CA2933793A1 - Methods, products and uses for the diagnosis of a neuroautoimmune disease by detecting autoantibodies to flotillin1 and/or flotillin2 - Google Patents

Abstract

The present invention relates to method for diagnosing a disease comprising the step detecting in a sample from a patient an autoantibody binding to flotillin1 and/or flotillin2, a polypeptide comprising flotillin1 and/or flotillin2 or a variant thereof, which is preferably immobilized, a use of said polypeptide for the diagnosis of a disease, an autoantibody binding to flotillin1 and/or flotillin2, a method for isolating said autoantibody, and a pharmaceutical composition, medical device or test kit comprising said polypeptide.

Description

Diagnosis of a neuroautoimmune disease The present invention relates to method for diagnosing a disease comprising the step detecting in a sample from a patient an autoantibody binding to flotillinl and/or flotillin2, a polypeptide comprising flotillinl and/or flotillin2 or a variant thereof, which is preferably immobilized, a use of said polypeptide for the diagnosis of a disease, an autoantibody binding to flotillinl and/or flotillin2, a method for isolating said autoantibody, and a pharmaceutical composition, medical device or test kit comprising said polypeptide.
Developing diagnostic systems for neurological diseases is a continuing challenge in biomedical science, not in the least because many symptoms encountered may be accounted for by a huge variety of causes including genetically-inherited diseases, drug abuse, malnutrition, infection, injury, psychiatric illness, immunological defects and cancer.
Since a neurological disease is rarely associated with a unique characteristic pattern of clinical symptoms, it is often difficult to provide a reliable diagnosis solely based on the observation and examination of the patients affected or their medical history.
The importance of an early diagnosis cannot be overemphasized. Many neurological disorders, most prominently Alzheimer's and Parkinson's diseases as well as Multiple Sclerosis, cannot be cured, but drugs are available that may be used to slow down their progression. The earlier the diagnosis, the better the chances to exploit the spectrum of available drugs to the full benefit of the patient.
This holds all the more true in the case of neurological diseases associated with autoantibodies.
In some cases, the link between a specific detectable autoantibody and a condition is sufficiently strong to allow for an immediate diagnosis.
But even if it is not, the detection of autoantibodies may point the physician in charge to therapeutic means that may be used to ameliorate the patient's condition.
There is a variety of widely used immunosuppressants that may be used regardless of the nature of the autoantibody's target. Alternatively, apheresis may be used to remove autoantibodies from the patient's blood. In many cases, patients went on to lead a normal life following early diagnosis and treatment of a neurological autoimmune disease.
Diagnostic assays based on the detection of autoantibodies may also corroborate the diagnosis of diseases other than those associated with autoantibodies. If it turns out that a blood sample is devoid of specific autoantibodies, this is likely to help the physician in charge exclude a range of possibilities and thus narrow down the spectrum of plausible conditions.
Examples of neurological conditions coinciding with the emergence of autoantibodies include Neuromyelitis optica, a disease characterised by loss of vision and spinal cord function, and anti-NMDA receptor encephalitis, which is associated with autonomic dysfunction, hypoventilation, cerebellar ataxia, hemiparesis, loss of consciousness, or catatonia. Whilst the involvement of autoantibodies and the nature of these conditions as such was previously poorly understood, many of this disease can now be diagnosed and treated efficiently owing to the availability of assays based on the detection of autoantibodies.
Multiple Sclerosis is another disease that is widely believed to be associated with autoimmune destruction of vital bodily structures. Approximately 20 % of the patients suffer from visual impairment, more specifically optic neuritis. A specific diagnosis is difficult owing to the wide spectrum of symptoms and the nature of the disease, with attacks being followed sometimes by complete disappearance of the symptoms.
Therefore, it is paramount that new approaches to distinguish neurological conditions associated with autoantibodies from those that are not be developed.
The problem underlying the present invention is to provide novel reagents, devices and methods that may be used to support the diagnosis and treatment of a neurological disease, in particular a disease associated with vision impartment, headache and/or brain lesions.
Another problem underlying the present invention is to provide novel reagents, devices and methods that may be used to distinguish autoimmune diseases, in particular neurological autoimmune diseases, from diseases other than autoimmune diseases, not in the least to

2 determine the most promising treatment regimen, more specifically whether or not an immunosuppressive treatment is adequate.
The problem underlying the present invention is solved by the subject-matter of the attached independent and dependent claims.
In a 1st aspect, the problem underlying the present invention is solved by a method for diagnosing a disease comprising the step detecting in a sample from a patient an autoantibody binding to flotillin1 and/or flotillin2.
In a 2nd aspect, the problem underlying the present invention is solved by a polypeptide comprising flotillin1 and/or flotillin2 or a variant thereof, which is preferably immobilized, more preferably on a solid carrier.
In a 3rd aspect, the problem underlying the present invention is solved by a use of the inventive polypeptide for the diagnosis of a disease, preferably comprising the step detecting in a sample an autoantibody binding to flotillin1 and/or flotillin2.
In a 4th aspect, the problem underlying the present invention is solved by the inventive polypeptide for use in the treatment of a disease.
In a 5th aspect, the problem underlying the present invention is solved by an autoantibody, preferably an isolated autoantibody, binding to flotillin1 and/or flotillin2, preferably to a complex comprising flotillin1 and flotillin2, wherein the autoantibody is preferably in complex with the inventive polypeptide 2.
In a 6th aspect, the problem underlying the present invention is solved by a method for isolating an autoantibody binding to flotillin1 and/or flotillin2, preferably a complex comprising flotillin1 and flotillin2, comprising the steps a) contacting a sample comprising the autoantibody with the inventive polypeptide under conditions compatible with formation of a complex, wherein said autoantibody binds to said polypeptide,

3 b) isolating the complex formed in step a), c) dissociating the complex isolated in step b), and d) separating the autoantibody from the polypeptide.
In a 7th aspect, the problem underlying the present invention is solved by a pharmaceutical composition or medical device, preferably diagnostic device, comprising the inventive polypeptide.
In an 8th aspect, the problem underlying the present invention is solved by a test kit for the diagnosis of a disease, which test kit comprises the inventive polypeptide, wherein preferably the test kit comprises, in addition, a means for detecting a complex comprising the polypeptide according to claim 2 and an autoantibody binding to flotillinl and/or florilin2.
In a preferred embodiment, the patient has or the disease is associated with one or more, preferably two or more symptoms selected from the group comprising elevated cell number in CSF, intrathecal IgG synthesis, oligoclonal bands in CSF, MRZ (M-measles, R-rubella, Z-varicella zoster) reaction in CSF, impairment of vision, preferably impairment of visual acuity, optic neuritis, headache, spinal cord lesions and brain lesions, preferably impairment of vision, more preferably of visual acuity, headache, spinal cord lesions and brain lesions.
In a preferred embodiment, the disease is a neurological disease, preferably a demyelinating disease, preferably a disease of the CNS, preferably of the encephalon, preferably associated with inflammation of the optical nerve.
In a preferred embodiment, the polypeptide is provided in the form of a cell comprising a nucleic acid encoding said polypeptide or in the form of a tissue comprising said polypeptide.
In a preferred embodiment, the polypeptide is a recombinant and/or isolated polypeptide.
In a preferred embodiment, the sample is a bodily fluid comprising antibodies, preferably selected from the group comprising whole-blood, serum, cerebrospinal fluid and saliva.

4 In a preferred embodiment, both a polypeptide comprising flotillin1 or a variant thereof and a polypeptide comprising flotillin2 or a variant thereof are present and preferably part of a complex.
In a preferred embodiment, the autoantibody binds to a complex comprising flotililn1 and flotillin2.
The present invention is based on the inventors' surprising finding that a neurological autoimmune disease exists that is associated with an autoantibody to flotillin1 and/or flotillin2 and symptoms from the group comprising elevated cell number in CSF, intrathecal IgG
synthesis, oligoclonal bands in CSF, MRZ reaction in CSF, impairment of vision, optic neuritis, headache, spinal cord lesions and brain lesions.
Furthermore, the present invention is based on the inventors' surprising finding that autoantibodies to flotillin1 and/or flotillin2 exist and may be detected in samples from a number of patients suffering from neurological symptoms, but not in samples obtained from healthy subjects.
Furthermore, the present invention is based on the inventors' surprising finding that known neurological diseases of unknown etiology, in particular demyelinating diseases, are associated with the presence of an autoantibody to flotillin1 and/or flotillin2.
Without wishing to be bound to any theory, the presence of such autoantibodies suggests that activity and function of flotillin1 and/or flotillin2 and/or downstream effectors is impaired in patients having such autoantibodies to the effect that neurological symptoms, more specifically demyelinating diseases, in particular those associated with visual important, occur.
Flotillin1 and -2 (synonyms: reggie-2 and -1) were originally found as being upregulated in regenerating axons of goldfish retinal ganglion cells after traumatic injury of the optic nerve.
However, they are well conserved in many eukaryotes including mammals. In vertebrates both proteins are ubiquitously expressed and most abundant in striated muscle, adipose tissue and lung tissues. On the molecular level, flotillins are attached to the cytoplasmic side of lipid rafts and are therefore often used as markers of lipid microdomains. Similar to other lipid raft constituents they are largely insoluble in Triton X-100 but float after sucrose density centrifugation. Functionally, they are involved in protein interaction, cell signaling, clustering of the amyloid precursor protein and amyloidogenic processing in neurons (Stuermer CA, Plattner H. The 'lipid raft' microdomain proteins reggie-1 and reggie-2 (flotillins) are scaffolds for protein interaction and signalling. Biochem Soc Symp 2005;(72):109-18). Overexpression of flotillin-2 is seen in several cancers and generally associated with a more severe progression (Arkhipova KA, Sheyderman AN, Laktionov KK, Mochalnikova VV, Zborovskaya IB. Simultaneous expression of flotillin-1, flotillin-2, stomatin and caveolin-1 in non-small cell lung cancer and soft tissue sarcomas. BMC Cancer 2014;14:100).
The present invention relates to a polypeptide comprising a mammalian, preferably human flotillin1 or variants thereof and/or flotillin2 or variants thereof, preferably immunogenic variants reactive to autoantibodies binding to flotillin1 and flotillin2 or variants thereof. Examples of mammalian flotillin1 and/or flotillin2 include those from human, monkey, mouse, rat, rabbit, guinea pig or pig. In a most preferred embodiment, flotillin1 is the polypeptide encoded by the data base code 075955 or a variant thereof and/or flotillin2 is the polypeptide encoded by the data base code Q14254 or a variant thereof. Throughout this application, any data base codes cited refer to the Uniprot data base, more specifically the version accessible on-line on May 29, 2015. NP_005794 and NP 004466 represent a nucleotide sequence encoding flotillin1 and flotillin2, respectively.
In a more preferred embodiment, the inventive polypeptide comprises both flotillin1 or a variant thereof and flotillin 2 or variants thereof, fused to each other directly or via a linker. In a most preferred embodiment, a complex comprising both polypeptide comprising flotillin1 or a variant thereof and a polypeptide comprising flotillin 2 or a variant thereof, binding directly to each other or via another molecule is used. Preferably the complex comprises no proteins other than a polypeptide comprising flotillin1 or a variant thereof and a polypeptide comprising flotillin 2 or a variant thereof. Such complex may be used to capture autoantibodies binding to said complex for the diagnosis of a disease.
If a complex comprising a first polypeptide comprising flotillin1 or a variant thereof and a second polypeptide comprising flotillin2 or a variant thereof is used, the first and the second polypeptide may be expressed in the same cell to form such a complex. Alternatively, the first and the second polypeptide may be expressed separately in different cells and reconstituted following expression, optionally in a purified form.
The teachings of the present invention may not only be carried out using polypeptides, in particular a polypeptide comprising the native sequence of flotillin1 and/or flotillin2, or nucleic acids having the exact sequences referred to in this application explicitly, for example by function, name, sequence or accession number, or implicitly, but also using variants of such polypeptides or nucleic acids.
In a preferred embodiment, the term "variant", as used herein, may refer to at least one fragment of the full length sequence referred to, more specifically one or more amino acid or nucleic acid sequence which is, relative to the full-length sequence, truncated at one or both termini by one or more amino acids. Such a fragment comprises or encodes for a peptide having at least 6, 7, 8, 10, 12, 15, 20, 25, 50, 75, 100, 150 or 200 successive amino acids of the original sequence or a variant thereof. The total length of the variant may be at least 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids.
In another preferred embodiment, the term "variant" relates not only to at least one fragment, but also to a polypeptide or a fragment thereof comprising amino acid sequences that are at least 40, 50, 60, 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99 % identical to the reference amino acid sequence referred to or the fragment thereof, wherein amino acids other than those essential for the biological activity, for example the ability of an antigen to bind to an (auto)antibody, or the fold or structure of the polypeptide are deleted or substituted and/or one or more such essential amino acids are replaced in a conservative manner and/or amino acids are added such that the biological activity of the polypeptide is preserved.
The state of the art comprises various methods that may be used to align two given nucleic acid or amino acid sequences and to calculate the degree of identity, see for example Arthur Lesk (2008), Introduction to bioinformatics, Oxford University Press, 2008, 3rd edition. In a preferred embodiment, the ClustalW software (Larkin, M. A., Blackshields, 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 2Ø
Bioinformatics, 23, 2947-2948) is used using default settings.

In a preferred embodiment, the polypeptide and variants thereof may, in addition, comprise chemical modifications, for example isotopic labels or covalent modifications such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, methylation, hydroxylation and the like. The person skilled in the art is familiar with methods to modify polypeptides. Any modification is designed such that it does not abolish the biological activity of the variant.
Moreover, variants may also be generated by fusion with other known polypeptides or variants thereof and comprise active portions or domains, preferably having a sequence identity of at least 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99 % when aligned with the active portion of the reference sequence, wherein the term "active portion", as used herein, refers to an amino acid sequence, which is less than the full length amino acid sequence or, in the case of a nucleic acid sequence, codes for less than the full length amino acid sequence, respectively, and/or is a variant of the natural sequence, but retains at least some of the biological activity.
In a preferred embodiment, the term "variant" of a nucleic acid comprises nucleic acids the complementary strand of which hybridizes, preferably under stringent conditions, to the reference or wild type nucleic acid. Stringency of hybridization reactions is readily determinable by one of ordinary skilled in the art, and in general is an empirical calculation dependent on probe length, washing temperature and salt concentration. In general longer probes require higher temperatures for proper annealing, while shorter probes less so.
Hybridization generally depends on the ability of denatured DNA to reanneal to complementary strands present in an environment below their melting temperature: The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which may be used. As a result, higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperature less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel, F. M. (1995), Current Protocols in Molecular Biology. John Wiley & Sons, Inc. Moreover, the person skilled in the art may 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 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 for any hybridization, i.e. hybridization occurs only if the probe is 70 % or more identical to the target sequence.
Probes having a lower degree of identity with respect to the target sequence may hybridize, but such hybrids are unstable and will be removed in a washing step under stringent conditions, for example lowering the concentration of salt to 2 x SSC or, optionally and subsequently, to 0,5 x SSC, while the temperature is, in order of increasing preference, 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, approximately 66 C - 68 C. In a particularly preferred embodiment, the temperature is approximately 64 C - 68 C or approximately 66 C - 68 C. It is possible to adjust the concentration of salt to 0.2 x SSC or even 0.1 x SSC. Nucleic acid sequences having a degree of identity with respect to the reference or wild type sequence of at least 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % may be isolated. In a preferred embodiment, the term variant of a nucleic acid sequence, as used herein, refers to any nucleic acid sequence that encodes the same amino acid sequence and variants thereof as the reference nucleic acid sequence, in line with the degeneracy of the genetic code.
The variant of the polypeptide has biological activity. In a preferred embodiment, such biological activity is the ability to bind specifically to the autoantibodies of interest, preferably those binding to flotillin1 and/or flotillin2 found in patients suffering from the disease identified by the inventors.
The inventive polypeptide, which comprises flotillin1 and/or flotillin2 or a variant thereof, or the inventive autoantibody, when used to carry out the teachings of the present invention, may be provided in any form and at any degree of purification, from liquid samples, tissues or cells comprising said polypeptide in an endogenous form, more preferably cells overexpressing the polypeptide, crude or enriched lysates of such cells, to purified and/or isolated polypeptide which is optionally essentially 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 tissues or cells, more preferably from mammalian cells or tissues, optionally from non-recombinant tissues or cells. In another preferred embodiment, the polypeptide is a recombinant protein, wherein the term "recombinant", as used herein, refers to a polypeptide produced using genetic engineering approaches at any stage of the production process, for example by fusing a nucleic acid encoding the polypeptide to a strong promoter for overexpression in cells or tissues or by engineering the sequence of the polypeptide itself. The person skilled in the art is familiar with methods for engineering nucleic acids and polypeptides encoded (for example, described in Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989), Molecular Cloning, CSH
or in Brown T. A.
(1986), Gene Cloning ¨ an introduction, Chapman & Hall) and for producing and purifying native or recombinant polypeptides (for example Handbooks õStrategies for Protein Purification", õAntibody Purification", õPurifying Challenging Proteins" (2009/2010), published by GE
Healthcare Life Sciences, and in Burgess, R. R., Deutscher, M. P. (2009), Guide to Protein Purification). In a preferred embodiment, a polypeptide is pure if at least 60, 70, 80, 90, 95 or 99 percent 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 inspection.
If the inventive polypeptide is provided in the form of tissue, it is preferred that the tissue is mammalian tissue, for example human, rat, primate, donkey, mouse, goat, horse, sheep, pig or cow, more preferably brain tissue, most preferably cerebellum. If a cell lysate is used, it is preferred that the cell lysate comprises the membranes associated with the surface of the cell. If said 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 from a mammal, for example rat, human, primate, donkey, mouse, goat, horse, sheep, pig or cow.
The polypeptide used to carry out the inventive teachings, including any variants, is preferably designed such that it comprises epitopes recognized by and/or binds specifically to autoantibodies binding to flotillin1 and/or flotillin2. In one embodiment, such polypeptide comprises a stretch of 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more, preferably at least 9 but no more than 16, consecutive amino acids from flotillin1 and/or flotillin2.
The person skilled in the art is familiar with guidelines used to design peptides having sufficient immunogenicity, for example those described in Jackson, D. C., Fitzmaurice, C.
J., Brown, L. E., Zeng, W. (1999), Preparation and properties of totally synthetic immunogenes, Vaccine Volume 18, Issues 3-4, September 1999, Pages 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, 2010 February; 9(2): 157-173.
Briefly, it is desirable that the peptide meets as many as possible of the following requirements: (a) it has a high degree of hydrophilicity, (b) it comprises one or more residues selected from the group comprising aspartate, proline, tyrosine and phenylalanine, (c) is has, for higher specificity, no or little homology with other known peptides or polypeptides, (d) it needs to be sufficiently soluble and (e) it comprises no glycosylation or phosphorylation sites unless required for specific reasons. Alternatively, bioinformatics approaches may be followed, for example those described by 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.
The inventive polypeptide, which comprises flotillin1 and/or flotillin2 or a variant thereof, preferably one or polypeptide or a complex of two or more polypeptides comprising flotillina and flotillin2, when used according to the present invention, may be provided in any kind of conformation. For example, the polypeptide may be an essentially unfolded, a partially or a fully folded polypeptide. In a preferred embodiment, the polypeptide is folded in the sense that the epitopes essential for the binding to the inventive autoantibody, or the protein or variant thereof in its entirety, adopt the fold adopted by the native protein in its natural environment. The person skilled in the art is familiar with methods suitable to determine whether or not a polypeptide is folded and if it is, which structure it has, for example limited proteolysis, NMR spectroscopy, CD
spectroscopy or X-ray crystallography (see for example Banaszak L. J. (2008), Foundations of Structural Biology, Academics Press, or Teng Q. (2013), Structural Biology:
Practical Applications, Springer), preferably CD spectroscopy is used.
The inventive polypeptide may be a fusion protein which comprises amino acid sequences other than those taken from flotillin1 and/or flotillin2, in particular a C-terminal or N-terminal tag, preferably a C-terminal tag, which is, in a preferred embodiment, as used herein, an additional sequence motif or polypeptide having a function that has some biological or physical function and may, for example, be used to purify, immobilize, precipitate or identify the inventive polypeptide. In a more preferred embodiment, the tag is a sequence or domain capable of binding specifically to a ligand, for example a tag selected from the group comprising His tags, thioredoxin, maltose binding protein, glutathione-S-transferase, a fluorescence tag, for example from the group comprising green fluorescent protein. SEQ ID NO 1, 2 and SEQs ID NO 5, 6 represent exemplary fusion polypeptides comprising flotillin1 and flotillin2, respectively.
The inventive polypeptide may be an immobilized polypeptide. In a preferred embodiment, the term "immobilized", as used herein, refers to a molecule bound to a solid carrier insoluble in an aqueous solution, more preferably via a covalent bond, electrostatic interactions, encapsulation or entrapment, for example by denaturing a globular polypeptide in a gel, or via hydrophobic interactions, most preferably via one or more covalent bonds. Such carrier is preferably an artificial carrier, which is not predominantly biological material such as a tissue section. Various suitable carriers, for example 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 Herr, A. E. (2013), Protein immobilizsation techniques for microfluidic assays, Biomicrofluidics 7(4), 041501. This way, the immobilized molecule, together with the insoluble carrier, may be separated from an aqueous solution in a straightforward manner, for example by filtration, centrifugation or decanting. An immobilized molecule may be immobilized in a reversible or irreversible manner. For example, the immobilization is reversible if the molecule interacts with the carrier via ionic interactions that can be masked by addition of a high concentration of salt or if the molecule is bound via a cleavable covalent bond such as a disulphide bridge which may be cleaved by addition of thiol-containing reagents. By contrast, the immobilization is irreversible if the molecule is tethered to the carrier via a covalent bond that cannot be cleaved in aqueous solution, for example a bond formed by reaction of an epoxide group and an amine group as frequently used to couple lysine side chains to affinity columns. The protein may be indirectly immobilized, for example by immobilizing an antibody or other entity having affinity to the molecule, followed by formation of a complex to the effect that the molecule-antibody complex is immobilized. Various ways to immobilize molecules are described in the literature, for example in Kim, D., Herr, and A. E. (2013), Protein immobilizsation techniques for microfluidic assays, Biomicrofluidics 7(4), 041501. In addition, various reagents and kits for immobilization reactions are commercially available, for example from Pierce Biotechnology.
It is essential that the sample used for the diagnosis in line with the present invention comprises antibodies, also referred to as immunoglobulins. Typically the sample of a bodily fluid comprises a representative set of the entirety of the subject's immunoglobulins.
However, the sample, once provided, may be subjected to further processing which may include fractionation, centrifugation, enriching or isolating the entirety of immunoglobulins or any immunoglobulin class of the subject, which may affect the relative distribution of immunoglobulins of the various classes.
The reagents, devices, methods and uses described throughout this application may be used for the diagnosis of a 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, more preferably an element of the nervous system essential for vision, more preferably the optical nerve.
In a preferred embodiment, the disease, more preferably a neurological disease, is associated with one or more symptoms, more preferably two or more, most preferably three or more from the group comprising cancer, elevated cell number in CSF, intrathecal IgG
synthesis, oligoclonal bands in CSF, MRZ reaction in CSF, impairment of vision, preferably of visual acuity, optic neuritis, headache, spinal cord lesions and brain lesions. Preferably the disease is responsive to immunomodulatory, preferably immunosuppressive therapy.
In another preferred embodiment, the disease is a neurological disease selected from the group comprising Alzheimer's Disease, Autism, Aspergers's Syndrome, Apraxia, Aphasia, Cerebellar syndrome, Cerebellitis, Chorea, Encephalitis, Movement disorder, spinocerebellar ataxia, preferably a non-progressive form, Paralysis, Paraplegia, Gaucher's disease, Myopathy, Myasthenia gravis, Multiple Sclerosis, Parkinsons's disease, Polyneuropathy and Dementia, preferably Cerebellar syndrome, Cerebellitis, Multiple Sclerosis, Movement disorder and Dementia, more preferably Multiple Sclerosis. In a more preferred embodiment, the disease is a demyelinating disease, more preferably a demyelinating disease affecting the CNS, more preferably Multiple Sclerosis, most preferably associated with optic neuritis.
In another preferred embodiment, the disease is a cancer or, preferably paraneoplastic neurological syndrome, which is associated both with one or more neurological symptoms, preferably from the group comprising elevated cell number in CSF, intrathecal IgG synthesis, oligoclonal bands in CSF, MRZ reaction in CSF, impairment of vision, optic neuritis, headache, spinal cord lesions and brain lesions and is furthermore associated with a cancer. Detection of autoantibodies to flotillin1 and/or flotillin2 may indicate an increased likelihood that a cancer is present which cannot be detected using other methods, or will appear as the disease progresses. In a preferred embodiment, the cancer is a cancer of tumor selected from the group comprising tumor of the lung, tumor of the thymus, thymic tumor, testicular tumor, head and neck cancer tumor, breast cancer tumor, ano-genital cancer tumor, melanoma, sarcoma, carcinoma, lymphoma, leukemia, mesothelioma, glioma, germ cell tumor, choriocarcinoma, pancreatic cancer, ovarian cancer, gastric cancer, carcinomatous lesion of the pancreas, pulmonary adenocarcinoma, colorectal adenocarcinoma, pulmonary squamous adenocarcinoma, gastric adenocarcinoma, ovarian surface epithelial neoplasm (e.g. a benign, proliferative or malignant variety thereof), oral squamous cell carcinoma, non small-cell lung carcinoma, endometrial carcinoma, a bladder cancer, prostate carcinoma, acute myelogenous leukemia (AML), myelodysplasia syndrome (MDS), non-small cell lung cancer (NSCLC), Wilms' tumor, leukemia, lymphoma, desmoplastic small round cell tumor, mesothelioma (e.g. malignant mesothelioma), a gastric cancer, colon cancer, lung cancer, breast cancer, germ cell tumor, ovarian cancer, uterine cancer, thyroid cancer, hepatocellular carcinoma, thyroid cancer, liver cancer, renal cancer, kaposis, sarcoma, and another carcinoma or sarcoma.
In a preferred embodiment, the term õdiagnosis", as used herein, refers to any kind of procedure aiming to obtain information instrumental in the assessment whether a patient suffers or is likely or more likely than the average or a comparative subject, the latter preferably having similar symptoms, to suffer from certain a disease or disorder in the past, at the time of the diagnosis or in the future, to find out how the disease is progressing or is likely to progress in the future or to evaluate the responsiveness of a patient with regard to a certain treatment, for example the administration of immunosuppressive drugs. In other words, the term "diagnosis" comprises not only diagnosing, but also prognosticating and/or monitoring the course of a disease or disorder.
In many cases the mere detection, in other words determining whether or not detectable levels of the antibody are present in the sample, is sufficient for the diagnosis. If the autoantibody can be detected, this will be information instrumental for the clinician's diagnosis and indicates an increased likelihood that the patient suffers from a disease. In a preferred embodiment, the relative concentration of the antibody in the serum, compared to the level that may be found in the average healthy subject, may be determined. While in many cases it may be sufficient to determine whether or not autoantibodies are present or detectable in the sample, the method carried out to obtain information instrumental for the diagnosis may involve determining whether the concentration is at least 0.1, preferably 0.2, 0.5, 1, 2, 5, 10, 20, 25, 50, 100, 200, 500, 1000, 10000 or 100000 times higher than the concentration found in the average healthy subject.
The person skilled in the art will appreciate that a clinician does usually not conclude whether or not the patient suffers or is likely to suffer from a disease, condition or disorders solely on the basis of a single diagnostic parameter, but needs to take into account other aspects, for example the presence of other autoantibodies, markers, 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 conclusive diagnosis. See Baenkler H. W.
(2012), General aspects of autoimmune diagnostics, in Renz, H., Autoimmune diagnostics, 2012, de Gruyter, page 3. The value of a diagnostic agent or method may also reside the possibility to rule out one disease, thus allowing for the indirect diagnosis of another. In a preferred embodiment, the meaning of any symptoms or diseases referred to throughout this application is in line with the person skilled in the art's understanding as of May 29, 2015 as evidenced by text books and scientific publications. In a preferred embodiment, the polypeptide according to the invention or the method may be used to determine whether a patient suffers from a disease characterized by symptoms similar to those of MS, more preferably for the distinction between MS and NMO.
Therefore, the term "diagnosis" does preferably not imply that the diagnostic methods or agents according to the present invention will be definitive and sufficient to finalize the diagnosis on the basis of a single test, let alone parameter, but may refer to a contribution to what is referred to as a "differential diagnosis", i. e. a systematic diagnostic procedure considering the likelihood of a range of possible conditions on the basis of a range of diagnostic parameters. Consequently, the inventive method, polypeptide or use, optionally for determining whether a patient suffers from the a disease, may comprise obtaining a sample from a patient, preferably a human patient, determining whether an autoantibody binding to flotillin1 and/or flotillin2 is present in said sample, wherein said determining is performed by contacting the sample with the inventive polypeptide and detecting whether binding occurs between said polypeptide and said autoantibody, preferably using a labeled secondary antibody, wherein said autoantibody binds to said polypeptide if present in the sample, and diagnosing the patient as suffering or being more likely to suffer from said neurological disorder or cancer if the autoantibody was determined to be present in the sample. In a preferred embodiment, the inventive method may contemplate the steps detecting antibody to a) Aquaporin-4 and b) flotillin1 and/or -2, preferably in that order.
The term "diagnosis" may also refer to a method or agent used to distinguish between two or more conditions associated with similar or identical symptoms.
The term "diagnosis" may also refer to a method or agent used to choose the most promising treatment regime for a patient. In other words, the method or agent may relate to selecting a treatment regimen for a subject. For example, the detection of autoantibodies may indicate that an immunosuppressive therapy is to be selected, which may include administrating to the patient one or more immunosuppressive drugs.

The present invention relates to a complex comprising an antibody, preferably autoantibody, binding to the inventive polypeptide. Such a complex may be used or detected as part of a method for diagnosing a disease. A liquid sample comprising antibodies from a subject may be used to practice the method. Such a liquid sample may be any bodily fluid comprising a representative set of antibodies from the subject, preferably a sample comprising antibodies of the IgG immunoglobulin class from the subject. For example, a sample may be cerebrospinal fluid (CSF), blood or blood serum, lymph, insterstitial fluid and is preferably serum or CSF, more preferably serum.
The step contacting a liquid sample comprising antibodies with the inventive polypeptide may be carried out by incubating an immobilized form of said polypeptide in the presence of the sample comprising antibodies under conditions that are compatible with the formation of the complex comprising said polypeptide and an antibody, preferably an autoantibody, binding to the inventive polypeptide. The liquid sample, then depleted of antibodies binding to the inventive polypeptide may be removed subsequently, followed by one or more washing steps. Finally the complex comprising the antibody and the polypeptide may be detected. In a preferred embodiment, the term "conditions compatible with the formation of the complex"
are conditions that allow for the specific antigen-antibody interactions to build up the complex comprising the polypeptide an the antibody. In a preferred embodiment such conditions may comprise incubating the polypeptide in sample diluted 1:100 in PBS buffer for 30 minutes at 25 C. In a preferred embodiment, the term õautoantibody", as used herein, refers to an antibody binding specifically to an endogenous molecule of the animal, preferably mammal, which produces said autoantibody, wherein the level of such antibody is more preferably elevated compared the average of any other antibodies binding specifically to such an endogenous molecule. In a most preferred embodiment, the autoantibody is an autoantibody binding to flotillin1 and/or flotillin2.
In a preferred embodiment, the detection of the complex for the prognosis, diagnosis, methods or test kit according to the present invention comprises the use of a method selected from the group comprising immunodiffusion techniques, immunoelectrophoretic techniques, light scattering immunoassays, light scattering immunoassays, agglutination techniques, labeled immunoassays such as those from the group comprising radiolabeled immunoassay, enzyme immunoassays, chemiluminscence immunoassays, and immunofluorescence techniques. The person skilled in the art is familiar with these methods, which are also described in the state of the art, for example in Zane, H. D. (2001), Immunology ¨ Theoretical &
Practical Concepts in Laboratory Medicine, W. B. Saunders Company, in particular in Chapter 14.
Alternatively, a sample comprising tissue comprising the inventive polypeptide rather than a liquid sample may be used. The tissue sample is preferably from a tissue expressing endogenous flotillin1 and/or flotillin2. Such a sample, which may be in the form of a tissue section fixed on a carrier, for example a glass slide for microscopic analysis, may then be contacted with the inventive antibody, preferably autoantibody, binding to the inventive polypeptide. The antibody is preferably labeled to allow for distinction from endogenous antibodies binding to the inventive polypeptide, so that newly formed complexes may be detected and, optionally, quantified. If the amount of complexes formed is lower than the amount found in a sample taken from a healthy subject, the subject from whom the sample examined has been taken is likely to suffer from a disease.
Any data demonstrating the presence or absence of the complex comprising the antibody and the inventive polypeptide may be correlated with reference data. For example, detection of said complex indicates that the patient who provided the sample analyzed has suffered, is suffering or is likely to suffer in the future from a disease. If a patient has been previously diagnosed and the method for obtaining diagnostically relevant information is run again, the amount of complex detected in both runs may be correlated to find out about the progression of the disease and/or the success of a treatment. For example, if the amount of complex is found to increase, this suggests that the disorder is progressing, likely to manifest in the future and/or that any treatment attempted is unsuccessful.
In a preferred embodiment, a microplate, membrane ELISA, dot blot, or line blot is used to carry out the diagnostic method according to the invention. The person skilled in the art is familiar with the experimental setup, which is described in the state of the art (Raoult, D., and Dasch, G. A.
(1989), The line blot: an immunoassay for monoclonal and other antibodies. Its application to the serotyping of gram-negative bacteria. J. lmmunol. Methods, 125 (1- 2), 57-65;
W02013041540).
In another preferred embodiment, the prognosis, diagnosis, methods or test kit in line with the inventive teachings contemplate 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 state of the art (US4647543; Voigt, J., Krause, C., Rohwader, E, Saschenbrecker, S., Hahn, M., Danckwardt, M., Feirer, C., Ens, K, Fechner, K, Barth, E, Martinetz, T., and Stocker, 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 systemic lupus erythematosus patients, Clinical Immunology, vol. 124, no. 1, pp. 18-21, 2007).
Suitable reagents, devices and software packages are commercially available, for example from EUROIMMUN, Lubeck, Germany.
A sample subjected to a test determining only whether an autoantibody binding to flotillin1 and/or flotillin2 is present, but it is preferred that diagnostic methods, tests, devices and the like contemplate determining the presence of autoantibodies against a variety of antigens relating to neurological autoimmune disease or variants thereof, preferably selected from the group comprising Hu, Yo, Ri, CV2, PNMA1, PNMA2, DNER/Tr, ARHGAP26, ITPR1, ATP1A3, NBC1, Neurochrondrin, CARPVIII, Zic4, Sox1, Ma, MAG, MPO, MBP, GAD65, amphiphysin, recoverin, GABA A receptor, GABA B receptor, glycine receptor, gephyrin, IgLON5, DPPX, aquaporin-4, MOG, NMDA receptor, AMPA receptors, GRM1, GRM5, LGI1, VGCC und mGluR1 and CASPR2, which antigens are preferably immobilized, for example on a medical device such as a line blot. The diagnostically relevant markers Neurochrondrin, (EP15001186), (EP14003703.7), NBC1 (EP14003958.7) and ATP1A3, also referred to as alpha 3 subunit of human neuronal Na(+)/K(+) ATPase (EP14171561.5) have been described in the state of the art.
According to the teachings of the present invention, an antibody, preferably an autoantibody binding to the inventive polypeptide is used for the diagnosis of a disease is provided. The person skilled in the art is familiar with methods for purifying antibodies, for example those described in Hermanson, G. T., MaIlia, A. K., and Smith, P. K. (1992), Immobilized Affinity Ligand Techniques, San Diego: Academic Press. Briefly, an antigen binding specifically to the antibody of interest, which antigen is the inventive polypeptide, is immobilized and used to purify, via affinity chromatography, the antibody of interest from an adequate source. A liquid sample comprising antibodies from a patient suffering from the neurological disorder identified by the inventors may be used as the source.

According to the invention, an antibody, for example an autoantibody, is provided that is capable of binding specifically to the inventive polypeptide. In a preferred embodiment, the term "antibody", as used herein, refers to any immunoglobulin-based binding moieties, more preferably one comprising at least one immunoglobulin heavy chain and one immunoglobulin light chain, including, but not limited to monoclonal and polyclonal antibodies as well as variants of an antibody, in particular fragments, which binding moieties are capable of binding to the respective antigen, more preferably binding specifically to it. In a preferred embodiment, the term "binding specifically", as used herein, means that the binding is stronger than a binding reaction characterized by a dissociation constant of 1 x 10-5 M, more preferably 1 x 10-7 M, more preferably 1 x 10' M, more preferably 1 x 10-9 M, more preferably 1 x 10-1 M, more preferably 1 x 10-11 M, more preferably 1 x 10-12 M, as determined by surface plasmon resonance using Biacore equipment at 25 C in PBS buffer at pH 7. The antibody may be part of an autoantibody preparation which is heterogeneous or may be a homogenous autoantibody, wherein a heterogeneous preparation comprises a plurality of different autoantibody species as obtainable by preparation from the sera of human donors, for example by affinity chromatography using the immobilized antigen to purify any autoantibody capable of binding to said antigen. The antibody may be glycosylated or non-glycosylated. The person skilled in the art is familiar with methods that may be used for the identification, production and purification of antibodies and variants thereof, for examples those described in EP 2 423 226 A2 and references therein. The antibody may be used as a diagnostic agent, by itself, or in combination, for example in complex with the inventive polypeptide.
The present invention provides a method for isolating an antibody, preferably an autoantibody, binding to the inventive polypeptide, comprising the steps a) contacting a sample comprising the antibody with the inventive polypeptide such that a complex is formed, b) isolating the complex formed in step a), c) dissociating the complex isolated in step b), and d) separating the antibody from the inventive polypeptide. A sample from a patient suffering from the novel neurological disorder identified by the inventors may be used as the source of antibody.
Suitable methods are described in the state of the art, for example in the Handbooks "Affinity chromatography", õStrategies for Protein Purification" and õAntibody Purification" (2009/2010), published by GE
Healthcare Life Sciences, and in in Philips, Terry, M., Analytical techniques in immunochemistry, 1992, Marcel Dekker, Inc.

The invention provides a pharmaceutical composition comprising the inventive polypeptide, which composition is preferably suitable for administration to a subject, preferably a mammalian subject, more preferably to a human. Such a pharmaceutical composition may comprise a pharmaceutically acceptable carrier. The pharmaceutical composition may, for example, be administered orally, parenterally, by inhalation spray, topically, by eyedrops, rectally, nasally, buccally, vaginally or via an implanted reservoir, wherein the term "parentally", as used herein, comprises subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, instrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition may be provided in suitable dosage forms, for example capsules, tablets and aqueous suspensions and solutions, preferably in sterile form. It may be used in a method of treatment of a disease, which method comprises administering an effective amount of the inventive polypeptide to a subject. In a preferred embodiment, the invention provides a vaccine comprising the inventive polypeptide, optionally comprising an auxiliary agent such as an adjuvans or a buffer, and the use of the inventive polypeptide for the preparation of a vaccine.
Within the scope of the present invention, a medical or diagnostic device comprising, preferably coated with the inventive (auto)antibody and/or the inventive polypeptide is provided. Preferably such a medical or diagnostic device comprises the inventive polypeptide in a form that allows contacting it with an aqueous solution, more preferably the liquid human sample, in a straightforward manner. In particular, the inventive polypeptide comprising may be immobilized on the surface of a carrier such as an artificial carrier, preferably selected from the group comprising glass plates or slides, biochips, microtiter plates, beads, for example magnetic beads, apharesis devices, chromatography columns, membranes or the like.
Exemplary medical devices include line blots, microplates, glass slides for microscopy, beads and biochips. In addition to the inventive polypeptide, the medical or diagnostic device may comprise additional polypeptides, preferably in an enriched, isolated and/or recombinant form, for example positive or negative controls or known other antigens binding to autoantibodies of diagnostic value, particularly those related other diseases associated with one or more identical or similar symptoms. The medical device, preferably comprising one diagnostically useful carrier comprising one or more antigens, preferably more than one antigen, or more than one diagnostically useful carriers each comprising one or more antigens, preferably one antigen, may comprise, in addition to the inventive polypeptide, one or more antigens from the group comprising Hu, Yo, Ri, CV2, PNMA1, PNMA2, DNER/Tr, ARHGAP26, ITPR1, ATP1A3, NBC1, Neurochrondrin, CARPVIII, Zic4, Sox1, Ma, MAC, MPO, MBP, GAD65, amphiphysin, recoverin, GABA A receptor, GABA B receptor, glycine receptor, gephyrin, IgLON5, DPPX, aquaporin-4, MOG, NMDA receptor, AMPA receptors, GRM1, GRM5, LGI1, VGCC und mGluR1 and CASPR2, preferably a combination comprising at least the polypeptide according to the present invention, aquaporin-4 and MOG. Variants of each antigen having, as biological activity, the ability to bind to the respective autoantibody to the antigen, may be used instead of the antigen.
The inventive teachings provide a kit, preferably for diagnosing a disease.
Such a kit may comprise instructions detailing how to use the kit and a means for contacting the inventive polypeptide with a bodily fluid sample from a subject, preferably a human subject, for example a line blot, wherein the inventive polypeptide is immobilized on the line blot.
Furthermore, the kit may comprise a positive control, for example a batch of autoantibody or recombinant antibody known to bind to the inventive polypeptide and a negative control, for example a protein having no detectable affinity to the inventive polypeptide such as bovine serum albumin. Finally, such a kit may comprise a standard solution of the antibody or antigen for preparing a calibration curve.
In a preferred embodiment, the kit comprises a means for detecting an antibody, more preferably an autoantibody, binding to the inventive polypeptide, preferably by detecting a complex comprising the inventive polypeptide and an antibody binding to the inventive polypeptide. Such means is preferably an agent that binds to said complex and modifies the complex or carries a label such that makes the complex detectable. For example, said means may be a labeled antibody binding to said polypeptide, at a binding site other than the binding site recognized by the primary antibody or to a constant region of the primary antibody.
Alternatively, said means may be a secondary antibody binding to the constant region of the autoantibody, preferably a secondary antibody specific for mammalian IgG class of antibodies.
A multitude of methods and means for detecting such a complex have been described in the state of the art, for example in Philips, Terry, M., Analytical techniques in immunochemistry, 1992, Marcel Dekker, Inc.
The inventive polypeptide comprising flotillin1 and/or flotillin2 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 that encodes for the inventive polypeptide or 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, as such, does not occur in nature and comprises, compared to natural nucleic acid, at least one modification, for example an isotopic content or chemical modifications, for example a methylation, sequence modification, label or the like indicative of synthetic origin. In a preferred embodiment, the nucleic acid is a recombinant nucleic acid or part or a nucleic acid, and is, in a more preferred embodiment, part of a vector, in which it may be functionally linked with a promoter that allows for expression, preferably overexpression of the nucleic acid. The person skilled in the art is familiar with a variety of suitable vectors, of which are commercially available, for example from Origene. For example, a vector encoding for fusion constructs with a C-terminal GFP may be used. The cell may be a eukaryotic or prokaryotic cell, preferably of eukaryotic cell, such as a yeast cell, and is more preferably a mammalian, more preferably a human cell such as a HEK293 cell. Examples of a mammalian cell include a HEK293, CHO or COS-7 cell. The cell comprising the nucleic acid encoding for the inventive polypeptide may be a recombinant cell or an isolated cell wherein the term "isolated" means that the cell is enriched such that, compared to the environment of the wild type of said cell, fewer cells of other differentiation or species or in fact no such other cells are present.
The inventive teachings may not only be used for a diagnosis, but also for preventing or treating a disease, more specifically a method for preventing or treating a disease, comprising the steps a) reducing the concentration of autoantibodies binding to the inventive polypeptide in the subject's blood and/or b) administering one or more immunosuppressive pharmaceutical substances, preferably selected from the group comprising rituximab, prednisone, methylprednisolone, cyclophosphamide, mycophenolatemofetil, intravenous immunoglobulin, tacrolimus, cyclosporine, methotrexate, azathioprine and/or the pharmaceutical composition.
Fig. 1 shows MRI of the head demonstrating multiple demyelinating lesions in the anti-flotillin positive patient. The female patient presented with optic neuritis of suspected autoimmune origin. Lesions which remained stable under immunomodulatory therapy over 5 month with interferone beta. T2 weighted magnetic resonance imaging (A, C), and flair imaging (B, D) of the same female 34 year old patient at initial presentation (A, B), and after 6 month (C, D).
Fig. 2 shows Immunofluorescence staining of central nervous tissues.
Cryosections were incubated with patient serum (1:100) or CSF (undiluted) in the first step, and with FITC labelled goat anti-human IgG in the second step. Nuclei were counterstained by incubation with TO-PRO-3 iodide (blue). A fine-granular staining of the molecular layers (ML) and a patchy staining of the granular layer was obtained. On Hippocampus the outer ML was more intense than the inner ML. A) Hippocampus rat, B) cerebellum rat, C) cerebellum monkey.
Fig. 3 shows Histo-immunoprecipitation and antigen identification.
Cryosections of rat or pig cerebellum were incubated with the patient's serum (1:200), washed in PBS
and solubilized using detergents. The solution was incubated with protein-G-coated magnetic beads. The immunocomplexes were eluted by SDS and subjected to SDS-PAGE analysis and Western blot.
Fig. 3A: Left: Staining with colloidal Coomassie. Right: Western blot after incubation with anti-flotillin2. Lane 1: molecular weight marker, lane 2 & 3: histo-immunoprecipitates of patient's sera from rat cerebellum, lanes 4 & 5: histo-immunoprecipitates of control samples.
The arrow indicates the position of the immunoprecipitated antigen 50 kDa while dotted arrows indicate the position of IgG heavy chain at 52 kDa.
Fig. 3B: Immunofluorescence staining of rat hippocampus (1-3) and cerebellum (4-6), monkey intestinal (7-9) and optic nerve (10-12) tissue sections with patient's serum (1 & 4) and anti-flotillin2 antibody (2 & 5). The merged pictures show co-localization of both reactivities including the more intense staining of the outer molecular layer on hippocampus (3 & 6).
Fig. 4 shows immunofluorescence staining of recombinant flotillin and its neutralization of the antibody reaction on tissue.
Fig. 4A: Immunofluorescence analysis of transfected HEK293 cells. Patient or control sera (1:1000) (green) were incubated on acetone-fixed recombinant HEK293 cells expressing flotillin1 (A), flotillin2 (B), flotillin1 and -2 (C) or a mock-transfected control (D).
Fig. 4B: Neutralization of immunofluorescence reaction on neuronal tissues.
Patient serum (green) was pre-incubated with extracts of HEK293 cells transfected with empty vector as control (1-4) or with flotillin1/2 (5-8). The extract containing flotillin1/2 greatly reduced or abolished the immune reaction. Nuclei were counterstained by incubation with TO-PRO-3 iodide (blue). Hippocampus rat (1 & 5), cerebellum rat (2 & 6), cerebellum monkey (3 & 7), HEK293-flotillin1/2 (4 & 8).

A number of sequences are disclosed in this application, more specifically SEQ
ID NO 1, which represents the nucleotide sequence of expression vector pTriEX-1-Flotillin-1[human]-His, SEQ
ID NO 2, which represents the polypeptide sequence of human flotillin1 attached to a C-terminal His tag, SEQ ID NO 3, which represents the nucleotide sequence of expression vector pTriEX-1-Flotillin-1[human], SEQ ID NO 4, which represents the polypeptide sequence of human flotillin1, SEQ ID NO 5, which represents the nucleotide sequence of expression vector pTriEX-1-Flotillin-2[human]-His, SEQ ID NO 6, which represents the polypeptide sequence of human flotillin2 attached to a C-terminal His tag, SEQ ID NO 7, which represents the nucleotide sequence of expression vector pTriEX-1-Flotillin-2[human], and SEQ ID NO 8, which represents the polypeptide sequence of human flotillin2.

Examples Summary:
The following examples demonstrate that a patient suffering from optic neuritis, more specifically vision impairment, headache and brain. lesions, was clinically assessed, but the molecular basis of their disease remained unknown. Their blood was screened, but contained no autoantibodies to known neurological markers. An autoantibody was isolated based on a characteristic staining pattern following reaction with several mammalian tissues, more specifically cerebellum and hippocampus from rat and pig.
lmmunoprecipitation, mass spectrometry and competitive binding studies using recombinant flotillin1 and flotillin2 revealed flotillin1 and flotillin2 as the targets of said antibody. 34 sera from patients with various neural autoantibodies (anti-NMDAR, anti-Hu, anti-Yo, anti-Ri, anti-AQP4, anti-LGI1, anti-CASPR2) and from 226 healthy controls showed no reaction when contacted with recombinant flotillin 1/2, confirming that the inventive method is a specific assay.
The patient responded positively to immunosuppressive treatment to the effect.
The visual symptoms completely vanished.
Characterization of the patient An in other respects healthy female patient (34 y) initially presented with blurred vision, local retrobulbar pain, and with reduced intensity of red color vision of the right eye which started five days before presentation. Except there was an unremarkable medical history without hints for any autoimmune disease in the near family except suspicion of multiple sclerosis in a grand mother on the mother's side. Clinical-neurological examination revealed an impaired vision on the right eye (0.6; left: 1.0). Testing of visual evoked potentials revealed a severe disturbation of processing of optical afferences. Moreover, further electrophysiological testing also revealed a slight disturbance of sensitive afferences from the left leg, but normal responses after transcranial magnetic stimulation to all four extremities. Cranial MRI
revealed signs of inflammation (contrast enhancement) of the right optical nerve as well as several small subcortical white matter lesions (without signs of blood brain barrier damage), typical for a demyelinating disease such as multiple sclerosis (Fig. 1). Blood testing showed normal values for renal and hepatic function, normal electrolytes, and a normal number and distribution of red and white cells. Testing for autoantibodies in the serum revealed no significant findings:
rheumatoid factor, pANCA, AMA, anti-phospholid, anti-Borrelia (IgG/IgM) and anti-Treponema were negative; ANA were present at a titer of 1:320. CSF revealed a mild pleocytosis (9 cells/pL), normal protein (269 mg/L), local IgG synthesis (53%), and oligoclonal bands that were absent in serum. Anti-AQP4 was neither detectable in CSF nor in serum.
The patient was treated with intravenous glucocorticoid pulse therapy under suspicion of autoimmune neuritis of the optical nerve. A clinically isolated syndrome was assumed and an immunomodulation with betaferone was initiated. 5 months later, the visual symptoms had vanished completely.
22 month after start of the initial symptoms, the patient presented with a stable clinical situation without further progress of the neurological symptoms. A control lumbar puncture revealed an autochthonous antibody production with oligoclonal bands in the CSF and a mild pleocytosis of cells / pL CSF.
Indirect immunofluorescence assay (IFA) Slides with a biochip mosaic including brain tissue cryosections (hippocampus of rat, cerebellum of rat, monkey, and pig) and HEK293 cells individually expressing 30 recombinant brain antigens was used for IFA. The slides were incubated with 70 pL of sample diluted in PBS, 0.2% Tween-20 (IFA buffer) at room temperature for 30 min, flushed with IFA
buffer and immersed in IFA buffer for 5 min. Subsequently, polyclonal goat anti-human pan-IgG
(EUROIMMUN) or monoclonal murine anti-human IgG1, IgG2, IgG3, or IgG4) (Sigma-Aldrich), each labelled with fluorescein isothiocyanate (FITC), were incubated at room temperature for 30 min. Slides were then washed again, embedded in PBS-buffered, DABCO containing glycerol (approximately 20 pL per mosaic) and examined by two independent observers using a laser scanning microscope (LSM700, Zeiss, Jena, Germany). Positive and negative controls were included. Samples were categorized based on tissue patterns and fluorescence intensity of transfected cells in direct comparison with non-transfected cells and control samples. Endpoint titers refer to the highest dilution showing visible fluorescence. Live-cell IFA with primary hippocampal neurons was conducted (Dalmau J, Gleichman AJ, Hughes EG, Rossi JE, Peng X, Lai M, et al. Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies. Lancet Neurol 2008 Oct 11).

A polyclonal rabbit antibody against flotillin2 (Sigma-Aldrich, dilution 1:100) was used in some experiments in the first step followed by incubation with anti-rabbit IgG-Cy3 (Jackson Research, Suffolk, United Kingdom). Cell nuclei were visualized by DNA staining with TO-PRO3 Iodide (dilution 1:2000) (ThermoFisher Scientific, Schwerte, Germany). Recombinant antigens were mixed with diluted serum sample 1 h prior to IFA (see Stocker W, Otte M, Ulrich S, Normann D, Finkbeiner H, Stocker K, et al. Autoimmunity to pancreatic juice in Crohn's disease. Results of an autoantibody screening in patients with chronic inflammatory bowel disease.
Scand J
Gastroenterol Suppl 1987;139:41-52) for neutralization experiments.
Histo-immunoprecipitation and identification of the antigen Cerebellum from rat or pig was dissected and shock-frozen in -160 C
isopentane. The tissue was then cryosected (4 pm) with a SM2000R microtome (Leica Microsystems, Nussloch, Germany), placed on glass slides, dried and stored at -196 C. For HIP, slides were incubated with patient's serum (diluted 1:100) at 4 C for 3 hours followed by 3 washing steps with IFA
buffer. The tissue was then extracted with solubilization buffer (100 mmol/L
tris-HCI pH 7.4, 150 mmol/L sodium chloride, 2.5 mmol/L EDTA, 0.5% (w/v) deoxycholate, 1% (w/v) Triton X-100 containing protease inhibitors) at room temperature for 30 minutes. The resulting suspension was homogenized and centrifuged at 16,000 x g at 4 C for 15 minutes.
Immunocomplexes were precipitated from the clear supernatant with Protein G Dynabeads (ThermoFisher Scientific, Dreieich, Germany) at 4 C overnight, washed 3 times with PBS, and eluted with PBS, 5 mmol/L
dithiothreitol, 1% (w/v) sodium dodecylsulfate at 95 C for 10 minutes. The eluates were analyzed by SDS-PAGE and mass spectrometry or Western blot.
Recombinant expression of flotillinl and flotillin2 in HEK293 Cloning of the expression vectors (SEQs ID NO 1 and 3 encoding a polypeptide comprising flotillin1; SEQs ID NO 5 and 7 encoding a polypeptide comprising flotillin2) was performed using standard methods. In order to prepare substrates for IFA, HEK293 were seeded on sterile cover glasses, transfected, and allowed to express flotillin1 and flotillin2 either individually or in conjunction for 48 hours. Cover glasses were washed with PBS, fixed with either acetone for 10 minutes at room temperature, air-dried, cut into millimeter-sized biochips and used as substrates in IFA as described. Alternatively, cells were transfected in standard T-flasks and the cells were harvested after 5 days. The cell sediment was extracted with solubilization buffer.
The extracts were stored in aliquots at -80 C until further use.

Studies with a larger cohort of patients Sera from 49 patients with various neural autoantibodies, including 20 with autoantibodies against AQP4 (titers up to 1:3,200), and from 226 healthy controls were analyzed by IFA. None of the sera reacted with HEK293-flotillin-1, HEK293-flotillin-2, and HEK293-flotillin-1/2.
Anti-flotillin-1/2 status was then determined retrospectively in 224 samples for which a general broad neural autoantibody screening, including the aforementioned parameters, had been conducted in the Clinical Immunological Laboratory Lubeck, and for which a neural tissue-reactive IgG autoantibody without known antigen-specificity had been reported.
Serum anti-flotillin-1/2 was revealed in four patients (P2 ¨ P5 in Table 1, titers:
1:1,000, 1:1,000, 1:10,000, 1:10,000). Patients P2, P3, and P5 also showed anti-flotillin-1/2 in CSF
(titers: 1:3.2, 1:100, 1:1,000). For P4, CSF was not available. Specific antibody indices >4 were calculated for P1, P3, and P5. Additional follow-up sera of the patients were analyzed when available and showed that the anti-flotillin-1/2 titers of P1, P2, and P4 were stable over a period of 18, 24, and 72 months, respectively. Serum of P5 showed a reduction to 1:320 seven weeks after plasma exchange.
HEK293-flotillin-1/2 was then integrated in the broad autoantibody screening regimen of the reference laboratory. In a cohort of 521 consecutive patients for whom a determination of anti-AQP4 was requested, eighteen were positive for anti-AQP4 while three exhibited anti-flotillin-1/2. For one of the latter medical records were accessible (serum: 1:1,000, no CSF, P6 in Table 1). An additional patient was identified during the diagnostic work-up of 150 consecutive unselected neurological patients (serum: 1:10, CSF negative, P7 in Table 1), and a further patient by screening of 57 anonymized, anti-AQP4- and anti-MOG-negative sera from patients with isolated ON (1:10, CSF not available).
In summary, all seven patients for whom medical records could be evaluated had radiological signs of disseminated demyelination, mild pleocytosis and OCB in CSF
consistent with MS or a CIS suggestive of MS. Six of them presented with optic neuritis. In all cases, the autoantibodies were of subclass IgG1 and bound to flotillin-1/2 but not to individual flotillin-1 or flotillin-2. None of the patients displayed anti-AQP4 or anti-MOG antibodies SEQUENCES
<210> 1 <211> 6895 <212> DNA
<213> artificial sequence <220>
<223> pTriEx-1-Flotillin-1[humani-His <400> 1 taatacgact cactataggg gaattgtgag cggataacaa ttccccggag ttaatccggg 60 acctttaatt caacccaaca caatatatta tagttaaata agaattatta tcaaatcatt 120 tgtatattaa ttaaaatact atactgtaaa ttacatttta tttacaatca aaggagatat 180 accatgtttt tcacttgtgg cccaaatgag gccatggtgg tctccgggtt ctgccgaagc 240 cccccagtca tggtggctgg agggcgtgtc tttgtcctgc cctgcatcca acagatccag 300 aggatctctc tcaacacact gaccctcaat gtcaagagtg aaaaggttta cactcgccat 360 ggggtcccca tctcagtcac tggcattgcc caggtaaaaa tccaggggca gaacaaggag 420 atgttggcgg ccgcctgtca gatgttcctg gggaagacgg aggctgagat tgcccacatt 480 gccctggaga cgttagaggg ccaccagagg gccatcatgg cccacatgac tgtggaggag 540 atctataagg acaggcagaa attctcagaa caggttttca aagtggcctc ctcagacctg 600 gtcaacatgg gcatcagtgt ggttagctac actctgaagg acattcacga tgaccaggac 660 tatttgcact ctttggggaa ggctcgaaca gctcaagtcc aaaaagatgc acggattgga 720 gaagcagagg ccaagagaga tgctgggatc cgggaagcta aagccaagca ggaaaaggtg 780 tctgctcagt acctgagtga gatcgagatg gccaaggcac agagagatta cgaactgaag 840 aaggccgcct atgacatcga ggtcaacacc cgccgagcac aggctgacct ggcctatcag 900 cttcaggtgg ccaagactaa gcagcagatt gaggagcagc gggtgcaggt gcaggtggtg 960 gagcgggccc agcaggtggc agtgcaggag caggagatcg cccggcggga gaaggagctg 1020 gaggcccggg tgcggaagcc agcggaagcg gagcgctaca agctggagcg cctagccgag 1080 gcagagaagt cccaactaat tatgcaggcg gaggcagaag ccgcgtctgt gcggatgcgt 1140 ggggaagctg aggcctttgc cataggggcc cgagcccgag ccgaggctga gcagatggcc 1200 aagaaggcag aagccttcca gctgtaccaa gaggctgctc agctggacat gctgctagag 1260 aagctgcccc aggtggcaga ggagatcagt ggtcccttga cttcagccaa taagatcaca 1320 ctggtgtcca gcggcagtgg gaccatgggg gcagccaaag tgactgggga agtactggac 1380 attctaactc gcctgccaga gagtgtggaa agactcacag gcgtgagcat ctcccaggtg 1440 aatcacaagc ctttgagaac agccatcgag caccaccatc accatcacca tcactaagtg 1500 attaacctca ggtgcaggct gcctatcacla aggtggtogc tggtgtogcc aatgccctgg 1560 ctcacaaata ccactgagat cgatcttttt ccctctgcca aaaattatgg ggacatcatg 1620 aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg 1680 tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa 1740 catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgta actagcataa 1800 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaagcatg cggaggaaat 1860 tctccttgaa gtttccctgg tgttcaaagt aaaggagttt gcaccagacg cacctctgtt 1920 cactggtccg gcgtattaaa acacgataca ttgttattag tacatttatt aagcgctaga 1980 ttctgtgcgt tgttgattta cagacaattg ttgtacgtat tttaataatt cattaaattt 2040 ataatcttta gggtggtatg ttagagcgaa aatcaaatga ttttcagcgt ctttatatct 2100 gaatttaaat attaaatcct caatagattt gtaaaatagg tttcgattag tttcaaacaa 2160 gggttgtttt tccgaaccga tggctggact atctaatgga ttttcgctca acgccacaaa 2220 acttgccaaa tcttgtagca gcaatctagc tttgtcgata ttcgtttgtg ttttgttttg 2280 taataaaggt tcgacgtcgt tcaaaatatt atgcgctttt gtatttcttt catcactgtc 2340 gttagtgtac aattgactcg acgtaaacac gttaaataga gcttggacat atttaacatc 2400 gggcgtgtta gctttattag gccgattatc gtcgtcgtcc caaccctcgt cgttagaagt 2460 tgcttccgaa gacgattttg ccatagccac acgacgccta ttaattgtgt cggctaacac 2520 gtccgcgatc aaatttgtag ttgagctttt tggaattatt tctgattgcg ggcgtttttg 2580 ggcgggtttc aatctaactg tgcccgattt taattcagac aacacgttag aaagcgatgg tgcaggcggt ggtaacattt cagacggcaa atctactaat ggcggcggtg gtggagctga tgataaatct accatcggtg gaggcgcagg cggggctggc ggcggaggcg gaggcggagg tggtggcggt gatgcagacg gcggtttagg ctcaaatgtc tctttaggca acacagtcgg cacctcaact attgtactgg tttcgggcgc cgtttttggt ttgaccggtc tgagacgagt gcgatttttt tcgtttctaa tagcttccaa caattgttgt ctgtcgtcta aaggtgcagc gggttgaggt tccgtcggca ttggtggagc gggcggcaat tcagacatcg atggtggtgg tggtggtgga ggcgctggaa tgttaggcac gggagaaggt ggtggcggcg gtgccgccgg tataatttgt tctggtttag tttgttcgcg cacgattgtg ggcaccggcg caggcgccgc tggctgcaca acggaaggtc gtctgcttcg aggcagcgct tggggtggtg gcaattcaat attataattg gaatacaaat cgtaaaaatc tgctataagc attgtaattt cgctatcgtt taccgtgccg atatttaaca accgctcaat gtaagcaatt gtattgtaaa gagattgtct caagctcgga acgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccacma accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 3600 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 3660 tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 3720 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 3780 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 3840 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 3900 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 3960 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 4020 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttgt taccaatgct 4080 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 4140 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 4200 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 4260 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 4320 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 4380 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 4440 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 4500 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgtccgcgcg tttcctgcat cttttaatca aatcccaaga tgtgtataaa ccaccaaact gccaaaaaat gaaaactgtc gacaagctct gtccgtttgc tggcaactgc aagggtctca atcctatttg taattattga ataataaaac aattataaat gtcaaatttg ttttttatta acgatacaaa ccaaacgcaa caagaacatt tgtagtatta tctataattg aaaacgcgta gttataatcg ctgaggtaat atttaaaatc attttcaaat gattcacagt taatttgcga caatataatt ttattttcac ataaactaga cgccttgtcg tcttcttctt cgtattcctt ctctttttca tttttctctt cataaaaatt aacatagtta ttatcgtatc catatatgta tctatcgtat agagtaaatt ttttgttgtc ataaatatat atgtcttttt taatggggtg tatagtaccg ctgcgcatag tttttctgta atttacaaca gtgctatttt ctggtagttc ttcggagtgt gttgctttaa ttattaaatt tatataatca atgaatttgg gatcgtcggt tttgtacaat atgttgccgg catagtacgc agcttcttct agttcaatta caccattttt tagcagcacc ggattaacat aactttccaa aatgttgtac gaaccgttaa acaaaaacag ttcacctccc ttttctatac tattgtctgc gagcagttgt ttgttgttaa aaataacagc cattgtaatg agacgcacaa actaatatca caaactggaa atgtctatca atatatagtt gctctagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggactattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatgca tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct 6480 gcgacgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct 6540 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc cttcgggctg 6600 taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg aaagccttga 6660 ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggctgtccg cggggggacg 6720 gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct 6780 ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc ctgggcaacg 6840 tgctggttat tgtgctgtct catcattttg gcaaagaatt ggatcggacc gaaat 6895 <210> 2 <211> 437 <212> PRT
<213> artificial sequence <220>
<223> Flotillin-1[human]-His <400> 2 Met Phe Phe Thr Cys Gly Pro Asn Glu Ala Met Val Val Ser Gly Phe Cys Arg Ser Pro Pro Val Met Val Ala Gly Gly Arg Val Phe Val Leu Pro Cys Ile Gin Gin Ile Gin Arg Ile Ser Leu Asn Thr Leu Thr Leu Asn Val Lys Ser Glu Lys Val Tyr Thr Arg His Gly Val Pro Ile Ser Val Thr Gly Ile Ala Gin Val Lys Ile Gin Gly Gin Asn Lys Glu Met Leu Ala Ala Ala Cys Gin Met Phe Leu Gly Lys Thr Glu Ala Glu Ile Ala His Ile Ala Leu Glu Thr Leu Glu Gly His Gln Arg Ala Ile Met Ala His Met Thr Val Glu Glu Ile Tyr Lys Asp Arg Gin Lys Phe Ser Glu Gin Val Phe Lys Val Ala Ser Ser Asp Leu Val Asn Met Gly Ile Ser Val Val Ser Tyr Thr Leu Lys Asp Ile His Asp Asp Gin Asp Tyr Leu His Ser Leu Gly Lys Ala Arg Thr Ala Gin Val Gin Lys Asp Ala Arg Ile Gly Glu Ala Glu Ala Lys Arg Asp Ala Gly Ile Arg Glu Ala Lys Ala Lys Gin Glu Lys Val Ser Ala Gin Tyr Leu Ser Glu Ile Glu Net Ala Lys Ala Gin Arg Asp Tyr Glu Leu Lys Lys Ala Ala Tyr Asp Ile Glu Val Asn Thr Arg Arg Ala Gin Ala Asp Leu Ala Tyr Gin Leu Gin Val Ala Lys Thr Lys Gin Gin Ile Glu Glu Gin Arg Val Gin Val Gin Val Val Glu Arg Ala Gin Gin Val Ala Val Gin Glu Gin Glu Ile Ala Arg Arg Glu Lys Glu Leu Glu Ala Arg Val Arg Lys Pro Ala Glu Ala Glu Arg Tyr Lys Leu Glu Arg Leu Ala Glu Ala Glu Lys Ser Gin Leu Ile Met Gin Ala Glu Ala Glu Ala Ala Ser Val Arg Met Arg Gly Glu Ala Glu Ala Phe Ala Ile Gly Ala Arg Ala Arg Ala Glu Ala Glu Gin Met Ala Lys Lys Ala Glu Ala Phe Gin Leu Tyr Gin Glu Ala Ala Gin Leu Asp Met Leu Leu Glu Lys Leu Pro Gin Val Ala Glu Glu Ile Ser Gly Pro Leu Thr Ser Ala Asn Lys Ile Thr Leu Val Ser Ser Gly Ser Gly Thr Met Gly Ala Ala Lys Val Thr Gly Glu Val Leu Asp Ile Leu Thr Arg Leu Pro Glu Ser Val Glu Arg Leu Thr Gly Val Ser Ile Ser Gin Val Asn His Lys Pro Leu Arg Thr Ala Ile Glu His His His His His His His His <210> 3 <211> 6897 <212> DNA
<213> artificial sequence <220>
<223> pTriEx-1-Flotillin-l[human]
<400> 3 taatacgact cactataggg gaattgtgag cggataacaa ttccccggag ttaatccggg 60 acctttaatt caacccaaca caatatatta tagttaaata agaattatta tcaaatcatt 120 tgtatattaa ttaaaatact atactgtaaa ttacatttta tttacaatca aaggagatat 180 accatgtttt tcacttgtgg cccaaatgag gccatggtgg tctccgggtt ctgccgaagc 240 cccccagtca tggtggctgg agggcgtgtc tttgtcctgc cctgcatcca acagatccag 300 aggatctctc tcaacacact gaccctcaat gtcaagagtg aaaaggttta cactcgccat 360 ggggtcccca tctcagtcac tggcattgcc caggtaaaaa tccaggggca gaacaaggag 420 atgttggcgg ccgcctgtca gatgttcctg gggaagacgg aggctgagat tgcccacatt 480 gccctggaga cgttagaggg ccaccagagg gccatcatgg cccacatgac tgtggaggag 540 atctataagg acaggcagaa attctcagaa caggttttca aagtggcctc ctcagacctg 600 gtcaacatgg gcatcagtgt ggttagctac actctgaagg acattcacga tgaccaggac 660 tatttgcact ctttggggaa ggctcgaaca gctcaagtcc aaaaagatgc acggattgga 720 gaagcagagg ccaagagaga tgctgggatc cgggaagcta aagccaagca ggaaaaggtg 780 tctgctcagt acctgagtga gatcgagatg gccaaggcac agagagatta cgaactgaag 840 aaggccgcct atgacatcga ggtcaacacc cgccgagcac aggctgacct ggcctatcag 900 cttcaggtgg ccaagactaa gcagcagatt gaggagcagc gggtgcaggt gcaggtggtg 960 gagcgggccc agcaggtggc agtgcaggag caggagatcg cccggcggga gaaggagctg gaggcccggg tgcggaagcc agcggaagcg gagcgctaca agctggagcg cctagccgag gcagagaagt cccaactaat tatgcaggcg gaggcagaag ccgcgtctgt gcggatgcgt ggggaagctg aggcctttgc cataggggcc cgagcccgag ccgaggctga gcagatggcc aagaaggcag aagccttcca gctgtaccaa gaggctgctc agctggacat gctgctagag aagctgcccc aggtggcaga ggagatcagt ggtcccttga cttcagccaa taagatcaca ctggtgtcca gcggcagtgg gaccatgggg gcagccaaag tgactgggga agtactggac attctaactc gcctgccaga gagtgtggaa agactcacag gcgtgagcat ctcccaggtg aatcacaagc ctttgagaac agcctgatcg agcaccacca tcaccatcac catcactaag tgattaacct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag atcgatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt tggcaacata tgcccatatg taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagca tgcggaggaa attctccttg aagtttccct ggtgttcaaa gtaaaggagt ttgcaccaga cgcacctctg ttcactggtc cggcgtatta aaacacgata cattgttatt agtacattta ttaagcgcta gattctgtgc gttgttgatt tacagacaat tgttgtacgt attttaataa ttcattaaat ttataatctt tagggtggta tgttagagcg aaaatcaaat gattttcagc gtctttatat ctgaatttaa atattaaatc ctcaatagat ttgtaaaata ggtttcgatt agtttcaaac 210 aagggttgtt tttccgaacc gatggctgga ctatctaatg gattttcgct caacgccaca 2220 aaacttgcca aatcttgtag cagcaatcta gctttgtcga tattcgtttg tgttttgttt tgtaataaag gttcgacgtc gttcaaaata ttatgcgctt ttgtatttct ttcatcactg tcgttagtgt acaattgact cgacgtaaac acgttaaata gagcttggac atatttaaca tcgggcgtgt tagctttatt aggccgatta tcgtcgtcgt cccaaccctc gtcgttagaa gttgcttccg aagacgattt tgccatagcc acacgacgcc tattaattgt gtcggctaac acgtccgcga tcaaatttgt agttgagctt tttggaatta tttctgattg cgggcgtttt tgggcgggtt tcaatctaac tgtgcccgat tttaattcag acaacacgtt agaaagcgat ggtgcaggcg gtggtaacat ttcagacggc aaatctacta atggcggcgg tggtggagct gatgataaat ctaccatcgg tggaggcgca ggcggggctg gcggcggagg cggaggcgga ggtggtggcg gtgatgcaga cggcggttta ggctcaaatg tctctttagg caacacagtc ggcacctcaa ctattgtact ggtttcgggc gccgtttttg gtttgaccgg tctgagacga gtgcgatttt tttcgtttct aatagcttcc aacaattgtt gtctgtcgtc taaaggtgca gcgggttgag gttccgtcgg cattggtgga gcgggcggca attcagacat cgatggtggt ggtggtggtg gaggcgctgg aatgttaggc acgggagaag gtggtggcgg cggtgccgcc ggtataattt gttctggttt agtttgttcg cgcacgattg tgggcaccgg cgcaggcgcc gctggctgca caacggaagg tcgtctgctt cgaggcagcg cttggggtgg tggcaattca atattataat tggaatacaa atcgtaaaaa tctgctataa gcattgtaat ttcgctatcg tttaccgtgc cgatatttaa caaccgctca atgtaagcaa ttgtattgta aagagattgt ctcaagctcg gaacgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 4140 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgtccgcg cgtttcctgc atcttttaat caaatcccaa gatgtgtata aaccaccaaa ctgccaaaaa atgaaaactg tcgacaagct ctgtccgttt gctggcaact gcaagggtct caatcctatt tgtaattatt gaataataaa acaattataa atgtcaaatt tgttttttat 5160 taacgataca aaccaaacgc aacaagaaca tttgtagtat tatctataat tgaaaacgcg 5220 tagttataat cgctgaggta atatttaaaa tcattttcaa atgattcaca gttaatttgc 5280 gacaatataa ttttattttc acataaacta gacgccttgt cgtcttcttc ttcgtattcc 5340 ttctcttttt catttttctc ttcataaaaa ttaacatagt tattatcgta tccatatatg 5400 tatctatcgt atagagtaaa ttttttgttg tcataaatat atatgtcttt tttaatgggg 5460 tgtatagtac cgctgcgcat agtttttctg taatttacaa cagtgctatt ttctggtagt 5520 tcttcggagt gtgttgcttt aattattaaa tttatataat caatgaattt gggatcgtcg 5580 gttttgtaca atatgttgcc ggcatagtac gcagcttctt ctagttcaat tacaccattt 5640 tttagcagca ccggattaac ataactttcc aaaatgttgt acgaaccgtt aaacaaaaac 5700 agttcacctc ccttttctat actattgtct gcgagcagtt gtttgttgtt aaaaataaca 5760 gccattgtaa tgagacgcac aaactaatat cacaaactgg aaatgtctat caatatatag 5820 ttgctctagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga 5880 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg 5940 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg 6000 acgtcaatgg gtggactatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca 6060 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgacgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc tccttcgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggctgtc cgagggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttggatcgga ccgaaat <210> 4 <211> 427 <212> PRT

<213> human <400> 4 Met Phe Phe Thr Cys Gly Pro Asn Glu Ala Met Val Val Ser Gly Phe Cys Arg Ser Pro Pro Val Met Val Ala Gly Gly Arg Val Phe Val Leu Pro Cys Ile Gin Gin Ile Gin Arg Ile Ser Leu Asn Thr Leu Thr Leu Asn Val Lys Ser Glu Lys Val Tyr Thr Arg His Gly Val Pro Ile Ser Val Thr Gly Ile Ala Gin Val Lys Ile Gin Gly Gin Asn Lys Glu Met Leu Ala Ala Ala Cys Gin Met Phe Leu Gly Lys Thr Glu Ala Glu Ile Ala His Ile Ala Leu Glu Thr Leu Glu Gly His Gin Arg Ala Ile Met Ala His Met Thr Val Glu Glu Ile Tyr Lys Asp Arg Gin Lys Phe Ser Glu Gin Val Phe Lys Val Ala Ser Ser Asp Leu Val Asn Met Gly Ile Ser Val Val Ser Tyr Thr Leu Lys Asp Ile His Asp Asp Gin Asp Tyr Leu His Ser Leu Gly Lys Ala Arg Thr Ala Gin Val Gin Lys Asp Ala Arg Ile Gly Glu Ala Glu Ala Lys Arg Asp Ala Gly Ile Arg Glu Ala Lys Ala Lys Gin Glu Lys Val Ser Ala Gin Tyr Leu Ser Glu Ile Glu Met Ala Lys Ala Gin Arg Asp Tyr Glu Leu Lys Lys Ala Ala Tyr Asp Ile Glu Val Asn Thr Arg Arg Ala Gin Ala Asp Leu Ala Tyr Gin Leu Gin Val Ala Lys Thr Lys Gin Gin Ile Glu Glu Gin Arg Val Gin Val Gin Val Val Glu Arg Ala Gin Gin Val Ala Vol Gin Glu Gin Glu Ile Ala Arg Arg Glu Lys Glu Leu Glu Ala Arg Val Arg Lys Pro Ala Glu Ala Glu Arg Tyr Lys Leu Glu Arg Leu Ala Glu Ala Glu Lys Ser Gln Leu Ile Met Gin Ala Clu Ala Glu Ala Ala Ser Val Arg Met Arg Gly Glu Ala Glu Ala Phe Ala Ile Gly Ala Arg Ala Arg Ala Glu Ala Glu Gin Met Ala Lys Lys Ala Glu Ala Phe Gin Leu Tyr Gin Glu Ala Ala Gin Leu Asp Met Leu Leu Glu Lys Leu Pro Gin Vol Ala Glu Glu Ile Ser Gly Pro Leu Thr Ser Ala Asn Lys Ile Thr Leu Val Ser Ser Gly Ser Gly Thr Met Gly Ala Ala Lys Val Thr Gly Glu Val Leu Asp Ile Leu Thr Arg Leu Pro Glu Ser Val Glu Arg Leu Thr Gly Val Ser Ile Ser Gln Val Asn His Lys Pro Leu Arg Thr Ala <210> 5 <211> 6898 <212> DNA
<213> artificial sequence <220>
<223> pTriEx-1-Flotillin-2[human]-His <400> 5 taatacgact cactataggg gaattgtgag cggataacaa ttccccggag ttaatccggg 60 acctttaatt caacccaaca caatatatta tagttaaata agaattatta tcaaatcatt 120 tgtatattaa ttaaaatact atactgtaaa ttacatttta tttacaatca aaggagatat 180 accatgggca attgccacac ggtgggcccc aacgaggcac tggtggtctc agggggctgt 240 tgtggttctg actacaagca gtatgtgttt ggcggctggg cttgggcctg gtggtgtatc 300 tcggacactc agaggatttc cctagagatt atgacgttgc agccccgctg tgaggacgta 360 gagacggccg agggggtagc tttaactgtg acgggtgtcg cccaggtgaa gatcatgacg 420 gagaaggagc tcctggctgt agcctgtgaa cagttcctgg gcaagaacgt gcaggacatt 480 aagaacgtcg tactgcagac cctggagggg catctacgct ccatccttgg gactctgact 540 gtggagcaga tttatcagga ccgagaccag tttgccaagc tggtgcggga agtggcagcc 600 cctgatgttg gccgtatggg catcgagatc ctcagcttca ccatcaagga tgtctatgac 660 aaagtagact atctgagctc cctgggcaag acacagactg ccgtggtaca gagagatgca 720 gacatcggtg tggcagaggc agagcgggac gcaggcatcc gggaagccga gtgcaagaag 780 gaaatgctag atgtgaagtt catggcagac accaagattg ctgactccaa gagagccttt 840 gagctgcaaa agtcagcctt cagtgaggag gtcaacatca agacagctga ggcccagttg 900 gcctatgagc tacaaggggc cagagagcaa cagaagatcc ggcaggaaga gattgagatt 960 gaggtagtac agcgcaagaa gcagatcgcc gtggaggcgc aggagatcct gcgcacagac 1020 aaggagctca tcgccacagt gcgccgccct gcagaggcag aggcccaccg catccagcag 1080 attgctgaag gcgaaaaggt gaaacaagtc ctcttggcac aagcagaagc tgagaagatt 1140 cgcaaaatcg gggaggcaga ggcagcagtc attgaggcca tgggcaaggc cgaggccgag cggatgaagc ttaaagctga ggcctaccag aagtacgggg atgcggccaa gatggccctg gtgctggagg ccctgcccca gattgctgcc aagatcgccg cacccctgac taaagtcgat gagattgtgg ttctcagtgg ggacaacagc aaggtgacat cagaagtgaa ccggctgcta gcagaactgc ctgcttctgt tcatgccctc actggtgtgg acctctcaaa gataccactg atcaagaaag ccactggtgt gcaggtgatc gagcaccacc atcaccatca ccatcactaa gtgattaacc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc tggctcacaa ataccactga gatcgatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat atgcccatat gtaactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagc atgcggagga aattctcctt gaagtttccc tggtgttcaa agtaaaggag tttgcaccag acgcacctct gttcactggt ccggcgtatt aaaacacgat acattgttat tagtacattt attaagcgct agattctgtg cgttgttgat ttacagacaa ttgttgtacg tattttaata attcattaaa tttataatct ttagggtggt atgttagagc gaaaatcaaa tgattttcag cgtctttata 2100 tctgaattta aatattaaat cctcaataga tttgtaaaat aggtttcgat tagtttcaaa 2160 caagggttgt ttttccgaac cgatggctgg actatctaat ggattttcgc tcaacgccac 2220 aaaacttgcc aaatcttgta gcagcaatct agctttgtcg atattcgttt gtgttttgtt 2260 ttgtaataaa ggttcgacgt cgttcaaaat attatgcgct tttgtatttc tttcatcact 2340 gtcgttagtg tacaattgac tcgacgtaaa cacgttaaat agagcttgga catatttaac 2400 atcgggcgtg ttagctttat taggccgatt atcgtcgtcg tcccaaccct cgtcgttaga 2460 agttgcttcc gaagacgatt ttgccatagc cacacgacgc ctattaattg tgtoggctaa 2520 cacgtccgcg atcaaatttg tagttgagct ttttggaatt atttctgatt gcgggcgttt 2580 ttgggcgggt ttcaatctaa ctgtgcccga ttttaattca gacaacacgt tagaaagcga 2640 tggtgcaggc ggtggtaaca tttcagacgg caaatctact aatggcggcg gtggtggagc 2700 tgatgataaa tctaccatcg gtggaggcgc aggcggggct ggcggcggag gcggaggcgg 2760 aggtggtggc ggtgatgcag acggcggttt aggctcaaat gtctotttag gcaacacagt 2820 cggcacctca actattgtac tggtttcggg cgccgttttt ggtttgaccg gtctgagacg 2880 agtgcgattt ttttcgtttc taatagcttc caacaattgt tgtctgtcgt ctaaaggtgc 2940 agcgggttga ggttccgtcg gcattggtgg agcgggcggc aattcagaca tcgatggtgg 3000 tggtggtggt ggaggcgctg gaatgttagg cacgggagaa ggtggtggcg gcggtgccgc 3060 cggtataatt tgttctggtt tagtttgttc gcgcacgatt gtgggcaccg gcgcaggcgc cgctggctgc acaacggaag gtcgtctgct tcgaggcagc gcttggggtg gtggcaattc aatattataa ttggaataca aatcgtaaaa atctgctata agcattgtaa tttcgctatc gtttaccgtg ccgatattta acaaccgctc aatgtaagca attgtattgt aaagagattg tctcaagctc ggaacgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 4200 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 4260 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 4320 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 4380 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 4440 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 4500 cottoggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 4560 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 4620 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 4680 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 4740 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 4800 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 4860 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 4920 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 4980 tcatgtccgc gcgtttcctg catcttttaa tcaaatccca agatgtgtat aaaccaccaa actgccaaaa aatgaaaact gtcgacaagc tctgtccgtt tgctggcaac tgcaagggtc tcaatcctat ttgtaattat tgaataataa aacaattata aatgtcaaat ttgtttttta ttaacgatac aaaccaaacg caacaagaac atttgtagta ttatctataa ttgaaaacgc gtagttataa tcgctgaggt aatatttaaa atcattttca aatgattcac agttaatttg cgacaatata attttatttt cacataaact agacgccttg tcgtcttctt cttcgtattc cttctctttt tcatttttct cttcataaaa attaacatag ttattatcgt atccatatat gtatctatcg tatagagtaa attttttgtt gtcataaata tatatgtctt ttttaatggg gtgtatagta ccgctgcgca tagtttttct gtaatttaca acagtgctat tttctggtag ttcttcggag tgtgttgctt taattattaa atttatataa tcaatgaatt tgggatcgtc ggttttgtac aatatgttgc cggcatagta cgcagcttct tctagttcaa ttacaccatt ttttagcagc accggattaa cataactttc caaaatgttg tacgaaccgt taaacaaaaa cagttcacct cccttttcta tactattgtc tgcgagcagt tgtttgttgt taaaaataac 5760 agccattgta atgagacgca caaactaata tcacaaactg gaaatgtcta tcaatatata 5820 gttgctctag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatataa 5880 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 5940 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggactat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat gcatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt ctccttcggg ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggq cggggttcgg cttctggcgt gtgaccggcg 6780 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 6840 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attggatcgg accgaaat 6898 <210> 6 <211> 438 <212> PRT
<213> artificial sequence <220>
<223> Flotillin-2[human]-His <400> 6 Met Gly Asn Cys His Thr Val Gly Pro Asn Glu Ala Leu Val Val Ser Gly Gly Cys Cys Gly Ser Asp Tyr Lys Gin Tyr Val Phe Gly Gly Trp Ala Trp Ala Trp Trp Cys Ile Ser Asp Thr Gin Arg Ile Ser Leu Glu Ile Met Thr Leu Gin Pro Arg Cys Glu Asp Val Glu Thr Ala Glu Gly Val Ala Leu Thr Val Thr Gly Val Ala Gin Val Lys Ile Met Thr Glu Lys Glu Leu Leu Ala Val Ala Cys Glu Gin Phe Leu Gly Lys Asn Val Gin Asp Ile Lys Asn Val Val Leu Gin Thr Leu Glu Gly His Leu Arg Ser Ile Leu Gly Thr Leu Thr Val Glu Gin Ile Tyr Gin Asp Arg Asp Gin Phe Ala Lys Leu Val Arg Glu Val Ala Ala Pro Asp Val Gly Arg Met Gly Ile Glu Ile Leu Ser Phe Thr Ile Lys Asp Val Tyr Asp Lys Val Asp Tyr Leu Ser Ser Leu Gly Lys Thr Gin Thr Ala Val Val Gin Arg Asp Ala Asp Ile Gly Val Ala Glu Ala Glu Arg Asp Ala Gly Ile Arg Glu Ala Glu Cys Lys Lys Glu Met Leu Asp Val Lys Phe Met Ala Asp Thr Lys Ile Ala Asp Ser Lys Arg Ala Phe Glu Leu Gin Lys Ser Ala Phe Ser Glu Glu Val Asn Ile Lys Thr Ala Glu Ala Gin Leu Ala Tyr Glu Leu Gin Gly Ala Arg Glu Gin Gin Lys Ile Arg Gin Glu Glu Ile Glu Ile Glu Val Val Gin Arg Lys Lys Gin Ile Ala Val Glu Ala Gin Glu Ile Leu Arg Thr Asp Lys Glu Leu Ile Ala Thr Val Arg Arg Pro Ala Glu Ala Glu Ala His Arg Ile Gin Gin Ile Ala Glu Gly Glu Lys Val Lys Gin Val Leu Leu Ala Gin Ala Glu Ala Glu Lys Ile Arg Lys Ile Gly Glu Ala Glu Ala Ala Val Ile Glu Ala Met Gly Lys Ala Glu Ala Glu Arg Met Lys Leu Lys Ala Glu Ala Tyr Gin Lys Tyr Gly Asp Ala Ala Lys Met Ala Leu Val Leu Glu Ala Leu Pro Gin Ile Ala Ala Lys Ile Ala Ala Pro Leu Thr Lys Val Asp Glu Ile Val Val Leu Ser Gly Asp Asn Ser Lys Val Thr Ser Glu Val Asn Arg Leu Leu Ala Glu Leu Pro Ala Ser Val His Ala Leu Thr Gly Val Asp Leu Ser Lys Ile Pro Leu Ile Lys Lys Ala Thr Gly Val Gin Val Ile Glu His His His His His His His His <210> 7 <211> 6900 <212> DNA
<213> artificial sequence <220>
<223> pTriEx-1-Flotillin-2[human]
<400> 7 taatacgact cactataggg gaattgtgag cggataacaa ttccccggag ttaatccggg 60 acctttaatt caacccaaca caatatatta tagttaaata agaattatta tcaaatcatt 120 tgtatattaa ttaaaatact atactgtaaa ttacatttta tttacaatca aaggagatat 180 accatgggca attgccacac ggtgggcccc aacgaggcac tggtggtctc agggggctgt 240 tgtggttctg actacaagca gtatgtgttt ggcggctggg cttgggcctg gtggtgtatc 300 tcggacactc agaggatttc cctagagatt atgacgttgc agccccgctg tgaggacgta 360 gagacggccg agggggtagc tttaactgtg acgggtgtcg cccaggtgaa gatcatgacg 420 gagaaggagc tcctggctgt agcctgtgaa cagttcctgg gcaagaacgt gcaggacatt 480 aagaacgtcg tactgcagac cctggagggg catctacgct ccatccttgg gactctgact 540 gtggagcaga tttatcagga ccgagaccag tttgccaagc tggtgcggga agtggcagcc 600 cctgatgttg gccgtatggg catcgagatc ctcagcttca ccatcaagga tgtctatgac 660 aaagtagact atctgagctc cctgggcaag acacagactg ccgtggtaca gagagatgca 720 gacatcggtg tggcagaggc agagcgggac gcaggcatcc gggaagccga gtgcaagaag 780 gaaatgctag atgtgaagtt catggcagac accaagattg ctgactccaa gagagccttt 840 gagctgcaaa agtcagcctt cagtgaggag gtcaacatca agacagctga ggcccagttg 900 gcctatgagc tacaaggggc cagagagcaa cagaagatcc ggcaggaaga gattgagatt 960 gaggtagtac agcgcaagaa gcagatcgcc gtggaggcgc aggagatcct gcgcacagac 1020 aaggagctca tcgccacagt gcgccgccct gcagaggcag aggcccaccg catccagcag attgctgaag gcgaaaaggt gaaacaagtc ctcttggcac aagcagaagc tgagaagatt cgcaaaatcg gggaggcaga ggcagcagtc attgaggcca tgggcaaggc cgaggccgag cggatgaagc ttaaagctga ggcctaccag aagtacgggg atgcggccaa gatggccctg 1260 gtgctggagg ccctgcccca gattgctgcc aagatcgccg cacccctgac taaagtcgat 1320 gagattgtgg ttctcagtgg ggacaacagc aaggtgacat cagaagtgaa ccggctgcta 1380 gcagaactgc ctgcttctgt tcatgccctc actggtgtgg acctctcaaa gataccactg 1440 atcaagaaag ccactggtgt gcaggtgtaa tcgagcacca ccatcaccat caccatcact 1500 aagtgattaa cctcaggtgc aggctgccta tcagaaggtg gtggctggtg tggccaatgc 1560 cctggctcac aaataccact gagatcgatc tttttccctc tgccaaaaat tatggggaca 1620 tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt ttcattgcaa 1680 tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt 1740 taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgcccat atgtaactag 1800 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa gcatgcggag 1860 gaaattctcc ttgaagtttc cctagtattc aaaataaagg agtttgcacc agacgcacct 1920 ctgttcactg gtccggcgta ttaaaacacg atacattgtt attagtacat ttattaagcg 1980 ctagattctg tgcgttgttg atttacagac aattgttgta cgtattttaa taattcatta aatttataat ctttagggtg gtatgttaga gcgaaaatca aatgattttc agcgtcttta tatctgaatt taaatattaa atcctcaata gatttgtaaa ataggtttcg attagtttca aacaagggtt gtttttccga accgatggct ggactatcta atggattttc gctcaacgcc acaaaacttg ccaaatcttg taggagcaat ctagctttgt cgatattcgt ttgtgttttg ttttgtaata aaggttcgac gtcgttcaaa atattatgcg cttttgtatt tctttcatca ctgtcgttag tgtacaattg actcgacgta aacacgttaa atagagcttg gacatattta acatcgggcg tgttagcttt attaggccga ttatcgtcgt cgtcccaacc ctcgtcgtta gaagttgctt ccgaagacga ttttgccata gccacacgac gcctattaat tgtgtcggct aacacgtccg cgatcaaatt tgtagttgag ctttttggaa ttatttctga ttgcgggcgt ttttgggcgg gtttcaatct aactgtgccc gattttaatt cagacaacac gttagaaagc gatggtgcag gcggtggtaa catttcagac ggcaaatcta ctaatggcgg cggtggtgga gctgatgata aatctaccat cggtggaggc gcaggcgggg ctggcggcgg aggcggaggc ggaggtggtg gcggtgatgc agacggcggt ttaggctcaa atgtctcttt aggcaacaca 2820 gtcggcacct caactattgt actggtttcg ggcgccgttt ttggtttgac cggtctgaga 2880 cgagtgcgat ttttttcgtt tctaatagct tccaacaatt gttgtctgtc gtctaaaggt 2940 gcagcgggtt gaggttccgt cggcattggt ggagcgggcg gcaattcaga catcgatggt ggtggtggtg gtggaggcgc tggaatgtta ggcacgggag aaggtggtgg cggcggtgcc gccggtataa tttgttctgg tttagtttgt tcgcgcacga ttgtgggcac cggcgcaggc gccgctggct gcacaacgga aggtcgtctg cttcgaggca gcgcttgggg tggtggcaat tcaatattat aattggaata caaatcgtaa aaatctgcta taagcattgt aatttcgcta tcgtttaccg tgccgatatt taacaaccgc tcaatgtaag caattgtatt gtaaagagat tgtctcaagc tcggaacgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tottoggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgtcc gcgcgtttcc tgcatctttt aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa ctgtcgacaa gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt attgaataat aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa cgcaacaaga acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag gtaatattta aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt ttcacataaa ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt ctcttcataa aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt aaattttttg ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg catagttttt ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc tttaattatt aaatttatat aatcaatgaa tttgggatcg 5580 tcggttttgt acaatatgtt gccggcatag tacgcagctt cttctagttc aattacacca 5640 ttttttagca gcaccggatt aacataactt tccaaaatgt tgtacgaacc gttaaacaaa 5700 aacagttcac ctcccttttc tatactattg tctgcgagca gttgtttgtt gttaaaaata 5760 acagccattg taatgagacg cacaaactaa tatcacaaac tggaaatgtc tatcaatata 5820 tagttgctct agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atgcatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctccttcg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctagggoct gtccgcggag ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattggatc ggaccgaaat <210> 8 <211> 428 <212> PRT
<213> human <400> 8 Met Gly Asn Cys His Thr Val Gly Pro Asn Glu Ala Leu Val Val Ser Gly Gly Cys Cys Gly Ser Asp Tyr Lys Gln Tyr Val Phe Gly Gly Trp Ala Trp Ala Trp Trp Cys Ile Ser Asp Thr Gln Arg Ile Ser Leu Glu Ile Met Thr Leu Gln Pro Arg Cys Glu Asp Val Glu Thr Ala Glu Gly Val Ala Leu Thr Val Thr Gly Val Ala Gln Val Lys Ile Met Thr Glu Lys Glu Leu Leu Ala Val Ala Cys Glu Gin Phe Leu Gly Lys Asn Val Gin Asp Ile Lys Asn Val Val Leu Gln Thr Leu Glu Gly His Leu Arg Ser Ile Leu Gly Thr Leu Thr Val Glu Gin Ile Tyr Gin Asp Arg Asp Gin Phe Ala Lys Leu Val Arg Glu Val Ala Ala Pro Asp Val Gly Arg Met Gly Ile Glu Ile Leu Ser Phe Thr Ile Lys Asp Val Tyr Asp Lys Val Asp Tyr Leu Ser Ser Leu Gly Lys Thr Gin Thr Ala Val Val Gin Arg Asp Ala Asp Ile Gly Val Ala Glu Ala Glu Arg Asp Ala Gly Ile Arg Glu Ala Glu Cys Lys Lys Glu Met Leu Asp Val Lys Phe Met Ala Asp Thr Lys Ile Ala Asp Ser Lys Arg Ala Phe Glu Leu Gin Lys Ser Ala Phe Ser Glu Glu Val Asn Ile Lys Thr Ala Glu Ala Gin Leu Ala Tyr Glu Leu Gin Gly Ala Arg Glu Gin Gin Lys Ile Arg Gin Glu Glu Ile Glu Ile Glu Val Val Gin Arg Lys Lys Gin Ile Ala Val Glu Ala Gin Glu Ile Leu Arg Thr Asp Lys Glu Leu Ile Ala Thr Val Arg Arg Pro Ala Glu Ala Glu Ala His Arg Ile Gin Gin Ile Ala Glu Gly Glu Lys Val Lys Gin Val Leu Leu Ala Gin Ala Glu Ala Glu Lys Ile Arg Lys Ile Gly Glu Ala Glu Ala Ala Val Ile Glu Ala Met Gly Lys Ala Glu Ala Glu Arg Met Lys Leu Lys Ala Glu Ala Tyr Gln Lys Tyr Gly Asp Ala Ala Lys Met Ala Leu Val Leu Glu Ala Leu Pro Gin Ile Ala Ala Lys Ile Ala Ala Pro Leu Thr Lys Val Asp Glu Ile Val Val Leu Ser Gly Asp Asn Ser Lys Val Thr Ser Glu Val Asn Arg Leu Leu Ala Glu Leu Pro Ala Ser Val His Ala Leu Thr Gly Val Asp Leu Ser Lys Ile Pro Leu Ile Lys Lys Ala Thr Gly Val Gin Val

Claims (15)

Claims

1. A method for diagnosing a disease comprising the step detecting in a sample from a patient an autoantibody binding to flotillin1 and/or flotillin2.

2. A polypeptide comprising flotillin1 and/or flotillin2 or a variant thereof, which is preferably immobilized, more preferably on a solid carrier.

3. A use of the polypeptide according to claim 2 for the diagnosis of a disease, preferably comprising the step detecting in a sample an autoantibody binding to flotillin1 and/or flotillin2.

4. The polypeptide according to claim 2 for use in the treatment of a disease.

5. An autoantibody, preferably an isolated autoantibody, binding to flotillin1 and/or flotillin2, wherein the autoantibody is preferably in complex with the polypeptide according to claim 2.

6. A method for isolating an autoantibody binding to flotillin1 and/or flotillin2, comprising the steps a) contacting a sample comprising the autoantibody with the polypeptide according to claim 2 under conditions compatible with formation of a complex, wherein said autoantibody binds to said polypeptide, b) isolating the complex formed in step a), c) dissociating the complex isolated in step b), and d) separating the autoantibody from the polypeptide.

7. A pharmaceutical composition or medical device, preferably diagnostic device, comprising the polypeptide according to claim 2.

8. A test kit for the diagnosis of a disease, which test kit comprises the polypeptide according to claim 2, wherein preferably the test kit comprises, in addition, a means for detecting a complex comprising the polypeptide according to claim 2 and an autoantibody binding to flotillin1 and/or flotillin2.

9. The method, polypeptide use, autoantibody, pharmaceutical composition, device or test kit according to any of claims 1, 3, 4 and 8, wherein the patient has or the disease is associated with one or more, preferably two or more symptoms selected from the group comprising elevated cell number in CSF, intrathecal lgG synthesis, oligoclonal bands in CSF, MRZ reaction in CSF, impairment of vision, preferably of visual acuity, optic neuritis, headache, spinal cord lesions and brain lesions.

10. The method, polypeptide, use, autoantibody, pharmaceutical composition, device or test kit according to any of claims 1, 3, 4, 8 and 9, wherein the disease is a neurological disease, preferably a demyelinating disease, preferably a disease of the CNS, preferably of the encephalon, preferably associated with inflammation of the optical nerve.

11. The polypeptide, autoantibody, method, pharmaceutical composition, device or test kit according to any of claims 1 to 10, wherein the polypeptide is provided in the form of a cell comprising a nucleic acid encoding said polypeptide or in the form of a tissue comprising said polypeptide.

12. The polypeptide, autoantibody, method, pharmaceutical composition, device or test kit according to any of claims 1 to 11, wherein the polypeptide is a recombinant and/or isolated polypeptide.

13. The method according to claim 1, 3, 6 and 9 to 12, wherein the sample is a bodily fluid comprising antibodies, preferably selected from the group comprising whole-blood, serum, cerebrospinal fluid and saliva.

14. The method, polypeptide, use, autoantibody, pharmaceutical composition, device or test kit according to any of claims 6 to 13, wherein both a polypeptide comprising flotillin- 1 or a variant thereof and a polypeptide comprising flotillin- 2 or a variant thereof are present and preferably part of a complex.

15. The method, polypeptide, use, autoantibody, pharmaceutical composition, device or test kit according to any of claims 1, 5, 6 and 9 to 14, wherein the autoantibody binds to a complex comprising flotililn1 and flotillin2.