Autoimmune Encephalitis Antibody Transient and Stable Transfection Detection Methods and Their Applications

Abstract

The present invention provides a method for detecting transient and stable transfection of autoimmunity encephalitis antibodies and its application. Specifically, the present invention provides a recombinant cell expressing mutant NMDAR, wherein the recombinant cell expresses a fusion protein of exogenous mutant NMDAR and a fluorescent protein, and has a high transfection survival rate. The recombinant cell of the present invention can be used to detect antibodies related to autoimmunity encephalitis, including NMDAR, AMPAR1, AMPAR2, LGI1, Caspr2, GABA B R. The present invention also provides a recombinant transfected cell line based on the present invention, and uses the transfected cell line to detect the method for anti-autoimmunity encephalitis by indirect immunofluorescence method. The method of the present invention has high sensitivity and specificity, and can accurately detect the types of pathogenic antibodies, so as to help patients implement targeted immunotherapy and recover health.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to a method for detecting transient transfection and stable transfection of autoimmune encephalitis antibodies and its application. Background Art

[0002] Autoimmune encephalitis (AE) is a type of central nervous system autoimmune disease newly recognized in the past decade or so. Its characteristic is that there are autoantibodies against neuronal surface proteins, ion channels, and receptors in the patient's body. These autoantibodies are considered to be pathogenic and can be used as markers for disease diagnosis. AE patients often present with severe clinical symptoms such as abnormal mental behavior, epileptic seizures, memory disorders, cognitive abnormalities, movement abnormalities, autonomic nervous system dysfunction, and decreased consciousness. However, early immunotherapy can achieve good curative effects and disease outcomes. Generally speaking, the common specific autoantibodies mediating AE are NMDAR, AMPAR1, AMPAR2, LGI1, Caspr2, GABA B R six kinds, and there are significant differences in the tumors and prognoses associated with AE patients mediated by different antibodies. Therefore, timely and accurate detection of relevant autoantibodies is of extremely important significance for the early diagnosis and treatment of AE.

[0003] In 2005, a group of symptoms including memory loss, mental symptoms, decreased consciousness level, and hypoventilation were first discovered in four young female patients with ovarian teratomas. [1] Subsequently, a specific antibody against N-methyl-D-aspartate receptor (NMDAR) was detected in these patients and several other patients with similar neurological symptoms. Since then, many of the reported cases have been children or young male patients, with or without tumors. This disease was named anti-NMDAR encephalitis.

[0004] Studies have shown that autoimmune encephalitis antibodies are one of the causes of autoimmune encephalitis. However, there is still a lack of a detection method for AE autoantibodies with both high sensitivity and specificity in this field.

[0005] Although attempts have been made to develop a detection method based on the interaction between AE autoantibodies and NMDAR, the conformation of recombinant expressed and purified NMDAR is different from that of NMDAR in the natural state, resulting in poor sensitivity and specificity.

[0006] Another detection method is based on cell-based assay (CBA), and a representative method is indirect immunofluorescence assay. In this method, generally, NMDAR subunits (NR1 and NR2A) are expressed by recombinant transfection of cell lines, and then anti-NMDAR antibodies are detected by indirect immunofluorescence assay, which has a certain sensitivity. However, this method still has disadvantages. For example, NMDAR subunits (NR1 and NR2A) can cause a large number of cell deaths, resulting in inaccurate measurement and poor repeatability.

[0007] Therefore, there is an urgent need in the art to develop new detection methods for AE autoantibodies with high sensitivity and high specificity. Summary of the Invention

[0008] The object of the present invention is to provide a detection method for AE autoantibodies with high sensitivity and high specificity.

[0009] Another object of the present invention is to provide a recombinant transfected cell line based on the present invention, and to detect the method for autoimmune encephalitis by using the transfected cell line through indirect immunofluorescence assay.

[0010] In the first aspect of the present invention, a method for preparing recombinant cells is provided, and the method includes the steps of:

[0011] (a) Providing cells to be transfected, a first plasmid and a second plasmid for transfection; wherein, the cells to be transfected are renal cells,

[0012] The first plasmid contains a first expression cassette, and the first expression cassette is used to express the fusion protein P0 shown in Formula I,

[0013] Z1-Z2 (I)

[0014] In the formula,

[0015] Z1 is the mutant NMDAR subunit NR1, and the sequence of the mutant NMDAR subunit NR1 is shown in SEQ ID No: 2;

[0016] Z2 is a GFP protein element;

[0017] The second plasmid contains a second expression cassette, and the second expression cassette is used to express the NMDAR subunit NR2A;

[0018] (b) Transfecting the first plasmid and the second plasmid into the cells to be transfected, thereby obtaining the recombinant cells C0, wherein the recombinant cells C0 express recombinant NMDAR protein.

[0019] (c) Detecting the survival of the recombinant cells obtained in step (b).

[0020] In another preferred embodiment, the recombinant NMDAR protein is composed of the fusion protein P0 shown in Formula I and the NMDAR subunit NR2A.

[0021] In another preferred embodiment, the first plasmid and the second plasmid are the same plasmid or different plasmids.

[0022] In another preferred embodiment, the method further comprises:

[0023] In another preferred embodiment, the cells to be transfected are human kidney cells.

[0024] In another preferred embodiment, the cells to be transfected are HEK-293 cells.

[0025] In the second aspect of the present invention, there is provided a recombinant cell (i.e., recombinant cell C0), and the recombinant cell is prepared by the method described in the first aspect.

[0026] In another preferred embodiment, the recombinant cell expresses an NMDAR mutant protein, and the NMDAR mutant protein is composed of an NR1 subunit and an NR2A subunit, wherein the NR1 subunit is a mutant subunit, and the 815th amino acid of the NR1 subunit is mutated from the wild-type glycine to arginine. (Note: The 815th amino acid is located in the M4 segment of the NMDAR transmembrane region).

[0027] In another preferred embodiment, the NR1 subunit exists in the form of the fusion protein shown in Formula I.

[0028] In another preferred embodiment, the recombinant cell expresses an NMDAR mutant protein, and the NMDAR mutant protein exists on the cell membrane of the recombinant cell in the form of a membrane protein.

[0029] In another preferred embodiment, the mutant NMDAR mutant protein exists on the cell membrane of the recombinant cell in the form of a transmembrane protein.

[0030] In another preferred embodiment, at 12-24 hours after transfection, the cell viability of the recombinant cell is ≥50%, preferably 50%-85%, more preferably 60%-75%.

[0031] In another preferred embodiment, the transfection refers to transfection with the first plasmid and the second plasmid.

[0032] In the third aspect of the present invention, there is provided the use of the recombinant cell described in the second aspect of the present invention or a transfection reagent for preparing the recombinant cell, which is used for preparing a detection reagent or kit for detecting autoimmune encephalitis.

[0033] In another preferred embodiment, the kit contains: (Y0) the recombinant cell C0 described in the second aspect of the present invention, or a transfection reagent for preparing the recombinant cell C0.

[0034] In another preferred embodiment, the kit further includes an instruction manual, which describes the method of use for detecting whether there is an antibody against the NMDAR protein in a test sample.

[0035] In another preferred embodiment, the kit further contains additional reagents selected from the following group:

[0036] (Y1) a recombinant cell C1 for expressing the AMPAR1 protein, or a transfection reagent for preparing the recombinant cell C1;

[0037] (Y2) a recombinant cell C2 for expressing the AMPAR2 protein, or a transfection reagent for preparing the recombinant cell C2;

[0038] (Y3) a recombinant cell C3 for expressing the LGI1 protein, or a transfection reagent for preparing the recombinant cell C3;

[0039] (Y4) a recombinant cell C4 for expressing the Caspr2 protein, or a transfection reagent for preparing the recombinant cell C4;

[0040] (Y5) a recombinant cell C5 for expressing GABA B R protein, or a transfection reagent for preparing the recombinant cell C5;

[0041] (Y6) any combination of Y1 to Y5.

[0042] In another preferred embodiment, the recombinant cells C0, C1, C2, C3, C4, and C5 are the same cell or different cells.

[0043] In another preferred embodiment, for the reagent, the recombinant cell C1 expresses a fusion protein P1 of the AMPAR1 protein and the GFP green fluorescent protein;

[0044] The recombinant cell C2 expresses a fusion protein P2 of the AMPAR2 protein and the GFP green fluorescent protein;

[0045] The recombinant cell C3 expresses a fusion protein P3 of the LGI1 protein and the GFP green fluorescent protein;

[0046] The recombinant cell C4 expresses a fusion protein P4 of the Caspr2 protein and the GFP green fluorescent protein;

[0047] The recombinant cell C5 expresses a fusion protein P5 of GABA B R protein and the GFP green fluorescent protein.

[0048] In the fourth aspect of the present invention, a detection kit is provided, and the kit contains:

[0049] (a) The recombinant cell C0 described in the second aspect of the present invention, or a transfection reagent for preparing the recombinant cell C0;

[0050] (b) A secondary antibody against the antibody of the anti-NMDAR protein.

[0051] In another preferred embodiment, the kit further contains an additional reagent selected from the following group:

[0052] (Y1) A recombinant cell C1 for expressing the AMPAR1 protein, or a transfection reagent for preparing the recombinant cell C1;

[0053] (Y2) A recombinant cell C2 for expressing the AMPAR2 protein, or a transfection reagent for preparing the recombinant cell C2;

[0054] (Y3) A recombinant cell C3 for expressing the LGI1 protein, or a transfection reagent for preparing the recombinant cell C3;

[0055] (Y4) A recombinant cell C4 for expressing the Caspr2 protein, or a transfection reagent for preparing the recombinant cell C4;

[0056] (Y5) A recombinant cell C5 for expressing the GABA B R protein, or a transfection reagent for preparing the recombinant cell C5;

[0057] (Y6) Any combination of the above Y1 to Y5.

[0058] In the fifth aspect of the present invention, a method for detecting whether there is an antibody against the anti-NMDAR protein in a sample is provided, including the steps of:

[0059] (a) Providing the recombinant cell described in the second aspect of the present invention, that is, the recombinant cell C0;

[0060] (b) Mixing the recombinant cell C0 with the sample to be tested to obtain a first mixture, so that the recombinant cell C0 binds to the antibody against the anti-NMDAR protein, thereby forming a first complex of "recombinant cell C0 - antibody against the anti-NMDAR protein";

[0061] (c) Washing the first mixture to obtain a washed first complex of "recombinant cell C0 - antibody against the anti-NMDAR protein";

[0062] (d) Mix the washed first complex of "recombinant cell C0 - anti - NMDAR protein antibody" with a secondary antibody to form a second complex of "recombinant cell C0 - anti - NMDAR protein antibody - secondary antibody"; and

[0063] (e) Detect the presence and / or quantity of the second complex of "recombinant cell C0 - anti - NMDAR protein antibody - secondary antibody" to obtain the detection result of the anti - NMDAR protein antibody.

[0064] In another preferred example, the method is non - diagnostic and non - therapeutic.

[0065] In another preferred example, the method is an in vitro method.

[0066] In another preferred example, in step (e), it includes detecting fluorescence and / or chromophore.

[0067] In another preferred example, the secondary antibody is a secondary antibody with a detectable label.

[0068] In another preferred example, the detectable label includes a chromogenic enzyme.

[0069] It should be understood that within the scope of the present invention, the above - mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Shows HEK293 cells transfected to express wild - type NMDAR and mutant NMDAR. Among them, Figure A shows transfection with wild - type GRIN1 plasmid + GRIN2A plasmid; Figure B shows transfection with pCDNA3.1 - GRIN1 - 815R - GFP plasmid + GRIN2A plasmid.

[0071] Figure 2 Shows the mortality of HEK293 cells transfected to express wild - type NMDAR and mutant NMDAR. Among them, the N1 / 2A group: transfected with wild - type GRIN1 plasmid + GRIN2A plasmid; the N1 - 815R group: transfected with pCDNA3.1 - GRIN1 - 815R - GFP plasmid (for expressing the fusion protein shown in Formula I) + GRIN2A plasmid; n = 5, P < 0.05.

[0072] Figure 3 Shows the structure of the pCDNA3.1 - GRIN1 - 815R - GFP plasmid.

[0073] Figure 4Shows the structure of the pCDNA3.1-GRIN2A plasmid. Detailed implementation mode

[0074] After extensive and in-depth research, the present inventors have developed for the first time a method with high specificity and high sensitivity for detecting antibodies against the NMDAR protein. The method of the present invention uses recombinant cells expressing a fusion protein P0 of formula I with a specific structure. Experiments have shown that the recombinant cells of the present invention prepared with the specific transfection reagent of the present invention can not only efficiently express the fusion protein P0 containing NMDAR, but also unexpectedly reduce the cytotoxicity (such as transfection toxicity) and cell death caused by the expression of exogenous NMDAR in non-neural cells, so that the immunofluorescence method based on the recombinant cells can detect antibodies against the NMDAR protein more specifically, more sensitively and more accurately. In addition, the recombinant cells of the present invention can further be used in combination with detection reagents and methods for other autoimmune encephalitis-specific antibodies (such as AMPAR1, AMPAR2, LGI1, Caspr2, GABA B R antibody) to more comprehensively and accurately provide the detection results of autoimmune encephalitis antibodies. On this basis, the present invention has been completed.

[0075] In a preferred embodiment, the present invention provides a method for expressing autoimmune encephalitis-specific antibodies (NMDAR, AMPAR1, AMPAR2, LGI1, Caspr2, GABA B R antibody) through a recombinant transfected cell line, and then detecting anti-AE specific antibodies by indirect immunofluorescence method to accurately detect AE.

[0076] Preferably, the present invention provides a method for detecting autoimmune encephalitis by transfecting a cell with a cDNA encoding the subunit of NMDAR shown in SEQ ID No:2 to obtain a recombinant cell, and then detecting the anti-NMDAR antibody by indirect immunofluorescence method.

[0077] Term

[0078] As used herein, "the method of the present invention", "the detection method of the present invention", "the detection method of the transfected cell line of the present invention" can be used interchangeably, and all refer to the method for detecting autoimmune encephalitis by indirect immunofluorescence method using a transfected cell line.

[0079] NMDAR

[0080] NMDAR, namely N-methyl-D-aspartate receptor, is an ionotropic glutamate receptor and a common AE-specific autoantigen. Clinically, anti-NMDAR type AE is mostly manifested as cognitive impairment, language disorder, seizure, movement disorder, dyskinesia or myotonia / abnormal posture, autonomic dysfunction or central hypoventilation, etc.

[0081] The wild-type human GRIN1 sequence is shown in SEQ ID No: 1:

[0082]

[0083] In the present invention, the gene sequence of the mutant GRIN1 is shown in SEQ ID NO.: 2:

[0084]

[0085] AMPAR

[0086] AMPAR is the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, an ionotropic glutamate receptor, and an AE-specific autoantigen. AMPAR1 and AMPAR2 are AMPARs with different compositions. Anti-AMPAR type AE is more common in women and can present with typical limbic encephalitis manifestations, including subacute disturbance of consciousness, depression, irritability, recent memory loss, and epileptic seizures due to medial temporal lobe dysfunction, etc.

[0087] LGI1

[0088] LGI1 is leucine-rich glioma-inactivated 1 protein, a common AE-specific autoantigen. Anti-LGI1 type AE clinically presents with various forms of epileptic seizures, typical facio-brachial dystonic seizures, refractory hyponatremia, autonomic dysfunction, etc. Head MRI shows obvious involvement of the temporal lobe.

[0089] Caspr2

[0090] Caspr2 is contactin associated protein-like 2, an AE-specific autoantibody. Anti-Caspr2 type AE can clinically present as limbic system encephalitis, Morvan syndrome, and neuromyotonia, etc.

[0091] GABA B R

[0092] GABA B R is the gamma aminobutyric acid receptor type B, belonging to the C family of G protein-coupled receptors. It is a common AE-specific autoantigen. Anti-GABA B R type AE, epileptic seizures often serve as the initial and prominent symptom during the disease process. Most patients develop limbic system encephalitis characterized by mental and behavioral abnormalities, memory impairment, and epileptic seizures during the course of the disease.

[0093] Plasmid

[0094] Plasmids are DNA molecules outside the chromosome (or nucleoid) in organisms such as bacteria, yeast, and actinomycetes. They exist in the cytoplasm (except for yeast, where the 2μm plasmid of yeast exists in the nucleus). They have the ability to replicate autonomously, enabling them to maintain a constant copy number in daughter cells and express the genetic information they carry. They are closed circular double-stranded DNA molecules. Plasmids are not essential substances for bacterial growth and reproduction and can be lost spontaneously or eliminated through artificial treatment, such as high temperature, ultraviolet light, etc. The genetic information carried by plasmids can endow host bacteria with certain biological characteristics, which is beneficial for bacteria to survive under specific environmental conditions.

[0095] Similar to the bacterial genome, plasmids also belong to circular double-stranded DNA and are therefore often used as vectors in DNA recombinant technology. A vector refers to a tool that delivers a useful foreign gene into a recipient cell through genetic engineering means for proliferation and expression. A certain target gene fragment is recombined into a plasmid to form a recombinant gene or recombinant. Then, this recombinant is transferred into a recipient cell (such as Escherichia coli) through microbiological transformation technology, enabling the target gene in the recombinant to reproduce or express in the recipient bacterium, thereby changing the original traits of the host cell or producing new substances.

[0096] The plasmids that can be used as the target transfection gene vectors in the present invention can be constructed by conventional methods or modified based on existing plasmids. For example, commercially available plasmids can be used for modification: NMDAR gene vector plasmid: wild-type GRIN1 plasmid (product number: P6908, company: Miaoling Plasmid Platform) + GRIN2A plasmid (product number: P8212, company: Miaoling Plasmid Platform).

[0097] A preferred NMDAR gene vector plasmid containing a mutation at position 815 in the present invention is the pCDNA3.1-GRIN1-815R-GFP plasmid + GRIN2A plasmid (product number: P8212, company: Miaoling Plasmid Platform). Among them, the purchased human wild-type pcDNA 3.1-GRIN1 plasmid (product number: P6908, company: Miaoling Plasmid Platform) is introduced with nucleotide mutations through gene editing, so that the 815th amino acid of the encoded protein is mutated from glycine to arginine, and then a GFP tag (fused expression of green fluorescent protein) is inserted into the plasmid to obtain the plasmid pCDNA3.1-GRIN1-815R-GFP.

[0098] The structure of pCDNA3.1-GRIN1-815R-GFP is as Figure 3 shown, and some of its basic information is as follows:

[0099] Gene name: GRIN1

[0100] Species: Human

[0101] Gene size: 2769bp

[0102] Gene information: XM_005266071.3

[0103] Editing information: The 815th amino acid is mutated from glycine to arginine

[0104] TAG: GFP (carboxyl terminus)

[0105] Cloning site (5'): BamHI

[0106] Cloning site (3'): EcoRI

[0107] Vector name: pCDNA3.1

[0108] Vector type: Mammalian expression

[0109] Vector size: 5417bp

[0110] Eukaryotic resistance: Neomycin, G418

[0111] Prokaryotic resistance: Ampicillin

[0112] Cloning strain: DH5α

[0113] Growth temperature: 37°C.

[0114] The structure of pCDNA3.1-GRIN2A is as Figure 4 shown, and some of its basic information is as follows:

[0115] Gene name: GRIN2A

[0116] Species: Human

[0117] Gene size: 3846bp

[0118] Gene information: NM_001134408.2

[0119] Editing information: None

[0120] TAG: None

[0121] Cloning site (5'): BamHI

[0122] Cloning site (3'): EcoRI

[0123] Vector name: pCDNA3.1

[0124] Vector type: Mammalian expression

[0125] Vector size: 5417bp

[0126] Eukaryotic resistance: Neomycin, G418

[0127] Prokaryotic resistance: Ampicillin

[0128] Cloning strain: DH5α

[0129] Growth temperature: 37 °C

[0130] Detection method of the present invention

[0131] The present invention provides a detection method for autoimmune encephalitis, comprising the steps of:

[0132] (a) Providing cells to be transfected (such as HEK-293 cells) and plasmids containing the target gene (i.e., the first plasmid and the second plasmid);

[0133] (b) Preparing a transfection system containing the plasmid, adding the cells to be transfected (such as HEK-293 cells) into a 96-well plate, transfecting with the transfection system, and changing the medium after the cultivation time t0 to obtain a transfected cell line;

[0134] (c) Incubating the transfected cells with the test sample and the secondary antibody in sequence to obtain a mixture of transfected cell-sample antibody-secondary antibody, wherein the secondary antibody is a specific secondary antibody labeled with a chromogenic enzyme and capable of binding to the test sample antibody.

[0135] In another preferred example, the HEK-293 cells are taken out from liquid nitrogen, resuscitated and passaged.

[0136] In another preferred example, the passaging is carried out when the cell density grows to about 70%.

[0137] In another preferred example, the passaging is carried out on 6 96-well plates.

[0138] In another preferred example, the density of the 96-well plate is 30,000 - 50,000 cells / well.

[0139] In another preferred example, the passaging is carried out overnight in a 37 °C cell incubator.

[0140] In another preferred example, the transfection system is a mixture containing plasmid, DMEM medium, and PEI reagent;

[0141] Wherein the transfection system contains 5 - 15 μL of DMEM medium, preferably 7 - 12 μL, more preferably 10 μL;

[0142] Contains 0.01 - 1 μg of plasmid, 0.05 - 2 μg, more preferably 0.1 μg;

[0143] Contains 0.03 - 3 μL of PET reagent, 0.1 - 1 μL, 0.3 μL;

[0144] In another preferred example, t0 is 5 - 10 hours, preferably 6 - 9 hours, more preferably 8 hours.

[0145] In another preferred example, the chromogenic enzyme is a red fluorescent protease.

[0146] In another preferred example, the secondary antibody is an anti - human IgG secondary antibody.

[0147] The advantages of the present invention include:

[0148] (a) The method for stably transfecting cells of the present invention can be used as a cell matrix for detecting autoantibodies in AE, and has the characteristics of simpler operation and better stability, which is worthy of popularization.

[0149] (b) The present invention unexpectedly discovers for the first time that the mutant protein obtained by mutating the amino acid at position 815 from glycine to arginine can significantly reduce the cytotoxicity caused to the cells to be transfected (such as HEK293 cells) when transfected with the cDNA encoding the NMDA receptor subunit, resulting in a significant decrease in the mortality rate of the transfected cells (the relative decrease is about 30%), thus helping to improve the detection efficiency and accuracy.

[0150] (c) Based on the positive of anti - NMDAR, the method of the present invention can, in combination with other detection reagents, further successfully detect the specific autoantibodies of anti - AMPAR encephalitis, anti - LGI1 encephalitis, anti - Caspr2 encephalitis, and anti - GABAR encephalitis. B Therefore, the detection method of the present invention has high detection efficiency, and can clearly diagnose the pathogenic cause, so as to formulate an effective treatment plan according to the cause and help the patient recover quickly.

[0151] (d) The method of the present invention has high sensitivity and high specificity, can accurately detect the types of pathogenic antibodies, and help the patient take the right medicine and recover.

[0152] (e) The transfected cell line used in the detection method of the present invention can be an instantaneously transfected cell line or a stably transfected cell line, both of which have the characteristics of high transfection survival rate and accurate detection results.

[0153] (f) The detection method of the present invention has the characteristics of high repeatability and high efficiency.

[0154] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions, for example, the conditions described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) by Sambrook et al., or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0155] General method

[0156] Live cell transfection method

[0157] This method is used to prepare a live cell transfection system for detecting anti-NMDAR antibodies in human serum and / or cerebrospinal fluid, and includes the following steps:

[0158] (1) Take out HEK-293 cells (from the Cell Bank of the Chinese Academy of Sciences) from liquid nitrogen, resuscitate and passage culture;

[0159] (2) Passage and seed plates: When the cell density grows to about 70%, passage the cells and seed 6 96-well plates (seeding density: 30,000 - 50,000 cells / well); Place them in a 37°C cell culture incubator and culture overnight;

[0160] (3) Transfection:

[0161] 1) Preparation of the transfection system: For each well: 10 μL of DMEM culture medium (product number: 12660012, company: GIBCO) + 0.1 μg of plasmid + 0.3 μL of PEI reagent;

[0162] 2) Transfect the plasmids containing the target genes into 6 96-well plates respectively: pCDNA3.1-GRIN1-815R-GFP (fused expression of green fluorescent protein) and pCDNA3.1-GRIN2A;

[0163] (4) Change the medium after 8 hours;

[0164] (5) It can be used for detection when the cell transfection efficiency reaches more than 40%.

[0165] Example 1 Establishment of a method for detecting autoantibodies in AE based on transiently transfected cells

[0166] 1.1 Group transfection of HEK293 cells:

[0167] (1) N1 / 2A group: Wild-type GRIN1 plasmid (product number: P6908, company: Miaoling Plasmid Platform) + GRIN2A plasmid (product number: P8212, company: Miaoling Plasmid Platform).

[0168] (2) Group N1-815R / 2A: Plasmid pCDNA3.1-GRIN1-815R-GFP + Plasmid GRIN2A (Catalog No.: P8212, Company: Miaoling Plasmid Platform). Among them, the purchased human wild-type pcDNA 3.1-GRIN1 plasmid (Catalog No.: P6908, Company: Miaoling Plasmid Platform) was genetically edited to introduce nucleotide mutations, causing the amino acid at position 815 to mutate from glycine to arginine, and then a GFP tag (fused to express green fluorescent protein) was inserted into the plasmid to obtain the plasmid pCDNA3.1-GRIN1-815R-GFP.

[0169] (3) Blank control;

[0170] 4 μg of total DNA in 35-mm cell culture wells was used to transfect cells. In cells transfected with NR constructs, an equal amount of NR (2 ng each) was used. The cells were cultured in 3% CO2 at 37 °C for 24 hours, washed in PBS, cultured in fresh medium at 37 °C for 24 hours, and then cultured for another 24 hours under 5% conditions.

[0171] 1.2 Measurement of cell death

[0172] Cells were removed from the culture dish using a cell scraper and centrifuged at 1000 g at 4 °C using a Beckman benchtop centrifuge. The cells were resuspended in 100–1 PBS and 100 / μl trypan blue (0.4%). The cell concentration was 200 - 300 cells / 0.1 mm 3 。

[0173] The number of live cells was blindly counted in a hemocytometer. The number of live cells (i.e., cells excluding trypan blue) under each condition was divided by the number of live cells in the transfection control wells to obtain the percentage of cell death. The calculation formula is as follows:

[0174] Percentage of cell death (%) = (1 - (number of live cells under each condition / number of live cells in the transfection control wells)) × 100%.

[0175] 1.3 Cytotoxicity of HEK293 cells after transfection with NMDA receptor subunit cDNA

[0176] HEK293 cells were transfected with cDNA encoding NMDA receptor subunits, and the cytotoxicity to the cells was observed.

[0177] 1.4 Results

[0178] As Figure 1 shown, in the N1-815R / 2A group (transfected with plasmid pCDNA3.1-GRIN1-815R-GFP + plasmid GRIN2A), most cells survived well (Figure 1 B), while the N1 / 2A group (wild-type GRIN1 plasmid + GRIN2A plasmid) had more cell deaths ( Figure 1 A).

[0179] As Figure 2 shown, the cell death rate of the N1-815R / 2A group was 53.58 ± 8.75%; while the cell death rate of the N1 / 2A group was: 38.00 ± 9.70%.

[0180] The above results indicate that the coding method of GRIN1-815R, even when the amino acid at position 815 is mutated from glycine to arginine, can significantly reduce the cytotoxicity caused by the transfection of cDNA encoding NMDA receptor subunits to HEK293 cells, resulting in a relative decrease in the cell death rate of about 29% after transfection ((53.58 - 38.00) / 53.58 = 29.1%).

[0181] Example 2: Establishment of a method for detecting autoantibodies in AE based on transiently transfected cells

[0182] Objective of this example: To establish a highly efficient and reliable system for detecting common autoantibodies in AE by indirect immunofluorescence method based on transiently transfected HEK293 cells. The method is as follows:

[0183] Use the existing commercial cDNA library to obtain the target genes corresponding to 6 common autoantibodies (NMDAR, AMPAR1, AMPAR2, LGI1, Caspr2, GABA B R). Mutate the amino acid at position 815 of NMDAR (GluN1 protein) from glycine to arginine, and fuse LGI1 to express the transmembrane region of Caspr2, while keeping the genes of other autoantibody proteins unchanged. Then edit the cDNA of these autoantibodies so that they fuse and express the gene fragment of green fluorescent protein (group transfection).

[0184] Seed HEK293 cells in 96-well plates and perform group transfection on HEK293 cells by the PEI method to make HEK293 cells express the above 6 modified autoantigens respectively.

[0185] Each well of the 96-well plate can be used to detect 1 specimen. During detection, the transfected HEK293 cells are incubated successively with the specimen to be tested and the fluorescein-labeled anti-human IgG secondary antibody.

[0186] During the result interpretation, the self - antigen is labeled with green fluorescence and expressed on the cell membrane surface. The red fluorescence is produced by the fluorescein - labeled anti - human IgG secondary antibody, which is used to label the autoantibody. If the red fluorescence coincides with the green fluorescence, the specimen is defined as positive for antibody detection. When determining the titer, the serum and cerebrospinal fluid are diluted at a fixed multiple, and the titer level is determined as +, ++, +++ or ++++ according to the intensity of the red fluorescence.

[0187] A total of 102 specimens were selected for anti - NMDAR antibody detection and compared with the anti - glutamate receptor antibody detection kit of EUROIMMUN. For the anti - NMDAR encephalitis - positive specimens screened out, the same immunofluorescence method was used for the detection of anti - AMPAR encephalitis, anti - LGI1 encephalitis, anti - Caspr2 encephalitis, anti - GABA B R encephalitis antibody detection method.

[0188] Results:

[0189] The mutant NMDAR subunit pCDNA3.1 - GRIN1 - 815R obtained after the mutation of the 815th amino acid of GluN1 can reduce the cytotoxicity of NMDAR to HEK293 cells.

[0190] The gene encoding the fusion protein of the self - antigen and green fluorescent protein was transfected into HEK293 cells, and it was visible that the green fluorescent protein was expressed in the cytoplasm and cell membrane of HEK293 cells.

[0191] The test results and antibody titers can be interpreted according to the co - localization of red and green fluorescence and the intensity of red fluorescence. The sensitivity of anti - NMDAR antibody detection was calculated to be 92.9%, and the specificity was 87.5%.

[0192] This method can successfully detect the specific autoantibodies of anti - AMPAR encephalitis, anti - LGI1 encephalitis, anti - Caspr2 encephalitis, anti - GABA B R encephalitis on the basis of anti - NMDAR positivity. This indicates that the detection method of the present invention has high detection efficiency and can clearly diagnose the pathogenic causes, so as to prescribe the right medicine and design an effective treatment plan for the real pathogenic cause to help the patient recover quickly.

[0193] Example 3: Exploration of the method for detecting autoantibodies in AE based on stably transfected cells

[0194] The purpose of this example: To produce stably transfected cells as the cell matrix for detecting autoantibodies in AE.

[0195] Method:

[0196] By constructing two inverted repeat sequences in the PiggyBac transposon system on both sides of the sequences required for the tetracycline-inducible expression system, a tool plasmid of the PiggyBac transposon system for tetracycline-inducible expression was obtained. The LGI1 (fused with green fluorescent protein) gene was cloned into the obtained tool plasmid. After transfection of cells, 0 μg / ml, 1 μg / ml, and 2 μg / ml doxycycline were added successively to verify the tetracycline-inducible expression system. And HEK293 was screened with different concentrations of puromycin and G418 to determine the lowest concentration that could kill all cells within 6 - 8 days as the screening concentration. The cells were screened with puromycin and G418 for 2 weeks, then transferred to 96-well plates for monoclonal production. The obtained monoclonal cells were successively expanded in culture, and doxycycline was added to verify the correctness of the clones. Ten specimens with positive anti-LGI1 antibodies and 2 specimens with negative anti-LGI1 antibodies measured by transient transfection of cells were selected, and the anti-LGI1 antibodies were detected with the stably transfected cells that had been stably formed, and the results were compared.

[0197] Results

[0198] A tool plasmid of the PiggyBac transposon system for tetracycline-inducible expression was successfully constructed, and the LGI1 (fused with green fluorescent protein) gene was cloned into the obtained tool plasmid. After transfection of cells, 0 μg / ml, 1 μg / ml, and 2 μg / ml doxycycline were added successively, and the expression level of green fluorescent protein gradually increased. The lowest antibiotic concentrations that could kill all cells within 6 - 8 days were determined to be 0.9 μg / ml of puromycin and 600 μg / ml of G418 respectively. A total of 9 stably transfected cell lines were obtained. The stably transfected cells could detect the anti-LGI1 antibodies in 10 specimens, and the results were consistent with those detected by transient transfection of cells.

[0199] The above results indicate that stably transfected cells can be used as a cell matrix for the detection of autoantibodies in AE, and have the characteristics of simpler operation and better stability.

[0200] Example 4: Anti-GABA B Detection of anti-KCTD16 antibodies in patients with anti-GABA

[0201] Objective of this example: To detect anti-GABA B Anti-KCTD16 antibodies in patients with anti-GABA R encephalitis, and to explore the correlation between anti-KCTD16 antibodies and co-existing tumors and the antigen regions recognized by the antibodies.

[0202] Methods:

[0203] Twenty-eight cases of anti-GABA BClinical data of patients with R encephalitis. Indirect immunofluorescence and immunoblotting based on fixed cell method were used to detect anti-KCTD8, 12, and 16 antibodies. The antigen domains recognized by the antibodies were determined by transfecting HEK293 cells to express KCTD12 and KCTD16 domain replacement proteins.

[0204] Results

[0205] A total of 9 patients were detected positive for anti-KCTD16 antibody, and the positive rate was 32.1%. Among the 9 patients positive for anti-KCTD16 antibody, 8 patients (88.9%) had tumors, including 4 patients with small cell lung cancer, 3 patients highly suspected of having tumors but with unclear pathology, 1 patient with gastric cardia cancer, and 1 patient had no tumor after 14 months of follow-up. Among the 19 patients negative for anti-KCTD16, 6 patients (31.6%) had tumors, including 4 patients with small cell lung cancer and 2 patients with thymoma. Thirteen patients had no tumor after an average of 15 months of follow-up (P = 0.013). Anti-KCTD16 antibodies in all patients could recognize two or three domains without selectivity.

[0206] Thus, anti-KCTD16 antibody can be present in anti-GABA B R encephalitis patients and is associated with tumor co-occurrence. Anti-KCTD16 antibody has no specific antigen domain for recognition, suggesting that we need to further study the production mechanism of this antibody.

[0207] Discussion

[0208] NMDAR (N-methyl-D-aspartate receptor) is an important class of excitatory amino acid (EAA) receptors in the central nervous system and belongs to the ionotropic glutamate receptors. NMDAR plays an important role in regulating neuron survival, participating in synaptic signal transduction, and the formation of plasticity.

[0209] Overactivation of NMDAR can lead to excitotoxic manifestations, which may be the potential pathogenesis of epilepsy, dementia, and stroke; conversely, schizophrenia-like symptoms may occur.

[0210] NMDAR includes the NR1 subunit that binds glycine and the NR2 (A, B, C, D) subunits that bind glutamate. Since NR1 is more widely distributed in the brain and is more consistent with the clinical damage sites of anti-NMDAR encephalitis, experimental studies have also shown that NR1 is the pathogenic subunit of this disease.

[0211] Autoantibodies against the extracellular site of the NR1 subunit are present in the serum and cerebrospinal fluid (CSF) of patients with anti-NMDAR encephalitis. The IgG of patients with anti-NMDAR encephalitis can recognize the extracellular N-terminal domain of the GluN1 subunit, and the N-terminal domain can regulate the function of the NMDAR ion channel, including the opening frequency, inactivation rate, and allosteric regulation of the channel. Therefore, the clinical manifestations of anti-NMDAR encephalitis can include psychiatric symptoms, seizures, disturbance of consciousness, oromotor and manual movement disorders, and autonomic dysfunction, etc.

[0212] Currently, it is possible to detect whether anti-NMDAR antibodies are present in serum or cerebrospinal fluid by using hippocampal tissues of rat brain slices, cultured cells with surface-labeled hippocampal neurons, or NR1 / NR2 transfected human embryonic kidney cells (HEK).

[0213] The present invention for the first time provides recombinant cells with high viability for detecting anti-NMDAR antibodies, and provides an indirect immunofluorescence method based on cell-based assay (CBA). The method of the present invention is a simple and effective detection method for detecting anti-NMDAR antibodies by expressing NMDAR subunits (NR1 and NR2A) in a recombinant transfected cell line, and has high sensitivity and specificity.

[0214] The research of the present invention shows that the method and detection reagent of the present invention have high sensitivity and specificity, can accurately detect the types of pathogenic antibodies, and contribute to the diagnosis and treatment of diseases.

[0215] All the documents mentioned in the present invention are cited in this application as references, as if each document is cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0216] References

[0217] [1]Vitaliani R,Mason W,Ances B,et al.Paraneoplasticencephalitis,psychiatric symptoms,and hypoventilation in ovarian teratoma[J].Ann Neurol,2005,58:594-604

[0218] [2] Waxman EA, Lynch DR. N-methyl-D-aspartate receptor subtypes: multiple roles in excitotoxicity and neurological disease[J]. Neuroscientist, 2005, 11: 37-49

[0219] [3] Coyle JT. Glutamate and schizophrenia: beyond the dopamine hypothesis[J]. Cell Mol Neurobiol, 2006, 26: 365-384

[0220] [4] Gleichman AJ, Spruce LA, Dalmau J, Seeholzer SH, Lynch DR. Anti-NMDA receptor encephalitis antibody binding is dependent on amino acid identity of a small region within the GluN1 amino terminal domain. The Journal of neuroscience: the official journal of the Society for Neuroscience 2012;32(32): 11082-94.

[0221] [5] Dalmau J, Gleichman AJ, Hughes EG, et al. Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies[J]. Lancet Neurol, 2008, 7: 1091-1098

[0222] [6] Graus F, Titulaer MJ, Balu R, Benseler S, Bien CG, Cellucci T, et al. A clinical approach to diagnosis of autoimmune encephalitis. The Lancet Neurology 2016;15: 391-404.

[0223] [7] Crisp SJ, Kullmann DM, Vincent A. Autoimmune synaptopathies. Nature reviews Neuroscience 2016; 17: 103 - 17. Sequence Listing <110> Chen Xiangjun <120> Method for detecting transient transfection and stable transfection of autoimmune encephalitis antibodies and its application <130> P2020 - 0517 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 2769 <212> DNA <213> Homo sapiens <400> 1 atgagcacca tgcgcctgct gacgctcgcc ctgctgttct cctgctccgt cgcccgtgcc 60 gcgtgcgacc ccaagatcgt caacattggc gcggtgctga gcacgcggaa gcacgagcag 120 atgttccgcg aggccgtgaa ccaggccaac aagcggcacg gctcctggaa gattcagctc 180 aatgccacct ccgtcacgca caagcccaac gccatccaga tggctctgtc ggtgtgcgag 240 gacctcatct ccagccaggt ctacgccatc ctagttagcc atccacctac ccccaacgac 300 cacttcactc ccacccctgt ctcctacaca gccggcttct accgcatacc cgtgctgggg 360 ctgaccaccc gcatgtccat ctactcggac aagagcatcc acctgagctt cctgcgcacc 420 gtgccgccct actcccacca gtccagcgtg tggtttgaga tgatgcgtgt ctacagctgg 480 aaccacatca tcctgctggt cagcgacgac cacgagggcc gggcggctca gaaacgcctg 540 gagacgctgc tggaggagcg tgagtccaag gcagagaagg tgctgcagtt tgacccaggg 600 accaagaacg tgacggccct 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Claims

1. A method for preparing recombinant cells, characterized in that, The method includes the steps: (a) Providing cells to be transfected, a first plasmid and a second plasmid for transfection; wherein, the cells to be transfected are HEK293 cells, The first plasmid contains a first expression cassette, and the first expression cassette is used for expressing the fusion protein P0 shown in Formula I, Z1-Z2 (I) In the formula, Z1 is a mutant NMDAR subunit NR1, and the sequence of the mutant NMDAR subunit NR1 is as shown in SEQ ID No: 2; Z2 is a GFP protein element; The second plasmid contains a second expression cassette, and the second expression cassette is used for expressing the NMDAR subunit NR2A; (b) Transfecting the first plasmid and the second plasmid into the cells to be transfected, thereby obtaining recombinant cells C0, wherein the recombinant cells C0 express recombinant NMDAR protein; (c) Detecting the survival of the recombinant cells obtained in step (b).

2. The method according to claim 1, wherein The recombinant NMDAR protein is composed of the fusion protein P0 shown in Formula I and the NMDAR subunit NR2A.

3. The method according to claim 1, wherein The recombinant cells express an NMDAR mutant protein, and the NMDAR mutant protein is composed of an NR1 subunit and an NR2A subunit, wherein the NR1 subunit is a mutant subunit, and the 815th amino acid of the NR1 subunit is mutated from wild-type glycine to arginine.

4. The method according to claim 1, characterized in that, The recombinant cells express an NMDAR mutant protein, and the NMDAR mutant protein exists in the cell membrane of the recombinant cells in the form of a membrane protein.

5. The method according to claim 1, wherein At 12-24 hours after transfection, the cell survival rate of the recombinant cells is ≥50%.

6. The method according to claim 1, characterized in that At 12-24 hours after transfection, the cell survival rate of the recombinant cells is 50%-85%.

7. The method according to claim 1, characterized in that At 12-24 hours after transfection, the cell survival rate of the recombinant cells is 60%-75%.

8. The method according to claim 1, wherein The recombinant cells are used for preparing a detection reagent or kit for detecting autoimmune encephalitis.

9. The method according to claim 8, wherein The kit further contains additional reagents selected from the following group: (Y1) Recombinant cells C1 for expressing AMPAR1 protein, or a transfection reagent for preparing the recombinant cells C1; (Y2) Recombinant cells C2 for expressing AMPAR2 protein, or a transfection reagent for preparing the recombinant cells C2; (Y3) Recombinant cells C3 for expressing LGI 1 protein, or a transfection reagent for preparing the recombinant cells C3; (Y4) Recombinant cells C4 for expressing Caspr2 protein, or a transfection reagent for preparing the recombinant cells C4; (Y5) For expressing GABA B Recombinant cell C5 of R protein, or transfection reagent for preparing the recombinant cell C5; (Y6) Any combination of Y1 to Y5.

10. The method according to claim 9, wherein The recombinant cells C1 express a fusion protein P1 of AMPAR1 protein and GFP green fluorescent protein; The recombinant cells C2 express a fusion protein P2 of AMPAR2 protein and GFP green fluorescent protein; The recombinant cells C3 express a fusion protein P3 of LGI 1 protein and GFP green fluorescent protein; The recombinant cells C4 express a fusion protein P4 of Caspr2 protein and GFP green fluorescent protein; The recombinant cell C5 expresses GABA B The fusion protein P5 of the R protein and the GFP green fluorescent protein.

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