EP4028769A1 - Improved detection of nmda receptor autoantibodies - Google Patents

Abstract

The present invention relates to a carrier for microscopic immunofluorescence analysis coated with a eukaryotic cell overexpressing a polypeptide comprising human NMDA receptor or a variant thereof, a carrier comprising the cell and a mock-transfected cell, wherein the cell and the mock-transfected cell are spatially separated, a kit comprising the cell or the carrier and a use of the cell or the carrier or a polypeptide comprising human NMDA receptor or a variant thereof for producing an artefact-free signal for the detection of an autoantibody to human NMDA receptor.

Description

Improved detection of NMDA receptor autoantibodies

The present invention relates to a carrier for microscopic immunofluorescence analysis coated with a eukaryotic cell overexpressing a polypeptide comprising human NMDA receptor or a variant thereof, a carrier comprising the cell and a mock-transfected cell, wherein the cell and the mock-transfected cell are spatially separated, a kit comprising the cell or the carrier and a use of the cell or the carrier or a polypeptide comprising human NMDA receptor or a variant thereof for producing an artefact-free signal for the detection of an autoantibody to human NMDA receptor.

Antibodies against NMDA type glutamate receptor (NMDAR) are specific markers for NMDA receptor autoimmune encephalitis, an inflammatory encephalopathic autoimmune disease which was first described in 2007 and is currently still widely underdiagnosed.

NMDARs belong to the ionotropic glutamate receptors and were named according to their ability to be activated by the synthetic amino acid N-methyl-D-aspartate (NMDA). They are localized in the post-synaptic membrane and form cation canals with major significance 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 are autoantibodies directed against an extracellular epitope of the NR1 subunit. These can be determined by immunohistochemical detection procedures or recombinant assays.

For NMDA receptor autoimmune encephalitis a virtually stereotypical clinical course occurring in phases is typical. In 100% of affected persons a flu-like prodromal phase (subfebrile temperature, headache, and fatigue) is followed by a psychotic stage with severe behavioral and personality changes, delusions, disturbed thoughts and hallucinations. Because of these features a large proportion of patients end up in psychiatric therapy, and in many cases a drug-induced psychosis is initially diagnosed. In the following phase consciousness disorders, hypoventilation, epileptic attacks, autonomous instability and l dyskinesia develop. Due to the severity of this disease including symptoms such as coma, status epilepticus, etc. many affected individuals are treated in intensive care for long periods of time.

In the majority of young female patients ovarian tumors (teratoma) are found, which amongst other things contain nerve cells. These cases involve a paraneoplastic syndrome (PNS) in NMDA receptor autoimmune encephalitis. The probability of an associated tumor disease is, on average, around 60%, although this is dependent on age and gender. NMDA receptor autoimmune encephalitis is increasingly diagnosed not only in young women, but also in older patients, in women without teratoma, in men (some with teratoma of the testis) and in children.

Prognosis for patients is improved with appropriate immunomodulatory therapy, and, in PNS, tumor detection and resection as early as possible. In around 75% of cases a substantial regression of symptoms can be achieved. However, 25% of patients die or suffer from severe neurological deficits. Survivors have memory loss for the duration of the illness, and there is a risk of relapses of the encephalitis syndrome, the latter in particular when the tumor is removed too late or not at all or if no tumor could be found.

Diagnosis of NMDA receptor autoimmune encephalitis is based on a combination of the characteristic clinical picture, together with the supporting results from brain MRT, EEG and CSF analysis if necessary, and the detection of anti-glutamate receptor NMDA type antibodies. Infectious encephalitides, especially HSV and other autoimmune aetiologies (limbic encephalitis with autoantibodies against Hu, Ma2, CV2 and amphiphysin) must be excluded by differential diagnostics. In general, antibodies against NMDA receptors should be determined in all patients with encephalitis where no pathogen has been detected and in suspected cases of limbic encephalitis. When a positive serological result is obtained a comprehensive teratoma investigation should be undertaken.

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 may be used for the high-throughput-screening of hundreds of patient samples in an automated manner under high-throughput-conditions. An excellent sensitivity of 98.1% and a specificity of 100% have been reported for this assay format. However, discrepancies in the results between and results obtained using standardized immunofluorescence kit products and other assay formats have been observed. While it is unclear whether such discrepancies are due to flaws associated with these commercial kits or any reference assays, there remains the need for continuous optimization.

Dalmau et al. (2007) disclose that paraneoplastic NMDA receptor autoimmune encephalitis associated with ovarian teratoma is associated with autoantibodies to the NR1/NR2 heteromer of the receptor. HEK293 cells transfected with plasmids containing 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).

McCracken et al. (2017) claim that standard clinical laboratory kits are specific but in rare cases yield inconclusive results or turn out insensitive or false negative results. (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).

Probst et al. (2014) discuss technical challenges for 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).

US7,972,796 discloses methods of diagnosing or determining a cause of an autoimmune encephalitis comprising the step of testing a biological sample for an antibody to NR1 subunit of the NMDA receptor.

A problem underlying the present invention is to provide a new test and related creations, methods and uses for the diagnosis or aiding in the diagnosis of neurological autoimmune diseases, in particular NMDA receptor autoimmune encephalitis, at the utmost degree of diagnostic reliability, with special emphasis on sensitivity and for specificity.

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

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

In a 2^nd aspect, the problem underlying the present invention is solved by a carrier comprising the cell according to the present invention and preferably a mock-transfected cell, wherein the cell and the mock-transfected cell are spatially separated, wherein the carrier is preferably a slide for microscopic immunofluorescence analysis.

In a preferred embodiment, the cell is transiently transfected with human NMDA receptor or a variant thereof.

In a preferred embodiment the cell is permanently expressing human NMDA receptor or a variant thereof.

In a preferred embodiment the cell is a fixed cell.

In a preferred embodiment the cell is fixed using formalin or acetone.

In a preferred embodiment, the cell is bound to an autoantibody to human NMDA receptor which is preferably bound to a secondary antibody.

In a preferred embodiment, the carrier further comprises one or more additional cells, preferably all from the group comprising a cell overexpressing GABA B or a variant thereof, a cell overexpressing LGI1 or a variant thereof, a cell overexpressing AMPAR1/R2 or a variant thereof, a cell overexpressing CASPR2 or a variant thereof, a cell overexpressing DPPX or a variant thereof, a cell overexpressing Hu or a variant thereof, a cell overexpressing Ma2 or a variant thereof, a cell overexpressing CV2 or a variant thereof and a cell overexpressing amphiphysin or a variant thereof, which are spatially separated from the cell overexpressing human NMDA receptor and, if present, the mock-transfected cell.

In a 3^rd aspect, the problem underlying the present invention is solved by a kit comprising the cell or the carrier according to the present invention, further comprising one or more reagents from the group comprising a secondary antibody, preferably labeled with a detectable label, a wash solution, a positive control, a negative control, a detergent, a cover glass, a mounting medium and a physiological salt solution, preferably PBS.

In a 4^th aspect, the problem is solved by a use of a vector comprising a nucleic acid encoding for a polypeptide comprising human NMDA receptor or a variant thereof or the cell according to the present invention for the manufacture of a kit or carrier or composition for the diagnosis of NMDA receptor autoimmune encephalitis, preferably the kit according to the present invention.

In a 5^th aspect, the problem is solved by a method comprising the steps a) contacting the carrier according to the present invention with a sample, b) washing the cell and c) detecting an autoantibody to human NMDA receptor bound to the carrier in step a), optionally an autoantibody to a neurological autoantigen overexpressed by another cell if a carrier comprising a second cell is used.

In a preferred embodiment the autoantibody is detected in step c) using a secondary antibody labeled with a fluorescent dye.

In a 6^th embodiment, the problem is solved by a use of a polypeptide comprising human NMDA receptor or a variant thereof or the cell or the carrier for producing artefact-free signals for the detection of an autoantibody to NMDA receptor.

In a 7^th embodiment, the problem is solved by a use of a polypeptide comprising human NMDA receptor or a variant thereof or the cell or the carrier for the high specificity diagnosis of NMDA receptor autoimmune encephalitis. In a preferred embodiment, the variant of the NMDA receptor is the NR1 subunit or a variant thereof.

The present invention is based on the inventors’ surprising finding that overexpression of rodent NMDA receptor or subunits thereof or variants thereof in eukaryotic cells is associated with the emergence of artefacts, in particular long protrusions of cells and unusually symmetrical spherical artefacts all of which stain positive when contacted with secondary antibodies with a label. These artefacts obscure images of positive stained cells or may be confused with cells specifically recognized by NMDA receptor autoantibodies, thus confusing untrained medical staff and leading to false positive or false negative diagnoses.

The present invention is based on the inventors’ surprising finding that artefact-free signals, preferably images for immunofluorescence analysis may be obtained if cells overexpressing human NMDA receptor rather than rodent NMDA receptor are used.

Without wishing to be bound to this, the inventors theorize that rat NMDA receptor subunits, when overexpressed in a eukaryotic cell line, in particular a human cell line, have a higher propensity to accumulate outside the cell expressing the subunit in the form of protrusion-like or spherical aggregates made of unfolded protein which may be bound and stained by a secondary antibody with a detectable dye in a non-specific manner. Therefore, such aggregates may obscure stained cells or may be confused with stained cells the presence of which normally indicates that the sample is positive.

In a preferred embodiment, the mammalian cell is a cell line from a mammal, preferably from a human. Exemplary cells lines include, but are not limited to HEK293, T-Rex-HeLa, T-Rex- CHO and T-REx™-Jurkat cells and derivatives thereof.

In a preferred embodiment, the term “human NMDAR receptor”, as used herein, refers to a receptor comprising human NR1 subunits or the combination of an NR1 subunit with another receptor subunit such as NR2. It is essential that the NR1 subunit is present, since it comprises the autoantibody binding site. NR1 subunits, when expressed in a mammalian cell, preferably in the presence of a less than equivalent amount of another receptor subunit such as NR2, have the ability to form NR1 homodimers, which are also capable to binding to the autoantibody. In a preferred embodiment, the human NR1 subunit has the sequence shown in SEQ ID N01. In a preferred embodiment, the human NR2 subunit has the sequence shown in SEQ ID N02 or SEQ ID N04.

The teachings of the present invention may not only be carried out using the polypeptides, in particular a polypeptide comprising the native sequence of a polypeptide such as the NR1 subunit of the human NMDA receptor or nucleic acids having the exact sequences referred to in this application explicitly, for example by function, name, sequence or accession number, or implicitly, but also using variants of such polypeptides or nucleic acids.

In a preferred embodiment, the term “variant”, as used herein, may refer to at least one fragment of the full length sequence referred to, more specifically one or more amino acid or nucleic acid sequence which is, relative to the full-length sequence, truncated at one or both termini by one or more amino acids. Such a fragment comprises or encodes for a peptide having at least 6, 7, 8, 10, 12, 15, 20, 25, 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 850, 870, 890, 900, 910, 920 or 930 successive amino acids of the original sequence or a variant thereof. The total length of the variant may be at least 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 750, 850, 870, 890, 900, 910, 920 or 930 or more amino acids.

The term "variant" relates not only to at least one fragment, but also to a polypeptide or a fragment thereof comprising amino acid sequences that are at least 40, 50, 60, 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98, 99, 99,3, 99,4, 99,5, 99,6, 99,7, 99,8 or 99,9, preferably at least 99,3 % identical to the reference amino acid sequence referred to or the fragment thereof, wherein amino acids other than those essential for the biological activity, for example the ability of an antigen to bind to an (auto)antibody, or the fold or structure of the polypeptide are deleted or substituted and/or one or more such essential amino acids are replaced in a conservative manner and/or amino acids are added such that the biological activity of the polypeptide is preserved. The state of the art comprises various methods that may be used to align two given nucleic acid or amino acid sequences and to calculate the degree of identity, see for example Arthur Lesk (2008), Introduction to bioinformatics, Oxford University Press, 2008, 3^rd edition. In a preferred embodiment, the ClustalW software (Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., Higgins, D. G. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948) is used using default settings. In a preferred embodiment, the polypeptide and variants thereof may, in addition, comprise chemical modifications, for example isotopic labels or covalent modifications such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, methylation, hydroxylation and the like. The person skilled in the art is familiar with methods to modify polypeptides. Any modification is designed such that it does not abolish the biological activity of the variant.

Moreover, variants may also be generated by N- or/and C-terminal fusion of polypeptides, fragments or variants thereof with other known polypeptides or variants thereof and comprise active portions or domains, preferably having a sequence identity of at least 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99 % when aligned with the active portion of the reference sequence, wherein the term "active portion", as used herein, refers to an amino acid sequence, which is less than the full length amino acid sequence or, in the case of a nucleic acid sequence, codes for less than the full length amino acid sequence, respectively, and/or is a variant of the natural sequence, but retains at least some of the biological activity.

In a preferred embodiment, the term "variant" of a nucleic acid comprises nucleic acids the complementary strand of which hybridizes, preferably under stringent conditions, to the reference or wild type nucleic acid. Stringency of hybridization reactions is readily determinable by one of ordinary skilled in the art, and in general is an empirical calculation dependent on probe length, washing temperature and salt concentration. In general longer probes require higher temperatures for proper annealing, while shorter probes less so. Hybridization generally depends on the ability of denatured DNA to reanneal to complementary strands present in an environment below their melting temperature: The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which may be used. As a result, higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperature less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel, F. M. (1995), Current Protocols in Molecular Biology. John Wiley & Sons, Inc. Moreover, the person skilled in the art may follow the instructions given in the manual Boehringer Mannheim GmbH (1993) The DIG System Users Guide for Filter Hybridization, Boehringer Mannheim GmbH, Mannheim, Germany and in Liebl, W., Ehrmann, M., Ludwig, W., and Schleifer, K. H. (1991) International Journal of Systematic Bacteriology 41: 255-260 on how to identify DNA sequences by means of hybridization. In a preferred embodiment, stringent conditions are applied for any hybridization, i.e. hybridization occurs only if the probe is 70 % or more identical to the target sequence. Probes having a lower degree of identity with respect to the target sequence may hybridize, but such hybrids are unstable and will be removed in a washing step under stringent conditions, for example lowering the concentration of salt to 2 x SSC or, optionally and subsequently, to 0,5 x SSC, while the temperature is, in order of increasing preference, approximately 50°C - 68°C, approximately 52°C - 68°C, approximately 54°C - 68°C, approximately 56°C - 68°C, approximately 58°C - 68°C, approximately 60°C - 68°C, approximately 62°C - 68°C, approximately 64°C - 68°C, approximately 66°C - 68°C. In a particularly preferred embodiment, the temperature is approximately 64°C - 68°C or approximately 66°C - 68°C. It is possible to adjust the concentration of salt to 0.2 x SSC or even 0.1 x SSC. Nucleic acid sequences having a degree of identity with respect to the reference or wild type sequence of at least 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 % may be isolated. In a preferred embodiment, the term variant of a nucleic acid sequence, as used herein, refers to any nucleic acid sequence that encodes the same amino acid sequence and variants thereof as the reference nucleic acid sequence, in line with the degeneracy of the genetic code.

The variant of the polypeptide has biological activity. In a preferred embodiment, such biological activity is the ability to bind specifically to an autoantibody binding to the NMDA receptor, preferably human NMDA receptor, as found in a patient suffering from an autoimmune disease associated with such autoantibody, preferably associated with a neurological disease such as epilepsy, encephalitis and PNS. For example, whether or not a variant of the polypeptide has such biological activity may be checked by determining whether or not the variant of interest binds to an autoantibody from a sample of a patient which autoantibody binds to wild type human NMDA receptor, preferably as determined by indirect immunofluorescence as described in the experimental section of this application.

Various publications can be used to design variants of the receptor. Sharma et al. disclosed that the N-terminal domain is essential for the 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 is may be used in a fusion protein. It is important that at least one NR1 subunit remains that comprises amino acids 25-380 of the NR1 subunit, because the inventors of US7,972,976 have shown that this is the part of the antigen that is essential for recognition of the autoantibody. Further guidance based on site-directed mutagenesis may 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 a preferred embodiment, the person skilled in the art, when designing variants or fragments, will consider that one or more amino acid residues characteristic of the human sequences should be preserved. In a more preferred embodiment, the variant comprises one or more, preferably at least two, three, four, five, or six, more preferably all from the group comprising Arg5, Leu9, Val17, Ser 159, Lys212, Leu267 and Leu415, with reference to SEQ ID N01 and highlighted in Fig. 6., while preferably other amino acid residues, as far as not essential for the biological activity or use for the detection of an autoantibody to NR1, may be modified or may be absent.

In a preferred embodiment, the cell according to the present invention overexpresses a polypeptide comprising human NMDA receptor or a variant. In a preferred embodiment, the term “overexpressing”, as used herein, means that the cell has been transfected with a nucleic acid, either transiently or stably in the sense that the nucleic acid has been incorporated in the genome of the cell, that comprises a nucleic acid sequence encoding a polypeptide comprising the human NMDA receptor or a variant thereof under the control of a promotor. Consequently, the transfected cell expresses more polypeptide recognized by the autoantibody than the same type of cell normally would, probably at least 10, 20, 30, 50, 100, 200 or 500 % more as judged by quantitative Western Blot. The promotor may be an inducible promotor, which allows for the induction of expression by addition of an inducer. The person skilled in the art is familiar with protocols and vectors for transiently overexpressing a polypeptide in a eukaryotic cell, for example the pTriEx system from Novagen and with protocols and vectors for stably transfecting a eukaryotic cell, for example the pcDNA™4/TO vector system from Invitrogen.

In a preferred embodiment, a fixed mammalian cell may be used. In a preferred embodiment, the term “fixed” cell, as used herein, refers to a cell that has been treated with a reactive chemical compound to the effect that the cell is no longer metabolically active, but still presents its epitopes for immunostaining with antibodies and their subsequent detection, for example by fluorescence. More preferably, the reactive chemical 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 that may be used to prepare fixed cells. Essentially, the cell which is attached to a solid support is washed by using washing buffer, followed by contacting with the reactive compound, for example immersion. Pure acetone or formalin or aqueous dilutions of the reactive chemical compound may be used.

According to the present invention, the cell is on a carrier for microscopic immunofluorescence analysis. Such a carrier may be a glass slide. The cell on the glass slide may be covered with a mounting buffer. A mounting medium is a liquid which helps maintain a near physiological pH to maintain the molecular structure of any diagnostically relevant molecular and their epitopes, is compatible with the emission of a fluorescence signal and prevents a premature loss of fluorescence due to bleaching of the fluorophore. At the same time its optical properties are matched with other buffers used, in particular it’s the refractive index which allows for an efficient microscopic fluorescence analysis. The mounting medium comprises a base component, preferably selected from the group comprising water, glycerol, natural oil or plastic or a mixture thereof, preferably water and glycerol. It may further comprise an antifade constituent which may reduce bleaching, preferably selected from the group comprising NPG (N-propyl gallate), DABCO (1,4- diazabicyclo[2.2.2]octane), 4POBN ((4-Pirydyl-1-oxide)-N-tert-butyl nitrone) and PPD (P- phenylanediamine). Various compositions and methods are described in the state of the art, for example in “Mountants and Antifades”, published by Wright Cell Imaging Facility, Toronto Western Research Institute University Health Network,

(https://de.scribd.com/document/47879592/Mountants-Antifades), Krenek et at. (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.

A cover glass may be placed on top of the composition comprising the sample and the mounting medium. Slides with cover glasses (FB 112d-1005-1) are available from EUROIMMUN Medizinische Labordiagnostika, AG. However, any carrier compatible with microscopic analysis of the fluorescence pattern may be used. The carrier may comprise a mock-transfected cell, which has been transfected with the same vector as the cell overexpressing a polypeptide comprising NMDA receptor, but without the nucleic acid encoding for the latter. Such mock-transfected cell may serve as a negative control. The carrier is configured for analysis using an immunofluorescence microscope. In a preferred embodiment, the carrier may comprise a field comprising the cell according to the invention. In addition the carrier may comprise additional fields. The fields are preferably surrounded by a hydrophobic surface. Each of these fields may comprise a cell overexpressing another autoantigen, preferably selected from the group comprising GABA A, GABA B, Lgl 1 , AMPAR1/R2, CASPR2, DPPX, Hu, Ma2, CV2, amphiphysin or a variant thereof. Suitable autoantigens and their sequences and variants are described in US15/023131 (GABA A), US8685656 (GABA B), US9250250 (Lgl1), 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), US9188587 (CASPR2), US9719993 (DPPX), US6193948/US5603934/US5807705 (Hu), US7026450 (Ma2),

US7329499 (CV2) and Genbank data base code AAH34376.1. All data base codes represent the sequences online at the earliest priority date of this application. The cell according to the invention is preferably spatially separated from any of the mock-transfected cell or a cell expressing another autoantigen such that a fluorescence signal can be assigned to a specific autoantigen, for example the human NMDA receptor. In a preferred embodiment, the carrier may comprise one or more additional fields, each comprising a tissue from the group comprising mammalian hippocampus and/or mammalian cerebellum, more preferably rat hippocampus rat cerebellum, primate hippocampus and primate cerebellum, most preferably all of them.

In a preferred embodiment, the cell is transiently transfected, which preferably means that the cell is transfected with a vector comprising a nucleic acid encoding for a polypeptide comprising human NMDA receptor, but this nucleic acid is not integrated into the genome of the cell. Consequently, the cell may lose the vector when cultivated for a prolonged time to the effect that it no longer expresses said polypeptide.

In a preferred embodiment, the cell permanently expresses the human NMDA receptor, more preferably because a nucleic acid encoding for a polypeptide comprising the NMDA receptor has been integrated into the genome. Consequently, the cell is likely to express the polypeptide permanently. In a preferred embodiment, a secondary antibody is an antibody binding specifically to all antibodies from an antibody class, preferably preferably IgG. Secondary antibodies typically recognize the constant domain of said class, but may also recognize other epitopes shared by antibodies from the class of interest, for example a conformational epitope across the 3D structure. A wide range of them is commercially available, for example from Thermo Fisher. It may be a monoclonal or a polyclonal antibody. A secondary antibody may bind specifically to all isotypes from the antibody class. For example a secondary antibody to IgG class antibodies may bind to lgG1, lgG2, lgG3 and lgG4 isotypes. This may be achieved by using as a secondary antibody to the class, preferably to IgG class antibodies, a mixture comprising an antibody binding specifically to each IgG isotype or a single antibody which reacts with all isotypes of interest. The use of secondary antibodies is explained in Kruger, N. J., Detection of Polypeptides on Blots Using Secondary Antibodies, in The Protein Protocols Handbook (ed. J. M. Walker), page 967, volume 1996, Springer.

The autoantibody to be detected or a secondary antibody used binds preferably specifically to human NMDA receptor or the autoantibody to be detected, respectively. “Binding specifically” preferably means that the binding reaction is stronger than a binding reaction characterized by a dissociation constant of 1 x 10⁵ M, more preferably 1 x 10⁷ M, more preferably 1 x 10⁸ M, more preferably 1 x 10⁹ M, more preferably 1 x 10¹⁰ M, more preferably 1 x 10¹¹ M, more preferably 1 x 10¹² M, as determined by surface plasmon resonance using Biacore equipment at 25 °C in PBS buffer at pH 7.

In a preferred embodiment, the cell is bound to an autoantibody to human NMDA receptor, and a secondary antibody is bound to the antibody. In a more preferred embodiment, the secondary antibody recognizes IgG class antibodies. For immunofluorescence analysis, the secondary antibody may comprise a detectable fluorescent label, more preferably FITC (fluorescein isothiocyanate).

In a preferred embodiment, the method according to the present invention comprises the step providing the carrier according to the present invention. The carrier may then be contacted with the sample suspected of comprising the autoantibody under conditions allowing for binding of any autoantibodies to the cell and the NMDA receptor or variant thereof expressed by the cell. The sample may then be removed and the carrier with the cell may be washed to remove any remaining sample. A secondary autoantibody or similar reagent or means binding to the autoantibody and carrying a detectable label such as a fluorescent dye may then be contacted with the carrier under conditions allowing formation of a complex between any bound autoantibody and the secondary antibody. The carrier may be washed then to remove non-bound secondary antibody. Finally, the presence of the autoantibody is detected by checking whether the secondary antibody may be detected, preferably by immunofluorescence, more preferably emitted by fluorescein or a derivative thereof, most preferably FITC.

In a preferred embodiment, the cell, method, kit, polypeptide or carrier is used for the diagnosis of a disease, preferably neurological autoimmune disease, more preferably from the group comprising paraneoplastic syndrome, autoimmune encephalitis, limbic encephalitis and epilepsy. In addition, the cell, method, kit, polypeptide or carrier is used for the diagnosis of teratoma.

In a preferred embodiment, the term ..diagnosis", as used herein, is to be used in its broadest possible sense and may to any kind of procedure aiming to obtain information instrumental in the assessment whether a patient suffers or is likely or more likely than the average or a comparative subject, the latter preferably having similar symptoms, to suffer from certain a disease or disorder in the past, at the time of the diagnosis or in the future, to find out how the disease is progressing or is likely to progress in the future or to evaluate the responsiveness of a patient or patients in general with regard to a certain treatment, for example the administration of immunosuppressive drugs, or to find out whether a sample is from such a patient. Such information may be used for a clinical diagnosis, but may also be obtained by an experimental and/or research laboratory for the purpose of general research, for example to determine the proportion of subjects suffering from the disease in a patient cohort or in a population. In other words, the term “diagnosis” comprises not only diagnosing, but also prognosticating and/or monitoring the course of a disease or disorder, including monitoring the response of one or more patients to the administration of a drug or candidate drug, for example to determine its efficacy. While the result may be assigned to a specific patient for clinical diagnostic applications and may be communicated to a medical doctor or institution treating said patient, this is not necessarily the casefor other applications, for example in diagnostics for research purposes, where it may be sufficient to assign the results to an sample from an anonymized patient.

In a preferred embodiment, the methods and products according to the present invention may be used for interaction studies, including determining whether a drug candidate or other compound may interfere with the binding of an autoantibody to the NMDA receptor or may affect any downstream process or the strength of their binding to their target such as the NR1 subunit. In preferred embodiment, they may be used for monitoring the immune response, more preferably the emergence and/or titer of antibodies to NMDAR, following the administration of an immunogenic composition comprising NMDAR or an immunogenic variant thereof, for example to a mammal, which may be a mammal other than a human such as a laboratory animal.

In many cases the mere detection of the autoantibody, in other words determining whether or not detectable levels of the antibody are present in the sample, is sufficient for the diagnosis. If the autoantibody can be detected, this will be information instrumental for the clinician’s diagnosis and indicates an increased likelihood that the patient suffers from a disease.

The person skilled in the art will appreciate that a clinician does usually not conclude whether or not the patient suffers or is likely to suffer from a disease, condition or disorders solely on the basis of a single diagnostic parameter, but needs to take into account other aspects, for example the presence of other autoantibodies, markers, blood parameters, clinical assessment of the patient’s symptoms or the results of medical imaging or other non- invasive methods such as polysomnography, to arrive at a conclusive diagnosis. See Baenkler H. W. (2012), General aspects of autoimmune diagnostics, in Renz, H., Autoimmune diagnostics, 2012, de Gruyter, page 3. The value of a diagnostic agent or method may also reside the possibility to rule out one disease, thus allowing for the indirect diagnosis of another. In a preferred embodiment, the meaning of any symptoms or diseases referred to throughout this application is in line with the person skilled in the art’s understanding as of the filing date or, preferably, earliest priority date of this application as evidenced by text books and scientific publications. It should be mentioned that the inventive methods or uses or products, taken alone, cannot be used to arrive at a definite, final diagnosis.

In a preferred embodiment, the term “diagnosis” may also refer to a method or agent used to distinguish between two or more conditions associated with similar or identical symptoms, for example autoimmune encephalitis and an encephalitis caused by an infection. Optionally, the presence or absence of an antibody to a viral antigen may be determined according to the present invention, for example relating to a virus causing encephalitis. In a preferred embodiment, the term “diagnosis” may also refer to a method or agent used to choose the most promising treatment regime for a patient. In other words, the method or agent may relate to selecting a treatment regimen for a subject. For example, the detection of autoantibodies may indicate that an immunosuppressive therapy is to be selected, which may include administrating to the patient one or more immunosuppressive drugs.

In a preferred embodiment, any information or data demonstrating the presence of absence of the autoantibody may be communicated to the patient or a medical doctor treating the patient, preferably by telephone, in a written form or via the internet, for example as an email or text message.

In a preferred embodiment, the term „autoantibody“, as used herein, refers to an antibody binding specifically to an endogenous molecule of the animal, preferably mammal, more preferably human, which produces said autoantibody, wherein the level of such antibody is more preferably elevated compared to the average healthy subject. In a most preferred embodiment, the autoantibody is an autoantibody binding to mammalian, preferably human NMDA receptor. The autoantibody may have the sequence of an antibody’s constant regions from the animal, preferably human, making it, but the variable region is able to bind specifically to the endogenous molecule of the animal, more specifically NMDA receptor. In a preferred embodiment, the autoantibody is isolated and/or purified from a sample, preferably tissue, serum, plasma, blood or CSF from the animal, preferably human. The autoantibody is a polyclonal, native antibody from the animal rather than a synthetic or recombinant antibody.

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

According to the present invention, a kit is provided, comprising the cell or the carrier and further comprising one or more, preferably all reagents from the group comprising a secondary antibody, preferably labeled with a detectable label, a washing solution, a positive control, a negative control, a detergent, a cover glass, a mounting medium and a physiological salt solution, preferably PBS, or salt required to prepare it. In a preferred embodiment, the positive control is a diluted sample, preferably serum or CSF, from a patient suffering from NMDA receptor autoimmune encephalitis or a monoclonal antibody to human NMDA receptor, preferably binding to the NR1 subunit recognized by the secondary antibody. The negative control may be a diluted sample from a healthy subject, for example a blood donor. The kit may comprise instructions how to carry out the assay. Preferably, the secondary antibody is a secondary antibody to IgG class antibodies, preferably human IgG glass antibodies.

In a preferred embodiment, the present invention provides a use of the cell, the polypeptide, the carrier for the manufacture of kit a composition for the diagnosis of a disease.

In a preferred embodiment, any method or use according to the present invention may be intended for a non-diagnostic use, i.e. determining the presence of an autoantibody to binding to human NMDA receptor for a use other than diagnosing a patient. For example, the method or use may be for testing in vitro the efficiency of a medical device designed to remove an autoantibody from a patient’s blood, wherein the testing is performed on a liquid other than patient’s blood. After the use of the medical device with a patient, its capacity to remove autoantibody may be checked by running a solution comprising antibody to human NMDA receptor through the device, followed by use of the method according to the present invention to confirm that less or no antibody is in the solution that has been passed through the device, i.e. showing that the device has still the capacity to remove antibody from the solution.

In another preferred embodiment, the method may be for confirming the reliability of a diagnostic assay and may involve detecting an antibody to human NMDA receptor in a solution, which is not a sample from a patient who requires a diagnosis, but is known to comprise an antibody to human NMDA receptor, preferably at a known concentration. For example, it may be a recombinant antibody or a sample diluted in a dilution buffer such as PBS from an anonymous patient whose identity cannot be traced back. Alternatively, the solution may be a negative control not comprising the antibody to check the background. Such method may be run in parallel with, after or before a diagnostic method. In a preferred embodiment, any method or use according to the present invention may be intended for generating an autoantibody profile, preferably for detecting a disease in a mammal, preferably a human.

In a preferred embodiment, any method or use according to the present invention may be for identifying a subject at risk of suffering from or developing a disease and/or a tumor.

In a preferred embodiment, the method may be for detecting an antibody, preferably autoantibody in a solution which is not a sample from a mammal to be diagnosed or for the purpose of providing a diagnosis, in particular not a diagnosis of a neurological disease such as NMDA receptor encephalitis.

In a preferred embodiment, the present invention provides an apparatus for analyzing a sample from a patient to detect an autoantibody against human NMDA receptor, indicating an increased likelihood of a disease or of developing a disease, comprising: a. a carrier, which contains a means for capturing the autoantibody from the sample when the sample is contacted with the carrier, wherein the means is the cell and the carrier is the carrier according to the present invention, b. a detectable means capable of binding to the antibody captured by the carrier when the detectable means is contacted with the carrier, wherein the detectable means is preferably a labeled secondary antibody capable of binding to the autoantibody captured on the carrier, c. optionally a means for removing any sample from the carrier and the detectable means, preferably by washing; d. a detecting device for detecting the presence of the detectable means and converting the results into an electrical signal, for example a fluorescence reader connected with a software capable of recognizing a pattern characteristic of a stained cell overexpressing human NMDAR in an image of the cell taken by the fluorescence reader or camera, and optionally a means for receiving the electronical signal from the detecting device and determining if the level of the signal is indicative of an increased likelihood of having or developing a disease, by comparing with the patterns characteristic of wild type or non- stained cells, preferably by a mock-transfected cell or cells not positively stained by an autoantibody binding to human NMDA receptor on the same carrier, or an input reference value obtained with samples from healthy subjects or by comparing the level of signal obtained with one sample with the level of signal obtained with a second sample obtained at a later time point, preferably at least one month later. Fig. 1A shows immunofluorescence analysis of HEK293 cells transiently transfected with a vector encoding rat NR1 subunit of the NMDA receptor, stained with a sample from a patient suffering from NMDA receptor encephalitis. White arrows mark artefacts in the form of protrusions which are artefacts resulting from the immunostaining of NR1 aggregates.

Fig. 1B shows immunofluorescence analysis of HEK293 cells permanently transfected with the human NR1 subunit. These images are clearly free of artefacts.

Fig. 2A shows immunofluorescence analysis of HEK293 cells transiently transfected with a vector encoding NR1 subunit of the rat NMDA receptor, stained with a 1:100 diluted sample from a patient who does not suffer from NMDA receptor encephalitis. Spherical bodies comprising NR1 aggregates are stained and could be mistaken for positively stained cells.

Fig. 2B shows immunofluorescence analysis of HEK293 permanently transfected with a vector encoding NR1 subunit of the human NMDA receptor, stained with the same sample. No spherical artefacts can be observed.

Fig. 3A shows immunofluorescence analysis of HEK293 cells transiently transfected with a vector encoding NR1 subunit of the rat NMDA receptor, stained with a sample from a patient who suffers from NMDA receptor encephalitis. Spherical bodies comprising NR1 aggregates are stained and obscure stained positive cells, which are shown in Fig. 3B where the same sample was used to stain HEK293 cells permanently expressing NR1 of the human NMDA receptor.

Figs. 4A, 4B and 4C show immunofluorescence analysis of HEK293 cells stained with a 1:10 sample from a patient suffering from NMDA receptor encephalitis. The cells transiently express rat NR1 (Fig. 4A), transiently human NR1 (Fig. 4B) or permanently human NR1 (Fig. 4C). In Fig. 4A, protrusion-like and spherical artefacts can be observed. In Fig. 4B, there are spherical artefacts, but the protrusions are less pronounced. In Fig. 4C, there are no artefacts.

Figs. 5A, 5B and 5C show immunofluorescence analysis of HEK293 cells stained with a 1:10 sample from a patient not suffering from NMDA receptor encephalitis. The cells transiently express rat NR1 (Fig. 5A), transiently human NR1 (Fig. 5B) or permanently human NR1 (Fig. 5C). In Fig. 5A, spherical artefacts convey the impression of cells positively stained by autoantibodies to NMDAR. In Fig. 5B, the spherical artefacts are less pronounced. In Fig. 5C, there are none.

Fig. 6 shows an alignment of the amino acid sequences of human NR1 subunit („Query 1“), which is SEQ ID N01, and rat NR1 subunit („Sbjct 1“). Amino acids that differ are in bold in the human sequence.

Sequences:

The present invention comprises a range of novel nucleic acid and polypeptide sequences, more specifically

SEQ ID N01 (polypeptide sequence human NR1): mstmrlltlallfscsvaraacdpkivnigavlstrkheqmfreavnqankrhgswkiqlnatsvthkpnaiqma lsvcedlissqvyailvshpptpndhftptpvsytagfyripvlglttrmsiysdksihlsflrtvppyshqssv wfemmrvyswnhiillvsddhegraaqkrletlleereskaekvlqfdpgtknvtallmeakelearviilsase ddaatvyraaamlnmtgsgyvwlvgereisgnalryapdgilglqlingknesahisdavgvvaqavhelleken itdpprgcvgntniwktgplfkrvlmsskyadgvtgrvefnedgdrkfanysimnlqnrklvqvgiyngthvipn drkiiwpggetekprgyqmstrlkivtihqepfvyvkptlsdgtckeeftvngdpvkkvictgpndtspgsprht vpqccygfcidlliklartmnftyevhlvadgkfgtqervnnsnkkewngmmgellsgqadmivapltinneraq yiefskpfkyqgltilvkkeiprstldsfmqpfqstlwllvglsvhvvavmlylldrfspfgrfkvnseeeeeda ltlssamwfswgvllnsgigegaprsfsarilgmvwagfamiivasytanlaaflvldrpeeritgindprlrnp sdkfiyatvkqssvdiyfrrqvelstmyrhmekhnyesaaeaiqavrdnklhafiwdsavlefeasqkcdlvttg elffrsgfgigmrkdspwkqnvslsilkshengfmedldktwvryqecdsrsnapatltfenmagvfmlvaggiv agiflifieiaykrhkdarrkqmqlafaavnvwrknlqdrksgraepdpkkkatfraitstlassfkrrrsskdt stgggrgalqnqkdtvlprraiereegqlqlcsrhres

SEQ ID N02 (polypeptide sequence NMDAR subunit NR2a) mgrvgywtllvlpallvwrgpapsaaaekgppalniavmlghshdvterelrtlwgpeqaaglpldvnvvallmn rtdpkslithvcdlmsgarihglvfgddtdqeavaqmldfisshtfvpilgihggasmimadkdptstffqfgas iqqqatv lkimqdydwhvfslvttifpgyrefisfvkttvdnsfvgwdmqnvitldtsfedaktqvqlkkihss villycskdeavlilsearslgltgydffwivpslvsgntelipkefpsglisvsyddwdyslearvrdgigilt taassmlekfsyipeakascygqmerpevpmhtlhpfmvnvtwdgkdlsfteegyqvhprlvvivlnkdrewekv gkwenhtlslrhavwpryksfsdcepddnhlsivtleeapfvivedidpltetcvrntvpcrkfvkinnstnegm nvkkcckgfcidilkklsrtvkftydlylvtngkhgkkvnnvwngmigevvyqravmavgsltineersevvdfs vpfvetgisvmvsrsngtvspsaflepfsasvwvmmfvmllivsaiavfvfeyfspvgynrnlakgkaphgpsft igkaiwllwglvfnnsvpvqnpkgttskimvsvwaffaviflasytanlaafmiqeefvdqvtglsdkkfqrphd ysppfrfgtvpngsternirnnypymhqymtkfnqkgvedalvslktgkldafiydaavlnykagrdegcklvti gsgyifattgygialqkgspwkrqidlallqfvgdgemeeletlwltgichneknevmssqldidnmagvfymla aamalslitfiwehlfywklrfcftgvcsdrpgllfsisrgiyscihgvhieekkkspdfnltgsqsnmlkllrs aknissmsnmnssrmdspkraadfiqrgslimdmvsdkgnlmysdnrsfqgkesifgdnmnelqtfvanrqkdnl nnyvfqgqhpltlnesnpntvevavsteskansrprqlwkksvdsirqdslsqnpvsqrdeataenrthslkspr ylpeemahsdisetsnratchrepdnsknhktkdnfkrsvaskypkdcsevertylktksssprdkiytidgeke pgfhldppqfvenvtlpenvdfpdpyqdpsenfrkgdstlpmnrnplhneeglsnndqyklyskhftlkdkgsph setseryrqnsthcrsclsnmptysghftmrspfkcdaclrmgnlydidedqmlqetg SEQ ID N03 (plasmid used for transiently expressing human NR1 in cells) ggggaattgtgagcggataacaattccccggagttaatccgggacctttaattcaacccaacacaatatattata gttaaataagaattattatcaaatcatttgtatattaattaaaatactatactgtaaattacattttatttacaa tcaaaggagatataccatgtctaccatgcgcctgctgacgctcgccctgctgttctcctgctccgtcgcccgtgc cgcgtgcgaccccaagatcgtcaacattggcgcggtgctgagcacgcggaagcacgagcagatgttccgcgaggc cgtgaaccaggccaacaagcggcacggctcctggaagattcagctcaatgccacctccgtcacgcacaagcccaa cgccatccagatggctctgtcggtgtgcgaggacctcatctccagccaggtctacgccatcctagttagccatcc acctacccccaacgaccacttcactcccacccctgtctcctacacagccggcttctaccgcatacccgtgctggg gctgaccacccgcatgtccatctactcggacaagagcatccacctgagcttcctgcgcaccgtgccgccctactc ccaccagtccagcgtgtggtttgagatgatgcgtgtctacagctggaaccacatcatcctgctggtcagcgacga ccacgagggccgggcggctcagaaacgcctggagacgctgctggaggagcgtgagtccaaggcagagaaggtgct gcagtttgacccagggaccaagaacgtgacggccctgctgatggaggcgaaagagctggaggcccgggtcatcat cctttctgccagcgaggacgatgctgccactgtataccgcgcagccgcgatgctgaacatgacgggctccgggta cgtgtggctggtcggcgagcgcgagatctcggggaacgccctgcgctacgccccagacggcatcctcgggctgca gctcatcaacggcaagaacgagtcggcccacatcagcgacgccgtgggcgtggtggcccaggccgtgcacgagct cctcgagaaggagaacatcaccgacccgccgcggggctgcgtgggcaacaccaacatctggaagaccgggccgct cttcaagagagtgctgatgtcttccaagtatgcggatggggtgactggtcgcgtggagttcaatgaggatgggga ccggaagttcgccaactacagcatcatgaacctgcagaaccgcaagctggtgcaagtgggcatctacaatggcac ccacgtcatccctaatgacaggaagatcatctggccaggcggagagacagagaagcctcgagggtaccagatgtc caccagactgaagattgtgacgatccaccaggagcccttcgtgtacgtcaagcccacgctgagtgatgggacatg caaggaggagttcacagtcaacggcgacccagtcaagaaggtgatctgcaccgggcccaacgacacgtcgccggg 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cgtattaaaacacgatacattgttattagtacatttattaagcgctagattctgtgcgttgttgatttacagaca attgttgtacgtattttaataattcattaaatttataatctttagggtggtatgttagagcgaaaatcaaatgat tttcagcgtctttatatctgaatttaaatattaaatcctcaatagatttgtaaaataggtttcgattagtttcaa acaagggttgtttttccgaaccgatggctggactatctaatggattttcgctcaacgccacaaaacttgccaaat cttgtagcagcaatctagctttgtcgatattcgtttgtgttttgttttgtaataaaggttcgacgtcgttcaaaa tattatgcgcttttgtatttctttcatcactgtcgttagtgtacaattgactcgacgtaaacacgttaaatagag cttggacatatttaacatcgggcgtgttagctttattaggccgattatcgtcgtcgtcccaaccctcgtcgttag aagttgcttccgaagacgattttgccatagccacacgacgcctattaattgtgtcggctaacacgtccgcgatca aatttgtagttgagctttttggaattatttctgattgcgggcgtttttgggcgggtttcaatctaactgtgcccg attttaattcagacaacacgttagaaagcgatggtgcaggcggtggtaacatttcagacggcaaatctactaatg gcggcggtggtggagctgatgataaatctaccatcggtggaggcgcaggcggggctggcggcggaggcggaggcg gaggtggtggcggtgatgcagacggcggtttaggctcaaatgtctctttaggcaacacagtcggcacctcaacta ttgtactggtttcgggcgccgtttttggtttgaccggtctgagacgagtgcgatttttttcgtttctaatagctt ccaacaattgttgtctgtcgtctaaaggtgcagcgggttgaggttccgtcggcattggtggagcgggcggcaatt cagacatcgatggtggtggtggtggtggaggcgctggaatgttaggcacgggagaaggtggtggcggcggtgccg ccggtataatttgttctggtttagtttgttcgcgcacgattgtgggcaccggcgcaggcgccgctggctgcacaa cggaaggtcgtctgcttcgaggcagcgcttggggtggtggcaattcaatattataattggaatacaaatcgtaaa aatctgctataagcattgtaatttcgctatcgtttaccgtgccgatatttaacaaccgctcaatgtaagcaattg tattgtaaagagattgtctcaagctcggaacgctgcgctcggtcgttcggctgcggcgagcggtatcagctcact caaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaa aggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaa atcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccc tcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgc tttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaac cccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttat cgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagt ggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaa aaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcaga ttacgcgcagaaaaaaaggatctcaagaagatcctttgttaccaatgcttaatcagtgaggcacctatctcagcg atctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttacca tctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagcca gccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaa gctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgc tcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgc aaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggtt atggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaacc aagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgcca catagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctg ttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttct gggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcata ctcttcctttttcaatattattgaagcatttatcagggttattgtctcatgtccgcgcgtttcctgcatctttta atcaaatcccaagatgtgtataaaccaccaaactgccaaaaaatgaaaactgtcgacaagctctgtccgtttgct ggcaactgcaagggtctcaatcctatttgtaattattgaataataaaacaattataaatgtcaaatttgtttttt attaacgatacaaaccaaacgcaacaagaacatttgtagtattatctataattgaaaacgcgtagttataatcgc tgaggtaatatttaaaatcattttcaaatgattcacagttaatttgcgacaatataattttattttcacataaac tagacgccttgtcgtcttcttcttcgtattccttctctttttcatttttctcttcataaaaattaacatagttat tatcgtatccatatatgtatctatcgtatagagtaaattttttgttgtcataaatatatatgtcttttttaatgg ggtgtatagtaccgctgcgcatagtttttctgtaatttacaacagtgctattttctggtagttcttcggagtgtg ttgctttaattattaaatttatataatcaatgaatttgggatcgtcggttttgtacaatatgttgccggcatagt acgcagcttcttctagttcaattacaccattttttagcagcaccggattaacataactttccaaaatgttgtacg aaccgttaaacaaaaacagttcacctcccttttctatactattgtctgcgagcagttgtttgttgttaaaaataa cagccattgtaatgagacgcacaaactaatatcacaaactggaaatgtctatcaatatatagttgctctagttat taatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaat ggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgcca atagggactttccattgacgtcaatgggtggactatttacggtaaactgcccacttggcagtacatcaagtgtat catatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgacc ttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgcatggtcgaggtgagccc cacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattatt ttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcgg ggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcg gcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgacgctgccttcgccccgtgcccc gctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacg gcccttctccttcgggctgtaattagcgcttggtttaatgacggcttgtttcttttctgtggctgcgtgaaagcc ttgaggggctccgggagggccctttgtgcggggggagcggctcggggctgtccgcggggggacggctgccttcgg ggggga^cgggg^caggg^cggggttcggcttctggcgtgtgaccggcggctctagagcctctgetaaccatgtteat gccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattg gatcggaccgaaattaatacgactcactata SEQ ID N04 (polypeptide sequence of NMDAR subunit NR2b) mkpraeccspkfwlvlavlavsgsrarsqksppsigiavilvgtsdevaikdahekddfhhlsvvprvelvamne tdpksiitricdlmsdrkiqgvvfaddtdqeaiaqildfisaqtltpilgihggssmimadkdessmffqfgpsi eqqasvmlnimeeydwyifsivttyfpgyqdfvnkirstiensfvgweleevllldmslddgdskiqnqlkklqs piillyctkeeatyifevansvgltgygytwivpslvagdtdtvpaefptglisvsydewdyglparvrdgiaii ttaasdmlsehsfipepksscynthekriyqsnmlnrylinvtfegrnlsfsedgyqmhpklviillnkerkwer vgkwkdkslqmkyyvwprmcpeteeqeddhlsivtleeapfvivesvdplsgtcmrntvpcqkrivtenktdeep gyikkcckgfcidilkkisksvkftydlylvtngkhgkkingtwngmigevvmkraymavgsltineersevvdf svpfietgisvmvsrsngtvspsaflepfsadvwvmmfvmllivsavavfvfeyfspvgynrcladgrepggpsf tigkaiwllwglvfnnsvpvqnpkgttskimvsvwaffaviflasytanlaafmiqeeyvdqvsglsdkkfqrpn dfsppfrfgtvpngsternirnnyaemhaymgkfnqrgvddallslktgkldafiydaavlnymagrdegcklvt igsgkvfastgygiaiqkdsgwkrqvdlailqlfgdgemeelealwltgichneknevmssqldidnmagvfyml gaamalslitficehlfywqfrhcfmgvcsgkpgmvfsisrgiyscihgvaieerqsvmnsptatmnnthsnilr llrtaknmanlsgvngspqsaldfirressvydisehrrsfthsdcksynnppceenlfsdyisevertfgnlql kdsnvyqdhyhhhhrphsigsassidglydcdnppfttqsrsiskkpldiglpsskhsqlsdlygkfsfksdrys ghddlirsdvsdisthtvtygniegnaakrrkqqykdslkkrpasaksrrefdeielayrrrpprspdhkryfrd keglrdfyldqfrtkensphwehvdltdiykersddfkrdsvsgggpctnrshikhgtgdkhgvvsgvpapwekn ltnvewedrsggnfcrscpsklhnysttvtgqnsgrqacirceackkagnlydisednslqeldqpaapvavtsn asttkypqsptnskaqkknrnklrrqhsydtfvdlqkeeaalaprsvslkdkgrfmdgspyahmfemsagestfa nnkssvptaghhhhnnpgggymlskslypdrvtqnpfiptfgddqcllhgsksyffrqptvagaskarpdfralv tnkpvvsalhgavparfqkdicignqsnpcvpnnknprafngssnghvyeklssiesdv

SEQ ID N05 plasmid used for permanently expressing or stable transfection of human NR1 in cells gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatag ttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagc tacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgc ttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaatt acggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcc tggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaa tagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaa gtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgc ccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattacca tggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagt ctccaccccattgacgtcaatgggagtttgttttggaaccaaaatcaacgggactttccaaaatgtcg taacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagag ctctccctatcagtgatagagatctccctatcagtgatagagatcgtcgacgagctcgtttagtgaac cgtcagatcgcctgaagctgggtaccagctgctagcaagcttgccgccgacatgtctaccatgcgcct gctgacgctcgccctgctgttctcctgctccgtcgcccgtgccgcgtgcgaccccaagatcgtcaaca ttggcgcggtgctgagcacgcggaagcacgagcagatgttccgcgaggccgtgaaccaggccaacaag cggcacggctcctggaagattcagctcaatgccacctccgtcacgcacaagcccaacgccatccagat ggctctgtcggtgtgcgaggacctcatctccagccaggtctacgccatcctagttagccatccaccta cccccaacgaccacttcactcccacccctgtctcctacacagccggcttctaccgcatacccgtgctg gggctgaccacccgcatgtccatctactcggacaagagcatccacctgagcttcctgcgcaccgtgcc gccctactcccaccagtccagcgtgtggtttgagatgatgcgtgtctacagctggaaccacatcatcc tgctggtcagcgacgaccacgagggccgggcggctcagaaacgcctggagacgctgctggaggagcgt gagtccaaggcagagaaggtgctgcagtttgacccagggaccaagaacgtgacggccctgctgatgga ggcgaaagagctggaggcccgggtcatcatcctttctgccagcgaggacgatgctgccactgtatacc gcgcagccgcgatgctgaacatgacgggctccgggtacgtgtggctggtcggcgagcgcgagatctcg gggaacgccctgcgctacgccccagacggcatcctcgggctgcagctcatcaacggcaagaacgagtc ggcccacatcagcgacgccgtgggcgtggtggcccaggccgtgcacgagctcctcgagaaggagaaca tcaccgacccgccgcggggctgcgtgggcaacaccaacatctggaagaccgggccgctcttcaagaga gtgctgatgtcttccaagtatgcggatggggtgactggtcgcgtggagttcaatgaggatggggaccg gaagttcgccaactacagcatcatgaacctgcagaaccgcaagctggtgcaagtgggcatctacaatg gcacccacgtcatccctaatgacaggaagatcatctggccaggcggagagacagagaagcctcgaggg taccagatgtccaccagactgaagattgtgacgatccaccaggagcccttcgtgtacgtcaagcccac gctgagtgatgggacatgcaaggaggagttcacagtcaacggcgacccagtcaagaaggtgatctgca ccgggcccaacgacacgtcgccgggcagcccccgccacacggtgcctcagtgttgctacggcttttgc atcgacctgctcatcaagctggcacggaccatgaacttcacctacgaggtgcacctggtggcagatgg caagttcggcacacaggagcgggtgaacaacagcaacaagaaggagtggaatgggatgatgggcgagc tgctcagcgggcaggcagacatgatcgtggcgccgctaaccataaacaacgagcgcgcgcagtacatc gagttttccaagcccttcaagtaccagggcctgactattctggtcaagaaggagattccccggagcac gctggactcgttcatgcagccgttccagagcacactgtggctgctggtggggctgtcggtgcacgtgg tggccgtgatgctgtacctgctggaccgcttcagccccttcggccggttcaaggtgaacagcgaggag gaggaggaggacgcactgaccctgtcctcggccatgtggttctcctggggcgtcctgctcaactccgg catcggggaaggcgcccccagatcgttctcagcgcgcatcctgggcatggtgtgggccggctttgcca tgatcatcgtggcctcctacaccgccaacctggcggccttcctggtgctggaccggccggaggagcgc atcacgggcatcaacgaccctcggctgaggaacccctcggacaagtttatctacgccacggtgaagca gagctccgtggatatctacttccggcgccaggtggagctgagcaccatgtaccggcatatggagaagc acaactacgagagtgcggcggaggccatccaggccgtgagagacaacaagctgcatgccttcatctgg gactcggcggtgctggagttcgaggcctcgcagaagtgcgacctggtgacgactggagagctgttttt ccgctcgggcttcggcataggcatgcgcaaagacagcccctggaagcagaacgtctccctgtccatcc tcaagtcccacgagaatggcttcatggaagacctggacaagacgtgggttcggtatcaggaatgtgac tcgcgcagcaacgcccctgcgacccttacttttgagaacatggccggggtcttcatgctggtagctgg gggcatcgtggccgggatcttcctgattttcatcgagattgcctacaagcggcacaaggatgctcgcc ggaagcagatgcagctggcctttgccgccgttaacgtgtggcggaagaacctgcaggatagaaagagt ggtagagcagagcctgaccctaaaaagaaagccacatttagggctatcacctccaccctggcttccag cttcaagaggcgtaggtcctccaaagacacgagcaccgggggtggacgcggcgctttgcaaaaccaaa aagacacagtgctgccgcgacgcgctattgagagggaggagggccagctgcagctgtgttcccgtcat agggagagctgactcgagtctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctag ttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactg tcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggt ggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtggg ctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcg gcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcg cccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaa tcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagg gtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacg ttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttga tttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacg cgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaa gtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggc agaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatccc gcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcag aggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggc ttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcagcacgtgttgacaattaa tcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgac cagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcg ggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatc agcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacga gctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccg agatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttc gtggccgaggagcaggactgacacgtgctacgagatttcgattccaccgccgccttctatgaaaggtt gggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagt tcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaat ttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatctta tcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgt gaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggt gcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacct gtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctctt ccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactca aaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggcca gcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacg agcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcg tttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgc ctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtagg tcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggt aactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacag gattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctaca ctagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagc tcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgcag aaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaact cacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaa tgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcag tgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtaga taactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctca ccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaac tttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaata gtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttca ttcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttag ctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcag cactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaacc aagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataatac cgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaa ggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatct tttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataag ggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggtt attgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcaca tttccccgaaaagtgccacctgacgtc

SEQ ID N06 (plasmid used for transiently expressing rat NR1 in cells) ttcgagctcgcccgacattgattattgactagttattaatagtaatcaattacggggtcattagttca tagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacg acccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattga cgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaag tacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttat gggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttgg cagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgt caatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccat tgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgt cagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcct ccgcggccgggaacggtgcattggaacgcggattccccgtgccaagagtgacgtaagtaccgcctata gagtctataggcccacccccttggcttcgttagaacgcggctacaattaatacataaccttatgtatc atacacatacgatttaggtgacactatagaataacatccactttgcctttctctccacaggtgtccac tcccaggtccaactgcacctcggttctaagcttgatatcgaattccgttgctgtcgaatattggtttg ttaaggcagtttctgtagaggtttctaagagaccagtcgcgcagtccgcgctgctgtcctttccgcct tttccgcgcgggtgttcgagcagcgccaaacacgcttcagcacctcggacagcatccgccgcgctcgc ccggggctcctagagaacccgggggcgcttgaccgcgcgcgggcggcccgcgggtcgtacatcgcgag gtcgtcgcactcgcgcaacccagagccaggcccgctgtgcccggagctcatgagcaccatgcacctgc tgacattcgccctgcttttttcctgctccttcgcccgcgccgcctgcgaccccaagatcgtcaacatc ggcgcggtgctgagcacgcgcaagcatgaacagatgttccgcgaggcagtaaaccaggccaataagcg acacggctcttggaagatacagctcaacgccacttctgtcacccacaagcccaacgccatacagatgg ccctgtcagtgtgtgaggacctcatctctagccaggtctacgctatcctagttagccacccgcctact cccaacgaccacttcactcccacccctgtctcctacacagctggcttctacagaatccctgtcctggg actgactacccgaatgtccatctactctgacaagagtatccacctgagtttccttcgcacggtgccgc cctactcccaccagtccagcgtctggtttgagatgatgcgagtctacaactggaaccacatcatcctg ctggtcagcgacgaccacgagggacgggcagcgcagaagcgcttggagacgttgctggaggaacggga gtccaaggcagagaaggtgctgcagtttgacccaggaaccaagaatgtgacggctctgctgatggagg cccgggaactggaggcccgggtcatcatcctttctgcaagcgaggacgacgctgccacagtgtaccgc gcagccgcaatgctgaacatgacgggctctgggtacgtgtggctggtcggggaacgcgagatctctgg gaacgccctgcgctacgctcctgatggcatcatcggacttcagctcatcaatggcaagaatgagtcag cccacatcagtgacgccgtgggcgtggtggcacaggcagttcacgaactcctagagaaggagaatatc actgacccaccgcggggttgcgtgggcaacaccaacatctggaagacaggaccattgttcaagagggt gctgatgtcttctaagtatgcggacggagtgactggccgtgtggaattcaatgaggatggggaccgga agtttgccaactatagtatcatgaacctgcagaaccgcaagctggtgcaagtgggcatctacaatggt acccatgtcatcccaaatgacaggaagatcatctggccaggaggagagacagagaaacctcgaggata ccagatgtccaccagactaaagatagtgacaatccaccaagagcccttcgtgtacgtcaagcccacaa tgagtgatgggacatgcaaagaggagttcacagtcaatggtgacccagtgaagaaggtgatctgtacg gggcctaatgacacgtccccaggcagcccacgccacacagtgccccagtgctgctatggcttctgcat agacctgctcatcaagctggcgcggaccatgaattttacctatgaggtgcacctggtggcagatggca agtttggcacacaggagcgggtaaacaacagcaacaaaaaggagtggaacggaatgatgggcgagcta ctcagtggccaagcggacatgattgtggcaccactgaccatcaacaatgagcgtgcgcagtacataga gttctccaagcccttcaagtaccagggcctgaccattttggtcaagaaggagattcccaggagcacac tggactcatttatgcagccttttcagagcacactgtggttgctagtaggactgtcagttcatgtggtg gctgtgatgctgtacctgctggaccgcttcagtccctttggccgattcaaggtgaacagtgaggagga ggaggaagatgcactgaccctgtcctctgccatgtggttttcctggggcgtcctgctcaactccggca ttggggaaggtgccccccggagtttctctgcacgtatcctaggcatggtgtgggctggtttcgccatg atcatagtggcttcctacactgccaacttggcagctttcctggtgctggatcggcctgaggagcgcat cacgggcatcaatgaccccaggctcagaaacccctcagacaagttcatctacgcaactgtaaagcaga gctccgtggacatctacttccggaggcaggtggagttgagtaccatgtaccggcacatggaaaaacac aattacgagagcgcagctgaggccatccaggctgtgcgggacaacaagctgcacgcctttatctggga ctcggccgtgctggagtttgaggcttcacagaagtgcgatctggtgaccacgggtgagctgttcttcc gctcaggctttggcatcggcatgcgcaaggacagcccctggaagcagaacgtttccctgtccatactc aagtcccatgagaatggcttcatggaagatctggataagacatgggttcggtatcaggaatgcgactc ccgcagcaatgctcctgcaaccctcacttttgagaacatggcaggggtcttcatgctggtggctggag gcatcgtagctgggattttcctcattttcattgagatcgcctacaagcgacacaaggatgcccgtagg aagcagatgcagctggcttttgcagccgtgaacgtgtggaggaagaacctgcaggatagaaagagtgg tagagcagagcccgaccctaaaaagaaagccacatttagggctatcacctccaccctggcctccagct tcaagagacgtaggtcctccaaagacacgagcaccgggggtggacgcggcgctttgcaaaaccaaaaa gacacagtgctgccgcgacgcgctattgagagggaggagggccagctgcagctgtgttcccgtcatag ggagagctgagacgccccgcccgccctcctctgcccctcccccgcagacagacgcacgggacagcggc ctggcccacgcagagccccggagcacgacggggtcgggggaggagcactcccagcctcccccaggccg tgcccgcctgcccaccggtcggccggctggccggtccaccctgtcccggccccgcgcgtgcccccgac gtcggagctaacgggccgccttgtctgtgtatttctattttacagcagtaccatcccactgatatcac gggcccgctcaacctctcagatccctcggtcagcaccgtggtgtgaggccccccggaggcgcccacct gcccagttagcccggccaaggacactgatgagtcctgctgctcgggaaggcctgagggaagcccaccc gccccagagactgcccaccctgggcctcccgtccgcctgctctgctgcctggcgggcagcccctgcag gaccaaggtgcggaccagagcggctgaggatgggccagagctgagccggctgggcagggccacagggc gctccggcagaggcagggccctgaggtctctgagcagtggggtgaggggcctaagtggccccggtcgg aggagtctggagcagaaatggcagccccatccttcctccagccactaccccaagctacagtgggggcc tatggccccagcttgctaggtcacccccgacccttcctccagcgcctgctctctgcaacttgatttcc acctctctcctgctgcaccaccctcccacgacatttccccaccccattcactgggttgtctctgacct ttcccagggctagccttcactgccctagtggcagtgcttcaggggtgctttctggctcccagacatct agggctccagactccaagagggctgagccttctcttctgtccgcagccacaataggcttcctcagacg ctggctcgtgatgagtcccgcaccttgggcaccagggagcgccatctgcctcccagtccggtgtcact caccccactaccttgtacatgaccagctctcccagtgtcccagtgtctgccccagggacaccgggcgc gcacagccacccctaatcccggtattcagtggtgatgcctaaaggaatgtcagaaaaaaaaaaaaaaa gcggccgctcgagcatgcatctagaggatccccggggaattcaatcgatggccgccatggcccaactt gtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttt tttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatcgatcgg gaattaattcggcgcagcaccatggcctgaaataacctctgaaagaggaacttggttaggtaccttct gaggcggaaagaaccagctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagca ggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctcccc agcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgc ccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatt tatgcagaggccgaggccgcctcggcctgtgagctattccagaagtagtgaggaggcttttttggagg cctaggcttttgcaaaaagctgttaacagcttggcactggccgtcgttttacaacgtcgtgactggga aaaccctggcgttacccaacttaatcgccttgcagcacatccccccttcgccacctggcgtaatagcg aagaggcccgcaccgatcgcccttcccaacagttgcgtagcctgaatggcgaatggcgcctgatgcgg tattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccc tgtacgggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgc cctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaag ctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaactt gatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttgga gtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctatt cttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaa tttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctc tgatgccgcatagttaagccaactccgctatcgctacgtgactgggtcatggctgcgccccgacaccc gccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtga ccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagtattct tgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttctta gacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacatt caaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagt atgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgc tcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatgg aactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagc acttttaaagttctgctatgtggcgcggtattatcccgtgatgacgccgggcaagagcaactcggtcg ccgcatacactattctcagaatgacttggttgagtactcaccagccacagaaaagcatcttacggatg gcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttactt ctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcg ccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgccag cagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaa ttaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctg gtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccag atggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaat agacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcata tatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgata atctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatc aaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgct accagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagca gagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgta gcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtg tcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggtt cgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattga gaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacagg agagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacc tctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaac gcggcctttttacggttcctggccttttggtggccttttgctcacatgttctttcctgcgttatcccc tgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccg agcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgt tggccgattcattaatccagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgc aattaatgtgagttacctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgt tgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattac

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

Example:

HEK 293 cells

Using comparative slides comprising fixed HEK 293 cells transiently overexpressing rat NR1, transiently overexpressing human NR1 and permanently overexpressing human NR1, various patient samples were analyzed. Cells were grown in DMEM medium comprising 10% heat inactivated fetal calf serum and 1 X antibiotic-antimycotic (Invitrogen # 15240) at 37 °C and 5% C02. For a transient transfection with rat or human NR1, plasmids having the sequences SEQ ID N06 or SEQ ID N03, respectively, were used. For a stable transfection with human NR1, the plasmid having sequence SEQ ID N05 was used. The protocol provided by Invitrogen for the T-Rex™ System (Catalog number K1020-01, from 2011) was used for stable transfection. However, ExGen500 (Catalog number 12783652, Thermofisher) was used rather than Lipofectamin. For transient transfection the ExGen500 protocol was used. Fixed cells were prepared by contacting cells grown on microscopy slides with acetone by washing the cells in PBS followed by incubation in 100% acetone.

Methodology and reagents

The methodology and reagents used are according to the manufacturer’s instruction in the 11 FT Neurology Mosaics (EUROIMMUN Medizinische Labordiagnostika AG, product number FA112d-1. The test system centers around the incubation of combinations of substrates with diluted patient sample. If the reaction is positive, specific antibodies of classes IgA, IgG and IgM attach to the antigens. In a second step, the attached antibodies are stained with FITC- labelled anti-human antibodies and made visible with a fluorescence microscope. Briefly, human serum samples were diluted 1:10 or 1:100 in PBS-Tween, followed by vortexing for 2 seconds. 30 pi samples per field are incubated for 30 mins using the TITERPLANE technology, followed by washing in PBS-Tween for 1 s, followed by incubation in PBS-Tween for 5 minutes in a cuvette for thorough washing. 25 mI of secondary antibody conjugate are then applied and incubated for 30 minutes using the TITERPLANE technology, followed by washing in PBS-Tween for 1 s, followed by incubation in PBS-Tween for 5 minutes in a cuvette for thorough washing.

After the incubations, the carrier with the fields is covered with up to 10 mI of mounting medium per field and a glass cover slide, followed by fluorescence microscopy analysis using a EUROSTAR microscope (EUROIMMUN Medizinische Labordiagnostika AG, Lubeck.

Results:

The results are shown in Figs. 1 to 5, as described in detail in the legends relating to these figures.

Overall, it is obvious that the use of HEK293 cells transiently expressing NR1 subunit from the rat NMDA receptor produces artefacts in the form of protrusions and spherical bodies, which obscure the images of stained cells and can even be confused with them.

The use of HEK293 cells transiently expressing NR1 subunit from the human receptor produces spherical bodies, but considerably less pronounced protrusions.

The use of HEK293 cells permanently overexpression NR1 from the human receptor produces neither artefact. Confusion of the staff analyzing the images and false-positive results can be avoided by using these cells.

Claims

Claims

1. A carrier for microscopic immunofluorescence analysis coated with a eukaryotic, preferably mammalian cell overexpressing a polypeptide comprising human NMDA receptor or a variant thereof.

2. The carrier according to claim 1 , wherein the cell is transiently transfected with human NMDA receptor or a variant thereof.

3. The carrier according to claim 2, wherein the cell is permanently overexpressing human NMDA receptor or a variant thereof of an NMDA receptor NR1 subunit or a variant thereof.

4. The carrier according to any of claims 1 to 3, wherein the cell is a fixed cell.

5. The carrier according to any of claims 1 to 3, wherein the cell is fixed using formalin or acetone.

6. The carrier according to any of claims 1 to 5, wherein the cell is bound to an autoantibody to human NMDA, which is preferably bound to a secondary antibody.

7. A carrier comprising the cell according to any of claims 1 to 6 and preferably a mock- transfected cell, wherein the cell and the mock-transfected cell are spatially separated.

8. The carrier according to any of claims 1 to 7, further comprising one or more additional cells, preferably all from the group comprising a cell overexpressing GABA B or a variant thereof, a cell overexpressing LGI1 or a variant thereof, a cell overexpressing AMPAR1/R2 or a variant thereof, a cell overexpressing CASPR2 or a variant thereof, a cell overexpressing DPPX or a variant thereof, a cell overexpressing Hu or a variant thereof, a cell overexpressing Ma2 or a variant thereof, a cell overexpressing CV2 or a variant thereof and a cell overexpressing amphiphysin or a variant thereof, which are spatially separated from the cell overexpressing human NMDA receptor and, if present, the mock-transfected cell.

9. The carrier according to any of claims 1 to 8, wherein the carrier comprises mammalian hippocampus and/or mammalian cerebellum, preferably spatially separated from the cell or cells, wherein the mammal is preferably rodent or primate.

10. A kit comprising the carrier according to any of claims 1 to 9, further comprising one or more, preferably all reagents from the group comprising a secondary antibody, preferably labeled with a detectable label, a wash solution, a positive control, a negative control, a detergent, a cover glass, a mounting medium and a physiological salt solution, preferably PBS.

11. A method comprising the steps a) contacting carrier according to any of claims 1 to 10 with a sample, b) washing the cell and c) detecting an autoantibody to human NMDA receptor bound to the cell in step a).

12. The method according to claim 11, wherein the sample is a sample from a human.

13. The method according to any of claims 11 or 12, wherein the autoantibody is detected in step c) using a secondary autoantibody labeled with a fluorescent dye.

14. A use of a polypeptide comprising human NMDA receptor or a variant thereof or the carrier according to any of claims 1 to 10 for producing artefact-free signals for the detection of an autoantibody to NMDA receptor.

15. A use of a polypeptide comprising human NMDA receptor or a variant thereof or the carrier according to any of claims 1 to 10 for the high specificity diagnosis of NMDA receptor autoimmune encephalitis.