VAMP2, Hu, Ri, Ma, Anna-3, Zic-4, SOX1, Yo, PCA2, Tr and glutamate decarboxylase.

[0121] Additional background and definitions relating to neurological syndromes and symptoms and their diagnosis or differential diagnosis (including antibody testing) may be derived from neurology textbooks available at the earliest priority date or filing date of this disclosure, such as Simon, Greenberg, Aminoff, Clinical Neurology, 7th edition, 2009, McGraw; Shoeenfeld, Meroni, and Gershwin, Autoantibodies 3rd edition, Elsevier, particularly including Part 11 of Chapter 76; and Darnell and Posner, Paraneoplastic Syndromes, Oxford University Press, 2011.

[0122] The methods described herein for determining the presence of antibodies binding to NECAB1 or its variants in a sample can be combined with methods for determining the presence of other antibodies associated with the occurrence of the disease or similar diseases described herein. For example, one or more polypeptides selected from NECAB1 or its variants, as described herein, can be used in combination with one or more polypeptides or their variants that bind to other antibodies associated with the occurrence of the disease or similar diseases described herein. For example, devices or carriers comprising polypeptides containing NECAB1 or its epitopes or variants of NECAB1 or its epitopes, such as the devices or carriers of the present invention, may also comprise polypeptides or their variants that bind to other antibodies associated with the occurrence of the disease or similar diseases described herein. Such devices can be used to analyze the presence of any antibodies from samples taken from subjects, such as patients, associated with the occurrence of the disease or similar diseases described herein. In the case of using such an apparatus or a method using several apparatuses, wherein each apparatus comprises at least one polypeptide or variant thereof, said polypeptide or variant thereof binding to an antibody that binds to NECAB1, or to the presence of another antibody associated with the occurrence of the disease or similar disease described herein, determining the presence of any antibody that binds to any assayed polypeptide or variant thereof may be sufficient for a person skilled in the art to conclude the presence of a disease, or to limit a suspected disease to certain diseases, or to assist in the diagnosis of a disease as described herein, or to obtain information about the risk of a subject having or developing a disease as described herein, currently, in the past, or in the future. Therefore, in some cases, determining the presence of an antibody that binds to NECAB1, or the presence of any other antibody associated with the occurrence of the disease or similar disease described herein, may be sufficient without obtaining information about which specific antibody is present in a given sample or drawing such a conclusion, as long as the presence of said antibody is associated with the occurrence of one or more of the same or similar diseases. Therefore, determining the presence of an antibody that binds to NECAB1 may include determining the presence of any antibody associated with the occurrence of the NECAB1-related disease or similar disease described herein.

[0123] Those skilled in the art will understand that, in certain circumstances, it can be helpful to first analyze the clinical symptoms of patients who have or are suspected of having a disease associated with the presence of antibodies binding to NECAB1. Based on the symptoms, severity of the damage, and other factors known in the art, a clinician or another medical or scientific person can then determine whether the antibody titer of the subject being analyzed is increased compared to a healthy subject, or compared to the titer of the subject being analyzed before the onset of symptoms, or compared to the mean. Subsequently or independently, one or more methods as described herein for determining the presence of antibodies binding to NECAB1 can be used to determine the presence of such antibodies and / or to determine the presence of any antibodies whose presence is associated with the disease described herein. Thus, determining the presence of antibodies binding to NECAB1 can be used as a pre-investigation or, among other things, investigation, allowing a clinician to conclude whether a subject currently, has, or may have a disease. Determining the presence of antibodies binding to NECAB1 can also be used to differentiate the presence of drug or alcohol use or abuse or infectious diseases from the presence of autoimmune diseases as described herein.

[0124] In some embodiments, determining the Ig class of the antibody that binds to NECAB1 can be helpful. Therefore, in one embodiment, determining the presence of an antibody that binds to NECAB1 includes determining the Ig class of said antibody. In other embodiments, determining the presence of an antibody that binds to NECAB1 does not include determining the Ig class of said antibody. Similarly, determining the presence of an antibody of a specific Ig class that binds to NECAB1 includes determining the presence of antibodies of other Ig classes that bind to NECAB1. Determining the presence of an antibody of a specific Ig class that binds to NECAB1 may also include determining the absence of said antibody.

[0125] As part of the diagnosis of neurological syndromes associated with NECAB1-related antibodies, clinicians will initially consider a range of tests and risk factors, which, for many conditions associated with the presence of antibodies that specifically bind to NECAB1, preferably encephalitis, can point to autoimmune or infectious diseases (Lancaster, J ClinNeurol. Jan 2016; 12(1) https: / / doi.org / 10.3988 / jcn.2016.12.1.1, Lee and Lee, The Laboratory Diagnosis of Autoimmune Encephalitis, Journal of EpilepsyResearch 6(2), 45). Detection of antibodies that specifically bind to NECAB1 will then confirm an autoimmune background, while the absence of antibodies will lead clinicians to consider autoimmune diseases associated with other antibodies. However, typically a range of antibody tests will then be performed to determine the presence or absence of antibodies, and a negative result will point to an infectious disease.

[0126] According to the present invention, a kit is provided comprising cells or a carrier, or a polypeptide and / or a tool for capture and / or a tool for detecting an antibody that specifically binds to NECAB1, and further comprising one or more, preferably all, reagents from the group consisting of: a secondary antibody (preferably labeled with a detectable marker), a washing solution, a positive control, a negative control, a detergent, coverslips, a mounting medium, and a physiological saline solution (preferably PBS or the salt required to prepare it). In a preferred embodiment, the positive control is a diluted sample (preferably serum or CSF) from a patient with a neurological autoimmune disease, or a monoclonal antibody that specifically binds to NECAB1. In a preferred embodiment, the kit includes a tool for capture and / or a tool for detecting an antibody that specifically binds to NECAB1. The negative control may be a diluted sample from a healthy subject, such as a blood donor. The kit may include instructions on how to perform the assay. Preferably, the secondary antibody is a secondary antibody that specifically binds to a constant region of an IgG antibody, preferably a human IgG antibody, more preferably a human IgG antibody. In other preferred embodiments, the secondary antibody may be labeled (as described above).

[0127] In a preferred embodiment, the present invention provides the use of cells, peptides, and carriers for preparing kits or compositions for diagnosing diseases, preferably related to the presence of mammalian antibodies, preferably autoantibodies, that specifically bind to NECAB1.

[0128] In a preferred embodiment, any method or use according to the invention can be intended for non-diagnostic purposes, i.e., determining the presence of antibodies that specifically bind to NECAB1, for use other than diagnosing a patient. For example, the method or use can be used to test the efficiency of a medical device designed to remove antibodies from a patient's blood in vitro, wherein the test is performed on a liquid other than the patient's blood. After the medical device is used on a patient, its ability to remove antibodies can be examined by passing a solution containing antibodies that specifically bind to NECAB1 through the device, and subsequently confirming, using the method according to the invention, that there are few or no antibodies in the solution that has passed through the device, i.e., indicating that the device still has the ability to remove antibodies from the solution.

[0129] According to the present invention, the method can be used to test the efficacy of a drug candidate that can be used to treat patients with or potentially with neurological autoimmune diseases. Such a drug candidate can be any molecule capable of interfering with the interaction between NECAB1 and its corresponding antibody.

[0130] In another preferred embodiment, the method can be used to confirm the reliability of a diagnostic assay and may involve detecting antibodies that specifically bind to NECAB1 in a solution not derived from a sample from a patient requiring diagnosis, but known to contain, preferably, a known concentration, of antibodies that specifically bind to NECAB1. For example, it could be a recombinant antibody, or a sample diluted in a dilution buffer (such as PBS) derived from an anonymous patient whose identity cannot be traced. Alternatively, the solution could be a negative control not containing the specifically binding antibody to check for background. Such a method can be run in parallel with, after, or before a diagnostic method. In a preferred embodiment, any method or use according to the invention can be intended for generating an antibody profile, preferably for detecting diseases in mammals, preferably humans.

[0131] In a preferred embodiment, any method or use according to the invention can be used to identify subjects at risk of having or developing a disease and / or tumor.

[0132] In a preferred embodiment, the method can be used to detect antibodies that specifically bind to NECAB1 in a solution that is not derived from a sample of a mammal to be diagnosed or for diagnostic purposes.

[0133] In a preferred embodiment, the problem upon which the present invention is based is solved by a method comprising the step of contacting a device containing a solid phase with a buffer solution, wherein a polypeptide comprising an antigen or at least one epitope thereof or a variant of the antigen or an epitope thereof is immobilized on the solid phase, wherein the antigen is NECAB1, and the buffer solution comprises an antibody that specifically binds to the antigen, wherein the solution is preferably not derived from a sample from a patient requiring diagnosis, wherein preferably:

[0134] a) The concentration of the antibody in the solution is known, and / or

[0135] b) The antibody is a recombinant antibody and / or

[0136] c) Contacting a medical device or diagnostic device with two or more solutions containing antibodies, wherein the two or more solutions have different antibody concentrations, and / or

[0137] d) The antibody is recognized by a secondary antibody that specifically binds to an IgG antibody.

[0138] The signal indicating whether the antibody has bound to the peptide is then detected, optionally a signal related to the antibody concentration in one or more solutions.

[0139] In a preferred embodiment, the present invention provides an apparatus for analyzing samples from a patient to detect antibodies that specifically bind to NECAB1, which indicate an increased likelihood of developing or progressing to a neurological autoimmune disease, the apparatus comprising:

[0140] a. A support containing tools for capturing antibodies from a sample when the sample comes into contact with the support, preferably said tools being cells or peptides, and said support being a support according to the invention.

[0141] b. A tool for detecting antibodies capable of binding to antibodies captured by the carrier when the detectable tool comes into contact with the carrier, wherein the tool is preferably a labeled secondary antibody capable of binding to antibodies captured on the carrier.

[0142] c. Optional tools for removing any sample from the carrier and the detectable tool, preferably by washing;

[0143] d. A detection device for detecting the presence of a detection instrument and converting the result into an electrical signal, such as a fluorescence reader or a fluorescence microscope with connected software capable of recognizing characteristic patterns of stained cells overexpressing an antigen or at least one epitope thereof, or a variant of said antigen or epitope, in cell images captured by the fluorescence reader or camera, wherein said antigen is NECAB1, and

[0144] Optional tools for receiving electronic signals from the detection device and determining whether the signal level indicates an increased likelihood of having or developing a disease by comparing it with a characteristic pattern of wild-type or unstained cells (preferably by simulated transfected cells on the same carrier or cells not stained with an antibody that specifically binds to NECAB1), or by input reference values ​​obtained from samples from healthy subjects, or by comparing the signal level obtained with one sample with the signal level obtained with a second sample at a subsequent time point (preferably at least one month later).

[0145] According to the present invention, an apparatus for removing antibodies specifically binding to NECAB1 from the blood, preferably serum, of a patient suffering from a neurological autoimmune disease is provided as part of an ex vivo method for removing antibodies specifically binding to NECAB1 from a patient. The apparatus comprises a carrier having a solid phase on which a polypeptide containing an antigen or at least one epitope thereof, or a variant of said antigen or epitope thereof, is immobilized, wherein said antigen is NECAB1. An apparatus may be used having a polypeptide containing an antigen or at least one epitope thereof, or a variant of said antigen or epitope thereof (where said antigen is NECAB1), or a secondary antibody or protein that captures all IgG antibodies, including IgG antibodies against NECAB1. A suitable method is described in Eisei Noiri and Noria Hanafusa, The Concise Manual of Apharesis Therapy, Springer Tokyo, 2014. Hamilton, P., Kanigicherla, D., Hanumapura, P., Walz, L., Kramer, D., Fischer, M., Brenchley, P., and Mitra, S. (2018) J.Clin. Aph. 33(3), 283-290. Another method is disclosed in EP3477300.

[0146] In some implementations, antibodies that specifically bind to NECAB1 and / or their detection are used as biomarkers, preferably wherein the biomarker indicates the health status of the subject, particularly whether the subject has an autoimmune disease (such as a neurological autoimmune disease) and / or cancer or is at risk of developing one.

[0147] In a preferred embodiment, the methods, uses, and products, such as peptides, carriers, or kits, can be used to diagnose or assist in the diagnosis of diseases, preferably as described herein, such as autoimmune diseases of the nervous system and / or cancer.

[0148] The methods, uses, and products described herein can also be used to differentiate between neurological diseases and / or cancers, such as distinguishing between autoimmune neurological diseases and diseases with different causes but similar symptoms (such as infectious neurological diseases or aseptic non-autoimmune neurological diseases), for example, distinguishing between autoimmune encephalitis (including limbic system encephalitis and brainstem encephalitis) and infectious and / or aseptic non-autoimmune encephalitis or meningitis, including encephalitis of infectious etiology (such as viral encephalitis, bacterial encephalitis, tick-borne encephalitis, herpes encephalitis, CMV encephalitis, Guillain-Barré syndrome, toxoplasmosis encephalitis), meningitis of infectious etiology (such as viral meningitis, bacterial meningitis), and aseptic non-autoimmune meningitis and encephalitis (such as Besnier-Boeck-Schaumann disease, Behcet disease, Mollaret meningitis).

[0149] In some embodiments, the term "one or more" includes "at least one". In some embodiments, "one or more" and "at least one" are interchangeable. In some embodiments, the term "preferredly" includes "optionally". In some embodiments, "preferredly" and "optionally" are interchangeable.

[0150] The present invention is further illustrated by the following non-limiting embodiments, from which other features, embodiments, aspects and advantages of the invention can be obtained. Attached Figure Description

[0151] Figure 1 Immunostaining was performed on neuronal tissue. Diluted patient serum or CSF samples were incubated on rat cerebellum (not shown), rat hippocampus, or monkey cerebellum (not shown) and analyzed by immunofluorescence microscopy. Immunoprecipitation assays were performed on patient serum, followed by SDS-PAGE. Specific bands were analyzed by mass spectrometry.

[0152] Figure 2 This demonstrates the identification of the target antigen. Immunoprecipitation assays were performed on patient serum, followed by SDS-PAGE and Western blotting. Specific bands were analyzed by mass spectrometry.

[0153] Figure 3 Antigen functionality was demonstrated in a recombinant cell-based immunofluorescence assay (RC-IFA). Recombinant HEK293 cells expressing the corresponding antigens were fixed in acetone and incubated with serum from index patients or control serum.

[0154] Figure 4 Demonstrating antigenic functionality in Western blots. Lysates of recombinant HEK293 cells expressing the corresponding antigens were analyzed by Western blot using patient serum.

[0155] Figure 5 The antigenic functionality was demonstrated in the Western blot. Recombinant human His-NECAB1 purified from Escherichia coli was analyzed by Western blot using patient serum and anti-His antibody.

[0156] Figure 6 The autoantibody binding to the human NECAB1 fragment fused to His-C3b is shown in the Western blot. NECAB1 epitopes were detected in all three fragments in all analyzed patient serology samples. Patient samples did not react with other His-C3b fusion proteins (not shown).

[0157] Figure 7 The NECAB1 peptide fragment is shown as determined by mass spectrometry. Detailed Implementation

[0158] sequence

[0159] This invention includes a series of novel nucleic acid and amino acid sequences, more specifically...

[0160] SEQ ID NO1: Human NECAB1

[0161] MEDSQETSPSSNNSSEELSSALHLSKGMSIFLDILRRADKNDDGKLSFEEFKAYFADGVLSGEELHELFHTIDTHNTNNLDTEELCEYFSQHLGEYENVLAALEDLNLSILKAMGKTKKDYQEASNLEQFVTRFLLKETLNQLQSLQNSLECAMETTEEQTRQERQGPAKPEVLS IQWPGKRSSRRVQRHNSFSPNSPQFNVSGPGLLEEDNQWMTQINRLQKLIDRLEKKDLKLEPPEEEIIEGNTKSHIMLVQRQMSVIEEDLEEFQLALKHYVESASSQSGCLRISIQKLSNESRYMIYEFWENSSVWNSHLQTNYSKTFQRSNVDFLETPELTSTMLVPASWWILNN

[0162] SEQ ID NO2-SEQ ID NO10: Only in the sequence list

[0163] SEQ ID NO11: Justice NECAB1

[0164] ATACGTCTCACATGGAAGATTCCCAGGAGACATCG

[0165] SEQ ID NO12: Antonym NECAB1

[0166] TATCGTCTCGTCGAGCTAGTTGTTCAGGATCCACCACG

[0167] SEQ ID NO13: NECAB1-His antithesis of no-stop codon

[0168] TATCGTCTCGTCGAGGTTGTTCAGGATCCACCACG

[0169] SEQ ID NO14: Antonym NECAB1_aa110

[0170] ATACGTCTCTCGATCATTAGATGGAAAGATTCAGGCCTTCAAG

[0171] SEQ ID NO15: Justice NECAB1_aa100

[0172] ATACGTCTCACATGCTAGCAGCACTTGAAGGCCTGAATC

[0173] SEQ ID NO16: Antonym NECAB1_aa209

[0174] ATACGTCTCTCGATCATTATTCTAATAAGCCTGGACCGCTGAC

[0175] SEQ ID NO17: Justice NECAB1_aa203

[0176] ATACGTCTCACATGAGCGGTCCAGGCTTATTAGAAGAAG

[0177] SEQ ID NO18: Amino acid residues 1-110 from human NECAB1

[0178] MEDSQETSPSSNNSSEELSSALHLSKGMSIFLDILRRADKNDDGKLSFEEFKAYFADGVLSGEELHELFHTIDTHNTNNLDTEELCEYFSQHLGEYENVLAALEDLNLSI

[0179] SEQ ID NO19: Amino acid residues 100-209 from human NECAB1

[0180] LAALEDLNLSILKAMGKTKKDYQEASNLEQFVTRFLLKETLNQLQSLQNSLECAMETTEEQTRQERQGPAKPEVLSIQWPGKRSSRRVQRHNSFSPNSPQFNVSGPGLLE

[0181] SEQ ID NO20: Amino acid residues 203 - 351 from human NECAB1

[0182] SGPGLLEEDNQWMTQINRLQKLIDRLEKKDLKLEPPEEEIIEGNTKSHIMLVQRQMSVIEEDLEEFQLALKHYVESASSQSGCLRISIQKLSNESRYMIYEFWENSSVWNSHLQTNYSKTFQRSNVDFLETPELTSTMLVPASWWILNN

[0183] SEQ ID NO21: Human NECAB1

[0184] MEDSQETSPSSNNSSEELSSALHLSKGMSIFLDILRRADKNDDGKLSFEEFKAYFADGVLSGEELHELFHTIDTHNTNNLDTEELCEYFSQHLGEYENVLAALEGLNLSILKAMGKTKKDYQEASNLEQFVTRFLLKETLNQLQSLQNSLECAMETTEEQTRQERQGPAKPEVLSIQWPGKRSSRRVQRHNSFSPNSPQFNVSGPGLLEEDNQWMTQINRLQKLIDRLEKKDLKLEPPEEEIIEGNTKSHIMLVQRQMSVIEEDLEEFQLALKHYVESASSQSGCLRISIQKLSNESRYMIYEFWENSSVWNSHLQTNYSKTFQRSNVDFLETPELTSTMLVPASWWILNN

[0185] SEQ ID NO22: Human NECAB1 (isoform)

[0186] MLVQRQMSVIEEDLEEFQLALKHYVESASSQSGCLRISIQKLSNESRYMIYEFWENSSVWNSHLQTNYSKTFQRSNVDFLETPELTSTMLVPASWWILNN

[0187] SEQ ID NO23 - 38: Only in the sequence listing

[0188] Example

[0189] Overview

[0190] method A patient with a neurological disorder (P4) underwent a serological survey. For this purpose, autoantibodies were screened in the patient's serum using hippocampal and cerebellar tissue sections via indirect immunofluorescence assay (IFA). Serum was analyzed using brain tissue homogenate in an immunoprecipitation assay followed by mass spectrometry. Identified candidate antigens were recombinantly expressed in HEK293 cells and applied to recombinant IFA.

[0191] result Neuronal tissue IFA screening from P4 serum revealed an IgG reactivity pattern corresponding to the antigen identified by immunoprecipitation and mass spectrometry: N-terminal EF-handled calcium-binding protein 1 (NECAB1). Serum IgG reactivity to the identified antigen was detected by recombinant cell-based IFA (RC-IFA). Healthy control serum was also analyzed by RC-IFA. Indexed samples showed positive results, while control serum was negative or showed a low positivity rate. The Western blot reactivity of indexed samples with the corresponding recombinant antigen was analyzed. Clinical data from indexed patients were available.

[0192] patient

[0193] The control group included serum from 100 healthy donors, 33 patients with chronic inflammatory demyelinating polyneuropathy, 30 patients with diabetic neuropathy, 30 patients with Guillain-Barré syndrome (GBS), 49 patients with SCLC but without PNS, and CSF from 89 patients with Alzheimer's disease, 15 patients with normal pressure hydrocephalus, 20 patients with psychosis, 12 patients with Lewy body dementia, and 25 patients with frontotemporal dementia.

[0194] The indexed patient had personality changes, cognitive impairment and mood disorders, breast cancer, and limbic encephalitis. Other patients, P1, P2, P3, and P5, had cancers (including breast and lung cancer) and neurological disorders (including neuronal neuropathy, limbic encephalitis, and cerebellar syndrome). A sixth patient (P6) with an autoantibody against NECAB1 was identified, but clinical information was unavailable.

[0195] Characterization of patient autoantibodies

[0196] Indirect immunofluorescence assays (IFA) of serum P1, P2, P3, and P4 using unfixed frozen sections of the rat hippocampus revealed unique staining patterns. Figure 1 ).

[0197] Immunoprecipitation

[0198] Immunoprecipitation was performed using 200 µl of rat or monkey brain tissue homogenate and 30 µl of patient or control serum. The homogenate was centrifuged at 16,000 x g for 10 min at 4 °C. The sediment was resuspended together with the patient or control serum in 500 µl of lysis buffer (pH 7.4, 100 mmol / L tris-HCl, 150 mmol / L sodium chloride, 2.5 mmol / L EDTA, 0.5% (w / v) deoxycholate, 1% (w / v) Triton X-100 containing protease inhibitors) and rotated at 4 °C for 1 h. The suspension was centrifuged at 16,000 x g for 10 min at 4 °C. The supernatant was then incubated with Protein G Dynabeads (ThermoFisher Scientific, Schwerte, Germany) at 4 °C for 3 h to capture immune complexes. The beads were washed three times with PBS and eluted for 10 min at 70°C with NuPage LDS sample buffer (ThermoFisher Scientific) containing 25 mmol / L dithiothreitol. Carbamoyl methylation was performed with 59 mM iodoacetamide (Bio-Rad, Hamburg, Germany), followed by SDS-PAGE (NuPAGE, ThermoFisher Scientific). The isolated proteins were visualized using Coomassie Brilliant Blue (G-250) and identified by mass spectrometry.

[0199] Mass spectrometry

[0200] Visible protein bands were cut from a gel stained with Coomassie Brilliant Blue G-250.

[0201] After decolorization and trypsin digestion, peptides were extracted, dried in SpeedVac, dissolved in water, and then analyzed by nano-LC-MS / MS.

[0202] Nanoscale LC-MS / MS measurements were performed using a Dionex Ultimate3000 RSLC nanoLC connected to a Bruker maXis II ETD QTOF, with a CaptiveSpray ion source, using an otofControl 5.2 and a Bruker CompassHyStar 5.1 (Bruker Daltonik GmbH, Bremen, Germany). Peptides were captured on an Acclaim PepMap 100 C18 trap column and separated using gradient elution on an Acclaim PepMap 100 C18 analytical column (Thermo Scientific, Waltham, MA).

[0203] Spectra were recorded in positive ion mode with a precursor ion scan rate of 4 Hz and dynamic acquisition of MS / MS spectra from 4 to 16 Hz, with a mass range of 150 to 2200 m / z. Dynamic CID energies ranging from 23 to 65 eV (depending on mass and charge state) were used to fragment ions via collision-induced dissociation (CID) with nitrogen.

[0204] Spectra were recalibrated and processed using Bruker Compass DataAnalysis software 5.2. Database searches were performed using Bruker BioPharma Compass software 3.1 with the Mascot search engine version 2.3 (Matrix Science, London, UK), targeting SwissProt and limited to mammals. A significance threshold of p < 0.05 was selected to evaluate protein hits. Peptides and proteins were accepted if their Mascot score exceeded 21. Figure 2 ).

[0205] Recombinant expression of antigen in HEK293 cells

[0206] The human NECAB1 antigen was cloned into the mammalian expression vector pTriEx-1 (Merck, Darmstadt, Germany), and the corresponding protein was transiently expressed in the human cell line HEK293 after PEI-mediated transfection (Exgene 500) according to the manufacturer's instructions (Biomol GmbH, Hamburg, Germany). Sequences SEQ ID NO1, 11, 12, and 13 were used for cloning.

[0207] To prepare the immunofluorescence substrate, cells were grown on coverslips and fixed with acetone two days after transfection.

[0208] For other purposes, cells were harvested 5 days post-transfection and lysed under high pressure. Lysates were stored in aliquots at -80°C until further use.

[0209] Recombinant expression of antigens in E. coli cells

[0210] The human NECAB1 antigen was cloned into the bacterial expression vector pET24d (Merck, Darmstadt, Germany), and the corresponding protein was transiently expressed in E. coli cells. Sequences SEQ ID NO1, 11, 12, 14, 15, 16, and 17 were used to clone the NECAB1 fragments aa_1-110, aa_100-209, and aa_203-351.

[0211] Indirect immunofluorescence assay (IFA)

[0212] IFA was performed using slides containing a biochip array of recombinant HEK293 cells expressing the identified antigen and control HEK293 cells transfected with an empty vector.

[0213] Each biochip mosaic array was incubated with 35 µL of sample diluted 1:100 in PBS at room temperature for 30 minutes, washed with PBS-Tween, and immersed in PBS-Tween for 5 minutes. In the second step, FITC-labeled goat anti-human IgG (EUROIMMUN Medizinische Labordiagnostika AG, Lübeck) was applied and incubated at room temperature for 30 minutes. The slides were washed again with PBS-Tween and then immersed in PBS-Tween for 5 minutes. The slides were embedded in PBS-buffered DABCO-containing glycerol (approximately 10 µL per region) and examined by fluorescence microscopy. Positive and negative controls were included. Samples were classified as positive or negative based on the fluorescence intensity of transfected cells compared directly with non-transfected cells and control samples. Figure 3 ).

[0214] The association between the immunostaining patterns observed in CNS tissues and the identified antigens was confirmed by pre-incubating patient serum with an extract containing the corresponding recombinant antigen one hour before incubation on frozen tissue sections. For patient serum, pre-incubation with an extract containing the corresponding antigen NECAB1 eliminated the specific immunostaining pattern, but this was not the case with the control extract.

[0215] Results were evaluated by two independent observers using a EUROStar II microscope (EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany). Unless otherwise specified, reagents were obtained from Merck, Darmstadt, Germany or Sigma-Aldrich, Heidelberg, Germany.

[0216] Specificity of autoantibodies

[0217] HEK293 cells transfected with the corresponding recombinant antigen or a simulated transfection were analyzed by IFA at a 1:100 dilution in 100 healthy control sera and 93 sera from patients with neuropathy (n=33 chronic inflammatory demyelinating polyneuropathy, n=30 diabetic neuropathy, and n=30 Guillain-Barré syndrome). None of the 100 healthy control sera reacted in a manner similar to that of the indexed patients. No reactivity was observed in sera from patients with SCLC but without PNS (n=49), nor in CSF from patients with Alzheimer's disease (n=89), patients with normal pressure hydrocephalus (n=15), patients with psychosis (n=20), patients with Lewy body dementia (n=12), and patients with frontotemporal dementia (n=25).

[0218] Immunoblotting

[0219] HEK cell lysates containing recombinant antigens, or purified NECAB1 recombinantly expressed in *E. coli*, or overlapping fragments expressing identified antigens (aa 1-110 (SEQ ID NO18), aa 100-209 (SEQ ID NO19), aa 203-351 (SEQ ID NO20)) of *E. coli*, were incubated with NuPage LDS sample buffer (ThermoFisher Scientific, Schwerte, Germany) containing 25 mmol / L dithiothreitol at 70°C for 10 min, followed by SDS-PAGE (NuPAGE, ThermoFisher Scientific). Following the manufacturer's instructions, the separated proteins were electrotransferred onto nitrocellulose membranes using transfer buffer (ThermoFisher Scientific) via blotting. The membrane was blocked with Universal Blot Buffer plus (EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany) for 15 minutes, and then incubated in Universal Blot Buffer plus with patient serum or control serum (diluted 1:200) or anti-His (1:2000, 70796-3, Merck) for 3 hours. This was followed by three washes with Universal Blot Buffer (EUROIMMUN), a second incubation with anti-human IgG-AP (1:10, EUROIMMUN) or anti-mouse IgG-AP (1:2000, Jackson immunoresearch, Suffolk, UK) for 30 minutes, three washes, and staining with NBT / BCIP substrate (EUROIMMUN). The results of the immunoblotting assay are shown below. Figure 4 , Figure 5 and Figure 6 middle.

Claims

1. A method comprising the step of detecting mammalian autoantibodies in a sample that specifically bind to NECAB1.

2. A method for isolating mammalian autoantibodies that specifically bind to NECAB1, comprising the following steps: a) Immobilizing a polypeptide containing NECAB1 or at least one epitope thereof, or a variant of NECAB1 or an epitope thereof, onto a support. b) Under conditions compatible with the formation of the complex, a sample containing an antibody is contacted with the polypeptide, wherein the antibody binds to the polypeptide. c) Separate the complex formed in step a) from the sample. d) Optionally, the antibody is separated from the polypeptide, and e) Optionally detect the antibody or complex.

3. A method comprising the following steps: a) Immobilizing a polypeptide containing NECAB1 or at least one epitope thereof, or a variant of NECAB1 or an epitope thereof, onto a support. b) Contact the carrier with a liquid containing a mammalian autoantibody that specifically binds to NECAB1, wherein the liquid does not contain a sample from the subject to be diagnosed. c) Contact the carrier with a tool for detecting the immobilized antibody, and d) Detect the presence of the antibody, preferably in a quantitative manner.

4. A mammalian autoantibody that specifically binds to NECAB1, preferably in a liquid comprising one or more, more preferably all, of a group consisting of artificial buffer, preservative and artificial anticoagulant.

5. A kit comprising a polypeptide containing NECAB1 or an epitope thereof or a variant of NECAB1 or an epitope thereof and a secondary antibody that specifically binds to human immunoglobulins.

6. The kit according to claim 5, wherein the polypeptide and / or the secondary antibody comprises a label.

7. The following items are used to determine the risk of developing disease in patients with an increased risk of developing and having a presence of mammalian autoantibodies that specifically bind to NECAB1: a) An antibody that specifically binds to NECAB1; or b) A combination of a polypeptide containing NECAB1 or at least one epitope thereof or a variant of NECAB1 or an epitope thereof with a tool for detecting or capturing antibodies, preferably IgG antibodies; or c) The kit according to any one of claims 5 to 6.

8. A kit comprising a polypeptide containing NECAB1 or at least one epitope thereof or a variant of NECAB1 or an epitope thereof, and a carrier, wherein... a) The polypeptide is immobilized on a carrier, and the kit further includes a secondary antibody that specifically binds to human immunoglobulins. b) The polypeptide and the carrier are configured to immobilize the polypeptide on the surface of the carrier, preferably via an affinity tag and a ligand binding to the affinity tag, and the kit further includes a secondary antibody that specifically binds to human immunoglobulins. c) The carrier is immobilized with a tool for capturing antibodies, preferably a secondary antibody that specifically binds to human immunoglobulins, and the kit further includes a tool for detecting the immobilized antibodies, preferably containing a detectable labeled peptide, or d) The carrier and the tool for capturing antibodies, preferably a secondary antibody that specifically binds to human immunoglobulins, are configured to immobilize the tool for capturing antibodies on the surface of the carrier, preferably via an affinity tag and a ligand binding to the affinity tag, and the kit further includes a tool for detecting the immobilized antibodies, preferably containing a detectable labeled peptide. And preferably from one or more, more preferably all, of the following: recombinant antibody bound to NECAB1, isolated antibody bound to NECAB1, chemical solution reacting with a detectable label, positive control, negative control, leak-proof container for incubating the sample with a carrier or reagent, wash buffer, and calibrators, preferably a set of calibrators.

9. Use of a polypeptide comprising NECAB1 or at least one epitope thereof or a variant of NECAB1 or an epitope thereof, or an autoantibody specifically binding to NECAB1, or a recombinant antibody specifically binding to NECAB1, for the preparation of an analytical kit or medical device for the diagnosis or auxiliary diagnosis of diseases related to the presence of mammalian autoantibodies specifically binding to NECAB1.

10. Use of mammalian autoantibodies or recombinant antibodies that specifically bind to NECAB1 as a positive control for detecting mammalian autoantibodies that specifically bind to NECAB1 in a sample, wherein the antibodies are preferably recognized by a secondary antibody against a human immunoglobulin.

11. An in vitro method for removing mammalian autoantibodies that specifically bind to NECAB1 from blood, preferably from the serum of a patient.

12. The method according to any one of claims 2-3, the kit according to any one of claims 5-6, the use according to claim 7 or 9, or the kit according to claim 8, wherein the polypeptide is a recombinant, isolated, and / or purified polypeptide.

13. The method according to any one of claims 1-3 and 11-12, the use according to any one of claims 7, 9, 10 and 12, or the kit according to any one of claims 5-6, 8 and 12, wherein the autoantibody is a human autoantibody or the sample is a mammalian, preferably a human sample, the sample comprising a group of representative autoantibodies, preferably selected from the group comprising whole blood, plasma, serum, cerebrospinal fluid and saliva.

14. The method according to any one of claims 1-3 and 11-13, the use according to any one of claims 7, 9, 10 and 12-13, or the kit according to any one of claims 5-6, 8 and 12-13, wherein the autoantibody or complex is detected using a detection method selected from the group consisting of: immunodiffusion, electrophoresis, light scattering immunoassay, agglutination, labeled immunoassay such as labeled immunoassay from the group comprising radiolabeled immunoassay, enzyme immunoassay, more preferably ELISA, chemiluminescent immunoassay, preferably electrochemiluminescent immunoassay, and immunofluorescence, preferably indirect immunofluorescence.

15. The method according to any one of claims 1-3 and 11-14, the use according to any one of claims 7, 9, 10 and 12-14, or the kit according to any one of claims 8 and 12-14, wherein the carrier is selected from the group consisting of: glass slides, preferably glass slides for microscopy, biochips, microtiter plates, side-flow devices, test strips, membranes, preferably line blots, chromatographic columns, and beads, preferably magnetic beads or fluorescent beads.

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