CN112147324A - Detection of autoantibodies - Google Patents

Abstract

The present invention relates to diagnostically useful carriers comprising means for specifically capturing antibodies that specifically bind to cytokinin-3 in a sample from a subject, kits comprising the carriers of the invention, detection methods, pharmaceutical compositions, use of the carriers of the invention for the preparation of a kit for diagnosing a disease and isolated human antibodies that specifically bind to cytokinin-3.

Description

Detection of autoantibodies

Technical Field

The present invention relates to diagnostically useful carriers comprising means for specifically capturing antibodies that specifically bind to lysenin-3 (Septin-3) in a sample from a subject, kits comprising the carriers of the invention, methods of detection, pharmaceutical compositions, use of the carriers of the invention in the preparation of kits for diagnosing a disease and isolated human antibodies that specifically bind to lysenin-3.

Background

Alleviation of neurological disorders is a continuing challenge in biomedical science, largely because of the many symptoms that can be encountered due to a wide variety of causes, including genetic disorders, drug abuse, malnutrition, infection, injury, psychiatric disorders, immunodeficiency and cancer.

Since neurological diseases are rarely associated with unique characteristic patterns of clinical symptoms, it is often difficult to provide a reliable assessment of an increased risk of a subject developing a neurological disease based solely on observations and examinations of affected subjects or medical histories thereof.

The importance of the early evaluation is not emphasized. Many neurological disorders (most notably alzheimer's and parkinson's diseases and multiple sclerosis) cannot be cured, but drugs are available that can be used to slow their progression. In addition, some rare types of cancer are associated with neurological symptoms. The earlier the assessment, the better the chance of utilizing the available therapeutic profile for the full benefit of the patient and ideally preventing the onset of disease.

This is especially true in the case of neurological diseases associated with autoantibodies. In some cases, the association between a specific detectable autoantibody and a condition is strong enough to allow immediate assessment. If autoantibodies are well detected before the onset of disease, this may allow physicians to apply immunosuppressive drugs, which may prevent the autoantibodies from interacting with the body's own structures and molecules and prevent disease.

Examples of neurological conditions consistent with the emergence of autoantibodies include: neuromyelitis optica (a disease characterized by loss of visual perception and spinal cord function) and anti-NMDA receptor encephalitis (which is associated with autonomic dysfunction), hypoventilation, cerebellar ataxia, hemiparesis, loss of consciousness or catatonic disorders. Although the involvement of autoantibodies and the nature of these conditions themselves have been poorly understood previously, the risk of many of these diseases can now be assessed and effectively addressed due to the availability of assays based on detection of autoantibodies.

Therefore, it is of utmost importance to develop new methods to distinguish between neurological conditions associated with autoantibodies and those associated with non-autoantibodies and to assess the risk of a patient to develop the disease.

Disclosure of Invention

The present invention relates to autoantibodies against mitogen-3 and assays based on their detection. To the best of the inventors' knowledge, the presence of autoantibodies against cytokinin-3 has not been reported in the prior art, let alone their usefulness. Recombinant antibodies that bind to cytokinin-3 have been commercialized by a number of companies, including Santa Cruz Biotechnology and Abcam.

The problem underlying the present invention is to provide novel agents, devices and methods which can be used to assess whether a subject is susceptible to developing an autoimmune disease, preferably a neurological disease or an autoimmune disease associated with a neurological disease or a neurological symptom or cancer, more preferably a paraneoplastic syndrome (PNS), and even more preferably one or more diseases selected from cerebellar ataxia, longitudinal myelitis, migraine, Amyotrophic Lateral Sclerosis (ALS), cerebellar syndrome, melanoma and cancer (carcinoma).

Another problem underlying the present invention is to provide novel agents, devices and methods which can be used to distinguish between autoimmune diseases, in particular neurological autoimmune diseases, more preferably selected from the group comprising PNS, cerebellar ataxia, longitudinal myelitis, migraine, Amyotrophic Lateral Sclerosis (ALS) and cerebellar syndrome, and diseases other than autoimmune diseases, such as infections associated with neurological symptoms, not only for the purpose of determining the most promising treatment options, more particularly for determining whether immunosuppressive treatment is moderate, preferably before the onset of the disease.

The problem underlying the present invention is solved by the subject matter of the independent and dependent claims appended hereto.

In a first aspect, the problem is solved by a diagnostically useful carrier comprising means for specifically capturing an antibody that specifically binds to cytokinin-3 in a sample from a subject.

In a second aspect, the problem is solved by a kit comprising a diagnostically useful carrier, wherein the kit comprises a calibrator, a wash buffer and/or means for detecting IgG antibodies.

In a third aspect, the problem is solved by a method comprising the step of detecting the presence or absence of an antibody to cytokinin-3 in a sample of a subject.

In a fourth aspect, the problem is solved by a pharmaceutical composition comprising cytokinin-3 or a variant thereof.

In a fifth aspect, the problem is solved by the use of a vehicle according to the invention for the manufacture of a kit for the diagnosis of a disease, wherein said disease is a neurological autoimmune disease, recurrent syncope and/or cancer (cancer).

In a sixth aspect, the problem is solved by an isolated human antibody that specifically binds to lysenin-3.

In a preferred embodiment, the antibody does not bind to dehiscin-1, dehiscin-2, dehiscin-4, dehiscin-5, dehiscin-6, dehiscin-7, dehiscin-8, dehiscin-9, dehiscin-10, dehiscin-11, and/or dehiscin-12.

In a preferred embodiment, the means for specifically capturing the antibody is recombinant and purified cytokinin-3 or a variant thereof or a eukaryotic cell expressing cytokinin-3 or a variant thereof.

In a preferred embodiment of the invention, the means to specifically capture antibodies that specifically bind to cytokinin-3 is (a) a recombinant and purified complex comprising cytokinin-3, cytokinin-5, cytokinin-6, cytokinin-7 and cytokinin-11 or variants of said cytokinin protein; or (b) a eukaryotic cell expressing a complex comprising dehiscence protein-3, dehiscence protein-5, dehiscence protein-6, dehiscence protein-7 and dehiscence protein-11 or a variant of said dehiscence protein.

In a preferred embodiment, the disease is PNS, preferably associated with a condition selected from the group comprising cerebellar ataxia, longitudinal spinosis, migraine, Amyotrophic Lateral Sclerosis (ALS) and/or cerebellar syndrome. In another preferred embodiment, the disease is selected from the group comprising PNS and tumors associated with cerebellar ataxia, longitudinal spinosis, migraine, Amyotrophic Lateral Sclerosis (ALS) and/or cerebellar syndrome, more preferably melanoma and/or carcinoma. In a preferred embodiment, the disease is cancer, preferably from the group comprising carcinoma, sarcoma, lymphoma, blastoma, melanoma, germ cell tumor and leukemia. In a more preferred embodiment, the cancer is melanoma or carcinoma, more preferably lung cancer.

In a preferred embodiment, the disease is recurrent syncope. The term "recurrent syncope" as used herein includes all types of syncope, such as nerve-mediated syncope (including but not limited to vascular vagal syncope and episodic syncope), heart-mediated syncope (including syncope caused by cardiac arrhythmias, obstructive cardiac pathology, structural cardiopulmonary disease and other cardiac causes such as sick sinus syndrome, adanas-stokes syndrome, subclavian blood theft syndrome, aortic dissection and pulmonary embolism), blood pressure-induced syncope (e.g., postural (postural) hypotensive syncope), central nervous system ischemia-induced syncope (e.g., syncope caused by vertebrobasal artery disease) and syncope caused by other causes such as motor epilepsy-mediated syncope and thermal syncope.

In a preferred embodiment, the invention is carried out using a sample which is or comprises a body fluid comprising antibodies, preferably selected from the group comprising whole blood, plasma, serum, cerebrospinal fluid and saliva.

In a preferred embodiment, the autoantibodies or complexes are detected using a technique selected from the group comprising: immunodiffusion techniques, immunoelectrophoresis techniques, light scattering immunoassays, agglutination techniques, labelled immunoassays, e.g. from immunoassays comprising a radioactive label, enzyme immunoassays, more preferably ELISA, preferably electrochemiluminescent immunoassays and immunofluorescence, preferably indirect immunofluorescence.

In a preferred embodiment, the carrier is selected from the group comprising: slides, preferably for microscopy, biochips, microtiter plates, lateral flow devices, test strips, membranes, preferably linear dot blots (line blots), chromatography columns and beads, preferably magnetic or fluorescent beads.

In a preferred embodiment, the variant comprises SEQ ID NO: 5.

The present invention is based on the surprising discovery by the inventors that autoantibodies to cytokinin-3 are present and can be detected in samples from many patients with neurological and/or cancer conditions, but not in samples obtained from healthy subjects.

Furthermore, the present invention is based on the surprising finding of the inventors that novel neurological diseases and/or cancers are associated with the detection of autoantibodies against cytokinin-3.

Without wishing to be bound by any theory, the presence of such autoantibodies indicates that the function of cytokinin-3 and/or downstream effectors is impaired in patients with such autoantibodies, resulting in development of neurological symptoms or cancer.

Mitogen-3 (40.704kDa, 358 amino acids) is a member of the mitogen family together with mitogen-1, mitogen-2, mitogen-4, mitogen-5, mitogen-6, mitogen-7, mitogen-8, mitogen-9, mitogen-10, mitogen-11 and mitogen-12. These proteins are fiber-forming cytoskeletal GTPases which have nucleotide binding and GTPase activity. The sequences of human, mouse, rat and pig cytokinin-3 as preferred targets for the autoantibodies of the invention and as preferred tools for the use and method of the invention are shown in SEQ ID Nos. 1 to 4, respectively.

Various studies have shown that different subsets of cytokinins can bind to form complexes with variable composition, and that these complexes assemble into filaments and higher order structures. Most cytokinins present a sequence at the C-terminus that is predicted to form a coiled coil and has been proposed to mediate cytokinin-cytokinin interactions, thereby controlling fibril formation. The central core of the cytokinin is formed by a guanine-nucleotide (GTP) binding domain, which shares at least three conserved motifs displaying the P-loop of the gtpase. In general, it has been shown that a multi-base region that binds membrane phospholipids is present at the interface between the N-terminus and the GTP-binding domain.

The present invention relates to a polypeptide comprising a mammalian, preferably human, polypeptide selected from the group consisting of mitogen-3 or an antigenic variant reactive with an autoantibody binding to mitogen-3. Mammalian cytokinin-3 includes homologues from human, monkey, mouse, rat, rabbit, guinea pig or pig, preferably human.

In a more preferred embodiment, mitogen-3 is a polypeptide encoded by SEQ ID NO.1(UniProtKB reference: Q9UH 03; encoded by NM-145734, NM-019106, NM-145733 or NM-001363845). Throughout this application, any database code referenced refers to the Uniprot database, and more specifically, the version of the application or its earliest priority application's filing date.

The teachings of the present invention can be practiced not only by using polypeptides, particularly polypeptides comprising the native sequence of a polypeptide such as cytokinin-3, or nucleic acids having the exact sequence explicitly (e.g., by function, name, sequence, or accession number) or implicitly referred to in this application, but also by using such polypeptides or variants of nucleic acids.

In a preferred embodiment, the term "variant" as used herein may refer to at least one fragment of the full-length sequence in question, more specifically one or more amino acid or nucleic acid sequences truncated at one or both ends by one or more amino acids relative to the full-length sequence. Such fragments comprise or encode a peptide having at least 6, 7, 8, 10, 12, 15, 20, 25, 50, 75, 100, 150, or 200 consecutive amino acids of the original sequence or a variant thereof. The total length of the variant may be at least or no more than 6, 7, 8,9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 350, 400, 500, 750, 1000 or more amino acids. In a more preferred embodiment, the variant is a polypeptide comprising the sequence shown in SEQ ID NO. 5.

The term "variant" refers not only to at least one fragment, but also to a polypeptide or fragment thereof comprising an amino acid sequence that is at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the referenced reference amino acid sequence or fragment thereof, wherein other amino acids than those essential for biological activity (e.g., the ability of an antigen to bind (auto) 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 conservative manner and/or amino acids are added, such that the biological activity of the polypeptide is retained. The prior art includes a variety of different methods that can be used to align two given nucleic acid or amino acid sequences and calculate the degree of identity, see, e.g., Arthur Lesk (2008), Introduction to bioinformatics, Oxford University Press,2008,3^rdThe edition. In a preferred embodiment, ClustalW software (Larkin, m.a., blackshelds, g., Brown, n.p., Chenna, r., McGettigan, p.a., McWilliam, h., Valentin, f., Wallace, i.m., Wilm, a., Lopez, r., Thompson, j.d., Gibson, t.j., Higgins, D.G. (2007). Clustal W and Clustal X version 2.0.Bioinformatics,23, 2947-.

In a preferred embodiment, the variant is a linear, unfolded polypeptide, which is optionally denatured.

In preferred embodiments, the polypeptides and variants thereof may also comprise chemical modifications, such as isotopic labelling, or covalent modifications such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, methylation, hydroxylation and the like. Those skilled in the art are familiar with methods for modifying polypeptides. Any modification is designed such that it does not abrogate the biological activity of the variant.

Furthermore, variants may also be produced by fusion with other known polypeptides or variants thereof and comprise active portions or domains, preferably having at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity when aligned with an active portion of a reference sequence, wherein the term "active portion" 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 encodes an amino acid sequence that is shorter than the full-length amino acid sequence, and/or is a variant of the native sequence, but retains at least some biological activity.

In a preferred embodiment, the term "variant" of a nucleic acid comprises a nucleic acid the complementary strand of which hybridizes, preferably under stringent conditions, to a reference nucleic acid or to a wild-type nucleic acid. The stringency of the hybridization reaction is readily determinable by one of ordinary skill in the art, and is generally an empirical calculation dependent on 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 reanneal with complementary strands present in the environment at temperatures below their melting temperature: the higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. Thus, higher relative temperatures will tend to make the reaction conditions more stringent, while lower reaction temperatures will be less stringent. For additional details and explanation of the stringency of hybridization reactions, see Ausubel, F.M (1995), Current Protocols in Molecular biology, john Wiley & Sons, Inc. Furthermore, The person skilled in The art can follow The guidance given in The handbook Boehringer Mannheim GmbH (1993) The DIG System Users Guide for Filter Hybridization, Boehringer Mannheim GmbH, Mannheim, Germany and in Liebl, W., Ehrmann, M., Ludwig, W., and Schleifer, K.H, (1991) International Journal of Systematic Bacteriology 41:255-260 on how to identify DNA sequences by means of Hybridization. In a preferred embodiment, stringent conditions are applied to any hybridization, i.e., hybridization only occurs if the probe has 70% or greater identity to the target sequence. Probes having a lower degree of identity relative to the target sequence can hybridize, but such hybrids are unstable and will be removed in a wash step under stringent conditions, e.g., reducing the salt concentration to 2 XSSC or, optionally and subsequently, to 0.5 XSSC, at temperatures (in increasing order of preference) of about 50 ℃ to 68 ℃, about 52 ℃ to 68 ℃, about 54 ℃ to 68 ℃, about 56 ℃ to 68 ℃, about 58 ℃ to 68 ℃, about 60 ℃ to 68 ℃, about 62 ℃ to 68 ℃, about 64 ℃ to 68 ℃, about 66 ℃ to 68 ℃. In particularly preferred embodiments, the temperature is from about 64 ℃ to 68 ℃ or from about 66 ℃ to 68 ℃. The salt concentration can be adjusted to 0.2 XSSC, or even 0.1 XSSC. Nucleic acid sequences can be isolated that have a degree of identity of at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% relative to a reference sequence or wild-type sequence. In a preferred embodiment, the term variant of a nucleic acid sequence as used herein refers to any nucleic acid sequence which, by virtue of the degeneracy of the genetic code, encodes the same amino acid sequence as encoded by the reference nucleic acid sequence, and variants thereof.

Variants of the polypeptides have biological activity. In a preferred embodiment, such biological activity is the ability to specifically bind to an autoantibody that binds to cytokinin-3, as found in a patient suffering from an autoimmune disease associated with such autoantibody, preferably associated with a neurological disease or condition such as PNS. For example, it can be examined whether a variant of mitogen-3 has such biological activity by determining whether the variant of interest binds to an autoantibody from a patient sample which autoantibody binds to wild-type mitogen-3, preferably as determined using indirect immunofluorescence of the rat hippocampus or cerebellum as described in the experimental part of this application.

In preferred embodiments, the variant of cytokinin-3 is a mammalian cytokinin-3 lacking at least 21, at least 20, at least 15, at least 10, at least 5, at least 4, at least 3, at least 2, or 1 amino acid of the C-terminus of the corresponding naturally occurring wild-type protein. In a more preferred embodiment, the variant of cytokinin-3 is the corresponding sequence of amino acids 1 to 337 of human cytokinin-3 according to SEQ ID No.1 or any other mammalian species determined by sequence alignment. In a more preferred embodiment, the variant is a polypeptide comprising the sequence shown in SEQ ID NO. 5.

When used to carry out the teachings of the present invention, any polypeptide according to the present invention may be provided in any form and with any degree of purification, from a liquid sample, tissue or cell comprising the polypeptide in endogenous form, more preferably a cell overexpressing the polypeptide, a crude or enriched lysate of such cells, to a purified and/or isolated polypeptide, which is optionally substantially pure. In a preferred embodiment, the polypeptide is a native polypeptide, wherein the term "native polypeptide" as used herein refers to a folded polypeptide, more preferably to a folded polypeptide purified from a tissue or cell, more preferably from a mammalian cell or tissue, optionally from a non-recombinant tissue or cell. In another preferred embodiment, the polypeptide is a recombinant protein, wherein the term "recombinant" as used herein refers to a polypeptide produced at any stage of the production process by using genetic engineering methods, e.g. by fusing a nucleic acid encoding the polypeptide with a strong promoter for overexpression in a cell or tissue or by engineering the sequence of the polypeptide itself. The person skilled in the art is familiar with methods for the engineering of nucleic acids and encoded polypeptides (e.g. described in Sambrook, j., Fritsch, e.f. and manitis, T. (1989), Molecular Cloning, CSH or in Brown T.A. (1986), Gene Cloning-an introduction, Chapman & Hall) and methods for the production and Purification of native or recombinant polypeptides (e.g. the handbook "Strategies for Protein Purification", "Antibody Purification", "Purification charging Proteins" (2009/2010) published by the GE Healthcare Sciences in Burgess, r.r., deutter, guto (M.P), Guide Protein Purification). In a preferred embodiment, a polypeptide is pure if at least 60%, 70%, 80%, 90%, 95% or 99% of the polypeptide in the respective sample consists of said polypeptide, as judged by SDS polyacrylamide gel electrophoresis followed by coomassie blue staining and visual assay.

If the polypeptide of the invention is provided in the form of a tissue, it is preferred that the tissue is a mammalian tissue, such as human, rat, primate, donkey, mouse, goat, horse, sheep, pig or cow, more preferably a brain tissue, most preferably the cerebellum. If a cell lysate is used, it is preferred that the cell lysate comprises a membrane associated with the cell surface or indeed a fraction enriched in the membrane. If the polypeptide is provided in the form of a recombinant cell, it is preferred that the recombinant cell is a eukaryotic cell, such as a yeast cell, more preferably a cell from a multicellular eukaryote, such as a plant, mammal, frog, or insect, most preferably a cell from a mammal, such as a rat, human, primate, donkey, mouse, goat, horse, sheep, pig, or cow.

The polypeptides (including any variants) used to carry out the teachings of the present invention are preferably designed such that they comprise at least one epitope that is recognized by and/or specifically binds to an autoantibody binding to mitogen-3. Any epitope is more preferably an epitope recognized only by an autoantibody to cytokinin-3, as compared to an antibody other than such an autoantibody. In one embodiment, such polypeptides comprise 6, 7, 8,9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more, preferably at least 9 but not more than 16 consecutive amino acids, respectively, from dehiscence protein-3. Those skilled in the art are familiar with guidelines for designing peptides with sufficient immunogenicity, for example in Jackson, d.c., fitzmacure, c.j., Brown, l.e., Zeng, W. (1999),

preparation and Properties of toilety synthetic microorganisms, Vaccine, Vol.18, pp.3-4, 9.1999, page 355-361; and those described in Black, M., Trent, A., Tirrell, M. and Olive, C. (2010), Advances in the design and delivery of peptide Subunit variations with a focus on Toll-like receptors indicators adhesives, Expert Rev vitamins, 2.2010, 9(2): 157-phase 173. In short, it is desirable that the peptide satisfies the following requirements as much as possible: (a) it has a high degree of hydrophilicity; (b) it comprises one or more residues selected from the group comprising aspartic acid, proline, tyrosine and phenylalanine; (c) for greater specificity, it has no or very low homology to other known peptides or polypeptides; (d) it needs to be sufficiently soluble; and (e) it does not contain glycosylation or phosphorylation sites, unless required for a particular reason. Alternatively, Bioinformatics approaches such as those described by Moreau, V., Fleury, C., Piquer, D., Nguyen, C., Novali, N., Villard, S., Lane, D., Granier, C., and Molina, F. (2008), PEPOP: computerized design of immunogenic peptides, BMC biologicals 2008,9:71 may be followed.

When used in accordance with the present invention, the polypeptides of the invention may be provided in any kind of conformation. For example, the polypeptide may be a substantially unfolded, partially folded or fully folded polypeptide. In a preferred embodiment, the polypeptide is folded in the sense that the epitope necessary for binding to the autoantibody of the invention, or the protein or variant thereof in its entirety, adopts the fold adopted by the native protein in its native environment. The person skilled in the art is familiar with methods suitable for determining whether a polypeptide is folded, and if so, which structure it has, for example limited proteolysis, nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy or X-ray crystallography (see, for example, Banaszak L.J (2008), Foundations of Structural Biology, academic Press; or Teng Q. (2013), Structural Biology: Practical Applications, Springer), preferably circular dichroism is used.

The polypeptide of the invention may be a fusion protein comprising other amino acid sequences than those taken from cytokinin-3, in particular a C-terminal or N-terminal tag, preferably a C-terminal tag, which in preferred embodiments as used herein is a functional additional sequence motif or polypeptide having some biological or physical function and which may be used, for example, for purification, immobilization, precipitation or identification of the polypeptide of the invention. In a more preferred embodiment, the tag is a sequence or domain capable of specifically binding to a ligand, e.g. selected from the group comprising: his-tag, thioredoxin, maltose binding protein, a tag of glutathione S-transferase, a fluorescent tag, for example selected from the group comprising green fluorescent protein.

The polypeptide of the present invention may be an immobilized polypeptide. In a preferred embodiment, the term "immobilized" as used herein refers to a molecule bound to a solid support that is insoluble in aqueous solution, more preferably by covalent bonds, electrostatic interactions, encapsulation or entrapment (e.g. by denaturing the globular polypeptide 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, and the like, have been described in the literature, for example in Kim, d, and her, A.E, (2013), Protein immobilization techniques for microfluidic assays, Biomicrofluidics7(4), 041501. In this way, the immobilized molecules together with the insoluble support can be separated from the aqueous solution in a straightforward manner, for example by filtration, centrifugation or decanting. The immobilized molecules may be immobilized in a reversible or irreversible manner. For example, immobilization is reversible if the molecule interacts with the carrier through ionic interactions that can be masked by the addition of high concentrations of salt, or if the molecule is bound through cleavable covalent bonds such as disulfide bridges (which can be cleaved by the addition of thiol-containing reagents). Conversely, if the molecule is tethered to the support by a covalent bond that cannot be cleaved in aqueous solution (e.g., a bond formed by the reaction of an epoxide group and an amine group often used to couple lysine side chains to affinity columns), then immobilization is irreversible. The protein may be immobilized indirectly, for example by immobilizing an antibody or other entity having affinity for the molecule, followed by complex formation, such that the molecule-antibody complex is immobilized. Various means for immobilizing molecules are described in the literature, for example in Kim, d., her and A.E (2013), Protein immobilization technologies for microfluidic assays, Biomicrofluidics7(4), 041501. In addition, various reagents and kits for immobilization reactions are commercially available, for example from Pierce Biotechnology.

It is essential that the solution or sample for detecting autoantibodies according to the invention comprises antibodies, also referred to as immunoglobulins. Typically, the body fluid sample comprises a representative set of immunoglobulin from a subject. However, once provided, the sample or solution may be subjected to further processing, which may include fractionation, centrifugation, enrichment, or isolation of the subject's ensemble of immunoglobulins or any immunoglobulin class, which may affect the relative distribution of the various different classes of immunoglobulins.

The reagents, devices, methods and uses described throughout this application can be used to identify subjects at increased risk of developing disease. In a preferred embodiment, the disease is a neurological disease. In a more preferred embodiment, the term "neurological disease" as used herein refers to any disease associated with a defect of the nervous system, in another preferred embodiment, as used herein, the term "PNS" (abbreviation for paraneoplastic neurological syndrome) refers to a systemic disorder caused indirectly by the presence of a tumor, e.g. due to the production and release of substances such as hormones or cytokines that are not normally produced by cells of tumor origin or at increased concentrations to produce or produce and release biologically active cells. Such systemic disorders may be revealed by a variety of conditions including cerebellar ataxia, longitudinal myelitis, migraine, Amyotrophic Lateral Sclerosis (ALS), or cerebellar syndrome. Any manifestation of PNS indicates that the patient should be thoroughly examined for the presence of a tumor, although the tumor may be too small to detect.

In many cases, a mere detection (in other words, determining whether a detectable level of antibody is present or not) is sufficient for the evaluation. If autoantibodies can be detected, this will be helpful information to the clinician and indicate an increased likelihood that the patient will suffer from the disease. In a preferred embodiment, an autoantibody is considered detectable if it can be detected using one or more methods selected from the group comprising immunoprecipitation, indirect immunofluorescence, ELISA or linear dot blot, preferably indirect immunofluorescence. In a preferred embodiment, the relative concentration of antibody in serum can be determined compared to levels that can be found in average healthy subjects. Although in many cases it may be sufficient to determine whether autoantibodies are present or detectable in a sample, a method carried out to obtain information helpful for diagnosis may comprise determining whether the concentration is at least 2 times, preferably 5, 10, 20, 25, 50, 100, 200, 500, 1000, 10000 or 100000 times the concentration found in an average healthy subject. In a preferred embodiment, the relative concentration of autoantibodies is determined using one or more methods selected from the group comprising semi-quantitative immunoprecipitation, semi-quantitative indirect immunofluorescence, ELISA or semi-quantitative linear dot blot, preferably ELISA. Experimental details are described in the experimental section of the present application or as described in textbooks or practice manuals available at the priority date of the present application.

One skilled in the art will appreciate that clinicians typically do not conclude whether a patient subject will likely suffer from a disease, condition, or disorder based solely on a single parameter, but need to consider other aspects, such as the presence of other autoantibodies, markers, blood parameters, clinical assessment of any symptoms, or medical imaging or other non-invasive methods (e.g., polysomnography). See Baenkler H.W (2012), General aspects of Autoimmune diagnostics, Renz, H., Autoimmune diagnostics,2012, de Gruyter, page 3. The value of the agent or method according to the invention may also be that it is possible to rule out the development of a disease. In preferred embodiments, the meaning of any symptom or disease mentioned throughout this application is consistent with the understanding of one skilled in the art, as filed date of this application or preferably at the earliest priority date, as evidenced by textbooks and scientific publications.

The methods, polypeptides or uses of the invention (which are optionally used to determine whether a disease is likely to develop) may include: obtaining a sample or solution comprising an antibody, preferably from a human patient, determining the presence or absence of autoantibodies which bind to cytokinin-3, wherein said determining is performed by contacting the sample with a polypeptide of the invention and detecting the presence or absence of binding between said polypeptide and said autoantibodies, preferably by using a labelled secondary antibody, wherein said autoantibodies, if present in the sample, bind to said polypeptide; and assessing the patient as more likely to suffer from the disease if the autoantibodies are determined to be present in the sample or solution.

The present invention relates to complexes comprising antibodies, preferably autoantibodies, bound to polypeptides of the invention. Such complexes can be used or detected as part of a method for identifying a subject at increased risk of developing a disease. If autoantibodies against cytokinin-3 are to be detected, a liquid sample comprising the antibodies from the subject may be used to carry out the method. Such a liquid sample may be any body fluid from a subject comprising a representative group of antibodies, preferably a sample from a subject comprising antibodies of an immunoglobulin class selected from the group comprising antibodies of the IgG, IgA and IgM classes, preferably IgG, more preferably IgG1 and IgG2, more preferably IgG 1. For example, the sample may be cerebrospinal fluid (CSF), blood or serum, lymph, interstitial fluid, preferably serum or CSF, more preferably serum. It is preferably an ex vivo sample.

The step of contacting a liquid sample or solution comprising an antibody with a polypeptide of the invention may be performed by: the polypeptides are incubated in immobilized form in the presence of a sample or solution comprising the antibodies under conditions compatible with the formation of a complex comprising the respective polypeptide and an antibody (preferably an autoantibody) which binds to a polypeptide of the invention. Subsequently, the liquid sample or solution depleted at that time of the antibodies bound to the polypeptides of the invention may be removed, followed by one or more washing steps. Finally, a complex comprising the antibody and the polypeptide may be detected. In a preferred embodiment, the term "conditions compatible with complex formation" are conditions that allow specific antigen-antibody interactions to establish a complex comprising the polypeptide and the antibody. In a preferred embodiment, such conditions may comprise incubating the polypeptide in a sample diluted 1:100 in PBS buffer for 30 minutes at 25 ℃. 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) producing said autoantibody, wherein the level of such antibody is more preferably elevated compared to the average of any other antibody specifically binding to such endogenous molecule. In a most preferred embodiment, the autoantibody is an autoantibody that binds to mitogen-3.

The method according to the invention is preferably an in vitro method.

In a preferred embodiment, the detection of the complex for the prognosis, evaluation, identification, method or test kit according to the invention comprises the use of a method selected from the group comprising: immunodiffusion techniques, immunoelectrophoresis techniques, light scattering immunoassays, agglutination techniques, labeled immunoassays such as those selected from the group comprising radiolabelled immunoassays, enzyme immunoassays, preferably ELISA, chemiluminescent immunoassays and immunofluorescence, preferably indirect immunofluorescence techniques. Those skilled in the art are familiar with these methods, which are also described in the prior art, for example in Zane, H.D, (2001), Immunology-clinical & Practical Concepts in Laboratory Medicine, W.B. Saunders Company, chapter 14.

Alternatively, a sample comprising a tissue comprising a polypeptide of the invention in addition to a liquid sample may be used. The tissue sample is preferably derived from a tissue expressing endogenous cytokinin-3, preferably expressing the polypeptide at an increased level compared to the average tissue in the corresponding organism, preferably a human. Such a sample, which may be in the form of a tissue section immobilized on a carrier, e.g. a slide for microscopic analysis, may then be contacted with an antibody of the invention, preferably an autoantibody, which binds to a polypeptide of the invention. Preferably, the antibodies are labeled to allow differentiation from endogenous antibodies that bind to the polypeptides of the invention, thereby allowing detection, and optionally quantification, of newly formed complexes. If the amount of complex formed is lower than that found in a sample taken from a healthy subject, the subject from which the sample under examination is taken may suffer from a disease.

Any data demonstrating the presence or absence of a complex comprising an antibody and a polypeptide of the invention can be correlated with reference data. For example, detection of the complex indicates that the patient providing the analyzed sample is likely to suffer from the disease in the future. If the patient is being treated and the method for obtaining diagnostically relevant information is run again, the amount of complex detected in the two runs can be correlated to learn information about disease progression and/or treatment success.

In another preferred embodiment, the prognosis, assessment, identification, method or test kit according to the teachings of the present invention contemplates the use of indirect immunofluorescence. The person skilled in the art is familiar with such techniques and the preparation of suitable samples, which are described in the prior art (US 4647543; Voigt, J., Krause, C.,

e, Saschenbrechker, S., Hahn, M., Danckwardt, M., Feirer, C., Ens, K, Fechner, K, Barth, E, Martinetz, T, and

W.(2012),Automated Indirect Immunofluorescence Evaluation of Antinuclear Autoantibodies on HEp-2 Cells,”Clinical and Developmental Immunology,vol.2012,doi:10.1155/2012/65105；Bonilla,E.,Francis,L.,Allam,F.et al.,Immuno-fluorescence microscopy is superior to fluorescent beads for detection of antinuclear antibody reactivity in systclinical Immunology, vol.124, No.1, pp.18-21,2007). Suitable reagents, devices and software packages are commercially available, for example from EUROIMMUN, lubeck, Germany.

The sample may be tested to determine only the presence of autoantibodies binding to cytokinin-3, but preferred methods, tests, devices etc. contemplate determining the presence or absence of autoantibodies against one or more other polypeptides, preferably associated with a neurological autoimmune disease, preferably selected from the group comprising, more preferably all from: hu, Yo, Ri, CV2, PNMA1, PNMA2, DNER/Tr, ARHGAP26, ITPR1, ATP1A3, NBC1, neurotensin, carp, Zic4, Sox1, Ma, MAG, MP0, MBP, GAD65, biliveroprotein, recoverin, GABA a receptor (EP13189172.3), GABA B receptor (EP2483417), glycine receptor, gephyrin, IgLON5(US2016/0349275), DPPX (US2015/0247847), aquaporin-4, MOG, NMDA receptor, AMPA receptor, GRM1, GRM5, LGI1, VGCC, and mGluR1 and CASPR2, the antigen preferably being immobilized, e.g. on a medical device such as a linear dot blot. It has been described in the prior art that one or more (preferably all) autoantibodies against the relevant markers, neurothlogin (EP15001186), ITPR1(EP14003703.7), NBC1(EP14003958.7), ATP1A3 (also known as the α 3 subunit of human neuron Na (+)/K (+) atpase (EP14171561.5)), florillin 1/2(EP3101424), NSF, STX1B and VAMP2(EP17001205.8) and RGS8(EP17000666.2) can additionally be detected.

According to the teachings of the present invention, antibodies, preferably autoantibodies, which bind to a polypeptide of the present invention are provided for evaluation or identification. The person skilled in the art is familiar with methods for purifying antibodies, such as those described in Hermanson, G.T., Mallia, A.K., and Smith, P.K, (1992), Immobilized Affinity Ligand technology, San Diego: Academic Press. Briefly, an antigen that specifically binds to an antibody of interest (the antigen of which is a polypeptide of the invention) is immobilized and used to purify the antibody of interest from an appropriate source via affinity chromatography. A liquid sample comprising antibodies from a patient suffering from a neurological disorder identified by the inventors can be used as a source.

According to the present invention, antibodies, such as autoantibodies, capable of specifically binding to mitogen-3 are provided. Vice versa, variants of cytokinin-3 specifically bind to autoantibodies that specifically bind to cytokinin-3. In a preferred embodiment, the term "antibody" as used herein refers to any immunoglobulin-based binding moiety, more preferably a binding moiety comprising at least one immunoglobulin heavy chain and at least one immunoglobulin light chain, including but not limited to monoclonal and polyclonal antibodies and variants, particularly fragments, of antibodies, which binding moiety is capable of binding, more preferably specifically binding, to the respective antigen. In a preferred embodiment, the term "specifically binds" or "specifically captures" as used herein means that the binding ratio is 1 × 10^-5M, more preferably 1X 10^-7M, more preferably 1X 10^-8M, more preferably 1X 10^-9M, more preferably 1X 10^-10M, more preferably 1X 10^-11M, more preferably 1X 10^-12The binding reaction, characterized by the dissociation constant of M, determined by surface plasmon resonance at 25 ℃ in PBS buffer at pH 7 using a Biacore instrument, was stronger. The antibody may be part of an autoantibody preparation which is a heterologous or may be a homologous autoantibody, wherein the heterologous preparation comprises a number of different autoantibody species, as obtainable by preparation from human donor serum, for example by affinity chromatography using an immobilised antigen to purify any autoantibody capable of binding to the antigen. The antibody may be glycosylated or non-glycosylated. The person skilled in the art is familiar with methods which can be used for identifying, generating and purifying antibodies and variants thereof, such as those described in EP 2423226 a2 and the references therein.

The present invention provides a method for isolating autoantibodies that bind to cytokinin-3, comprising the steps of: a) contacting a sample comprising the antibody with a polypeptide of the invention, thereby forming a complex, b) separating the complex formed in step a), c) dissociating the complex separated in step b), and d) separating the antibody from the polypeptide of the invention. Samples from patients suffering from new neurological disorders identified by the inventors can be used as a source of antibodies. Suitable methods are described in the prior art, for example in the handbooks "Affinity chromatography", "Strategies for Protein Purification" and "Antibody Purification" (2009/2010) published by GE Healthcare Life Sciences, and in Philips, Terry, M., Analytical techniques in immunology, 1992, Marcel Dekker, Inc.

The present invention provides a pharmaceutical composition comprising a polypeptide of the invention, said composition preferably being suitable for administration to a subject, preferably a mammalian subject, more preferably a human. Such pharmaceutical compositions may comprise a pharmaceutically acceptable carrier. The pharmaceutical composition may be administered, for example, by the oral route, by the parenteral route, by inhalation spray, by the topical route, by eye drops, by the rectal route, by the nasal route, by the buccal route, by the vaginal route or via an implanted reservoir, wherein the term "parenteral route" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions may be provided in suitable dosage forms, such as capsules, tablets, and aqueous suspensions and solutions, preferably in sterile form. It may be used in a method of treating a disease comprising administering to a subject an effective amount of a polypeptide of the invention. In a preferred embodiment, the invention provides a vaccine comprising a polypeptide of the invention, optionally comprising an auxiliary agent such as an adjuvant or buffer; and the use of the polypeptides of the invention for the preparation of vaccines.

Within the scope of the present invention, a medical device is provided comprising a reagent, preferably coated, for the detection of the (auto) antibodies of the invention and/or the polypeptides of the invention. Preferably, such a medical device comprises a polypeptide of the invention in a form that allows it to be brought into contact with an aqueous solution, more preferably a liquid human sample, in a direct manner. In particular, the comprised polypeptide of the invention may be immobilized on the surface of a carrier, preferably selected from the group comprising: glass plates or slides, biochips, microtiter plates, beads, e.g., magnetic beads, blood component separation (aphares) devices, chromatography columns, membranes, and the like. Exemplary medical devices include linear dot blots, microtiter plates, microscope slides, beads, preferably magnetic beads, and biochips. In addition to the polypeptides of the invention, the medical or diagnostic device may comprise additional polypeptides, such as positive or negative controls, e.g., samples comprising or not comprising antibodies that bind to the polypeptide of interest, or other antigens known to bind to diagnostic autoantibodies, particularly those related to other diseases associated with one or more of the same or similar symptoms.

The teachings of the present invention provide kits, preferably for identifying a subject at increased risk of developing a disease. Such a kit may comprise instructions detailing how to use the kit, and means for contacting the polypeptide of the invention with a sample of bodily fluid from a subject (preferably a human subject), such as a linear dot blot, wherein the polypeptide of the invention is immobilized on the linear dot blot. Further, the kit may comprise a positive control, such as a collection of autoantibodies or recombinant antibodies known to bind to a polypeptide of the invention; and a negative control, e.g., a protein that has no detectable affinity for the polypeptide of the invention, e.g., bovine serum albumin. Finally, such a kit may contain a standard solution of an antibody that binds to cytokinin-3 for preparing a calibration curve.

In a preferred embodiment, the kit comprises means for detecting autoantibodies bound to a polypeptide of the invention, preferably by detecting a complex comprising a polypeptide of the invention and an antibody bound to a polypeptide of the invention. Such means are preferably reagents that bind to the complex and modify the complex or carry a label to make the complex detectable. For example, the means may be a labelled antibody which binds to the polypeptide or to a constant region of a primary antibody at a binding site other than that recognized by the primary antibody. Alternatively, the means may be a secondary antibody that binds to the constant region of the autoantibody, preferably a secondary antibody specific for a mammalian class IgG antibody. More preferably, the labeled secondary antibody specifically binds human IgG. Many methods and tools for detecting such complexes have been described in the prior art, for example in Philips, Terry, M., Analytical techniques in biochemistry, 1992, Marcel Dekker, Inc.

The cytokinin-3 or a variant thereof may be produced or provided in the form of a cell comprising and/or expressing a nucleic acid encoding said polypeptide. If a nucleic acid comprising a sequence encoding a polypeptide of the invention or a variant thereof is used, such a nucleic acid may be an unmodified nucleic acid. In a preferred embodiment, the nucleic acid is a nucleic acid that does not itself occur in nature and comprises at least one modification, such as isotopic content or chemical modifications, such as methylation, sequence modifications, tags, etc., as compared to the native nucleic acid, which is indicative of a synthetic origin. In a preferred embodiment, the nucleic acid is a recombinant nucleic acid or a part of a nucleic acid, and in a more preferred embodiment, is a part of a vector in which it may be functionally linked to a promoter allowing expression (preferably overexpression) of the nucleic acid. The person skilled in the art is familiar with a wide variety of suitable vectors, which are commercially available, for example from Origene. For example, vectors encoding fusion constructs with C-terminal GFP can be used. The cell may be eukaryotic or prokaryotic, preferably eukaryotic, such as yeast, and more preferably mammalian, more preferably human, such as HEK 293. Examples of mammalian cells include HEK293, CHO or COS-7 cells. The cell comprising a nucleic acid encoding a polypeptide of the invention may be a recombinant cell or an isolated cell, wherein the term "isolated" means that the cell is enriched such that there are few or virtually no other differentiated or species of cells as compared to the wild-type environment of the cell.

In a preferred embodiment, the medical device according to the invention, preferably a slide suitable for microscopic examination, comprises one or more, preferably all, reagents selected from the group comprising a first eukaryotic cell expressing (preferably overexpressing) a cytokinin-3 or a variant thereof, preferably eukaryotic (preferably mammalian) tissue expressing endogenous cytokinin-3, such as rat or primate cerebellum, being the same type of cell as the first eukaryotic cell, but a second eukaryotic cell not expressing or not expressing cytokinin-3. The first and second eukaryotic cells are cultured cells derived from an isolated cell line such as HEK 293. Preferably, the first and second cells are each transfected with a vector (vector) sharing the same backbone, wherein the vector used to transfect the first cell comprises a nucleic acid encoding cytokinin-3 or a variant thereof and the vector used to transfect the second cell does not comprise cytokinin-3 or a variant thereof. The second cell can be used as a negative control. The agents may be spatially separated on the medical device such that they can be evaluated independently without antigens from one agent contaminating the other. In a more preferred embodiment, the first and/or second cells are fixed cells, for example using methanol or acetone. Protocols for immobilizing cells are described in the prior art. As further reagents, a labelled secondary antibody, preferably labelled with a fluorescent dye, may be provided. The reagents and medical devices may be part of a kit.

In a preferred embodiment, the diagnostic method according to the invention is carried out using microtiter plates, membranes, blots such as dot blots or linear dot blots. Those skilled in The art are familiar with The experimental set-up of linear dot blots, which are described in The prior art (Raoult, D. and Dasch, G.A. (1989), The line blot: an immunological for monoclonal and other antibodies.its application to The serotyping of gram-negative bacteria.J.immunological methods,125(1-2), 57-65; WO 2013041540). If the medical device is a linear dot blot, it may comprise a mitogen-3 or variant thereof immobilized on a membrane, preferably in the shape of a test strip. The membrane may comprise one or more additional antigens that are spatially separated from the cytokinin-3. The membrane may comprise a control strip indicating the addition of a sample, such as a blood sample, and/or a control strip indicating the addition of a secondary antibody. The kit may comprise any component, preferably all from the group comprising linear dot blots, secondary antibodies and wash solutions.

In another preferred embodiment, the medical device is a microtiter plate comprising at least 8 wells. At least one well is coated directly or indirectly with cytokinin-3 or a variant thereof. At least 3, preferably 4, more preferably 5 calibrators are provided which comprise antibodies to cytokinin-3 at defined concentrations and which can be used to set a calibration curve for semi-quantitative analysis. A secondary antibody comprising an enzyme activity marker may be provided. The kit may comprise any component, preferably all from the group comprising microtiter plates, calibrators, wash solutions and secondary antibodies.

In another preferred embodiment, the medical device is a bead coated directly or indirectly with cytokinin-3 or a variant thereof. The beads may be selected from the group consisting of magnetic beads and fluorescent beads. A secondary antibody comprising a label capable of chemiluminescence or fluorescence may be provided. A positive control comprising an antibody to cytokinin-3 may be provided. At least 3, preferably 4, more preferably 5 calibrators may be provided, which comprise an antibody against cytokinin-3 at a defined concentration and which may be used to establish a calibration curve for semi-quantitative analysis. If the label is capable of producing chemiluminescence, a solution may be provided containing other components necessary for the chemiluminescent reaction. For example, if the label is an enzyme, the solution comprises a substrate. If the label is a compound capable of generating chemiluminescence, such as an acridinium ester, the other compounds required for the reaction are provided in solution. The kit may comprise any component, preferably all from the group comprising beads, secondary antibodies, calibrators, wash solutions and solutions comprising other components.

The teachings of the present invention may be used not only for assessing or identifying, but also for preventing or treating a disease, more particularly, for a method of preventing or treating a disease, comprising the steps of: a) reducing the concentration of autoantibodies binding to the polypeptide of the invention in the blood of the subject, and/or b) administering one or more immunosuppressive pharmaceutical substances, preferably selected from the group comprising: rituximab, prednisone, methylprednisolone, cyclophosphamide, mycophenolate mofetil, intravenous immunoglobulin, tacrolimus, cyclosporine, methotrexate, azathioprine, and/or pharmaceutical compositions.

In a preferred embodiment, the invention provides the use of a reagent for detecting autoantibodies against cytokinin-3 or reagents binding to such antibodies, or a reagent for detecting nucleic acid encoding cytokinin-3 or variants or nucleic acid specifically hybridizing to nucleic acid encoding cytokinin-3, or a vector or cell comprising said nucleic acid for the manufacture of a kit for the diagnosis of a disease.

In a preferred embodiment, any of the methods or uses according to the present invention may be used for in vitro testing of the efficiency of a medical device designed to remove autoantibodies from patient blood, wherein the testing is performed on a liquid other than patient blood. After use of the medical device with a patient, it may be checked for the ability to remove autoantibodies, running a solution containing antibodies to lysenin-3 through the device and then using the method according to the invention to confirm that less or no antibodies are in solution that has passed through the device, i.e. to show that the device still has the ability to remove antibodies from solution. Alternatively, from a batch comprising a large number of devices, a small number of devices may be tested to confirm or test the quality of the entire batch in a quality control sense, wherein the sample or solution may comprise a known concentration of antibody against lysenin-3.

In another preferred embodiment, the method may be used to confirm the reliability of an antibody detection assay and may involve detecting antibodies to lysenin-3 in a solution that is not a sample from a patient but is known to contain antibodies to lysenin-3, preferably at a known concentration. Alternatively, the solution may be a negative control containing no antibody to check background. This method may be performed in parallel with, after or before the diagnostic method. In a preferred embodiment, any of the methods or uses according to the invention can be used for generating autoantibody profiles, preferably for detecting diseases in mammals, preferably humans. In a preferred embodiment, any method or use may be used for detecting a disease-associated marker in a sample from a patient with a neurological disease.

In a preferred embodiment, any of the methods or uses according to the invention may be used to identify a subject at risk of having or developing a neurological disease and/or tumor.

Brief description of the drawings

FIG. 1 shows the results of indirect immunofluorescence assays from sera of representative positive patients using permeabilized frozen sections of the rat hippocampus and rat cerebellum. Granular staining of the molecular layer was observed.

FIG. 2 shows SDS PAGE (A) or Western blot of cytolysin-3 immunoprecipitated from homogenized rat cerebellum using patient serum, followed by Coomassie staining using commercial recombinant antibodies that bind to cytolysin-3 (B).

FIG. 3 shows the immunofluorescence results of HEK293 cells expressing the indicated proteins and using patient sera. No background fluorescence signal was observed using mock transfected HEK293 cells and control sera.

FIG. 4 shows immunofluorescence results using patient serum against rat cerebellum, primate cerebellum, and rat hippocampus (A). The fluorescent signal (B) was eliminated by preincubation with HEK293 lysate containing lysenin-3.

FIG. 5 shows that bacterially expressed proteins (Split-protein-3-His and split-protein-5 without His-tag, split-protein-6, split-protein-7 and split-protein-11 were purified by IMAC via His-tag) were used to form a solid phase in an indirect ELISA for detection of human IgG antibodies. SDS-PAGE analysis of the purified antigen revealed several protein bands (A). All recombinantly expressed proteins (lysenin-3-His, lysenin-5, lysenin-6, lysenin-7 and lysenin-11) can be identified by mass spectrometry. (B) ELISA results with recombinant Split-protein-3 and co-expressed Split-protein-3, Split-protein-5, Split-protein-6, Split-protein-7 and Split-protein-11 and 20 healthy control sera in 2 patients sera positive in RC-IFA. The number and total number of positive sera are given below the figure.

FIG. 6 shows immunohistochemical staining of mouse cerebellum (A1, B1) and patient cancer specimens (A2-4, B2-4) incubated with 1:1,000(A1) or 1:500 dilution (A2-4) of rabbit commercial antibody against Split-3 and control rabbit serum (B1-4). (a2, B2) melanoma, patient 1; (a3, B3) lymph node metastasis, patient 1; (A4, B4) lymph node metastasis, patient 3. Magnification times 200.

FIG. 7 shows a summary of five patients (P1-P5) whose sera showed reactivity against cytokinin-3.

FIG. 8 shows Western blots with full-length lysenin-3-His (aa 1-358) expressed in HEK293 cells or His-GST-lysenin-3 fragments (aa 1-62, 51-261, 205-358) expressed in bacteria and patient serum 2(PS2) or control serum (CS1, 2).

Figure 9 shows a list of patients with migraine, Amyotrophic Lateral Sclerosis (ALS), and recurrent syncope, respectively, whose sera showed immunoreactivity to cytokinin-3. Further shown is the maximal serum dilution capable of detecting either the lysenin complex or lysenin-3 alone.

The present application comprises a series of sequences, more specifically:

SEQ ID No.1 (human mitogen-3)

MSKGLPETRTDAAMSELVPEPRPKPAVPMKPMSINSNLLGYIGIDTIIEQMRKKTMKTGFDFNIMVVGQSGLGKSTLVNTLFKSQVSRKASSWNREEKIPKTVEIKAIGHVIEEGGVKMKLTVIDTPGFGDQINNENCWEPIEKYINEQYEKFLKEEVNIARKKRIPDTRVHCCLYFISPTGHSLRPLDLEFMKHLSKVVNIIPVIAKADTMTLEEKSEFKQRVRKELEVNGIEFYPQKEFDEDLEDKTENDKIRQESMPFAVVGSDKEYQVNGKRVLGRKTPWGIIEVENLNHCEFALLRDFVIRTHLQDLKEVTHNIHYETYRAKRLNDNGGLPPGEGLLGTVLPPVPATPCPTAE

SEQ ID No.2 (mouse mitogen-3)

MSKGLPEARTDAAMSELVPEPRPKPAVPMKPVSINSNLLGYIGIDTIIEQMRKKTMKTGFDFNIMVVGQSGLGKSTLVNTLFKSQVSRKASSWNREEKIPKTVEIKAIGHVIEEGGVKMKLTVIDTPGFGDQINNENCWEPIEKYINEQYEKFLKEEVNIARKKRIPDTRVHCCLYFISPTGHSLRPLDLEFMKHLSKVVNIIPVIAKADTMTLEEKSEFKQRVRKELEVNGIEFYPQKEFDEDLEDKTENDKIRQESMPFAVVGSDKEYQVNGKRVLGRKTPWGIIEVENLNHCEFALLRDFVIRTHLQDLKEVTHNIHYETYRAKRLNDNGGLPPVSVDTEESHDSNP

SEQ ID No.3 (rat mitogen-3)

MSKGLPEARTDTAMSELVPEPRPKPAVPMKPVSINSNLLGCIGIDTIIEQMRKKTMKTGFDFNIMVVGQSGLGKSTLVNTLFKSQVSRKASSWNREEKIPKTVEIKAIGHVIEEGGVKMKLTVIDTPGFGDQINNENCWEPIEKYINEQYEKFLKEEVNIARKKRIPDTRVHCCLYFISPTGHSLRPLDLEFMKHLSKVVNVIPVIAKADTMTLEEKSEFKQRVRKELEVNGIEFYPQKEFDEDLEDKTENDKIRQESMPFAVVGSDKEYQVNGKRVLGRKTPWGIIEVENLNHCEFALLRDFVIRTHLQDLKEVTHNIHYETYRAKRLNDNGGLPPGEGLLGTVLPPVPATPCPTAE

SEQ ID No.4 (porcine cytoclasin-3)

MSKGLPEARTDTAMSELVPEPRPKPAVPMKPVSINSSLLGYIGIDTIIEQMRKKTMKTGFDFNIMVVGQSGLGKSTLVNTLFKSQVSRKASSWNREEKIPKTVEIKAIGHVIEEGGVKMKLTVIDTPGFGDQINNENCWEPIEKYINEQYEKFLKEEVNIARKKRIPDTRVHCCLYFISPTGHSLRPLDLEFMKHLSKVVNIIPVIAKADTMTLEEKSEFKQRVRKELEVNGIEFYPQKEFDEDLEDKTENDKIRQESMPFAVVGSDKEYQVNGKRVLGRKTPWGIIEVENLNHCEFALLRDFVIRTHLQDLKEVTHNIHYETYRAKRLNDNGGLPPCFEAVSGCGSLLPTAARRI

SEQ ID NO 5 (human mitogen-31-62 aa)

MSKGLPETRTDAAMSELVPEPRPKPAVPMKPMSINSNLLGYI GIDTIIEQMRKKTMKTGFD

The invention is further illustrated by the following non-limiting examples from which other features, embodiments, aspects and advantages of the invention may be derived.

Examples

Summary of the invention

Method of producing a composite material: two patients with neurological conditions (P1-P2) were serologically investigated. To this end, sera from two patients were subjected to a comprehensive autoantibody screen by indirect immunofluorescence assay (IFA) and immunoblotting. Immunoprecipitation with cerebellar lysates followed by Mass Spectrometry (MS) was used to identify autoantigens, which was verified by Western Blotting (WB) with monospecific animal antibodies against the respective target antigens, as well as by recombinant expression in HEK293 cells and use of recombinant proteins in immunoassays. In addition, 3 sera (P3-P5) with similar staining patterns as patients 1 and 2 but without known autoantibody reactivity were screened against anti-cytokinin-3 antibodies as well as negative control sera. lysenin-3-His and lysenin-5, without His tag-6. And (3) coexpressing the cytokinin-7 and the cytokinin-11. Purified proteins were analyzed in ELISA using anti-cytokinin-3 positive patient sera and healthy controls.

Results: IFA screening of sera from P1 to P5 revealed IgG reactivity with a molecular layer in rat cerebellum and hippocampus. In the rat sea horse, the outer molecular layer stained more deeply than the inner molecular layer. On rat cerebellum, in addition to particle staining of the molecular layer, pimple staining of the granular layer was also observed, whereas purkinje cells were negative. Furthermore, no IgG reactivity was found in a panel of 30 recombinantly expressed established neuroself antigens. Sera from P1 to P2 were immunoprecipitated with mitogen-3, mitogen-5, mitogen-6, mitogen-7 and mitogen-11 by Coomassie-stained SDS-PAGE followed by MALDI-TOF mass spectrometry. When the immunoprecipitates were analyzed by Western blotting using a monospecific animal antibody against mitogen-3, the anti-mitogen-3 showed reactivity with the immunoprecipitates of P1 to P3, but not in the immunoprecipitates of 3 control sera. Furthermore, in the sera of P1 to P2 and in the sera of 3 additional patients (P3 and P5) with similar staining patterns to rat cerebellum and rat hippocampus, anti-cytokinin-3 antibodies could be detected with recombinant proteins by RC-IFA. Screening of healthy control sera with neural tissue (n-150) without a specific response in IIFT revealed a positive sample for anti-cytokinin complex. Results obtained by RC-IFA were confirmed by ELISA using recombinant expression of lysenin-3-His + lysenin-5, lysenin-6, lysenin-7, and lysenin-11 (. DELTA.His).

Clinical data from four patients with anti-cytokinin-3 autoantibodies was available. P1 has malignant melanoma associated with cerebellar ataxia, P3 has Small Cell Lung Cancer (SCLC) associated with cerebellar syndrome, and P4 has longitudinal generalized myelitis. P5 had cerebellar ataxia with no tumor association.

Patient's health

The control group included 150 healthy donors.

Indirect immunofluorescence assay (IFA)

IFA was performed using a slide with a biochip array of frozen sections of brain tissue (hippocampus of rat, cerebellum of rat) in combination with recombinant HEK293 cells expressing 30 different brain antigens, Hu, Yo, Ri, CV2, PNMA2, ITPR1, Homer 3, CARP VIII, ARHGAP26, ZIC4, DNER/Tr, GAD65, GAD67, amphisarin, recoverin, GABA_BReceptors, glycine receptors, DPPX, IgLON5, glutamate receptors (type NMDA, AMPA, mGluR1, mGluR5, GLURD2), LGI1, CASPR2, AQP4(M1 and M23), MOG, ATP1A3, NCDN (EUROIMMUN, FA 111a-1003-51, FA 1112-1003-50, FA-1128-1003-50, FA112d-1003-1, FA 112M-1003-50, FA1151-1003-50, Misker, Hahn S, Rosenkranzanz T, Muller M, Dettmann IM, Mindorf S, Denno Y, Brakopp S, Scharf M, Teegen B, Probst C, Melzer N, MeinHM, Terborrg C,

W,Komorowski L.,2016,Autoantibodies against glutamate receptor2after allogenic stem cell transplantation.Neurol Neuroimmunol Neuroinflamm.,3(4):e255；Scharf M,Miske R,Heidenreich F,Giess R,Landwehr P,

IM,Begemann N,Denno Y,Tiede S,

C,Schlumberger W,Unger M,Teegen B,

W,Probst C,Komorowski L,2015,Neuronal Na+/K+ATPase is an autoantibody target in paraneoplastic neurologic syndrome,Neurology；84(16):1673-9；Miske R,Gross CC,Scharf M,Golombeck KS,Hartwig M,Bhatia U,Schulte-Mecklenbeck A,

K,Strippel C,L,Synofzik M,Lohmann H,Dettmann IM,Deppe M,Mindorf S,Warnecke T,Denno Y,Teegen B,Probst C,Brakopp S,Wandinger KP,Wiendl H,

w, Meuth SG, Komorowski L, Melzer N,2016, neuroBindin is a neuronal target antigen in autoimmune cerebellar de-generation, Neurol neuro-immumnol neuro-flum; 4(1) e 307)). Each biochip mosaic (biochip mosaic) was incubated with 70. mu.L of PBS-diluted sample at room temperature for 30 minutes, washed with PBS-Tween and immersed in PBS-Tween for 5 minutes. In the second step, Alexa 488-labeled goat anti-human IgG (Jackson Research, Suffolk, United Kingdom) or Fluorescein Isothiocyanate (FITC) -labeled goat anti-human IgG (EUROIMMUN Medizinische Labordiagnostika AG, Lubeck) was applied and incubated at room temperature for 30 minutes. The slides were washed again with PBS-Tween rinse and then immersed in PBS-Tween for 5 minutes. Slides were embedded in PBS-buffered glycerol-containing DABCO (approximately 20 μ L per field) 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, as compared directly to untransfected cells and control samples. The endpoint titer refers to the last dilution that was able to show visible fluorescence.

The results were evaluated by two independent observers using a EUROStar II microscope (EUROIMMUN Medizinische Labordiagnostika AG, Lubeck, Germany). Reagents were obtained from Merck, Darmstadt, Germany and Sigma-Aldrich, Heidelberg, Germany, if not otherwise specified.

Immunoblotting

Immunoprecipitated cerebellar lysates were incubated with NuPage LDS sample buffer (ThermoFisher Scientific, Schwerte, Germany) containing 25mmol/L dithiothreitol at 70 ℃ for 10 min, followed by SDS-PAGE (NuPAGE, ThermoFisher Scientific, Schwerte, Germany). The isolated proteins were electrotransferred onto nitrocellulose membranes by slot blotting with transfer buffer (ThermoFisher Scientific) according to the manufacturer's instructions. The membrane was blocked for 15 minutes with a Universal Block Buffer plus (EUROIMMUN Medizinische Labordiagnostika AG, Lubeck) and incubated with monospecific rabbit antibodies against cytokinin-3 (Sigma Aldrich, HPA003548,1:20,000) in the Universal Block Buffer plus for 3 hours, followed by 3 washing steps with a Universal Block Buffer (EUROIMMUN Medizinische Labordiagnostika AG, Lubeck), a second 30 minute washing step with anti-human IgG-AP (EUROIMN Medizinische Labordiagnostika AG, Lubeck, 1:10) or anti-rabbit IgG-AP (1:2,000) in the Universal Block Buffer plus, 3 washing steps and staining with NBIMT/MUN substrate (EUROIMMUN Medizinische AG, Lubeck). Reagents were obtained from Merck, Darmstadt, Germany or Sigma-Aldrich, Heidelberg, Germany, if not otherwise specified.

Identification of antigens

The cerebellum of rats and pigs was dissected and snap frozen in liquid nitrogen. The slurry was homogenized by Miccra D-8(Roth, Karlsruhe, Germany) and a manual homogenizer (Sartorius,

germany) the tissue was homogenized at 4 ℃ in a solubilization buffer (100mmol/L tris-HCl pH 7.4, 150mmol/L sodium chloride, 2.5mmol/L ethylenediaminetetraacetic acid, 0.5% (w/v) sodium deoxycholate, 1% (w/v) Triton X-100) containing protease inhibitors (Complete mini, Roche Diagnostics, Penzberg, Germany). The tissue lysate was centrifuged at 21,000 Xg for 15 minutes at 4 ℃ and the clear supernatant was incubated with patient serum (1:16.7 dilution) overnight at 4 ℃. The samples were then incubated with protein G Dynabeads (ThermoFisher Scientific, Dreieich, Germany) for 3 hours at 4 ℃ to capture the immune complexes. The beads were washed 3 times with PBS and eluted with NuPage LDS sample buffer (ThermoFisher Scientific, Schwerte, Germany) containing 25mmol/L dithiothreitol for 10 min at 70 ℃. Urea methylation (carbamidatholation) was performed with 59mM iodoacetamide (Bio-Rad, Hamburg, Germany) followed by SDS-PAGE (NuPAGE, ThermoFisher Scientific, Schwerte, Germany). The isolated protein was visualized with Coomassie Brilliant blue (G-250) (Merck) and identified by mass spectrometry.

Mass spectrometry

The visible protein band was cut from the Coomassie brilliant blue G-250 stained gel. Decolorization and trypsin digestionThen, the peptide is extracted and spotted onto MTP Anchor chip by using alpha-cyano-4-hydroxycinnamic acid^TM384TF target.

MALDI-TOF/TOF measurements were performed using the Autoflex III smartmean TOF/TOF200 system using flexControl 3.4 software. Mass spectra of Peptide Mass Fingerprints (PMFs) were recorded in the mass range 600Da-4,000Da and in the positive ion reflectron mode with 4,000 and 10,000 bombardments (shots). Mass spectra were externally calibrated with a commercially available Peptide Calibration Standard II, processed with flexAnalysis 3.4 and peak lists analyzed with BioTools 3.2.

Mascot search Engine Mascot Server 2.3(Matrix Science, London, UK) was used for protein identification by performing searches on NCBI or SwissProt databases restricted to mammals (Mammalia). The search parameters are as follows: the mass tolerance was set at 80ppm, a missed cleavage site was accepted, and urea methylation of cysteine residues and oxidation of methionine residues were set as fixed and variable modification, respectively. To evaluate protein hits, a significance threshold of p <0.05 was chosen.

To further confirm PMF hits, 2 to 5 peptides per identified protein were selected for MS/MS measurements using the WARP feedback mechanism of BioTools. The parent and fragment masses were recorded with 400 and 1000 shots, respectively. The mass spectra were processed and analyzed as described above with a fragment mass tolerance of 0.7 Da.

Recombinant expression of mitogen proteins in HEK293 cells

The split-cell protein was cloned into the mammalian expression vector pTriEx-1(Merck, Darmstadt, Germany) and the corresponding split-cell protein was transiently expressed in the human cell line HEK293, followed by PEI-mediated transfection (Exgene 500) according to the instructions of the manufacturer (Biomol GmbH, Hamburg, Germany).

For the generation of immunofluorescent substrates, cells were grown on coverslips and acetone fixed two days after transfection.

For other purposes, cells were harvested 5 days after transfection and lysed by high pressure. The lysate was stored in aliquots at-80 ℃ for further use.

And (3) purification:

the cytokinin-3-His was purified by metal affinity chromatography immobilized on nickel Rapid Transit (ABT). Protein analysis was performed by SDS-PAGE using the NuPAGE system according to the manufacturer's (Invitrogen) manual and by mass spectrometry. In addition to cytokinin-3-His, co-expressed cytokinin-5, cytokinin-6, cytokinin-7 and cytokinin-11 without a histidine tag could be detected.

Characterization of patient autoantibodies

Indirect immunofluorescence assays (IFA) of sera P1 to P5 using permeabilized frozen sections of rat cerebellum and hippocampus showed particle staining of the molecular layer (fig. 1). Further monospecific assays were performed using recombinant HEK293 cells expressing 30 neuro-autoantigens, Hu, Yo, Ri, CV2, PNMA2, SOX1, ITPR1, Homer 3, CARP VIII, ARHGAP26, ZIC4, DNER/Tr, GAD65, GAD67, biproteins, recoverin, GABAB receptors, glycine receptors, DPPX, IgLON5, glutamate receptors (type NMDA, AMPA, mGluR1, mGluR5, GLURD2), LGI1, CASPR2, AQP4(M1 and M23), MOG, ATP1A3 and NCDN. No specific reactivity was observed.

Identification of mitoproteins as target neuronal autoantigens

The immunoprecipitates from homogenized rat and monkey cerebellum obtained with P1 and P2 were analyzed by SDS-PAGE. In Coomassie-stained SDS-PAGE, a specific band of 40-50kDa was detected (FIG. 2A). Protein bands were identified as lysenin-3, lysenin-5, lysenin-6, lysenin-7 and lysenin-11 (UNIPROT accession numbers Q9WU34, Q9JJM9, NP _001166900.1, Q9WVC0, B3GNI6) using MALDI-TOF MS. As evidence of correct antigen identification, immunoprecipitates were tested by western blotting using antibodies against neuron-specific cytokinin-3. As shown by the specific band of 40-50kDa (FIG. 2B), the immunoprecipitates from patient sera contained cytokinin-3, which was not present in the immunoprecipitates from control sera. In addition, patient samples were tested by IFA using transfected HEK293 cells expressing lysenin-3 (SEQ ID No.1) or co-expressing lysenin-3, lysenin-5, lysenin-6, lysenin-7 and lysenin-11. The patient's serum reacted with cells expressing cytokinin-3 alone as well as cells co-expressing cytokinin-3, cytokinin-5, cytokinin-6, cytokinin-7 and cytokinin-11 (FIG. 3A). In contrast, mock-transfected cells did not exhibit any specific antibody binding (fig. 3B). When cytokinin-5, cytokinin-6, cytokinin-7, or cytokinin-11 were expressed alone, the patient sera (P1 to P3) did not show any antibody binding. Other combinations of cytokinin co-expression were analyzed by RC-IFA with patient sera: mitogen-3, mitogen-5, mitogen-6, mitogen-7; mitogen-3, mitogen-5, mitogen-7, mitogen-11; mitogen-5, mitogen-6, mitogen-7, mitogen-11; mitogen-3, mitogen-5, mitogen-7; mitogen-5, mitogen-6, mitogen-7; mitogen-3, mitogen-7, and mitogen-11. Three patients (P1-P3) showed reduced or no response to co-expressing cells without the combination of cytokinin-3. These results indicate the importance of neuron-specific cytokinin-3.

The patient autoantibody response to tissue was abrogated by pre-incubation with HEK293 lysate containing cytokinin-3 (SEQ ID No.1) (fig. 4). Antibody binding was unaffected when comparable fractions from mock-transfected HEK293 cells were used.

Specificity of anti-Sphaemagglutinin-3 autoantibodies

Up to 150 healthy control sera were analyzed by IFA in parallel with patient samples using HEK 293-dehiscein-3 or HEK 293-dehiscein-3, dehiscein-5, dehiscein-6, dehiscein-7, dehiscein-11 or other combinations. Only one of 150 healthy control sera showed a positive reaction with HEK 293-lysenin-3, lysenin-5, lysenin-6, lysenin-7, lysenin-11 cells diluted 1: 100.

Immunoassay for detection of autoantibodies with recombinant mitogen

A96-well plate (Nunc, Germany) was coated with 100. mu.l of recombinant protein in PBS at a concentration of 2.5. mu.g/ml for 2 hours at 25 ℃, washed 3 times with wash buffer (0.05% [ wt/vol ] Tween 20 in PBS), and then blocked with blocking buffer (0.1% [ wt/vol ] casein in PBS) for 1 hour. The success of antigen immobilization was confirmed by incubation with a 1:2,000 dilution of a murine monoclonal anti-hexahistidine-tagged antibody (Sigma-Aldrich, Germany). The experimental serum samples were diluted 1:100 in sample buffer (1% [ wt/vol ] casein in PBS, 0.05% [ wt/vol ] Tween 20) and incubated for 30 minutes at room temperature. After three washes, bound antibody was detected by incubation with 1:16,000 diluted anti-mouse IgG-HRP conjugate (Jackson Research, UK) or undiluted anti-human IgG-POD (Euroimmun, Germany) in sample buffer for 30 min, washed as described above, and incubated with Tetramethylbenzidine (TMB) substrate (Euroimmun, Germany) for 15 min. All incubation steps were performed at room temperature. The Optical Density (OD) at 450nm was read using an automated spectrophotometer (Tecan, Germany).

The 3 patient sera (P1, P2 and P4) and 20 healthy control sera were analyzed by ELISA using lysenin-3-His + lysenin-5 without His tag, lysenin-6, lysenin-7, lysenin-11 and purified by lysenin-3-His by IMAC (fig. 5). All patient sera analyzed showed positive reactions, while all control sera were negative. The results confirm the results obtained from RC-IFA. In addition, patient sera showed positive reactions in ELISA (P2, P3) and Western blot (P1-3) experiments using purified cytokinin-3-His as antigen.

Histopathology of patient tumor specimens

Formalin-fixed paraffin-embedded melanoma (P1) and lymph node metastasis (P1 and P3) from patient tissues were sectioned (4 μm). As a control for antibody functionality, mouse cerebellar tissue was sectioned. Sections were mounted on glass slides, deparaffinized, rehydrated, and heat-induced epitope Retrieval (heat-induced epitope-Retrieval) was performed using a Target Retrieval Solution (pH9, 3-in-1, Dako, Hamburg, Germany) according to the supplier's instructions. Subsequently, slides were washed with Tris Buffered Saline (TBS) containing 0.05% tween 20 at room temperature. Blocking was performed with Serum-free Protein Block (Thermo Fisher Scientific, Schwerte, Germany) for 10 minutes. Polyclonal rabbit anti-cytokinin-3 (HPA003548, Sigma-Aldrich, Taufkirchen, Germany) was diluted 1:500 or 1:1,1000 in Dako antibody diluent and then applied for 30 minutes. As negative control, the rabbit immunoglobulin fraction (X0936, Dako, Hamburg, Germany) was used. Incubations were performed using the Bond Polymer Refine Detection System (Leica Biosystems, Wetzlar, Germany) according to the manufacturer's instructions. After 10 minutes, the development of 3, 3-diaminobenzidine (DAB; Leica Biosystems, Wetzlar, Germany) was stopped. Hematoxylin (Leica Biosystems, Wetzlar, Germany) was used for counterstaining. Slides were mounted with Neo-Mount (water seal free tablet (VWR, Darmstadt, Germany)).

Incubation of mouse cerebellar sections with anti-lysenin-3 antibody showed the same pattern as observed with indexing patient sera in indirect immunofluorescence assays with rat cerebella (a1 of fig. 6). Incubation of patient tumor specimens revealed regions with high expression of cytokinin-3 in all three tissues analyzed (A2-A4 of FIG. 6). Therefore, it is very possible to presume Paraneoplastic Neuronal Syndrome (PNS) in these two patients.

FIG. 7 shows a summary of five patients (P1-P5) whose sera showed reactivity to cytokinin-3.

Characterization of the cleavage protein-3 epitope recognized by the patient autoantibodies

For epitope mapping, E.coli lysed as described above expressing either full-length lysenin-3-His HEK239 cells or His-GST-lysenin-3 fragments (aa 1-62, 51-261, 205-358) expressed in pET24d vector (Merck, Germany) were incubated with NuPage LDS sample buffer containing 25mmol/L dithiothreitol (ThermoFisher Scientific, Germany) for 10 min at 70 ℃. Lysates were immunoblotted as described above. Briefly, membranes were incubated with anti-His mouse monoclonal antibody (Merck, Germany, 1:2000), patient serum or control serum (1:200) in a Universal Blot Buffer plus for 3 hours and anti-mouse IgG-AP (Jackson ImmunoResearch, UK, 1:2000) or anti-human IgG-AP (1:10) in a Universal Blot Buffer plus for 30 minutes.

The full-length protein and the N-terminal split-3 fragment of cytokinin-3 (aa 1-62) were recognized by the patient serum (P2), but not by the control sera (CS1 and CS2) (FIG. 8), indicating that the patient autoantibodies recognize epitopes located in the first 62 amino acids of split-3. The presence of all fragments was confirmed by a positive anti-His tag reaction.

Patients with other clinical features of autoantibodies against cytokinin-3

Fig. 9 discloses additional sera showing immunoreactivity against cytokinin-3 from patients with migraine, Amyotrophic Lateral Sclerosis (ALS), and recurrent syncope, respectively.

Sequence listing

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Claims (15)

1. A diagnostically useful carrier comprising means for specifically capturing antibodies that specifically bind to cytokinin-3 in a sample from a subject.

2. The carrier according to claim 1, wherein the antibody does not bind to dehiscence protein-1, dehiscence protein-2, dehiscence protein-4, dehiscence protein-5, dehiscence protein-6, dehiscence protein-7, dehiscence protein-8, dehiscence protein-9, dehiscence protein-10, dehiscence protein-11 and/or dehiscence protein-12.

3. The carrier according to claim 1 or 2, wherein the carrier is selected from the group consisting of beads, test strips, microtiter plates, blots, glass surfaces, slides and membranes.

4. A vehicle according to claims 1 to 3, wherein the means for specifically capturing the antibody is

a) Recombinant and purified cytokinin-3 or a variant thereof; or

b) A eukaryotic cell expressing cytokinin-3 or a variant thereof.

5. A kit comprising a diagnostically useful carrier according to any one of claims 1 to 4, wherein the kit comprises a calibrator, a wash buffer and/or means for detecting IgG antibodies.

6. A method comprising the step of detecting the presence or absence of an antibody to cytokinin-3 in a sample from a subject.

7. A pharmaceutical composition comprising dehiscin-3 or a variant thereof.

8. Use of a vehicle according to any one of claims 1 to 4 in the manufacture of a kit for the diagnosis of a disease, wherein the disease is a neurological autoimmune disease, recurrent syncope and/or cancer.

9. Use according to claim 8, wherein the disease is paraneoplastic syndrome.

10. Use according to claim 8, wherein the autoimmune disease of the nervous system is cerebellar ataxia, longitudinal myelitis universalis, migraine, Amyotrophic Lateral Sclerosis (ALS) or cerebellar syndrome.

11. Use according to claim 8, wherein the cancer is melanoma or carcinoma.

12. An isolated human antibody that specifically binds to cytokinin-3.

13. The vehicle according to claim 1, the method according to claim 6 and the antibody according to claim 12, wherein the dehiscence protein-3 is human dehiscence protein-3.

14. A carrier according to claim 1, a method according to claim 6 and an antibody according to claim 12, wherein dehiscence protein-3 is combined with one or more dehiscence protein polypeptides.

15. A vehicle according to claim 4 or a pharmaceutical composition according to claim 7, wherein the variant comprises the amino acid sequence of SEQ ID NO: 5.

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