Improved detection of nmda receptor autoantibodies

Abstract

The present invention relates to a support coated with eukaryotic cells overexpressing a polypeptide comprising a human NMDA receptor or a variant thereof for use in a microscopic immunofluorescence assay; a support comprising said cells and mock transfected cells, wherein said cells and said mock transfected cells are spatially separated; a kit comprising said cells or said support, and the use of said cells or said support or a polypeptide comprising a human NMDA receptor or a variant thereof for producing a signal free of artefacts for detecting autoantibodies directed against a human NMDA receptor.

Description

[0001] The present invention relates to a support for microscopic immunofluorescence analysis coated with eukaryotic cells overexpressing a polypeptide comprising a human NMDA receptor or a variant thereof; a support comprising said cells and mock transfected cells, wherein said cells and said mock transfected cells are spatially separated; a kit comprising said cells or said support, and the use of said cells or said support or a polypeptide comprising a human NMDA receptor or a variant thereof for the production of a pseudosignal-free signal for the detection of autoantibodies against the human NMDA receptor.

[0002] Antibodies against NMDA-type glutamate receptors (NMDARs) are a specific marker of NMDA receptor autoimmune encephalitis, an inflammatory brain type autoimmune disease that was first described in 2007 and is still widely underdiagnosed.

[0003] NMDARs belong to the ionotropic glutamate receptors and are named after their ability to be activated by the synthetic amino acid N-methyl-D-aspartate (NMDA). They are located in the postsynaptic membrane and form cation channels of great importance for synaptic transmission and plasticity. The receptors consist of two subunits, NR1 and NR2. Their activity is regulated by the binding of ligands such as the neurotransmitter glutamate. In the serum of patients with NMDA receptor autoimmune encephalitis, autoantibodies against the extracellular epitope of the NR1 subunit are present. These can be determined by immunohistochemical detection procedures or recombinant assays.

[0004] The actual routine clinical course that occurs at various stages is typical in the case of NMDA receptor autoimmune encephalitis. In 100% of those affected, a flu-like prodromal phase (hypothermia, headache, and fatigue) is followed by a psychotic phase with severe behavioral and personality changes, delusions, thought disorder, and hallucinations. Due to these characteristics, most patients ultimately receive psychiatric treatment, and in many cases, the initial diagnosis is drug-induced psychosis. In the following stages, there is progression to consciousness disorders, hypoventilation, seizures, autonomic instability, and movement disorders. Due to the severity of this disease, including symptoms such as coma, status epilepticus, etc., many affected individuals receive long-term treatment under intensive care.

[0005] Ovarian tumors (teratomas) containing nerve cells are found in most young female patients. These cases involve paraneoplastic syndrome (PNS) in NMDA receptor autoimmune encephalitis. The probability of associated tumor disease is approximately 60% on average, although this depends on age and gender. NMDA receptor autoimmune encephalitis is increasingly diagnosed not only in young women but also in older patients, women without teratomas, men (some with testicular teratomas), and children.

[0006] The prognosis of the patient is improved by appropriate immunomodulatory therapy and, in PNS, the tumor should be detected as early as possible and resected. In about 75% of cases, a substantial regression of symptoms can be achieved. However, 25% of patients die or have severe neurologic deficits. Survivors lose memory during the disease and are at risk of relapse of the encephalitis syndrome, which occurs in particular if the tumor is resected too late or not at all or if the tumor cannot be found.

[0007] The diagnosis of NMDA receptor autoimmunity encephalitis is based on the combination of characteristic clinical presentation with supportive results of brain MRI, EEG and CSF analysis, if necessary, and detection of anti-glutamate receptor NMDA type antibodies. Infectious encephalitis, especially HSV and other autoimmune etiologies (limbic encephalitis with autoantibodies against Hu, Ma2, CV2 and doublecortin) must be excluded by differential diagnosis. In general, antibodies against NMDA receptors should be determined in all encephalitis patients without detection of a pathogen and in suspected cases of limbic encephalitis. When positive serological results are obtained, a thorough investigation for teratomas should be performed.

[0008] Indirect immunofluorescence (IFT) is a simple and modern method that enables highly sensitive monospecific detection of NMDA receptor autoantibodies. A kit comprising transfected cells for in vitro diagnostic use is commercially available (EUROIMMUN Medizinische Labordiagnostika AG, product number FA 112d-1003-51). It can be used for high-throughput screening of hundreds of patient samples in an automated manner under high-throughput conditions.

[0009] This assay format is reported to have an excellent sensitivity of 98.1% and a specificity of 100%. However, differences between the results obtained using the standardized immunofluorescence kit product and other assay formats have been observed. While it is not yet clear whether such differences are caused by a defect associated with these commercial kits or any reference assay, there is still a need for continuous optimization.

[0010] Dalmau et al. (2007) disclose that paraneoplastic NMDA receptor autoimmunity encephalitis associated with ovarian teratomas is associated with autoantibodies against the NR1 / NR2 heteromer of the receptor. Transfected HEK293 cells containing plasmids of the rodent NR1, NR2A or NR2B subunits of the NMDA receptor were used. (Dalmau, J., Tuzun, E., Wu, H., Masjuan, J., Rossi, J. E., Voloschin, A., Baehring, J. M., Shimazaki, H., Koide, R., King, D., Mason, W., Sansing, L. H., Dichter, M. A., Rosenfeld, M. R., Lynch, D. R. (2007) Paraneoplastic Anti-N-methyl-D-aspartate Receptor Encephalitis Associated with Ovarian Teratoma, Ann Neurol, 61(1): 25-36).

[0011] McCracken et al. (2017) claim that standard clinical laboratory kits have specificity but in rare cases produce inconclusive results or produce results that are not sensitive or false negative. (McCracken, L., Zhang, J., Greene, M., Crivaro, A., Gonzales, J., Kamoun, M., Lancaster, E. (2017) Improving the antibody-based evaluation of autoimmune encephalitis, Neurology.org / nn).

[0012] Probst et al. (2014) discuss the technical challenges of developing and improving standardized serological test systems for the detection of autoantibodies, including NMDA receptor autoantibodies (Probst, C., Saschenbrecker, S., Stoecker, W., Komorowski, L. (2014) Anti-neural autoantibodies: Current diagnostic challenges. Multiple Sclerosis and Related Disorders. 3, 303-320).

[0013] US 7,972,796 discloses a method of diagnosing or determining the cause of an autoimmune encephalitis comprising the step of testing a biological sample for antibodies against the NMDA receptor NR1 subunit.

[0014] The underlying problem of the present invention is to provide new tests and related creations, methods and uses for diagnosing or aiding in the diagnosis of a neurological autoimmune disease, in particular NMDA receptor autoimmune encephalitis, with maximum diagnostic reliability, with particular emphasis on sensitivity and specificity.

[0015] The underlying problem of the present invention is solved by the subject matter of the appended independent and dependent claims.

[0016] In a first aspect, the problem is solved by a support for microscopic immunofluorescence analysis coated with eukaryotic cells, preferably mammalian cells, overexpressing a polypeptide comprising a human NMDA receptor or a variant thereof.

[0017] In a second aspect, the underlying problem of the present invention is solved by a support comprising cells according to the invention and preferably mock transfected cells, wherein the cells and the mock transfected cells are spatially separated, wherein the support is preferably a glass slide for microscopic immunofluorescence analysis.

[0018] In a preferred embodiment, the cells are transiently transfected with a human NMDA receptor or a variant thereof.

[0019] In a preferred embodiment, the cells permanently express a human NMDA receptor or a variant thereof.

[0020] In a preferred embodiment, the cells are fixed.

[0021] In a preferred embodiment, the cells are fixed using formalin or acetone.

[0022] In a preferred embodiment, the cells bind to autoantibodies against the human NMDA receptor, which are preferably bound to a secondary antibody.

[0023] In a preferred embodiment, the support further comprises one or more additional cells, preferably all cells from the group comprising cells overexpressing GABAB or a variant thereof, cells overexpressing LGI1 or a variant thereof, cells overexpressing AMPAR1 / R2 or a variant thereof, cells overexpressing CASPR2 or a variant thereof, cells overexpressing DPPX or a variant thereof, cells overexpressing Hu or a variant thereof, cells overexpressing Ma2 or a variant thereof, cells overexpressing CV2 or a variant thereof and cells overexpressing Bigraphin or a variant thereof, which are spatially separated from the cells overexpressing the human NMDA receptor and, if present, the mock transfected cells.

[0024] In a third aspect, the underlying problem of the present application is solved by a kit comprising the cell or support according to the present application, said kit further comprising one or more reagents selected from the group comprising secondary antibodies, preferably labeled with a detectable marker, washing solutions, positive controls, negative controls, detergents, cover slips, sample mounting agents and physiological salt solutions, preferably PBS.

[0025] In a fourth aspect, the problem is solved by the manufacture of a kit or support or composition for the diagnosis of NMDA receptor autoimmun encephalitis using a vector comprising a nucleic acid encoding a polypeptide comprising a human NMDA receptor or a variant thereof or a cell according to the present application, preferably a kit according to the present application.

[0026] In a fifth aspect, the problem is solved by a method comprising the steps of

[0027] a) contacting the support according to the present application with a sample,

[0028] b) washing the cells and

[0029] c) detecting autoantibodies against the human NMDA receptor bound to the support in step a), and, if a support comprising a second cell is used, optionally detecting autoantibodies against a neural autoantigen overexpressed by the other cell.

[0030] In a preferred embodiment, in step c) the autoantibodies are detected using a secondary antibody labeled with a fluorescent dye.

[0031] In a sixth embodiment, the problem is solved by the production of a false- free signal for the detection of autoantibodies against the NMDA receptor using a polypeptide or cell or support comprising a human NMDA receptor or a variant thereof.

[0032] In a seventh embodiment, the problem is solved by the highly specific diagnosis of NMDA receptor autoimmun encephalitis using a polypeptide or cell or support comprising a human NMDA receptor or a variant thereof.

[0033] In a preferred embodiment, the variant of the NMDA receptor is the NR1 subunit or a variant thereof.

[0034] The present application is based on the surprising finding of the inventors that the overexpression of a rodent NMDA receptor or a subunit thereof or a variant thereof in eukaryotic cells is associated with the appearance of false signals, in particular long processes of the cells and abnormally symmetrical spherical false signals, all of which stain positive when contacted with a labeled secondary antibody. These false signals blur the image of the positively stained cells or can be confused with the cells specifically recognized by the NMDA receptor autoantibodies, thus confusing untrained medical personnel and leading to false positive or false negative diagnoses.

[0035] The present application is based on the surprising finding of the present inventors that if cells overexpressing human NMDA receptors instead of rodent NMDA receptors are used, no false positives can be obtained, preferably images for immunofluorescence analysis.

[0036] Without wishing to be bound by this, the present inventors hypothesize that when overexpressed in eukaryotic cell lines, in particular human cell lines, rat NMDA receptor subunits are more prone to accumulate in protrusion-like or globular aggregates consisting of unfolded proteins outside the cell expressing the subunit, which can be bound and stained in a non-specific manner with secondary antibodies having a detectable dye. Such aggregates can thus blur the stained cells or can be confused with the stained cells, the presence of which generally indicates that the sample is positive.

[0037] In preferred embodiments, the mammalian cells are cell lines from mammals, preferably from humans. Exemplary cell lines include, but are not limited to, HEK293, T-Rex-HeLa, T-Rex-CHO and T-REx TM - Jurkat cells and derivatives thereof.

[0038] In preferred embodiments, the term "human NMDAR receptor" as used herein refers to a receptor comprising a human NR1 subunit or a combination of a NR1 subunit with another receptor subunit such as NR2. The presence of a NR1 subunit is essential as it comprises the autoantibody binding site. When a NR1 subunit is expressed in a mammalian cell, preferably in the presence of less than equimolar amounts of another receptor subunit such as NR2, the NR1 subunit has the ability to form NR1 homodimers, which are also capable of binding the autoantibody. In preferred embodiments, the human NR1 subunit has the sequence shown in SEQ ID NO 1. In preferred embodiments, the human NR2 subunit has the sequence shown in SEQ ID NO 2 or SEQ ID NO 4.

[0039] The teachings of the present application can not only be used with polypeptides, in particular polypeptides comprising the natural sequence of a polypeptide such as a NR1 subunit of a human NMDA receptor, or nucleic acids having the exact sequence explicitly mentioned in the present application (e.g. by function, name, sequence or accession number) or implicitly mentioned, but also with variants of such polypeptides or nucleic acids.

[0040] In preferred embodiments, the term "variant" as used herein can refer to at least one fragment of the referenced full-length sequence, more specifically one or more amino acid or nucleic acid sequences that are truncated at one or both ends by one or more amino acids relative to the full-length sequence. Such fragments comprise or encode a peptide having at least 6, 7, 8, 10, 12, 15, 20, 25, 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 850, 870, 890, 900, 910, 920, or 930 contiguous amino acids of the original sequence or a variant thereof. The total length of the variant can be at least 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 750, 850, 870, 890, 900, 910, 920, or 930 or more amino acids.

[0041] The term "variant" not only relates to at least one fragment, but also to a polypeptide comprising an amino acid sequence having at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, preferably at least 99.3%, identity to the mentioned reference amino acid sequence or fragment thereof, wherein amino acids other than those necessary for the biological activity (e.g. the ability of an antigen to bind to an (auto)antibody or the folding or structure of a polypeptide) are deleted or substituted and / or one or more of such necessary amino acids are replaced in a conservative manner and / or amino acids are added such that the biological activity of the polypeptide is retained. The state of the art comprises various methods which can 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, 3rdedition. 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 2.0. Bioinformatics, 23, 2947-2948) is used with default settings.

[0042] In a preferred embodiment, the polypeptides and variants thereof can additionally comprise chemical modifications, such as isotopic labeling or covalent modifications, such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, methylation, hydroxylation, etc. The person skilled in the art is familiar with methods of modifying polypeptides. Any modification is designed so as not to eliminate the biological activity of the variant.

[0043] Furthermore, variants can also be generated by fusion of the polypeptide, fragments or variants thereof to the N- or / and C-terminus of other known polypeptides or variants thereof and comprise an active portion or domain which, when aligned with the active portion of the reference sequence, preferably has a sequence identity of up to 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99%, wherein the term "active portion" as used herein refers to an amino acid sequence which is shorter than the full-length amino acid sequence or, in the case of nucleic acid sequences, encodes a nucleic acid sequence which is smaller than the full-length amino acid sequence, respectively, and / or is a variant of the native sequence but retains at least a certain biological activity.

[0044] In preferred embodiments, the term "variant" of a nucleic acid includes a nucleic acid whose complementary strand hybridizes to the reference or wild-type nucleic acid, preferably under stringent conditions. Stringency of hybridization reactions can be readily determined by one of ordinary skill in the art and is typically an empirical calculation. In general, longer probes require higher temperatures for proper annealing, while shorter probes require milder temperatures. Hybridization generally depends on the ability of the complementary strands of DNA to anneal to each other under the appropriate conditions of temperature, ionic strength, and pH, and it is influenced by the base composition and sequence of the probe. As a general proposition, the more similar the sequence of the probe to the target sequence, the higher the ionic strength that can be tolerated. Thus, higher salt concentrations make conditions more stringent. Other details and explanations regarding stringency of hybridization reactions can be found in Ausubel, F. M. (1995), Current Protocols in Molecular Biology. John Wiley & Sons, Inc. In addition, one of skill in the art can follow the instructions given in the manuals Mannheim GmbH (1993) The DIG System Users Guide for Filter Hybridization, Boehringer Mannheim GmbH, Mannheim, Germany and Liebl, W., Ehrmann, M., Ludwig, W., and Schleifer, K. H. (1991) International Journal of Systematic Bacteriology 41 :255-260 regarding how to identify DNA sequences by hybridization means. In preferred embodiments, stringent conditions apply to any hybridization, i.e. hybridization occurs only if the probe has 70% or more identity to the target sequence. Probes with a lower degree of identity to the target sequence can hybridize, but such hybrids are unstable and will be removed in a washing step under stringent conditions, e.g. reducing the concentration of salt to 2x SSC, or optionally subsequently to 0.5x SSC, while the temperature is in increasing order of preference about 50°C-68°C, about 52°C-68°C, about 54°C-68°C, about 56°C-68°C, about 58°C-68°C, about 60°C-68°C, about 62°C-68°C, about 64°C-68°C, about 66°C-68°C. In particularly preferred embodiments, the temperature is about 64°C-68°C or about 66°C-68°C. The concentration of salt can be adjusted to 0.2x SSC or even 0.1x SSC. Nucleic acid sequences can be isolated which have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the reference or wild-type sequence.In preferred embodiments, the term variant of a nucleic acid sequence, as used herein, refers to any nucleic acid sequence encoding the same amino acid sequence as the reference nucleic acid sequence and variants thereof, in accordance with the degeneracy of the genetic code.

[0045] The variant of the polypeptide has a biological activity. In preferred embodiments, this biological activity is the ability to specifically bind to autoantibodies that bind to NMDA receptors, preferably human NMDA receptors, as found in patients having an autoimmune disease associated with such autoantibodies, preferably an autoimmune disease associated with neurological diseases such as epilepsy, encephalitis and PNS. For example, whether a variant of the polypeptide has this biological activity can be checked by determining whether the variant of interest binds to autoantibodies from a patient sample that bind to wild-type human NMDA receptors, preferably as determined by indirect immunofluorescence as described in the experimental section of the present application.

[0046] Various publications can be used to design variants of the receptor. Sharma et al. disclose that the N-terminal domain is necessary for epitope- autoantibody recognition (Sharma, R., A-Saleem, F. H., Puligedda, R. D., Rattelle, A., Lynch, D. R. and Dessain, S. K. (2018) Membrane-bound and soluble forms of an NMDA receptor extracellular domain retain epitopes targeted in auto-immune encephalitis, BMC Biotechnology 18: 41). They show that it can be used in fusion proteins. It is important to retain at least one NR1 subunit comprising amino acids 25-380 of the NR1 subunit as the inventors of US 7,972,976 have shown that this is the antigenic portion necessary for recognition of autoantibodies. Further guidance based on site-directed mutagenesis can be found by Gleichman et al. (2012) who identified an epitope comprising N368 and G369 (Gleichman, A. J., Spruce, L. A., Dalmau, J., Seeholzer, S. H. and Lynch, D. R. (2012) Anti-NMDA Receptor Encephalitis Antibody binding Is Dependent on Amino Acid Identity of a Small Region within the GluN1 Amino Terminal Domain, J. Neuroscience, 32(32) 11083-11094). In preferred embodiments, the skilled person will take into account that one or more amino acid residue features of the human sequence should be retained when designing variants or fragments. In more preferred embodiments, the variant comprises one or more, preferably at least two, three, four, five or six, more preferably all of the residues from the group comprising Arg5, Leu9, Val17, Ser 159, Lys212, Leu267 and Leu415 (referenced to SEQ ID NO 1 and highlighted in Figure 6 the sequence below), while preferably other amino acid residues can be modified or absent as long as they are not necessary for biological activity or detection purposes against autoantibodies to NR1.

[0047] In preferred embodiments, the cells according to the application overexpress a polypeptide comprising a human NMDA receptor or variant. In preferred embodiments, the term "overexpress" as used herein means that the cell has been transiently or stably (in the sense that the nucleic acid has been integrated into the genome of the cell) transfected with a nucleic acid comprising a nucleic acid sequence encoding a polypeptide comprising a human NMDA receptor or variant thereof under the control of a promoter. Thus, the transfected cell expresses more of the polypeptide recognized by the autoantibody than a cell of the same type would normally express, possibly at least 10%, 20%, 30%, 50%, 100%, 200% or 500% more as judged by quantitative Western blotting. The promoter can be an inducible promoter which allows induction of expression by addition of an inducer. The person skilled in the art is familiar with protocols and vectors for transient overexpression of polypeptides in eukaryotic cells, such as the pTriEx system of Novagen, as well as protocols and vectors for stable transfection of eukaryotic cells, such as the pcDNA TM 4 / TO vector system.

[0048] In preferred embodiments, fixed mammalian cells can be used. In preferred embodiments, the term "fixed" cell as used herein refers to a cell which has been treated with a reactive compound to obtain the effect that the cell no longer has metabolic activity, but still presents its epitopes for immunostaining with antibodies and their subsequent detection (e.g. by fluorescence). More preferably, the reactive compound is selected from the group comprising acetone, formalin, methanol and ethanol or mixtures thereof, preferably all of them. The person skilled in the art is familiar with protocols which can be used to prepare fixed cells. Basically, cells attached to a solid support are washed by using a washing buffer and then brought into contact, e.g. by immersion, with the reactive compound. Pure acetone or formalin or an aqueous dilution of the reactive compound can be used.

[0049] According to the present application, cells are on a support for microscopic immunofluorescence analysis. This support can be a glass slide. Cells on the glass slide can be covered with a mounting buffer. The sample mounting agent is a liquid which helps to maintain a close to physiological pH, to maintain the molecular structure of any diagnostic relevant molecule and its epitope, compatible with the emission of the fluorescent signal, and to prevent premature loss of fluorescence due to bleaching of the fluorophore. At the same time, its optical properties match the other buffers used, in particular its refractive index allows for efficient microscopic fluorescence analysis. The sample mounting agent comprises a matrix component, which is preferably selected from the group comprising water, glycerol, natural oils or plastics or mixtures thereof, preferably water and glycerol. It can further comprise an anti-fade component which reduces bleaching, which is preferably selected from the group comprising NPG (n-propyl gallate), DABCO (1,4-diazabicyclo[2.2.2]octane), 4POBN ((4-pyridyl-1-oxide)-N-tert-butyl nitrone) and PPD (p-phenylenediamine). Various compositions and methods are described in the prior art, for example in "Mountants and Antifades" (http: / / www.wrightcellimaging.com / mountants.html) published by Wright Cell Imaging Facility, Toronto Western Research Institute University Health Network, Krenek et al. (1989) Comparison of antifading agents used in immunofluorescence, J. Immunol. Meth 117, 91-97 and Nairn et al. (1969) Microphotometry in Immunofluorescence, Clin. Exp. Immunol. 4, 697-705. https: / / de.scribd.com / document / 47879592 / Mountants-Antifades

[0050] A coverslip can be placed on top of the composition comprising the sample and the sample mounting agent. Glass slides with coverslips (FB112d-1005-1) are available from EUROIMMUN Medizinische Labordiagnostika, AG. However, any support compatible with the microscopic analysis of the fluorescent pattern can be used. The support can comprise mock transfected cells which have been transfected with a vector identical to the one used to transfect the cells overexpressing the polypeptide comprising the NMDA receptor but without the nucleic acid encoding the latter. This mock transfected cells can serve as a negative control. The support is configured for analysis using an immunofluorescence microscope.

[0051] ​In a preferred embodiment, the carrier can comprise a region comprising cells according to the application. In addition, the carrier can comprise additional regions. The regions are preferably surrounded by a hydrophobic surface. Each of these regions can contain cells overexpressing another autoantigen, preferably selected from the group comprising GABAA, GABAB, Lgl l, AMPAR1 / R2, CASPR2, DPPX, Hu, Ma2, CV2, dual binding protein or variants thereof. Suitable autoantigens and their sequences and variants are described in US 15 / 023131 (GABAA), US 8685656 (GABAB), US 9250250 (Lgl l), Lai, M., Hughes, E.G., Peng, X., Zhou, L., Gleichman, A.J., Shu, H., Mata, S., Kremens, D., Vitaliani, R., Geschwind, M.D., Bataller, L., Kalb, R.G., Davis, R., Graus, F., Lynch, D.R., Balice-Gordon, R., Dalmau, J. (2009) AMPA receptor antibodies in limbic encephalitis alter synaptic receptor location, Ann. Neurol. 65(4), 424-434 (AMPAR1 / R2), US 9188587 (CASPR2), US 9719993 (DPPX), US 6193948 / US 5603934 / US 5807705 (Hu), US 7026450 (Ma2), US 7329499 (CV2) and in the Genbank database code AAH34376.1. All database codes represent the sequence online at the earliest priority date of the present application. The cells according to the application are preferably spatially separated from any mock transfected cells or cells expressing another autoantigen, such that the fluorescent signal can be assigned to a specific autoantigen, e.g. human NMDA receptor. In a preferred embodiment, the carrier can comprise one or more additional regions, each region comprising tissue from the group comprising tissue of mammalian hippocampus and / or mammalian cerebellum, more preferably rat hippocampus, rat cerebellum, primate hippocampus and primate cerebellum, most preferably all of them.

[0052] In a preferred embodiment, the cells are transiently transfected, which preferably means that the cells are transfected with a vector comprising a nucleic acid encoding a polypeptide comprising human NMDA receptor, but the nucleic acid is not integrated into the genome of the cells. Thus, when cultured for a long time, the cells can lose the vector and thus no longer express the polypeptide.

[0053] In preferred embodiments, the cell permanently expresses the human NMDA receptor, more preferably because the nucleic acid encoding the polypeptide comprising the NMDA receptor has been integrated into the genome. Thus, the cell can permanently express the polypeptide.

[0054] In preferred embodiments, the secondary antibody is an antibody that specifically binds to all antibodies of an antibody class, preferably IgG. Secondary antibodies usually recognize the constant domain of the class, but can also recognize other epitopes common to antibodies of the target class, such as conformational epitopes spanning the 3D structure. A large fraction of them is commercially available, e.g. from Thermo Fisher. They can be monoclonal or polyclonal antibodies. The secondary antibody can specifically bind to all isotypes of the antibody class. For example, a secondary antibody against IgG class antibodies can bind to IgGl, IgG2, IgG3 and IgG4 isotypes. This can be achieved by using a mixture comprising antibodies that specifically bind to each IgG isotype or a single antibody that reacts with all target isotypes as secondary antibody against the class of antibodies, preferably IgG class antibodies. The use of secondary antibodies is explained in Kruger, N.J., Detection of Polypeptides on Blots Using Secondary Antibodies, in The Protein Protocols Handbook (J.M. Walker ed.), p. 967, Vol. 1996, Springer.

[0055] The autoantibody to be detected or the secondary antibody used preferably specifically binds to the human NMDA receptor or the autoantibody to be detected, respectively. "Specifically binds" preferably means that the binding reaction is stronger than the binding reaction of the antibody to a non-specific binding partner with a dissociation constant of 1 x 10 -5 M, more preferably 1 x 10 -7 M, more preferably 1 x 10 -8 M, more preferably 1 x 10 -9 M, more preferably 1 x 10 -10 M, more preferably 1 x 10 -11 M, more preferably 1 x 10 -12 M (as determined by surface plasmon resonance using a Biacore device at 25°C in PBS buffer at pH 7).

[0056] In preferred embodiments, the cell binds to an autoantibody against the human NMDA receptor and the secondary antibody binds to said antibody. In more preferred embodiments, the secondary antibody recognizes an IgG class antibody. For immunofluorescence analysis, the secondary antibody can comprise a detectable fluorescent label, more preferably FITC (fluorescein isothiocyanate).

[0057] In a preferred embodiment, the method according to the application comprises the step of providing a support according to the application. The support can then be contacted with a sample suspected of comprising autoantibodies under conditions allowing any autoantibodies to bind to the cells and the NMDA receptor or variant thereof expressed by the cells. The sample can then be removed and the support with the cells can be washed to remove any remaining sample. A second autoantibody or similar agent or means that binds to said autoantibodies and carries a detectable label such as a fluorescent dye can then be contacted with the support under conditions allowing the formation of a complex between any bound autoantibodies and the secondary antibody. The support can then be washed to remove unbound secondary antibody. Finally, the presence of autoantibodies is detected by checking whether the secondary antibody can be detected, preferably by immunofluorescence, more preferably by fluorescence emitted by luciferin or a derivative thereof, most preferably FITC.

[0058] In a preferred embodiment, the cells, methods, kits, polypeptides or supports are used for the diagnosis of a disease, preferably a neurological autoimmune disease, more preferably from the group comprising paraneoplastic syndromes, autoimmune encephalitis, limbic encephalitis and epilepsy. In addition, the cells, methods, kits, polypeptides or supports are used for the diagnosis of teratomas.

[0059] In a preferred embodiment, the term "diagnosis" as used herein is used in its broadest sense and can refer to any type of procedure aimed at obtaining information that helps to assess whether a patient has or is likely to have or is more likely than an average subject or a comparative subject (the latter preferably having similar symptoms) to have a certain disease or condition in the past, at the time of diagnosis or in the future, to ascertain how a disease progresses or can develop in the future, or to assess the responsiveness of one or more patients to a certain treatment, such as the administration of an immunosuppressive drug, or to ascertain whether a sample is from such a patient. Such information can be obtained for clinical diagnostic purposes, but also for general research purposes, such as determining the proportion of subjects in a patient cohort or population that have the disease. In other words, the term "diagnosis" not only includes diagnosis, but also prediction and / or monitoring of a disease or condition, including monitoring of the response of one or more patients to the administration of a drug or a candidate drug, for example to determine its efficacy. While the results can be assigned to a particular patient for clinical diagnostic applications and can be communicated to the physician or institution that treats said patient, this is not necessarily the case for other applications, such as in diagnoses for research purposes, where it can be sufficient to assign the results to an anonymous sample of a patient.

[0060] In preferred embodiments, the methods and products according to the application can be used for interaction studies, including determining whether a candidate drug or other compound will interfere with the binding of autoantibodies to NMDA receptors, or whether it will affect any downstream processes or their binding strength to their targets such as the NR1 subunit. In preferred embodiments, they can be used to monitor the immune response, more preferably the appearance and / or titer of antibodies against NMDARs, after e.g. administration of an immunogenic composition comprising NMDARs or immunogenic variants thereof to a mammal, which can be a mammal other than a human, e.g. an experimental animal.

[0061] In many cases, it is sufficient for diagnosis to merely detect autoantibodies, in other words to determine whether a sample contains a detectable level of antibodies. If autoantibodies can be detected, this is information that helps the clinician to diagnose and indicates an increased likelihood that the patient has the disease.

[0062] The skilled person will understand that a clinician will not base his conclusion whether a patient has or can have a disease, condition or disorder on a single diagnostic parameter, but will need to consider other aspects, e.g. the presence of other autoantibodies, markers, blood parameters, a 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, p. 3. The value of a diagnostic agent or method also lies in excluding one disease, thereby allowing the possibility of indirectly diagnosing another disease. In preferred embodiments, the meaning of any symptom or disease referred to throughout this application is in line with the understanding of the skilled person at the date of filing of this application or, preferably, the earliest priority date of this application as evidenced by textbooks and scientific publications. It should be mentioned that the methods or uses or products of the present application cannot be used on their own to arrive at a definitive final diagnosis.

[0063] In preferred embodiments, the term “diagnosis” can also refer to a method or agent for distinguishing between two or more diseases associated with similar or identical symptoms (e.g. autoimmune encephalitis and encephalitis caused by an infection). Optionally, the presence or absence of antibodies against viral antigens can be determined according to the present application, e.g. in relation to viruses causing encephalitis.

[0064] In preferred embodiments, the term "diagnosis" can also refer to a method or agent for selecting the most promising treatment regimen for a patient. In other words, the method or agent can involve selecting a treatment regimen for a subject. For example, the detection of autoantibodies can indicate that an immunosuppressive therapy is to be selected, which can include administering one or more immunosuppressive drugs to the patient.

[0065] In preferred embodiments, any information or data demonstrating the presence or absence of autoantibodies can be communicated to the patient or to the physician treating the patient, preferably by telephone, in writing or via the internet (e.g. as an email or text message).

[0066] In preferred embodiments, the term "autoantibody" as used herein refers to an antibody that specifically binds to an endogenous molecule of the animal (preferably a mammal, more preferably a human) from which the autoantibody was produced, wherein the level of such antibody is more preferably elevated compared to an average healthy subject. In most preferred embodiments, the autoantibody is an autoantibody that binds to a NMDA receptor of a mammal (preferably a human). The autoantibody can have a sequence of the constant region of an antibody from the animal (preferably a human), making it an autoantibody, but the variable region is capable of specifically binding to an endogenous molecule of the animal, more specifically a NMDA receptor. In preferred embodiments, the autoantibody is isolated and / or purified from a sample, preferably from a tissue, serum, plasma, blood or CSF of the animal (preferably a human). The autoantibody is a polyclonal natural antibody from the animal, rather than a synthetic or recombinant antibody.

[0067] The method according to the present application is preferably an in vitro method.

[0068] According to the present application, a kit is provided comprising the cell or support and further comprising one or more, preferably all, reagents from the group comprising secondary antibodies (preferably labeled with a detectable label), washing solution, positive control, negative control, detergent, cover glass, sample mounting agent and physiological salt solution (preferably PBS) or salts necessary for the preparation thereof. In preferred embodiments, the positive control is a diluted sample, preferably serum or CSF, from a patient suffering from NMDA receptor autoimmunity encephalitis or a monoclonal antibody against the human NMDA receptor, which preferably binds to the NR1 subunit recognized by the secondary antibody. The negative control can be a diluted sample from a healthy subject (e.g. blood donor). The kit can comprise instructions on how to perform the assay. Preferably, the secondary antibody is a secondary antibody against IgG class antibodies (preferably human IgG class antibodies).

[0069] In preferred embodiments, the present application provides the use of the cell, polypeptide, support for the manufacture of a kit and composition for the diagnosis of a disease.

[0070] In a preferred embodiment, any method or use according to the application can be used for non-diagnostic purposes, i.e. to determine the presence of autoantibodies binding to human NMDA receptors for purposes other than diagnosing a patient. For example, the method or use can be used for testing in vitro the efficiency of a medical device designed to remove autoantibodies from a patient's blood, wherein said testing is performed on a liquid other than the patient's blood. After use of the medical device on the patient, its ability to remove autoantibodies can be checked by flowing a solution containing antibodies against human NMDA receptors through the device, and then confirming the absence or the presence of less antibodies in the solution that has flowed through the device using a method according to the application (i.e. showing that the device still has the ability to remove antibodies from the solution).

[0071] In another preferred embodiment, the method can be used to confirm the reliability of a diagnostic assay, and can comprise detecting antibodies against human NMDA receptors in a solution that is not a sample from a patient to be diagnosed, but that is known to contain antibodies against human NMDA receptors, preferably at a known concentration. For example, it can be a recombinant antibody or a sample from an anonymous patient whose identity cannot be traced, diluted in a dilution buffer such as PBS. Alternatively, the solution can be a negative control containing no antibodies, to check the background. Such a method can be run in parallel with, after or before the diagnostic method. In a preferred embodiment, any method or use according to the application can be intended for generating an autoantibody profile, preferably for detecting a disease in a mammal, preferably a human.

[0072] In a preferred embodiment, any method or use according to the application can be used for identifying a subject at risk of having or developing a disease and / or a tumor.

[0073] In a preferred embodiment, the method can be used to detect antibodies, preferably autoantibodies, in a solution that is not a sample from a mammal to be diagnosed, or that is not a sample for the purpose of providing a diagnosis, in particular a diagnosis of a neurological disease such as NMDA receptor encephalitis.

[0074] In a preferred embodiment, the application provides a device for analyzing a sample from a patient to detect autoantibodies against human NMDA receptors, said autoantibodies being indicative of an increased likelihood of a disease or of developing a disease, said device comprising:

[0075] a. a support comprising means to capture autoantibodies from the sample when the sample is in contact with the support, wherein the means are cells and the support is a support according to the application,

[0076] b. a detectable means capable of binding the antibodies captured by the support when the detectable means is in contact with the support, wherein the detectable means is preferably a labeled secondary antibody capable of binding the autoantibodies captured on the support,

[0077] c. optionally a means for removing (preferably by washing) any sample from the support and the detectable means;

[0078] d. a detection device for detecting the presence of the detectable means and transforming the result into an electrical signal, for example a fluorescence reader connected to a software capable of recognizing in the cell images taken by the fluorescence reader or by a camera the pattern features of the cells positively stained for human NMDAR overexpression, and

[0079] optionally a means for receiving the electrical signal from the detection device and determining whether the signal level is indicative of an increased likelihood of suffering or developing the disease by comparing it to the pattern features of wild type or unstained cells (preferably by mock transfected cells or cells not positively stained with the autoantibodies binding to the human NMDA receptor on the same support) or to an input reference value obtained with a sample of a healthy subject, or by comparing the signal level obtained with one sample to the signal level obtained with a second sample obtained at a later time point (preferably at least one month later).

[0080] Figure 1A Immunofluorescence analysis of HEK293 cells transiently transfected with a vector encoding the rat NR1 subunit of the NMDA receptor stained with a sample of a patient suffering from NMDA receptor encephalitis. White arrows mark artifacts in the form of protrusions, which are false landscapes generated by the immunostaining of NR1 aggregates.

[0081] Figure 1B Immunofluorescence analysis of HEK293 cells permanently transfected with the human NR1 subunit. These images are clearly free of artifacts.

[0082] Figure 2A Immunofluorescence analysis of HEK293 cells transiently transfected with a vector encoding the rat NR1 subunit of the NMDA receptor stained with a 1 : 100 dilution of a sample of a patient not suffering from NMDA receptor encephalitis. Spheres containing NR1 aggregates are stained, possibly mistaken for positively stained cells.

[0083] Figure 2B Immunofluorescence analysis of HEK293 permanently transfected with a vector encoding the human NR1 subunit of the NMDA receptor stained with the same sample. No spherical artifacts are observed.

[0084] Figure 3AImmunofluorescence analysis of HEK293 cells transiently transfected with a vector encoding the NR1 subunit of the rat NMDA receptor stained with samples from patients with NMDA receptor encephalitis. Spheres comprising NR1 aggregates are stained and the stained positive cells are blurred, which is shown in Figure 3B

[0085] Figure 4A Figure 4B Figure 4C Immunofluorescence analysis of HEK293 cells stained with 1 : 10 samples from patients with NMDA receptor encephalitis. The cells transiently express rat NR1 Figure 4A ), transiently express human NR1 Figure 4B ) or permanently express human NR1 Figure 4C In Figure 4A , both protrusions and spherical artifacts can be observed. In Figure 4B , there are spherical artifacts, but the protrusions are less evident. In Figure 4C , there are no artifacts.

[0086] Figure 5A Figure 5B Figure 5C Immunofluorescence analysis of HEK293 cells stained with 1 : 10 samples from patients without NMDA receptor encephalitis. The cells transiently express rat NR1 Figure 5A ), transiently express human NR1 Figure 5B ) or permanently express human NR1 Figure 5C In Figure 5A , spherical artifacts convey the impression of cells positively stained against autoantibodies against NMDAR. In Figure 5B , the spherical artifacts are less evident. In Figure 5C , there are no artifacts.

[0087] Figure 6 Alignment of the amino acid sequence of the human NR1 subunit ("Query 1") with the amino acid sequence of the rat NR1 subunit ("Sbjct 1"). In the human sequence, the different amino acids are shown in bold.

[0088] Sequence:

[0089] The present invention comprises a series of novel nucleic acid and polypeptide sequences, more specifically SEQ ID NO 1 (polypeptide sequence human NR1):

[0090]

[0091] ​​​​​SEQ ID NO 2 (polypeptide sequence NMDAR subunit NR2a)

[0092]

[0093] SEQ ID NO 3 (plasmid for transient expression of human NR1 in cells)

[0094]

[0095]

[0096] SEQ ID NO 4 (polypeptide sequence NMDAR subunit NR2b)

[0097]

[0098] SEQ ID NO 5 plasmid for permanent expression or stable transfection of human NR1 in cells

[0099]

[0100]

[0101]

[0102] SEQ ID NO 6 (plasmid for transient expression of rat NR1 in cells)

[0103]

[0104]

[0105]

[0106]

[0107] The application is further illustrated by the following non-limiting examples from which other characteristics, embodiments, aspects and advantages of the application can be derived. Example:

[0108] HEK 293 cells

[0109] Various patient samples were analyzed using comparative slides containing fixed HEK 293 cells transiently overexpressing rat NR1, transiently overexpressing human NR1 and permanently overexpressing human NR1. Cells were grown at 37°C and 5% C02 in DMEM medium containing 10% heat inactivated fetal bovine serum and IX antibiotic-antimycotic (Invitrogen #15240). For transient transfection with rat or human NR1, plasmids with sequence SEQ ID NO 6 or SEQ ID NO 3 respectively were used. To stably transfect with human NR1, a plasmid with sequence SEQ ID NO 5 was used. Invitrogen T-Rex TM The protocol provided by the system (catalog number K1020-01 from 2011) was used for stable transfection. However, ExGen500 (catalog number 12783652, Thermofisher) was used instead of Lipofectamin. For transient transfection, the ExGen500 protocol was used. Fixed cells were prepared by contacting cells grown on microscope slides with acetone, washing the cells in PBS and then incubating in 100% acetone.

[0110] Methods and reagents

[0111] The methods and reagents used were according to the manufacturer's instructions in the IIFT Neurology Mosaics (EUROIMMUN Medizinische Labordiagnostika AG, product number FA112d-1). This test system is centered on the incubation of a substrate with a diluted patient sample. If the reaction is positive, specific antibodies of the IgA, IgG and IgM classes attach to the antigen. In a second step, the attached antibodies are stained with FITC-labeled anti-human antibodies and made visible with a fluorescence microscope.

[0112] Briefly, human serum samples were diluted 1 : 10 or 1 : 100 in PBS-Tween and then vortexed for 2 seconds. 30 μΐ of sample per area was incubated for 30 minutes using the TITERPLANE technique, then washed for 1 s in PBS-Tween followed by incubation in PBS-Tween for 5 minutes in a cuvette for thorough washing. Then 25 μΐ of secondary antibody conjugate was applied and incubated for 30 minutes using the TITERPLANE technique, followed by washing for 1 s in PBS-Tween and then incubation in PBS-Tween for 5 minutes in a cuvette for thorough washing.

[0113] After incubation, the carrier with the field was covered with up to 10 μl of sample sealant per field and a glass coverslip, and then analysed using fluorescence microscopy with a EUROSTAR microscope (EUROIMMUN Medizinische Labordiagnostika AG, Lübeck).

[0114] Results:

[0115] The results are shown in Figures 1 to 5, as described in detail in the legends associated with these figures.

[0116] In general, it is clear that the use of HEK293 cells transiently expressing the NR1 subunit from the rat NMDA receptor produced artefacts in the form of protrusions and spheroids that blurred the image of the stained cells and even possibly confused them.

[0117] The use of HEK293 cells transiently expressing the NR1 subunit from the human receptor produced spheroids, but significantly fewer protrusions.

[0118] The use of HEK293 cells permanently overexpressing NR1 from the human receptor did not produce either type of artefact. The use of these cells avoids confusion for the staff analysing the images and false-positive results. SEQUENCE LISTING <110> EUROIMMUN Medizinische Labordiagnostika AG - a PerkinElmer company) <120> Improved detection of NMDA receptor autoantibodies <130> 19PP055EP <160> 6 <170> PatentIn 3.5 version <210> 1 <211> 938 <212> PRT <213> Homo sapiens <400> 1 Met Ser Thr Met Arg Leu Leu Thr Leu Ala Leu Leu Phe Ser Cys Ser 1 5 10 15 Val Ala Arg Ala Ala Cys Asp Pro Lys Ile Val Asn Ile Gly Ala Val 20 25 30 Leu Ser Thr Arg Lys His Glu Gin Met Phe Arg Glu Ala Val Asn Gin 35 40 45 Ala Asn Lys Arg His Gly Ser Trp Lys He Gin Leu Asn Ala Thr Ser 50 55 60 Val Thr His Lys Pro Asn Ala He Gin Met Ala Leu Ser Val Cys Glu 65 70 75 80 Asp Leu He Ser Ser Gin Val Tyr Ala He Leu Val Ser His Pro Pro 85 90 95 Thr Pro Asn Asp His Phe Thr Pro Thr Pro Val Ser Tyr Thr Ala Gly 100 105 110 Phe Tyr Arg He Pro Val Leu Gly Leu Thr Thr Arg Met Ser He Tyr 115 120 125 Ser Asp Lys Ser He His Leu Ser Phe Leu Arg Thr Val Pro Pro Tyr 130 135 140 Ser His Gin Ser Ser Val Trp Phe Glu Met Met Arg Val Tyr Ser Trp 145 150 155 160 Asn His He He Leu Leu Val Ser Asp Asp His Glu Gly Arg Ala Ala 165 170 175 Gln Lys Arg Leu Glu Thr Leu Leu Glu Glu Arg Glu Ser Lys Ala Glu 180 185 190 Lys Val Leu Gin Phe Asp Pro Gly Thr Lys Asn Val Thr Ala Leu Leu 195 200 205 Met Glu Ala Lys Glu Leu Glu Ala Arg Val Ile Ile Leu Ser Ala Ser 210 215 220 Glu Asp Asp Ala Ala Thr Val Tyr Arg Ala Ala Ala Met Leu Asn Met 225 230 235 240 Thr Gly Ser Gly Tyr Val Trp Leu Val Gly Glu Arg Glu Ile Ser Gly 245 250 255 Asn Ala Leu Arg Tyr Ala Pro Asp Gly Ile Leu Gly Leu Gin Leu Ile 260 265 270 Asn Gly Lys Asn Glu Ser Ala His Ile Ser Asp Ala Val Gly Val Val 275 280 285 Ala Gin Ala Val His Glu Leu Leu Glu Lys Glu Asn Ile Thr Asp Pro 290 295 300 Pro Arg Gly Cys Val Gly Asn Thr Asn Ile Trp Lys Thr Gly Pro Leu 305 310 315 320 Phe Lys Arg Val Leu Met Ser Ser Lys Tyr Ala Asp Gly Val Thr Gly 325 330 335 Arg Val Glu Phe Asn Glu Asp Gly Asp Arg Lys Phe Ala Asn Tyr Ser 340 345 350 Ile Met Asn Leu Gin Asn Arg Lys Leu Val Gin Val Gly lie Tyr Asn 355 360 365 Gly Thr His Val lie Pro Asn Asp Arg Lys lie lie Trp Pro Gly Gly 370 375 380 Glu Thr Glu Lys Pro Arg Gly Tyr Gin Met Ser Thr Arg Leu Lys lie 385 390 395 400 Val Thr lie His Gin Glu Pro Phe Val Tyr Val Lys Pro Thr Leu Ser 405 410 415 Asp Gly Thr Cys Lys Glu Glu Phe Thr Val Asn Gly Asp Pro Val Lys 420 425 430 Lys Val lie Cys Thr Gly Pro Asn Asp Thr Ser Pro Gly Ser Pro Arg 435 440 445 His Thr Val Pro Gin Cys Cys Tyr Gly Phe Cys lie Asp Leu Leu lie 450 455 460 Lys Leu Ala Arg Thr Met Asn Phe Thr Tyr Glu Val His Leu Val Ala 465 470 475 480 Asp Gly Lys Phe Gly Thr Gin Glu Arg Val Asn Asn Ser Asn Lys Lys 485 490 495 Glu Trp Asn Gly Met Met Gly Glu Leu Leu Ser Gly Gin Ala Asp Met 500 505 510 Ile Val Ala Pro Leu Thr Ile Asn Asn Glu Arg Ala Gin Tyr Ile Glu 515 520 525 Phe Ser Lys Pro Phe Lys Tyr Gin Gly Leu Thr Ile Leu Val Lys Lys 530 535 540 Glu Ile Pro Arg Ser Thr Leu Asp Ser Phe Met Gin Pro Phe Gin Ser 545 550 555 560 Thr Leu Trp Leu Leu Val Gly Leu Ser Val His Val Val Ala Val Met 565 570 575 Leu Tyr Leu Leu Asp Arg Phe Ser Pro Phe Gly Arg Phe Lys Val Asn 580 585 590 Ser Glu Glu Glu Glu Glu Glu Asp Ala Leu Thr Leu Ser Ser Ala Met Trp 595 600 605 Phe Ser Trp Gly Val Leu Leu Asn Ser Gly Ile Gly Glu Gly Ala Pro 610 615 620 Arg Ser Phe Ser Ala Arg Ile Leu Gly Met Val Trp Ala Gly Phe Ala 625 630 635 640 Met Ile Ile Val Ala Ser Tyr Thr Ala Asn Leu Ala Ala Phe Leu Val 645 650 655 Leu Asp Arg Pro Glu Glu Arg lie Thr Gly lie Asn Asp Pro Arg Leu 660 665 670 Arg Asn Pro Ser Asp Lys Phe lie Tyr Ala Thr Val Lys Gin Ser Ser 675 680 685 Val Asp lie Tyr Phe Arg Arg Gin Val Glu Leu Ser Thr Met Tyr Arg 690 695 700 His Met Glu Lys His Asn Tyr Glu Ser Ala Ala Glu Ala lie Gin Ala 705 710 715 720 Val Arg Asp Asn Lys Leu His Ala Phe lie Trp Asp Ser Ala Val Leu 725 730 735 Glu Phe Glu Ala Ser Gin Lys Cys Asp Leu Val Thr Thr Gly Glu Leu 740 745 750 Phe Phe Arg Ser Gly Phe Gly lie Gly Met Arg Lys Asp Ser Pro Trp 755 760 765 Lys Gin Asn Val Ser Leu Ser lie Leu Lys Ser His Glu Asn Gly Phe 770 775 780 Met Glu Asp Leu Asp Lys Thr Trp Val Arg Tyr Gin Glu Cys Asp Ser 785 790 795 800 Arg Ser Asn Ala Pro Ala Thr Leu Thr Phe Glu Asn Met Ala Gly Val 805 810 815 Phe Met Leu Val Ala Gly Gly lie Val Ala Gly lie Phe Leu lie Phe 820 825 830 lie Glu lie Ala Tyr Lys Arg His Lys Asp Ala Arg Arg Lys Gln Met 835 840 845 Gln Leu Ala Phe Ala Ala Val Asn Val Trp Arg Lys Asn Leu Gln Asp 850 855 860 Arg Lys Ser Gly Arg Ala Glu Pro Asp Pro Lys Lys Lys Ala Thr Phe 865 870 875 880 Arg Ala lie Thr Ser Thr Leu Ala Ser Ser Phe Lys Arg Arg Arg Ser 885 890 895 Ser Lys Asp Thr Ser Thr Gly Gly Gly Arg Gly Ala Leu Gln Asn Gln 900 905 910 Lys Asp Thr Val Leu Pro Arg Arg Ala lie Glu Arg Glu Glu Gly Gln 915 920 925 Leu Gln Leu Cys Ser Arg His Arg Glu Ser 930 935 <210> 2 <211> 1258 <212> PRT <213> Homo sapiens <400> 2 Met Gly Arg Val Gly Tyr Trp Thr Leu Leu Val Leu Pro Ala Leu Leu 1 5 10 15 Val Trp Arg Gly Pro Ala Pro Ser Ala Ala Ala Glu Lys Gly Pro Pro 20 25 30 Ala Leu Asn Ile Ala Val Met Leu Gly His Ser His Asp Val Thr Glu 35 40 45 Arg Glu Leu Arg Thr Leu Trp Gly Pro Glu Gln Ala Ala Gly Leu Pro 50 55 60 Leu Asp Val Asn Val Val Ala Leu Leu Met Asn Arg Thr Asp Pro Lys 65 70 75 80 Ser Leu Ile Thr His Val Cys Asp Leu Met Ser Gly Ala Arg Ile His 85 90 95 Gly Leu Val Phe Gly Asp Asp Thr Asp Gln Glu Ala Val Ala Gln Met 100 105 110 Leu Asp Phe Ile Ser Ser His Thr Phe Val Pro Ile Leu Gly Ile His 115 120 125 Gly Gly Ala Ser Met Ile Met Ala Asp Lys Asp Pro Thr Ser Thr Phe 130 135 140 Phe Gln Phe Gly Ala Ser Ile Gln Gln Gln Ala Thr Val Met Leu Lys 145 150 155 160 Ile Met Gln Asp Tyr Asp Trp His Val Phe Ser Leu Val Thr Thr Ile 165 170 175 Phe Pro Gly Tyr Arg Glu Phe lie Ser Phe Val Lys Thr Thr Val Asp 180 185 190 Asn Ser Phe Val Gly Trp Asp Met Gin Asn Val lie Thr Leu Asp Thr 195 200 205 Ser Phe Glu Asp Ala Lys Thr Gin Val Gin Leu Lys Lys lie His Ser 210 215 220 Ser Val lie Leu Leu Tyr Cys Ser Lys Asp Glu Ala Val Leu lie Leu 225 230 235 240 Ser Glu Ala Arg Ser Leu Gly Leu Thr Gly Tyr Asp Phe Phe Trp lie 245 250 255 Val Pro Ser Leu Val Ser Gly Asn Thr Glu Leu lie Pro Lys Glu Phe 260 265 270 Pro Ser Gly Leu lie Ser Val Ser Tyr Asp Asp Trp Asp Tyr Ser Leu 275 280 285 Glu Ala Arg Val Arg Asp Gly lie Gly lie Leu Thr Thr Ala Ala Ser 290 295 300 Ser Met Leu Glu Lys Phe Ser Tyr lie Pro Glu Ala Lys Ala Ser Cys 305 310 315 320 Tyr Gly Gin Met Glu Arg Pro Glu Val Pro Met His Thr Leu His Pro 325 330 335 Phe Met Val Asn Val Thr Trp Asp Gly Lys Asp Leu Ser Phe Thr Glu 340 345 350 Glu Gly Tyr Gln Val His Pro Arg Leu Val Val Ile Val Leu Asn Lys 355 360 365 Asp Arg Glu Trp Glu Lys Val Gly Lys Trp Glu Asn His Thr Leu Ser 370 375 380 Leu Arg His Ala Val Trp Pro Arg Tyr Lys Ser Phe Ser Asp Cys Glu 385 390 395 400 Pro Asp Asp Asn His Leu Ser Ile Val Thr Leu Glu Glu Ala Pro Phe 405 410 415 Val Ile Val Glu Asp Ile Asp Pro Leu Thr Glu Thr Cys Val Arg Asn 420 425 430 Thr Val Pro Cys Arg Lys Phe Val Lys Ile Asn Asn Ser Thr Asn Glu 435 440 445 Gly Met Asn Val Lys Lys Cys Cys Lys Gly Phe Cys Ile Asp Ile Leu 450 455 460 Lys Lys Leu Ser Arg Thr Val Lys Phe Thr Tyr Asp Leu Tyr Leu Val 465 470 475 480 Thr Asn Gly Lys His Gly Lys Lys Val Asn Asn Val Trp Asn Gly Met 485 490 495 Ile Gly Glu Val Val Tyr Gln Arg Ala Val Met Ala Val Gly Ser Leu 500 505 510 Thr Ile Asn Glu Glu Arg Ser Glu Val Val Asp Phe Ser Val Pro Phe 515 520 525 Val Glu Thr Gly Ile Ser Val Met Val Ser Arg Ser Asn Gly Thr Val 530 535 540 Ser Pro Ser Ala Phe Leu Glu Pro Phe Ser Ala Ser Val Trp Val Met 545 550 555 560 Met Phe Val Met Leu Leu Ile Val Ser Ala Ile Ala Val Phe Val Phe 565 570 575 Glu Tyr Phe Ser Pro Val Gly Tyr Asn Arg Asn Leu Ala Lys Gly Lys 580 585 590 Ala Pro His Gly Pro Ser Phe Thr Ile Gly Lys Ala Ile Trp Leu Leu 595 600 605 Trp Gly Leu Val Phe Asn Asn Ser Val Pro Val Gln Asn Pro Lys Gly 610 615 620 Thr Thr Ser Lys Ile Met Val Ser Val Trp Ala Phe Phe Ala Val Ile 625 630 635 640 Phe Leu Ala Ser Tyr Thr Ala Asn Leu Ala Ala Phe Met Ile Gln Glu 645 650 655 Glu Phe Val Asp Gln Val Thr Gly Leu Ser Asp Lys Lys Phe Gln Arg 660 665 670 Pro His Asp Tyr Ser Pro Pro Phe Arg Phe Gly Thr Val Pro Asn Gly 675 680 685 Ser Thr Glu Arg Asn Ile Arg Asn Asn Tyr Pro Tyr Met His Gln Tyr 690 695 700 Met Thr Lys Phe Asn Gln Lys Gly Val Glu Asp Ala Leu Val Ser Leu 705 710 715 720 Lys Thr Gly Lys Leu Asp Ala Phe Ile Tyr Asp Ala Ala Val Leu Asn 725 730 735 Tyr Lys Ala Gly Arg Asp Glu Gly Cys Lys Leu Val Thr Ile Gly Ser 740 745 750 Gly Tyr Ile Phe Ala Thr Thr Gly Tyr Gly Ile Ala Leu Gln Lys Gly 755 760 765 Ser Pro Trp Lys Arg Gln Ile Asp Leu Ala Leu Leu Gln Phe Val Gly 770 775 780 Asp Gly Glu Met Glu Glu Leu Glu Thr Leu Trp Leu Thr Gly Ile Cys 785 790 795 800 His Asn Glu Lys Asn Glu Val Met Ser Ser Gln Leu Asp Ile Asp Asn 805 810 815 Met Ala Gly Val Phe Tyr Met Leu Ala Ala Ala Met Ala Leu Ser Leu 820 825 830 Ile Thr Phe Ile Trp Glu His Leu Phe Tyr Trp Lys Leu Arg Phe Cys 835 840 845 Phe Thr Gly Val Cys Ser Asp Arg Pro Gly Leu Leu Phe Ser Ile Ser 850 855 860 Arg Gly Ile Tyr Ser Cys Ile His Gly Val His Ile Glu Glu Lys Lys 865 870 875 880 Lys Ser Pro Asp Phe Asn Leu Thr Gly Ser Gln Ser Asn Met Leu Lys 885 890 895 Leu Leu Arg Ser Ala Lys Asn Ile Ser Ser Met Ser Asn Met Asn Ser 900 905 910 Ser Arg Met Asp Ser Pro Lys Arg Ala Ala Asp Phe Ile Gln Arg Gly 915 920 925 Ser Leu Ile Met Asp Met Val Ser Asp Lys Gly Asn Leu Met Tyr Ser 930 935 940 Asp Asn Arg Ser Phe Gln Gly Lys Glu Ser Ile Phe Gly Asp Asn Met 945 950 955 960 Asn Glu Leu Gln Thr Phe Val Ala Asn Arg Gln Lys Asp Asn Leu Asn 965 970 975 Asn Tyr Val Phe Gln Gly Gln His Pro Leu Thr Leu Asn Glu Ser Asn 980 985 990 Pro Asn Thr Val Glu Val Ala Val Ser Thr Glu Ser Lys Ala Asn Ser 995 1000 1005 Arg Pro Arg Gln Leu Trp Lys Lys Ser Val Asp Ser Ile Arg Gln 1010 1015 1020 Asp Ser Leu Ser Gln Asn Pro Val Ser Gln Arg Asp Glu Ala Thr 1025 1030 1035 Ala Glu Asn Arg Thr His Ser Leu Lys Ser Pro Arg Tyr Leu Pro 1040 1045 1050 Glu Glu Met Ala His Ser Asp Ile Ser Glu Thr Ser Asn Arg Ala 1055 1060 1065 Thr Cys His Arg Glu Pro Asp Asn Ser Lys Asn His Lys Thr Lys 1070 1075 1080 Asp Asn Phe Lys Arg Ser Val Ala Ser Lys Tyr Pro Lys Asp Cys 1085 1090 1095 Ser Glu Val Glu Arg Thr Tyr Leu Lys Thr Lys Ser Ser Ser Pro 1100 1105 1110 Arg Asp Lys Ile Tyr Thr Ile Asp Gly Glu Lys Glu Pro Gly Phe 1115 1120 1125 His Leu Asp Pro Pro Gln Phe Val Glu Asn Val Thr Leu Pro Glu 1130 1135 1140 Asn Val Asp Phe Pro Asp Pro Tyr Gln Asp Pro Ser Glu Asn Phe 1145 1150 1155 Arg Lys Gly Asp Ser Thr Leu Pro Met Asn Arg Asn Pro Leu His 1160 1165 1170 Asn Glu Glu Gly Leu Ser Asn Asn Asp Gln Tyr Lys Leu Tyr Ser 1175 1180 1185 Lys His Phe Thr Leu Lys Asp Lys Gly Ser Pro His Ser Glu Thr 1190 1195 1200 Ser Glu Arg Tyr Arg Gln Asn Ser Thr His Cys Arg Ser Cys Leu 1205 1210 1215 Ser Asn Met Pro Thr Tyr Ser Gly His Phe Thr Met Arg Ser Pro 1220 1225 1230 Phe Lys Cys Asp Ala Cys Leu Arg Met Gly Asn Leu Tyr Asp Ile 1235 1240 1245 Asp Glu Asp Gln Met Leu Gln Glu Thr Gly 1250 1255 <210> 3 <211> 8431 <212> DNA <213> Artificial Sequence <220> <223> Plasmid for transient expression of human NR1 in cells <400> 3 ggggaattgt gagcggataa caattccccg gagttaatcc gggaccttta attcaaccca 60 acacaatata ttatagttaa ataagaatta ttatcaaatc atttgtatat taattaaaat 120 actatactgt aaattacatt ttatttacaa tcaaaggaga tataccatgt ctaccatgcg 180 cctgctgacg ctcgccctgc tgttctcctg ctccgtcgcc cgtgccgcgt gcgaccccaa 240 gatcgtcaac attggcgcgg tgctgagcac gcggaagcac gagcagatgt tccgcgaggc 300 cgtgaaccag gccaacaagc ggcacggctc ctggaagatt cagctcaatg ccacctccgt 360 cacgcacaag cccaacgcca tccagatggc tctgtcggtg tgcgaggacc tcatctccag 420 ccaggtctac gccatcctag ttagccatcc acctaccccc aacgaccact tcactcccac 480 ccctgtctcc tacacagccg gcttctaccg catacccgtg ctggggctga ccacccgcat 540 gtccatctac tcggacaaga gcatccacct gagcttcctg cgcaccgtgc cgccctactc 600 ccaccagtcc agcgtgtggt ttgagatgat gcgtgtctac agctggaacc acatcatcct 660 gctggtcagc gacgaccacg agggccgggc ggctcagaaa cgcctggaga cgctgctgga 720 ggagcgtgag tccaaggcag agaaggtgct gcagtttgac ccagggacca agaacgtgac ggccctgctg atggaggcga aagagctgga ggcccgggtc atcatccttt ctgccagcga 840 ggacgatgct gccactgtat accgcgcagc cgcgatgctg aacatgacgg gctccgggta 900 cgtgtggctg gtcggcgagc gcgagatctc ggggacgcc ctgcgctacg ccccagacgg 960 catcctcggg ctgcagctca tcaacggcaa gaacgagtcg gcccacatca gcgacgccgt gggcgtggtg gcccaggccg tgcacgagct cctcgagaag gagaacatca ccgacccgcc 1080. gcggggctgc gtgggcaaca ccaacatctg gaagaccggg ccgctcttca agagagtgct gatgtcttcc aagtatgcgg atggggtgac tggtcgcgtg gagttcaatg aggatgggga 1200. ccggaagttc gccaactaca gcatcatgaa cctgcagaac cgcaagctgg tgcaagtggg catctacaat ggcacccacg tcatccctaa tgacaggaag atcatctggc caggcggaga gacagagaag cctcgagggt accagatgtc caccagactg aagattgtga cgatccacca 1380 ggagcccttc gtgtacgtca agcccacgct gagtgatggg acatgcaagg aggagttcac 1440 agtcaacggc gacccagtca agaaggtgat ctgcaccggg cccaacgaca cgtcgccggg 1500 cagcccccgc cacacggtgc ctcagtgttg ctacggcttt tgcatcgacc tgctcatcaa 1560 gctggcacgg accatgaact tcacctacga ggtgcacctg gtggcagatg gcaagttcgg 1620 cacacaggag cgggtgaaca acagcaacaa gaaggagtgg aatgggatga tgggcgagct 1680 gctcagcggg caggcagaca tgatcgtggc gccgctaacc ataaacaacg agcgcgcgca 1740 gtacatcgag ttttccaagc ccttcaagta ccagggcctg actattctgg tcaagaagga 1800 gattccccgg agcacgctgg actcgttcat gcagccgttc cagagcacac tgtggctgct 1860 ggtggggctg tcggtgcacg tggtggccgt gatgctgtac ctgctggacc gcttcagccc 1920 cttcggccgg ttcaaggtga acagcgagga ggaggaggag gacgcactga ccctgtcctc 1980 ggccatgtgg ttctcctggg gcgtcctgct caactccggc atcggggaag gcgcccccag 2040 atcgttctca gcgcgcatcc tgggcatggt gtgggccggc tttgccatga tcatcgtggc 2100 ctcctacacc gccaacctgg cggccttcct ggtgctggac cggccggagg agcgcatcac 2160 gggcatcaac gaccctcggc tgaggaaccc ctcggacaag tttatctacg ccacggtgaa 2220 gcagagctcc gtggatatct acttccggcg ccaggtggag ctgagcacca tgtaccggca 2280 tatggagaag cacaactacg agagtgcggc ggaggccatc caggccgtga gagacaacaa 2340 gctgcatgcc ttcatctggg actcggcggt gctggagttc gaggcctcgc agaagtgcga 2400 cctggtgacg actggagagc tgtttttccg ctcgggcttc ggcataggca tgcgcaaaga 2460 cagcccctgg aagcagaacg tctccctgtc catcctcaag tcccacgaga atggcttcat 2520 ggaagacctg gacaagacgt gggttcggta tcaggaatgt gactcgcgca gcaacgcccc 2580 tgcgaccctt acttttgaga acatggccgg ggtcttcatg ctggtagctg ggggcatcgt 2640 ggccgggatc ttcctgattt tcatcgagat tgcctacaag cggcacaagg atgctcgccg 2700 gaagcagatg cagctggcct ttgccgccgt taacgtgtgg cggaagaacc tgcaggatag 2760 aaagagtggt agagcagagc ctgaccctaa aaagaaagcc acatttaggg ctatcacctc 2820 caccctggct tccagcttca agaggcgtag gtcctccaaa gacacgagca ccgggggtgg 2880 acgcggcgct ttgcaaaacc aaaaagacac agtgctgccg cgacgcgcta ttgagaggga 2940 ggagggccag ctgcagctgt gttcccgtca tagggagagc taactcgagc accaccatca 3000 ccatcaccat cactaagtga ttaacctcag gtgcaggctg cctatcagaa ggtggtggct 3060 ggtgtggcca atgccctggc tcacaaatac cactgagatc gatctttttc cctctgccaa 3120 aaattatggg gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt 3180 tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg 3240 gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc 3300 ccatatgtaa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct 3360 gaaagcatgc ggaggaaatt ctccttgaag tttccctggt gttcaaagta aaggagtttg 3420 caccagacgc acctctgttc actggtccgg cgtattaaaa cacgatacat tgttattagt 3480 acatttatta agcgctagat tctgtgcgtt gttgatttac agacaattgt tgtacgtatt 3540 ttaataattc attaaattta taatctttag ggtggtatgt tagagcgaaa atcaaatgat 3600 tttcagcgtc tttatatctg aatttaaata ttaaatcctc aatagatttg taaaataggt 3660 ttcgattagt ttcaaacaag ggttgttttt ccgaaccgat ggctggacta tctaatggat 3720 tttcgctcaa cgccacaaaa cttgccaaat cttgtagcag caatctagct ttgtcgatat 3780 tcgtttgtgt tttgttttgt aataaaggtt cgacgtcgtt caaaatatta tgcgcttttg 3840 tatttctttc atcactgtcg ttagtgtaca attgactcga cgtaaacacg ttaaatagag 3900 cttggacata tttaacatcg ggcgtgttag ctttattagg ccgattatcg tcgtcgtccc 3960 aaccctcgtc gttagaagtt gcttccgaag acgattttgc catagccaca cgacgcctat 4020 taattgtgtc ggctaacacg tccgcgatca aatttgtagt tgagcttttt ggaattattt 4080 ctgattgcgg gcgtttttgg gcgggtttca atctaactgt gcccgatttt aattcagaca 4140 acacgttaga aagcgatggt gcaggcggtg gtaacatttc agacggcaaa tctactaatg 4200 gcggcggtgg tggagctgat gataaatcta ccatcggtgg aggcgcaggc ggggctggcg 4260 gcggaggcgg aggcggaggt ggtggcggtg atgcagacgg cggtttaggc tcaaatgtct 4320 ctttaggcaa cacagtcggc acctcaacta ttgtactggt ttcgggcgcc gtttttggtt 4380 tgaccggtct gagacgagtg cgattttttt cgtttctaat agcttccaac aattgttgtc 4440 tgtcgtctaa aggtgcagcg ggttgaggtt ccgtcggcat tggtggagcg ggcggcaatt 4500 cagacatcga tggtggtggt ggtggtggag gcgctggaat gttaggcacg ggagaaggtg 4560 gtggcggcgg tgccgccggt ataatttgtt ctggtttagt ttgttcgcgc acgattgtgg 4620 gcaccggcgc aggcgccgct ggctgcacaa cggaaggtcg tctgcttcga ggcagcgctt 4680 ggggtggtgg caattcaata ttataattgg aatacaaatc gtaaaaatct gctataagca 4740 ttgtaatttc gctatcgttt accgtgccga tatttaacaa ccgctcaatg taagcaattg 4800 tattgtaaag agattgtctc aagctcggaa cgctgcgctc ggtcgttcgg ctgcggcgag 4860 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 4920 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 4980 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 5040 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 5100 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 5160 cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg 5220 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 5280 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 5340 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 5400 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 5460 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 5520 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 5580 atcctttgtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 5640 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 5700 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 5760 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 5820 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 5880 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 5940 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 6000 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 6060 tttcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 6120 tgctttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 6180 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 6240 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 6300 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 6360 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gtccgcgcgt ttcctgcatc ttttaatcaa atcccaagat gtgtataac caccaaactg ccaaaaaatg aaaactgtcg acaagctctg tccgtttgct ggcaactgca agggtctcaa tcctatttgt aattattgaa taataaaaca attaaatg tcaaatttgt tttttattaa cgatacaaac caaacgcaac aagaacattt gtagtattat ctaattga aaacgcgtag ttataatcgc tgaggtaata tttaaatca ttttcaaatg attcacagtt aatttgcgac aatataattt tattttcaca taaactagac gccttgtcgt cttcttcttc gtattccttc tctttttcat ttttctcttc ataaaatta acatagttat tatcgtatcc fathers ctatcgtata gagtaattt tttgttgtca fathers tgtctttttt aatggggtgt atagtaccgc tgcgcatagt ttttctgtaa tttacaacag tgctattttc tggtagttct tcggagtgtg ttgctttaat father's father tgaatttggg atcgtcggtt ttgtacaata tgttgccggc atagtacgca gcttcttcta 7140 gttcaattac accatttttt agcagcaccg gattaacata actttccaaa atgttgtacg 7200 aaccgttaaa caaaaacagt tcacctccct tttctatact attgtctgcg agcagttgtt 7260 tgttgttaaa aataacagcc attgtaatga gacgcacaaa ctaatatcac aaactggaaa 7320 tgtctatcaa tatatagttg ctctagttat taatagtaat caattacgggg gtcattagtt 7380 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga 7440 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca 7500 atagggactt tccattgacg tcaatgggtg gactatttac ggtaaactgc ccacttggca 7560 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg 7620 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc 7680 tacgtattag tcatcgctat taccatgcat ggtcgaggtg agccccacgt tctgcttcac 7740 tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt 7800 ttgtgcagcg atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg 7860 cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct 7920 ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc 7980 gcggcgggcg ggagtcgctg cgacgctgcc ttcgccccgt gccccgctcc gccgccgcct 8040 cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg 8100 gcccttctcc ttcgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt 8160 ggctgcgtga aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg 8220 ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc 8280 tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc 8340 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattg 8400 gatcggaccg aaattaatac gactcactat a 8431 <210> 4 <211> 1484 <212> PRT <213> Homo sapiens <400> 4 Met Lys Pro Arg Ala Glu Cys Cys Ser Pro Lys Phe Trp Leu Val Leu 1 5 10 15 Ala Val Leu Ala Val Ser Gly Ser Arg Ala Arg Ser Gln Lys Ser Pro 20 25 30 Pro Ser Ile Gly Ile Ala Val Ile Leu Val Gly Thr Ser Asp Glu Val 35 40 45 Ala Ile Lys Asp Ala His Glu Lys Asp Asp Phe His His Leu Ser Val 50 55 60 Val Pro Arg Val Glu Leu Val Ala Met Asn Glu Thr Asp Pro Lys Ser 65 70 75 80 Island Island Thr Arg Island Cys Asp Leu Met Ser Asp Arg Lys Island Gln Gly 85 90 95 Val Val Phe Ala Asp Asp Thr Asp Gln Glu Ala Ile Ala Gln Ile Leu 100 105 110 Asp Phe Ile Ser Ala Gln Thr Leu Thr Pro Ile Leu Gly Ile His Gly 115 120 125 Gly Ser Ser Met Ile Met Ala Asp Lys Asp Glu Ser Ser Met Phe Phe 130 135 140 Gln Phe Gly Pro Ser Ile Glu Gln Gln Ala Ser Val Met Leu Asn Ile 145 150 155 160 Met Glu Glu Tyr Asp Trp Tyr Ile Phe Ser Ile Val Thr Thr Tyr Phe 165 170 175 Pro Gly Tyr Gln Asp Phe Val Asn Lys Ile Arg Ser Thr Ile Glu Asn 180 185 190 Ser Phe Val Gly Trp Glu Leu Glu Glu Val Leu Leu Leu Asp Met Ser 195 200 205 Leu Asp Asp Gly Asp Ser Lys Ile Gln Asn Gln Leu Lys Lys Leu Gln 210 215 220 Ser Pro Ile Ile Leu Leu Tyr Cys Thr Lys Glu Glu Ala Thr Tyr Ile 225 230 235 240 Phe Glu Val Ala Asn Ser Val Gly Leu Thr Gly Tyr Gly Tyr Thr Trp 245 250 255 Ile Val Pro Ser Leu Val Ala Gly Asp Thr Asp Thr Val Pro Ala Glu 260 265 270 Phe Pro Thr Gly Leu Ile Ser Val Ser Tyr Asp Glu Trp Asp Tyr Gly 275 280 285 Leu Pro Ala Arg Val Arg Asp Gly Ile Ala Ile Ile Thr Thr Ala Ala 290 295 300 Ser Asp Met Leu Ser Glu His Ser Phe Ile Pro Glu Pro Lys Ser Ser 305 310 315 320 Cys Tyr Asn Thr His Glu Lys Arg Ile Tyr Gln Ser Asn Met Leu Asn 325 330 335 Arg Tyr Leu Ile Asn Val Thr Phe Glu Gly Arg Asn Leu Ser Phe Ser 340 345 350 Glu Asp Gly Tyr Gln Met His Pro Lys Leu Val Ile Ile Leu Leu Asn 355 360 365 Lys Glu Arg Lys Trp Glu Arg Val Gly Lys Trp Lys Asp Lys Ser Leu 370 375 380 Gln Met Lys Tyr Tyr Val Trp Pro Arg Met Cys Pro Glu Thr Glu Glu 385 390 395 400 Gln Glu Asp Asp His Leu Ser Ile Val Thr Leu Glu Glu Ala Pro Phe 405 410 415 Val Ile Val Glu Ser Val Asp Pro Leu Ser Gly Thr Cys Met Arg Asn 420 425 430 Thr Val Pro Cys Gln Lys Arg Ile Val Thr Glu Asn Lys Thr Asp Glu 435 440 445 Glu Pro Gly Tyr Ile Lys Lys Cys Cys Lys Gly Phe Cys Ile Asp Ile 450 455 460 Leu Lys Lys Ile Ser Lys Ser Val Lys Phe Thr Tyr Asp Leu Tyr Leu 465 470 475 480 Val Thr Asn Gly Lys His Gly Lys Lys Ile Asn Gly Thr Trp Asn Gly 485 490 495 Met Ile Gly Glu Val Val Met Lys Arg Ala Tyr Met Ala Val Gly Ser 500 505 510 Leu Thr Ile Asn Glu Glu Arg Ser Glu Val Val Asp Phe Ser Val Pro 515 520 525 Phe Ile Glu Thr Gly Ile Ser Val Met Val Ser Arg Ser Asn Gly Thr 530 535 540 Val Ser Pro Ser Ala Phe Leu Glu Pro Phe Ser Ala Asp Val Trp Val 545 550 555 560 Met Met Phe Val Met Leu Leu Ile Val Ser Ala Val Ala Val Phe Val 565 570 575 Phe Glu Tyr Phe Ser Pro Val Gly Tyr Asn Arg Cys Leu Ala Asp Gly 580 585 590 Arg Glu Pro Gly Gly Pro Ser Phe Thr Ile Gly Lys Ala Ile Trp Leu 595 600 605 Leu Trp Gly Leu Val Phe Asn Asn Ser Val Pro Val Gln Asn Pro Lys 610 615 620 Gly Thr Thr Ser Lys Ile Met Val Ser Val Trp Ala Phe Phe Ala Val 625 630 635 640 Ile Phe Leu Ala Ser Tyr Thr Ala Asn Leu Ala Ala Phe Met Ile Gln 645 650 655 Glu Glu Tyr Val Asp Gln Val Ser Gly Leu Ser Asp Lys Lys Phe Gln 660 665 670 Arg Pro Asn Asp Phe Ser Pro Pro Phe Arg Phe Gly Thr Val Pro Asn 675 680 685 Gly Ser Thr Glu Arg Asn Ile Arg Asn Asn Tyr Ala Glu Met His Ala 690 695 700 Tyr Met Gly Lys Phe Asn Gln Arg Gly Val Asp Asp Ala Leu Leu Ser 705 710 715 720 Leu Lys Thr Gly Lys Leu Asp Ala Phe Ile Tyr Asp Ala Ala Val Leu 725 730 735 Asn Tyr Met Ala Gly Arg Asp Glu Gly Cys Lys Leu Val Thr Ile Gly 740 745 750 Ser Gly Lys Val Phe Ala Ser Thr Gly Tyr Gly Ile Ala Ile Gln Lys 755 760 765 Asp Ser Gly Trp Lys Arg Gln Val Asp Leu Ala Ile Leu Gln Leu Phe 770 775 780 Gly Asp Gly Glu Met Glu Glu Leu Glu Ala Leu Trp Leu Thr Gly Ile 785 790 795 800 Cys His Asn Glu Lys Asn Glu Val Met Ser Ser Gln Leu Asp Ile Asp 805 810 815 Asn Met Ala Gly Val Phe Tyr Met Leu Gly Ala Ala Met Ala Leu Ser 820 825 830 Leu Ile Thr Phe Ile Cys Glu His Leu Phe Tyr Trp Gln Phe Arg His 835 840 845 Cys Phe Met Gly Val Cys Ser Gly Lys Pro Gly Met Val Phe Ser Ile 850 855 860 Ser Arg Gly Ile Tyr Ser Cys Ile His Gly Val Ala Ile Glu Glu Arg 865 870 875 880 Gln Ser Val Met Asn Ser Pro Thr Ala Thr Met Asn Asn Thr His Ser 885 890 895 Asn Ile Leu Arg Leu Leu Arg Thr Ala Lys Asn Met Ala Asn Leu Ser 900 905 910 Gly Val Asn Gly Ser Pro Gln Ser Ala Leu Asp Phe Ile Arg Arg Glu 915 920 925 Ser Ser Val Tyr Asp Ile Ser Glu His Arg Arg Ser Phe Thr His Ser 930 935 940 Asp Cys Lys Ser Tyr Asn Asn Pro Pro Cys Glu Glu Asn Leu Phe Ser 945 950 955 960 Asp Tyr Ile Ser Glu Val Glu Arg Thr Phe Gly Asn Leu Gln Leu Lys 965 970 975 Asp Ser Asn Val Tyr Gln Asp His Tyr His His His His Arg Pro His 980 985 990 Ser Ile Gly Ser Ala Ser Ser Ile Asp Gly Leu Tyr Asp Cys Asp Asn 995 1000 1005 Pro Pro Phe Thr Thr Gln Ser Arg Ser Ile Ser Lys Lys Pro Leu 1010 1015 1020 Asp Ile Gly Leu Pro Ser Ser Lys His Ser Gln Leu Ser Asp Leu 1025 1030 1035 Tyr Gly Lys Phe Ser Phe Lys Ser Asp Arg Tyr Ser Gly His Asp 1040 1045 1050 Asp Leu Ile Arg Ser Asp Val Ser Asp Ile Ser Thr His Thr Val 1055 1060 1065 Thr Tyr Gly Asn Ile Glu Gly Asn Ala Ala Lys Arg Arg Lys Gln 1070 1075 1080 Gln Tyr Lys Asp Ser Leu Lys Lys Arg Pro Ala Ser Ala Lys Ser 1085 1090 1095 Arg Arg Glu Phe Asp Glu Ile Glu Leu Ala Tyr Arg Arg Arg Pro 1100 1105 1110 Pro Arg Ser Pro Asp His Lys Arg Tyr Phe Arg Asp Lys Glu Gly 1115 1120 1125 Leu Arg Asp Phe Tyr Leu Asp Gln Phe Arg Thr Lys Glu Asn Ser 1130 1135 1140 Pro His Trp Glu His Val Asp Leu Thr Asp Ile Tyr Lys Glu Arg 1145 1150 1155 Ser Asp Asp Phe Lys Arg Asp Ser Val Ser Gly Gly Gly Pro Cys 1160 1165 1170 Thr Asn Arg Ser His Ile Lys His Gly Thr Gly Asp Lys His Gly 1175 1180 1185 Val Val Ser Gly Val Pro Ala Pro Trp Glu Lys Asn Leu Thr Asn 1190 1195 1200 Val Glu Trp Glu Asp Arg Ser Gly Gly Asn Phe Cys Arg Ser Cys 1205 1210 1215 Pro Ser Lys Leu His Asn Tyr Ser Thr Thr Val Thr Gly Gln Asn 1220 1225 1230 Ser Gly Arg Gln Ala Cys Ile Arg Cys Glu Ala Cys Lys Lys Ala 1235 1240 1245 Gly Asn Leu Tyr Asp Ile Ser Glu Asp Asn Ser Leu Gln Glu Leu 1250 1255 1260 Asp Gln Pro Ala Ala Pro Val Ala Val Thr Ser Asn Ala Ser Thr 1265 1270 1275 Thr Lys Tyr Pro Gln Ser Pro Thr Asn Ser Lys Ala Gln Lys Lys 1280 1285 1290 Asn Arg Asn Lys Leu Arg Arg Gln His Ser Tyr Asp Thr Phe Val 1295 1300 1305 Asp Leu Gln Lys Glu Glu Ala Ala Leu Ala Pro Arg Ser Val Ser 1310 1315 1320 Leu Lys Asp Lys Gly Arg Phe Met Asp Gly Ser Pro Tyr Ala His 1325 1330 1335 Met Phe Glu Met Ser Ala Gly Glu Ser Thr Phe Ala Asn Asn Lys 1340 1345 1350 Ser Ser Val Pro Thr Ala Gly His His His His Asn Asn Pro Gly 1355 1360 1365 Gly Gly Tyr Met Leu Ser Lys Ser Leu Tyr Pro Asp Arg Val Thr 1370 1375 1380 Gln Asn Pro Phe Ile Pro Thr Phe Gly Asp Asp Gln Cys Leu Leu 1385 1390 1395 His Gly Ser Lys Ser Tyr Phe Phe Arg Gln Pro Thr Val Ala Gly 1400 1405 1410 Ala Ser Lys Ala Arg Pro Asp Phe Arg Ala Leu Val Thr Asn Lys 1415 1420 1425 Pro Val Val Ser Ala Leu His Gly Ala Val Pro Ala Arg Phe Gln 1430 1435 1440 Lys Asp Ile Cys Ile Gly Asn Gln Ser Asn Pro Cys Val Pro Asn 1445 1450 1455 Asn Lys Asn Pro Arg Ala Phe Asn Gly Ser Ser Asn Gly His Val 1460 1465 1470 Tyr Glu Lys Leu Ser Ser Ile Glu Ser Asp Val 1475 1480 <210> 5 <211> 7779 <212> DNA <213> Artificial Sequence <220> <223> Plasmids used for permanent expression or stable transfection of human NR1 in cells. <400> 5 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttaggttag gcgttttgcg ctgcttcgcg atgtacggc cagatatacg cgttgacatt 240 gattattgac tagttatta tagtaatca ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggag tatttacggt aactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taatggccc gcctggcatt 540 atgcccagta catgacctta tgggacttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatca tggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtca tgggagttg tttggaacc 720 aaaatcaacg ggactttcca aaatgtcgta acactccgc cccattgacg caatggggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc 840 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgaa 900 gctgggtacc agctgctagc aagcttgccg ccgacatgtc taccatgcgc ctgctgacgc 960 tcgccctgct gttctcctgc tccgtcgccc gtgccgcgtg cgaccccaag atcgtcaaca 1020 ttggcgcggt gctgagcacg cggaagcacg agcagatgtt ccgcgaggcc gtgaaccagg 1080 ccaacaagcg gcacggctcc tggaagattc agctcaatgc cacctccgtc acgcacaagc 1140 ccaacgccat ccagatggct ctgtcggtgt gcgaggacct catctccagc caggtctacg 1200 ccatcctagt tagccatcca cctaccccca acgaccactt cactcccacc cctgtctcct 1260 acacagccgg cttctaccgc atacccgtgc tggggctgac cacccgcatg tccatctact 1320 cggacaagag catccacctg agcttcctgc gcaccgtgcc gccctactcc caccagtcca 1380 gcgtgtggtt tgagatgatg cgtgtctaca gctggaacca catcatcctg ctggtcagcg 1440 acgaccacga gggccgggcg gctcagaaac gcctggagac gctgctggag gagcgtgagt 1500 ccaaggcaga gaaggtgctg cagtttgacc cagggaccaa gaacgtgacg gccctgctga 1560 tggaggcgaa agagctggag gcccgggtca tcatcctttc tgccagcgag gacgatgctg 1620 ccactgtata ccgcgcagcc gcgatgctga acatgacggg ctccgggtac gtgtggctgg 1680 tcggcgagcg cgagatctcg gggaacgccc tgcgctacgc cccagacggc atcctcgggc 1740 tgcagctcat caacggcaag aacgagtcgg cccacatcag cgacgccgtg ggcgtggtgg 1800 cccaggccgt gcacgagctc ctcgagaagg agaacatcac cgacccgccg cggggctgcg 1860 tgggcaacac caacatctgg aagaccgggc cgctcttcaa gagagtgctg atgtcttcca 1920 agtatgcgga tggggtgact ggtcgcgtgg agttcaatga ggatggggac cggaagttcg 1980 ccaactacag catcatgaac ctgcagaacc gcaagctggt gcaagtgggc atctacaatg 2040 gcacccacgt catccctaat gacaggaaga tcatctggcc aggcggagag acagagaagc 2100 ctcgagggta ccagatgtcc accagactga agattgtgac gatccaccag gagcccttcg 2160 tgtacgtcaa gcccacgctg agtgatggga catgcaagga ggagttcaca gtcaacggcg 2220 acccagtcaa gaaggtgatc tgcaccgggc ccaacgacac gtcgccgggc agcccccgcc 2280 acacggtgcc tcagtgttgc tacggctttt gcatcgacct gctcatcaag ctggcacgga 2340 ccatgaactt cacctacgag gtgcacctgg tggcagatgg caagttcggc acacaggagc 2400 gggtgaacaa cagcaacaag aaggagtgga atgggatgat gggcgagctg ctcagcgggc 2460 aggcagacat gatcgtggcg ccgctaacca taaaacga gcgcgcgcag tacatcgagt 2520 tttccaagcc cttcaagtac cagggcctga ctattctggt caagaaggg attccccgga 2580 gcacgctgga ctcgttcatg cagccgttcc agagcacact gtggctgctg gtggggctgt 2640 cggtgcacgt ggtggccgtg atgctgtacc tgctgggaccg cttcagcccc ttcggccggt 2700 tcaaggtgaa cagcgaggag gaggaggag acgcactgac cctgtcctcg gccatgtggt 2760 tctcctgggg cgtcctgctc aactccggca tcgggggaagg cgcccccaga tcgttctcag 2820 cgcgcatcct gggcatggtg tgggccggct ttgccatgat catcgtggcc tcctacaccg 2880 ccaacctggc ggccttcctg gtgctggacc ggccggagga gcgcatcacg ggcatcaacg 2940 accctcggct gaggaaccc tcggacaagt ttatctacgc cacggtgaag cagagctccg 3000 tggatatcta cttccggcgc caggtggagc tgagcaccat gtaccggcat atggagaagc 3060 acaactacga gagtgcggcg gaggccatcc aggccgtgag agacaacaag ctgcatgcct 3120 tcatctggga ctcggcggtg ctggagttcg aggcctcgca gaagtgcgac ctggtgacga 3180 ctggagagct gtttttccgc tcgggcttcg ccataggcat gcgcaaagac agcccctgga 3240 agcagaacgt ctccctgtcc atcctcaagt cccacgagaa tggcttcatg gaagacctgg 3300 acaagacgtg ggttcggtat caggaatgtg actcgcgcag caacgcccct gcgaccctta 3360 cttttgagaa catggccggg gtcttcatgc tggtagctgg gggcatcgtg gccgggatct 3420 tcctgatttt catcgagatt gcctacaagc ggcacaagga tgctcgccgg aagcagatgc 3480 agctggcctt tgccgccgtt aacgtgtggc ggaagaacct caggataga aagagtggta 3540 gagcagagcc tgaccctaaa aagaaagcca catttagggc tatcacctcc accctggctt 3600 ccagcttcaa gaggcgtagg tcctccaaag acacgagcac cggggtgga cgcggcgctt 3660 tgcaaacca aaaagacaca gtgctgccgc gacgcgctat tgagagggag gagggccagc 3720 tgcagctgtg ttcccgtcat agggagagct gactcgagtc tagagggccc gtttaaaccc 3780 gctgatcagc ctcgactgtg cttctagtt gccagccatc tgttgtttgc cctcccccg 3840 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa 3900 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca 3960 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg 4020 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg 4080 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 4140 ccctagcgcc cgctcctttc gctttcttc cttcctttct cgccacgttc gcggctttc 4200 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 4260 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga 4320 cggttttcg cccttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 4380 ctggaacaac actcaaccct atctcggtct attctttga tttataaggg atttgccga 4440 ttcggccta ttggttaaaa aatgagctga tttaacaaaa attaacgcg aattaattct 4500 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 4560 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc 4620 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac 4680 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact 4740 aattttttt attatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta 4800 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccggggag cttgtatatc 4860 cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata 4920 gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg 4980 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc 5040 cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc 5100 agcgcggtcc aggaccaggt ggtgccggac aaccctgg cctgggtgtg ggtgcgcggc 5160 ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc 5220 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac 5280 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat 5340 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc 5400 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg 5460 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 5520 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 5580 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct 5640 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 5700 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 5760 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 5820 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 5880 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 5940 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 6000 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 6060 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 6120 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 6180 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 6240 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 6300 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 6360 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 6420 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt 6480 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 6540 aaacaaacca ccgctggtag cggttttttt gtttgcaagc agcagattac gcgcagaaaa 6600 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 6660 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 6720 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 6780 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 6840 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 6900 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 6960 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 7020 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 7080 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 7140 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 7200 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 7260 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 7320 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 7380 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 7440 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 7500 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 7560 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 7620 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 7680 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 7740 gttccgcgca catttccccg aaaagtgcca cctgacgtc 7779 <210> 6 <211> 8967 <212> DNA <213> Artificial Sequence <220> <223> Plasmid for transient expression of rat NR1 in cells <400> 6 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca 60 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 120 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 180 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 240 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 300 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 360 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 420 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccacccat tgacgtcaat 480 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 540 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt 600 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccataagaaga 660 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt 720 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta 780 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac 840 tagagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc 900 acctcggttc taagcttgat atcgaattcc gttgctgtcg aatattggtt tgttaaggca 960 gtttctgtag aggtttctaa gagaccagtc gcgcagtccg cgctgctgtc ctttccgcct 1020 tttccgcgcg ggtgttcgag cagcgccaaa cacgcttcag cacctcggac agcatccgcc 1080 gcgctcgccc ggggctccta gagaacccgg gggcgcttga ccgcgcgcgg gcggcccgcg 1140 ggtcgtacat cgcgaggtcg tcgcactcgc gcaacccaga gccaggcccg ctgtgcccgg 1200 agctcatgag caccatgcac ctgctgacat tcgccctgct tttttcctgc tccttcgccc 1260 gcgccgcctg cgaccccaag atcgtcaaca tcggcgcggt gctgagcacg cgcaagcatg 1320 aacagatgtt ccgcgaggca gtaaaccagg ccaataagcg acacggctct tggaagatac 1380 agctcaacgc cacttctgtc acccacaagc ccaacgccat acagatggcc ctgtcagtgt 1440 gtgaggacct catctctagc caggtctacg ctatcctagt tagccacccg cctactccca 1500 acgaccactt cactcccacc cctgtctcct acacagctgg cttctacaga atccctgtcc 1560 tgggactgac tacccgaatg tccatctact ctgacaagag tatccacctg agtttccttc 1620 gcacggtgcc gccctactcc caccagtcca gcgtctggtt tgagatgatg cgagtctaca 1680 actggaacca catcatcctg ctggtcagcg acgaccacga gggacgggca gcgcagaagc 1740 gcttggagac gttgctggag gaacgggagt ccaaggcaga gaaggtgctg cagtttgacc 1800 caggaaccaa gaatgtgacg gctctgctga tggaggcccg ggaactggag gcccgggtca 1860 tcatcctttc tgcaagcgag gacgacgctg ccacagtgta ccgcgcagcc gcaatgctga 1920 acatgacggg ctctgggtac gtgtggctgg tcggggaacg cgagatctct gggaacgccc 1980 tgcgctacgc tcctgatggc atcatcggac ttcagctcat caatggcaag aatgagtcag 2040 cccacatcag tgacgccgtg ggcgtggtgg cacaggcagt tcacgaactc ctagagaagg 2100 agaatatcac tgacccaccg cggggttgcg tgggcaacac caacatctgg aagacaggac 2160 cattgttcaa gagggtgctg atgtcttcta agtatgcgga cggagtgact ggccgtgtgg 2220 aattcaatga ggatggggac cggaagtttg ccaactatag tatcatgaac ctgcagaacc 2280 gcaagctggt gcaagtgggc atctacaatg gtacccatgt catcccaaat gacaggaaga 2340 tcatctggcc aggaggag acagagaaac ctcgaggata ccagatgtcc accagacta 2400 agatagtgac aatccaccaa gagccctcg tgtacgtcaa gcccacaatg agtgatggga 2460 catgcaaga ggagttcaca gtcaatggtg acccagtgaa gaaggtgatc tgtacggggc 2520 ctaatgacac gtccccaggc agcccacgcc accagtgcc ccagtgctgc tatggctct 2580 gcatagacct gctcatcaag ctggcgcgga ccatgaattt tacctatgg gtgcacctgg 2640 tggcagatgg caagtttggc accaggagc gggtaaaaca cagcaacaa aaggagtgga 2700 acggaatgat gggcgagcta ctcagtggcc aagcggacat gattgtggca ccactgacca 2760 tcaacaatga gcgtgcgcag tacatagagt tctccaagcc cttcaagtac cagggcctga 2820 ccattttggt caagaaggag attcccagga gcacactgga ctcatttag cagccttttc 2880 agagcacact gtggttgcta gtaggactgt cagttcatgt gtggctgtg atgctgtacc 2940 tgctgaccg cttcagtccc tttggccgat tcaggtga tgtgaggag gaggaggag 3000 atgcactgac cctgtcctct gccatgtggt ttcctgggg cgtcctgctc aactccggca 3060 ttggggaagg tgccccccgg agtttctctg cacgtatcct aggcatggtg tgggctggtt 3120 tcgccatgat catagtggct tcctacactg ccaacttggc agctttcctg gtgctggatc 3180 ggcctgagga gcgcatcacg ggcatcaatg accccaggct cagaaacccc tcagacaagt 3240 tcatctacgc aactgtaaag cagagctccg tggacatcta cttccggagg caggtggagt 3300 tgagtaccat gtaccggcac atggaaaaac acaattacga gagcgcagct gaggccatcc 3360 aggctgtgcg ggacaacaag ctgcacgcct ttatctggga ctcggccgtg ctggagtttg 3420 aggcttcaca gaagtgcgat ctggtgacca cgggtgagct gttcttccgc tcaggctttg 3480 gcatcggcat gcgcaaggac agcccctgga agcagaacgt ttccctgtcc atactcaagt 3540 cccatgagaa tggcttcatg gaagatctgg ataagacatg ggttcggtat caggaatgcg 3600 actcccgcag caatgctcct gcaaccctca cttttgagaa catggcaggg gtcttcatgc 3660 tggtggctgg aggcatcgta gctgggattt tcctcatttt cattgagatc gcctacaagc 3720 gacacaagga tgcccgtagg aagcagatgc agctggcttt tgcagccgtg aacgtgtgga 3780 ggaagaacct gcaggataga aagagtggta gagcagagcc cgaccctaaa aagaaagcca 3840 catttagggc tatcacctcc accctggcct ccagcttcaa gagacgtagg tcctccaaag 3900 acacgagcac cgggggtgga cgcggcgctt tgcaaaacca aaaagacaca gtgctgccgc 3960 gacgcgctat tgagagggag gagggccagc tgcagctgtg ttcccgtcat agggagagct 4020 gagacgcccc gcccgccctc ctctgcccct cccccgcaga cagacgcacg ggacagcggc 4080 ctggcccacg cagagccccg gagcacgacg gggtcggggg aggagcactc ccagcctccc 4140 ccaggccgtg cccgcctgcc caccggtcgg ccggctggcc ggtccaccct gtcccggccc 4200 cgcgcgtgcc cccgacgtcg gagctaacgg gccgccttgt ctgtgtattt ctattttaca 4260 gcagtaccat cccactgata tcacgggccc gctcaacctc tcagatccct cggtcagcac 4320 cgtggtgtga ggccccccgg aggcgcccac ctgcccagtt agcccggcca aggacactga 4380 tgagtcctgc tgctcgggaa ggcctgaggg aagcccaccc gccccagaga ctgcccaccc 4440 tgggcctccc gtccgcctgc tctgctgcct ggcgggcagc ccctgcagga ccaaggtgcg 4500 gaccagagcg gctgaggatg ggccagagct gagccggctg ggcagggcca cagggcgctc 4560 cggcagaggc agggccctga ggtctctgag cagtggggtg aggggcctaa gtggccccgg 4620 tcggaggagt ctggagcaga aatggcagcc ccatccttcc tccagccact accccaagct 4680 acagtggggg cctatggccc cagcttgcta ggtcaccccc gacccttcct ccagcgcctg 4740 ctctctgcaa cttgatttcc acctctcc tgctgcacca ccctcccacg acatttcccc 4800 accccattca ctgggttgtc tctgaccttt cccagggcta gccttcactg ccctagtggc 4860 agtgcttcag gggtgctttc tgctcccag acatctaggg ctccagactc caagagggct 4920 gagccttctc ttctgtccgc agccacaata ggcttcctca gacgctggct cgtgatgagt 4980 cccgcacctt gggcaccagg gagcgccatc tgcctcccag tccggtgtca ctcaccccac 5040 taccttgtac atgaccagct ctcccagtgt cccagtgtct gccccaggga caccgggcgc 5100 gcacagccac ccctaatccc ggtattcagt ggtgatgcct aaaggaatgt cagaaaaaaa 5160 aaaaaaagc ggccgctcga gcatgcatct agaggatccc cggggaattc aatcgatggc 5220 cgccatggcc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 5280 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 5340 tcaatgtatc ttatcatgtc tggatcgatc gggaattaat tcggcgcagc accatggcct 5400 gaaataacct ctgaaagagg aacttggtta ggtaccttct gaggcggaaa gaaccagctg 5460 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg 5520 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca 5580 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact 5640 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta 5700 atttttttta tttatgcaga ggccgaggcc gcctcggcct gtgagctatt ccagaagtag 5760 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tgttaacagc ttggcactgg 5820 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg 5880 cagcacatcc ccccttcgcc acctggcgta atagcgaaga ggcccgcacc gatcgccctt 5940 cccaacagtt gcgtagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc 6000 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtacggg 6060 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 6120 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 6180 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 6240 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac 6300 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 6360 tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat 6420 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa 6480 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata 6540 gttaagccaa ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca 6600 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct 6660 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg 6720 aggcagtatt cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc 6780 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc 6840 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc 6900 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc 6960 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg 7020 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat ggaactggat 7080 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc 7140 acttttaaag ttctgctatg tggcgcggta ttatcccgtg atgacgccgg gcaagagcaa 7200 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agccacagaa 7260 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt 7320 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct 7380 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat 7440 gaagccatac caaacgacga gcgtgacacc acgatgccag cagcaatggc aacaacgttg 7500 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg 7560 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt 7620 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg 7680 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg 7740 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg 7800 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa 7860 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt 7920 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 7980 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 8040 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 8100 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta 8160 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 8220 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 8280 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 8340 agatacctac agcgtgagca ttgagaaagc gccacgcttc ccgaagggag aaaggcggac 8400 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 8460 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 8520 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 8580 cggttcctgg ccttttggtg gccttttgct cacatgttct ttcctgcgtt atcccctgat 8640 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg 8700 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct 8760 ctccccgcgc gttggccgat tcattaatcc agctggcacg acaggtttcc cgactggaaa 8820 gcgggcagtg agcgcaacgc aattaatgtg agttacctca ctcattaggc accccaggct 8880 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac 8940 acaggaaaca gctatgacca tgattac 8967

Claims

1. A support for microscopic immunofluorescence analysis coated with eukaryotic cells that permanently overexpress a polypeptide comprising a human NMDA receptor, wherein the human NMDA receptor comprises a human NMDA receptor NR1 subunit, wherein the NR1 subunit comprises Arg5, Leu9, Val17, Ser 159, Lys212, Leu267, and Leu415 with reference to SEQ ID NO 1, wherein the eukaryotic cells are mammalian cells.

2. The support of claim 1, wherein the cells are fixed cells.

3. The support of claim 1, wherein the cells are fixed using formalin or acetone.

4. The support of claim 1, wherein the cells bind to autoantibodies against human NMDA.

5. The support of claim 4, wherein the autoantibodies against human NMDA bind to a secondary antibody.

6. The support of claim 1, further comprising mock transfected cells, wherein the cells and the mock transfected cells are spatially separated.

7. The support of claim 1, further comprising one or more additional cells from the group consisting of cells that overexpress GABAB, cells that overexpress LGI1, cells that overexpress AMPAR1 / R2, cells that overexpress CASPR2, cells that overexpress DPPX, cells that overexpress Hu, cells that overexpress Ma2, cells that overexpress CV2, and cells that overexpress Bigraphin, the cells being spatially separated from the cells that overexpress human NMDA receptor.

8. The support of claim 1, further comprising all cells from the group consisting of cells that overexpress GABAB, cells that overexpress LGI1, cells that overexpress AMPAR1 / R2, cells that overexpress CASPR, cells that overexpress DPPX, cells that overexpress Hu, cells that overexpress Ma2, cells that overexpress CV2, and cells that overexpress Bigraphin, the cells being spatially separated from the cells that overexpress human NMDA receptor.

9. The support of claim 6, further comprising one or more cells from the group consisting of cells that overexpress GABAB, cells that overexpress LGI1, cells that overexpress AMPAR1 / R2, cells that overexpress CASPR, cells that overexpress DPPX, cells that overexpress Hu, cells that overexpress Ma2, cells that overexpress CV2, and cells that overexpress Bigraphin, the cells being spatially separated from the cells that overexpress human NMDA receptor and mock transfected cells.

10. The support according to claim 6, further comprising all cells from the group comprising cells overexpressing GABAB, cells overexpressing LGI1, cells overexpressing AMPAR1 / R2, cells overexpressing CASPR, cells overexpressing DPPX, cells overexpressing Hu, cells overexpressing Ma2, cells overexpressing CV2 and cells overexpressing Bigraphin, which are spatially separated from the cells overexpressing human NMDA receptor and mock transfected cells.

11. The support according to claim 1, wherein the support comprises mammalian hippocampus and / or mammalian cerebellum.

12. The support according to claim 11, wherein the mammalian hippocampus and / or mammalian cerebellum are spatially separated from the cells.

13. The support according to claim 11, wherein the mammal is a rodent or a primate.

14. A kit comprising the support according to any one of claims 1 to 13, further comprising one or more reagents from the group comprising secondary antibodies labeled with a detectable marker, washing solution, positive control, negative control, detergent, cover glass, sample mounting medium and physiological salt solution.

15. The kit according to claim 14, wherein the physiological salt solution is PBS.

16. A method comprising the steps of: a) contacting the support according to any one of claims 1 to 13 with a sample, b) washing the cells and c) detecting autoantibodies against human NMDA receptor bound to cells in step a).

17. The method according to claim 16, wherein the sample is a sample from a human.

18. The method according to claim 16, wherein the autoantibodies are detected in step c) using a second autoantibody labeled with a fluorescent dye.

19. The method according to claim 17, wherein the autoantibodies are detected in step c) using a second autoantibody labeled with a fluorescent dye.

20. Use of a eukaryotic cell permanently overexpressing a polypeptide comprising a human NMDA receptor or a support according to any one of claims 1 to 13 for producing a false- free signal for detecting autoantibodies against NMDA receptor, wherein the human NMDA receptor comprises an NMDA receptor NR1 subunit, wherein the NMDA receptor NR1 subunit comprises Arg5, Leu9, Val17, Ser 159, Lys212, Leu267 and Leu415 with reference to SEQ ID NO 1.

21. Use of a eukaryotic cell permanently overexpressing a polypeptide comprising a human NMDA receptor, wherein the human NMDA receptor comprises an NMDA receptor NR1 subunit, wherein the NMDA receptor NR1 subunit comprises Arg5, Leu9, Val17, Ser 159, Lys212, Leu267 and Leu415 with reference to SEQ ID NO 1, or a support according to any one of claims 1 to 13, for the manufacture of a kit for detecting the presence or absence of autoantibodies against a human NMDA receptor.

22. The use according to claim 21, wherein the polypeptide comprises an NMDA receptor NR1 subunit, wherein the NMDA receptor NR1 subunit comprises Arg5, Leu9, Val17, Ser 159, Lys212, Leu267 and Leu415 with reference to SEQ ID NO 1.

23. The use according to claim 21 or 22, wherein the polypeptide comprises an NMDA receptor NR1 subunit, wherein the NMDA receptor NR1 subunit comprises Arg5, Leu9, Val17, Ser 159, Lys212, Leu267 and Leu415 with reference to SEQ ID NO 1,

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