CN118483432A - Application of reagent for detecting anti-IDE autoantibody in detection and/or diagnosis of nervous system autoimmune disease - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a reagent for detecting an anti-IDE autoantibody in detecting and/or diagnosing an autoimmune disease of a nervous system. The present invention compares patient serum with healthy human serum suffering from autoimmune diseases of the nervous system, and finds signals present in patient serum but not in healthy human serum: the anti-IDE autoantibody can be used as a marker for diagnosing the autoimmune diseases of the nervous system, enriches the biomarkers for distinguishing the autoimmune diseases of the nervous system, improves the diagnosis accuracy of the autoimmune diseases of the nervous system, and can especially realize the auxiliary diagnosis of the autoimmune diseases of the nervous system.

Description

Application of reagent for detecting anti-IDE autoantibody in detection and/or diagnosis of nervous system autoimmune disease

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of a reagent for detecting an anti-IDE autoantibody in diagnosis of autoimmune diseases of a nervous system.

Background

Autoimmune diseases of the nervous system are diseases in which the human immune system erroneously attacks the nervous system, resulting in damage to the tissue structure and function of the nervous system. The diseases have wide injury range, complex and various clinical manifestations, and can involve the central nervous system, the peripheral nervous system and nerve-muscle joints to cause pathological changes such as neuronal or axonal injury, demyelination, nerve-muscle joint damage and the like. Common autoimmune diseases of the nervous system include: autoimmune encephalitis (autoimmune encephalitis, AE), demyelinating diseases of the central nervous system (central nervous system, CNS), stiff syndrome (stiffperson syndrome, SPS), immune-mediated peripheral neuropathy, autoimmune cerebellar ataxia, myasthenia syndrome, etc. Autoantibodies play a very important role in the diagnosis and differential diagnosis of autoimmune diseases of the nervous system, particularly autoimmune encephalitis and CNS inflammatory demyelinating diseases.

At present, although progress is made in the recognition of autoantibody-mediated immune mechanisms in patients with autoimmune encephalitis, a part of patients showing encephalitis symptoms still have negative serological detection results, so that searching and identifying new encephalitis autoantibodies and improving the existing diagnosis and treatment level remain a difficult task.

Insulin degrading enzymes (insulin-degradingenzyme, IDE) are mainly localized to the cytoplasm, but in certain cell types are localized to the cell membrane, peroxisomes and mitochondria or secreted extracellularly. Insulin is a hormone critical to glucose homeostasis, and IDE as an insulin level regulator, can significantly degrade intracellular insulin without breaking disulfide bonds linking the a and B chains of insulin, thereby terminating insulin activity. IDE also degrades various proteins with various physiological and pathological functions, such as various peptides of beta-amyloid (aβ), glucagon, islet amyloid polypeptide (IAPP), and insulin-like growth factor, and is involved in intercellular peptide signaling, with extensive substrate promiscuity. Among them, β -amyloid (aβ) can lead to the formation of senile plaques and apoptosis of nerve cells in the brain, whereas alzheimer's disease is characterized by the presence of neurotoxic aβ deposits in the brain, the production and deposition of aβ being important factors affecting the progression and prognosis of Alzheimer's Disease (AD). IDE also performs other non-proteolytic functions, such as: chaperones/dead-end chaperones, E1-ubiquitin activating enzymes and proteasome modulators. It also reacts as a heat shock protein, regulating cellular protein homeostasis. At present, the existence of anti-IDE autoantibodies in patients with nervous system autoimmune diseases is not reported.

Disclosure of Invention

The invention aims to provide the application of the reagent for detecting the anti-IDE autoantibody in detecting and/or diagnosing the autoimmune diseases of the nervous system, specifically detect and/or diagnose the autoimmune diseases of the nervous system, improve the accuracy of diagnosing the autoimmune diseases of the nervous system, and especially realize the auxiliary diagnosis of the autoimmune diseases of the nervous system.

The invention provides application of a reagent for detecting anti-IDE autoantibodies in preparing products for detecting and/or diagnosing nervous system autoimmune diseases.

Preferably, the agent for detecting an anti-IDE autoantibody comprises one or more of IDE protein, a homolog of IDE protein, a derivative of IDE protein, cells expressing IDE protein, tissues expressing IDE protein, and a lysate of IDE protein.

Preferably, the amino acid sequence of the IDE protein comprises any one of a) to c):

a) An amino acid sequence shown in SEQ ID NO. 1;

b) 10% -80% of the amino acid sequence shown in SEQ ID NO.1, and can recognize the amino acid sequence of the anti-IDE autoantibody;

c) The amino acid sequence of a) or b) is modified or mutated and the amino acid sequence of an anti-IDE autoantibody can be recognized.

Preferably, the modification comprises one or more of a glycosylation modification, a phosphorylation modification, an acetylation modification, a methylation modification and a ubiquitination modification.

Preferably, the nucleotide sequence encoding the amino acid sequence of the IDE protein comprises any one of i) to iii):

i) the nucleotide sequence shown in SEQ ID NO. 3;

II) 10% -80% of the nucleotide sequence shown in SEQ ID NO.3, and the coded amino acid sequence can recognize an anti-IDE autoantibody;

The encoded amino acid sequence recognizes an anti-IDE autoantibody after modification or mutation of the nucleotide sequence of III) I) or II).

Preferably, the symptoms of the neurological autoimmune disease include one or more of headache, fever, muscle soreness, vomiting, insomnia, snoring, abnormal gait, decreased memory, dysphagia, abnormal spirit, seizures, cognitive dysfunction, language disorder, disturbance of consciousness and movement disorder.

Preferably, the symptoms of the neurological autoimmune disease include one or more of headache, fever, muscle soreness, vomiting, insomnia, snoring, abnormal gait, decreased memory, dysphagia, abnormal spirit, seizures, cognitive dysfunction, language disorder, disturbance of consciousness and movement disorder.

Preferably, the neurological autoimmune disease is autoimmune encephalitis.

The invention also provides a kit for detecting and/or diagnosing the autoimmune diseases of the nervous system, which comprises a reagent for detecting the anti-IDE autoantibody and a labeled antibody.

Preferably, the agent for detecting anti-IDE autoantibodies comprises one or more of IDE proteins, cells expressing IDE proteins, tissues expressing IDE proteins, and lysates containing IDE proteins.

Preferably, the amino acid sequence of the IDE protein comprises any one of a) to c):

a) An amino acid sequence shown in SEQ ID NO. 1;

b) 10% -80% of the amino acid sequence shown in SEQ ID NO.1, and can recognize the amino acid sequence of the anti-IDE autoantibody;

c) The amino acid sequence of a) or b) is modified or mutated and the amino acid sequence of an anti-IDE autoantibody can be recognized.

Advantageous effects

The invention uses patient serum and healthy human serum with nervous system autoimmune disease to incubate rat brain tissue slice, uses fluorescent secondary antibody to amplify signal and uses the mode of co-staining with neuron specific Marker antibody, finds that compared with healthy human serum, the patient serum has autoantibody, uses the modes of immune co-precipitation and mass spectrum identification to screen out the signal on the patient serum as autoantibody for recognizing IDE antigen, uses serum neutralization experiment to verify the reality of target antigen, uses the mode of incubating patient serum with over-expressed target antigen cell and recovering serum effective antibody component to prepare experiment elution antibody containing IDE autoantibody and control elution antibody without IDE autoantibody, incubates over-expressed cell climbing slice, rat brain tissue slice and cytotoxicity experiment to verify the reality and specificity of target antigen, which shows that IDE protein is expressed in rat brain tissue and rat neuron and has obvious signal.

The invention also provides a kit for detecting and/or diagnosing the autoimmune diseases of the nervous system, which comprises a reagent for detecting the anti-IDE autoantibody and a labeled antibody. The invention takes the reagent for detecting the anti-IDE autoantibody as the main component for the first time, and establishes a kit for detecting and/or diagnosing the autoimmune disease of the nervous system. The kit can be used for qualitatively or quantitatively analyzing the anti-IDE autoantibody, is simple and convenient to operate, and can be used for detecting and/or diagnosing the autoimmune diseases of the nervous system, in particular for assisting in diagnosing the autoimmune diseases of the nervous system.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.

FIG. 1 is the result of staining of patient 1 serum and normal control serum of example 1 on rat brain tissue sections;

FIG. 2 shows the co-staining of patient 1 serum and Neun antibodies of example 1 on rat brain tissue sections;

FIG. 3 shows the immunoprecipitation results of patient 1 serum from example 2 with normal control serum;

FIG. 4 shows Western Blot results of immunoprecipitates of patient 1 serum from example 2 and normal control serum;

FIG. 5 shows Western Blot results of commercial antibodies to IDE in example 3 in overexpressing cells and rat brain tissue;

FIG. 6 shows the staining results of patient 1 neutralizing serum on overexpressing cells;

FIG. 7 shows the Western Blot results of patient 1 and serum from example 4 on over-expressed IDE proteins;

FIG. 8 is a graph showing the staining of the over-expressed cells with patient 1 serum recovered antibodies of example 5;

FIG. 9 shows the Western Blot results of patient 1 serum recovery antibodies from example 5 over-expressing IDE protein and rat brain tissue protein;

FIG. 10 is a graph showing the co-staining results of patient 1 serum and IDE antibodies on over-expressed cell slide of example 6;

FIG. 11 is a graph of 3 positive patients screened for anti-IDE autoantibodies in example 6.

Detailed Description

The invention provides application of a reagent for detecting anti-IDE autoantibodies in preparing products for diagnosing autoimmune diseases of a nervous system.

In the present invention, the reagent for detecting an anti-IDE autoantibody preferably comprises one or more of IDE protein, cells expressing IDE protein, tissues expressing IDE protein and lysate containing IDE protein, and more preferably IDE protein. The amino acid sequence of the IDE protein of the present invention preferably includes any one of a) to c): a) An amino acid sequence shown in SEQ ID NO. 1; b) 10% -80% of the amino acid sequence shown in SEQ ID NO.1, and can recognize the amino acid sequence of the anti-IDE autoantibody; c) The amino acid sequence in a) or b) is modified or mutated, and the amino acid sequence of the anti-IDE autoantibody can be identified, and is further shown as SEQ ID NO. 2.

In the present invention, the nucleotide sequence encoding the IDE protein preferably includes any one of i) to iii): i) the nucleotide sequence shown in SEQ ID NO. 3; II) 10% -80% of the nucleotide sequence shown in SEQ ID NO.3, and the coded amino acid sequence can recognize an anti-IDE autoantibody; the encoded amino acid sequence recognizes an anti-IDE autoantibody after modification or mutation of the nucleotide sequence of III) I) or II).

In the present invention, the modification preferably includes one or more of glycosylation modification, phosphorylation modification, acetylation modification, methylation modification, and ubiquitination modification.

The specific sequences of SEQ ID NO. 1-3 of the present invention are as follows:

SEQ ID NO.1：MRYRLAWLLHPALPSTFRSVLGARLPPPERLCGFQKKTYSKM NNPAIKRIGNHITKSPEDKREYRGLELANGIKVLLISDPTTDKSSAALDVHIGSLSDPPNIAGLSHFCEHMLFLGTKKYPKENEYSQFLSEHAGSSNAFTSGEHTNYYFDVSHEHLEGALDRFAQFFLCPLFDESCKDREVNAVDSEHEKNVMNDAWRLFQLEKATGNPKHPFSKFGTGNKYTLETRPNQEGIDVRQELLKFHSAYYSSNLMAVCVLGRESLDDLTNLVVKLFSEVENKNVPLPEFPEHPFQEEHLKQLYKIVPIKDIRNLYVTFPIPDLQKYYKSNPGHYLGHLIGHEGPGSLLSELKSKGWVNTLVGGQKEGARGFMFFIINVD LTEEGLLHVEDIILHMFQYIQKLRAEGPQEWVFQECKDLNAVAFRFKDKERPRGYTSKIAGILHYYPLEEVLTAEYLLEEFRPDLIEMVLDKLRPENVRVAIVSKSFEGKTDRTEEWYGTQYKQEAIPDEVIKKWQNADLNGKFKLPTKNEFIPTNFEILPLEKEATPYPALIKDTAMSKLWFKQDDKFFLPKACLNFEFFSPFAYVDPLHCNMAYLYLELLKDSLNEYAYAAELAGLSYDLQNTIYGMYLSVKGYNDKQPILLKKIIEKMATFEIDEKRFEIIKEAYMRSLNNFRAEQPHQHAMYYLRLLMTEVAWTKDELKEALDDVTLPRLKAFIPQLLSRLHIEALLHGNITKQAALGIMQMVEDTLIEHAHTKPLLPSQLVRYREVQLPDRGWFVYQQRNEVHNNCGIEIYYQTDMQSTSENMFLELFCQIISEPCFNTLRTKEQLGYIVFSGPRRANGIQGLRFIIQSEKPPHYLESRVEAFLITMEKSIEDMTEEAFQKHIQALAIRRLDKPKKLSAECAKYWGEIISQQYNFDRDNTEVAYLKTLTKEDIIKFYKEMLAVDAPRRHKVSVHVLAREMDSCPVVGEFPCQNDINLSQAPALPQPEVIQNMTEFKRGLPLFPLVKPHINFMAAKL.

SEQ ID NO.2：MRYRLAWLLHPALPSTFRSVLGARLPPPERLCGFQKKTYSKM NNPAIKRIGNHITKSPEDKREYRGLELANGIKVLLISDPTTDKSSAALDVHIGSLSDPPNIAGLSHFCEHMLFLGTKKYPKENEYSQFLSEHAGSSNAFTSGEHTNYYFDVSHEHLEGALDRFAQFFLCPLFDESCKDREVNAVDSEHEKNVMNDAWRLFQLEKATGNPKHPFSKFGTGNKYTLETRPNQEGIDVRQELLKFHSAYYSSNLMAVCVLGRESLDDLTNLVVKLFSEVENKNVPLPEFPEHPFQEEHLKQLYKIVPIKDIRNLYVTFPIPDLQKYYKSNPGHYLGHLIGHEGPGSLLSELKSKGWVNTLVGGQKEGARGFMFFIINVDLTEEGLLHVEDIILHMFQYIQKLRAEGPQEWVFQECKDLNAVAFRFKDKERPRGYTSKIAGILHYYPLEEVLTAEYLLEEFRPDLIEMVLDKLRPENVRVAIVSKSFEGKTDRTEEWYGTQYKQEAIPDEVIKKWQNADLNGKFKLPTKNEFIPTNFEILPLEKEATPYPALIKDTAMSKLWFKQDDKFFLPKLKDSLNEYAYAAELAGLSYDLQNTIYGMYLSVKGYNDKQPILLKKIIEKMATFEIDEKRFEIIKEAYMRSLNNFRAEQPHQHAMYYLRLLMTEVAWTKDELKEALDDVTLPRLKAFIPQLLSRLHIEALLHGNITKQAALGIMQMVEDTLIEHAHTKPLLPSQLVRYREVQLPDRGWFVYQQRNEVHNNCGIEIYYQTDMQSTSENMFLELFCQIISEPCFNTLRTKEQLGYIVFSGPRRANGIQGLRFIIQSEKPPHYLESRVEAFLITMEKSIEDMTEEAFQKHIQALAIRRLDKPKKLSAECAKYWGEIISQQYNFDRDNTEVAYLKTLTKEDIIKFYKEMLAVDAPRRHKVSVHVLAREMDSCPVVGEFPCQNDINLSQAPALPQPEVIQNMTEFKRGLPLFPLVKPHINFMAAKL.

wherein the bolded parts in SEQ ID NO.1 and SEQ ID NO.2 are different amino acid residues.

SEQ ID NO.3：5'-atgcggtaccggctagcgtggcttctgcaccccgcactgcccagcaccttccgctcagtcct cggcgcccgcctgccgcctccggagcgcctgtgtggtttccaaaaaaagacttacagcaaaatgaataatccagccatcaagagaataggaaatcacattaccaagtctcctgaagacaagcgagaatatcgagggctagagctggccaatggtatcaaagtacttcttatcagtgatcccaccacggataagtcatcagcagcacttgatgtgcacataggttcattgtcggatcctccaaatattgctggcttaagtcatttttgtgaacatatgctttttttgggaacaaagaaataccctaaagaaaatgaatacagccagtttctcagtgagcatgcaggaagttcaaatgcctttactagtggagagcataccaattactattttgatgtttctcatgaacacctagaaggtgccctagacaggtttgcacagttttttctgtgccccttgttcgatgaaagttgcaaagacagagaggtgaatgcagttgattcagaacatgagaagaatgtgatgaatgatgcctggagactctttcaattggaaaaagctacagggaatcctaaacaccccttcagtaaatttgggacaggtaacaaatatactctggagactagaccaaaccaagaaggcattgatgtaagacaagagctactgaaattccattctgcttactattcatccaacttaatggctgtttgtgttttaggtcgagaatctttagatgacttgactaatctggtggtaaagttattttctgaagtagagaacaaaaatgttccattgccagaatttcctgaacaccctttccaagaagaacatcttaaacaactttacaaaatagtacccattaaagatattaggaatctctatgtgacatttcccatacctgaccttcagaaatactacaaatcaaatcctggtcattatcttggtcatctcattgggcatgaaggtcctggaagtctgttatcagaacttaagtcaaagggctgggttaatactcttgttggtgggcagaaggaaggagcccgaggttttatgttttttatcattaatgtggacttgaccgaggaaggattattacatgttgaagatataattttgcacatgtttcaatacattcagaagttacgtgcagaaggacctcaagaatgggttttccaagagtgcaaggacttgaatgctgttgcttttaggtttaaagacaaagagaggccacggggctatacatctaagattgcaggaatattgcattattatcccctagaagaggtgctcacagcggaatatttactggaagaatttagacctgacttaatagagatggttctcgataaactcagaccagaaaatgtccgggttgccatagtttctaaatcttttgaaggaaaaactgatcgcacagaagagtggtatggaacccagtacaaacaagaagctataccggatgaagtcatcaagaaatggcaaaatgctgacctgaatgggaaatttaaacttcctacaaagaatgaatttattcctacgaattttgagattttaccgttagaaaaagaggcgacaccataccctgctcttattaaggatacagctatgagcaaactttggttcaaacaagatgataagttttttttgccgaaggcttgtctcaactttgaatttttcagcccatttgcttatgtggaccccttgcactgtaacatggcctatttgtaccttgagctcctcaaagactcactcaacgagtatgcatatgcagcagagctagcaggcttgagctatgatctccaaaataccatctatgggatgtatctttcagtgaaaggttacaatgacaagcagccaattttactaaagaagattattgagaaaatggctacctttgagattgatgaaaaaagatttgaaattatcaaagaagcatatatgcgatctcttaacaatttccgggctgaacagcctcaccagcatgccatgtactacctccgcttgctgatgactgaagtggcctggactaaagatgagttaaaagaagctctggatgatgtaacccttcctcgccttaaggccttcatacctcagctcctgtcacggctgcacattgaagcccttctccatggaaacataacaaagcaggctgcattaggaattatgcagatggttgaagacaccctcattgaacatgctcataccaaacctctccttccaagtcagctggttcggtatagagaagttcagctccctgacagaggatggtttgtttatcagcagagaaatgaagttcacaataactgtggcatcgagatatactaccaaacagacatgcaaagcacctcagagaatatgtttctggagctcttctgtcagattatctcggaaccttgcttcaacaccctgcgcaccaaggagcagttgggctatatcgtcttcagcgggccacgtcgagctaatggcatacagggcttgagattcatcatccagtcagaaaagccacctcactacctagaaagcagagtggaagctttcttaattaccatggaaaagtccatagaggacatgacagaagaggccttccaaaaacacattcaggcattagcaattcgtcgactagacaaaccaaagaagctatctgctgagtgtgctaaatactggggagaaatcatctcccagcaatataattttgacagagataacactgaggttgcatatttaaagacacttaccaaggaagatatcatcaaattctacaaggaaatgttggcagtagatgctccaaggagacataaggtatccgtccatgttcttgccag ggaaatggattcttgtcctgttgttggagagttcccatgtcaaaatgacataaatttgtcacaagcaccagccttgccacaacctgaagtgattcagaacatgaccgaattcaagcgtggtctgccactgtttccccttgtgaaaccacatattaacttcatggctgcaaaactctga-3'.

In the present invention, the symptom of the autoimmune disease of the nervous system preferably includes one or more of headache, fever, muscular soreness, vomiting, insomnia, snoring, abnormal gait, memory decline, dysphagia, mental abnormality, seizures, occurrence of cognitive dysfunction, language disorder, conscious disturbance and dyskinesia; the neurological autoimmune disease is preferably autoimmune encephalitis. The product of the invention preferably comprises a kit. The diagnosis according to the invention is preferably an auxiliary diagnosis.

The anti-IDE autoantibody is closely related to the autoimmune diseases of the nervous system, can distinguish the autoimmune diseases of the nervous system from other autoimmune diseases, and provides a basis for diagnosing the autoimmune diseases of the nervous system. According to the invention, by taking the anti-IDE autoantibody as a marker, a doctor can judge whether the patient suffers from the related diseases of the nervous system autoimmunity or excludes the possibility of suffering from the autoimmune diseases of another nervous system according to the comprehensive conditions of clinical symptoms, physiological and biochemical detection indexes, disease marker detection results and the like of the patient by detecting the anti-IDE autoantibody, and the doctor is helped to select a more promising treatment scheme or treatment medicament for the patient.

The invention also provides a kit for diagnosing the autoimmune diseases of the nervous system, which comprises a reagent for detecting the anti-IDE autoantibody and a labeled antibody.

The relevant content of the reagent for detecting anti-IDE autoantibodies in the present invention is described above, and is not described herein. The kit of the invention preferably further comprises one or more of FITC-labeled goat anti-human IgG antibodies, reaction buffers, positive controls, negative controls, and sample dilutions. The positive control of the present invention preferably comprises serum or antibodies that specifically bind to the IDE cell slide; the reaction buffer preferably comprises a surfactant-containing phosphate, further preferably a PBST solution; the sample diluent preferably comprises a surfactant-containing phosphate, and more preferably is a PBST solution.

The invention uses the reagent for detecting the anti-IDE autoantibody as the main component, establishes the reagent kit for detecting and/or diagnosing the autoimmune diseases of the nervous system, can qualitatively or quantitatively analyze the anti-IDE autoantibody, thereby detecting and/or diagnosing the autoimmune diseases of the nervous system, and has the auxiliary diagnosis function in particular. When the kit is used for detection, the detection mode adopted is not strictly required, and the well-known modes of the kit can be adopted, such as CBA, TBA, ELISA, an immune colloidal gold method, an immunoblotting, an immune spot, a membrane strip method, chemiluminescence, a radioimmunoassay, a liquid chip method, lateral chromatography or flow cytometry.

For further explanation of the present invention, the use of the anti-IDE autoantibodies provided herein in the preparation of products for detecting and/or diagnosing autoimmune diseases of the nervous system is described in detail below with reference to the accompanying drawings and examples, which are not to be construed as limiting the scope of the present invention.

The patient serum in the embodiment of the invention is given by a hospital, the serum of a healthy subject comes from the serum of a healthy physical examination person given by a physical examination center of the hospital after the consent of the person, the patient information is as follows, and the discovery process of the anti-IDE autoantibody is described in detail by taking an experiment carried out by one patient sample as an example:

Patient 1: sex women, 56 years old, have no obvious causes before half a year of continuous dizziness, rotation of vision accompanying objects, unstable walking, recent fever, absentmindedness, poor spirit, slurred speech, and accompanied by memory decline of nearby events, and seek medical attention in advance. The normal functions of blood, urine and feces, liver and kidney, blood sedimentation, hypersensitive C reaction protein, thyroid gland, lumbar puncture pressure and cerebral spinal fluid leucocyte number are all normal; no history of drug and poison contact, healthy spouse and child spouse, and no detailed cause of the parent's age, and family history of hypertension. Doctors suspected of having other autoimmune diseases send their samples to clinical laboratory, and NMDAR, LGI1, AMPA1, LGI1, GABAR, DPPX, ri, yo, hu, amphiphysin, CV2, AQP4, MBP, MOG, GFAP antibodies in cerebrospinal fluid and serum were negative.

Example 1

Screening of patient serum by indirect immunofluorescence

1. Preparing a rat brain tissue frozen climbing tablet:

Adult rats are selected for anesthesia, the abdominal cavity is opened after the four limbs of the rats harden, the apex of the heart is exposed, and PBS is infused from the left apex of the heart so as to be convenient for systemic circulation; then taking out the brain tissue, and fixing the brain tissue with methanol for 10-30 min; the sample is transferred into 30wt.% sucrose solution for dehydration, and is placed at 4 ℃ until the tissue mass is settled; dripping a small amount of embedding medium OCT onto a sample table, placing into a freezing table of a frozen microtome (manufacturer: LEICA model: CM 1950) at-20deg.C, coating a thin layer on the surface of the sample with OCT when the tissue is slightly whitened, continuously freezing for 20min, and slicing to obtain frozen climbing slices of rat brain tissue.

2. Serum incubation:

Patient 1 and normal control serum were each run at 1:10, respectively incubating on rat brain tissue frozen slices, incubating for 1h at room temperature, and washing with PBST for 3 times, each time for 5min; diluted FITC-labeled goat anti-human secondary antibody (manufacturer: jackson cat# 109-095-170) was added, incubated at room temperature for 30min, PBST was washed 3 times, each time for 5min, and observed under a fluorescence microscope, positive signals were found on the hippocampal sites on frozen sections of rat brain tissue in patient 1 serum, and positive signals were not found on the normal control serum at the sites (FIG. 1).

3. Antibody co-staining

The climbing slices incubated with patient 1 serum from step2 were co-stained with a neuron-specific markerNeuN antibody (manufacturer: wuhan Sanying cat # 26975-1-AP), neuN antibody 1:200 dilution, PBST washing 3 times, each time for 5min; alexaFluor 594-labeled goat anti-rabbit IgG (manufacturer: jackson cat. Number: 115-585-144) was incubated for 30min at room temperature, and PBST was washed 3 times for 5min each; DAPI dyes the cell nucleus, dyes for 10min at room temperature, washes for 3 times by PBST, each time for 5min; microscopic observation revealed that the signal from patient 1 serum overlapped the signal from NeuN antibodies on frozen sections of rat brain tissue (fig. 2), indicating that the antigen recognized by the antibodies in patient 1 serum was present on neuronal cells.

4. Screening of target antigens for autoantibodies known to be related to the nervous system

Searching 60 reported related nervous system autoantibody target antigens (comprising DPPX、IgLON5、GlyR、GABAARα1、GABAARγ2、GABAARβ3、mGluR5、D2R、Neurexin-3α、AQP4、MBP、MOG、GFAP、AQP1、Flotillin1/2、homer-3、ITPR1、mGluR5、ARHGAP26、KCNA4、ATP1A3、NCDN、Septin-5、NrCAM、Gliomedin、KLHL11、gephyrin、CARP-VIII、TGM2、TGM6、Trib2、TPO、AK5、GRIA3、MUNC18-1、KCTD16、mGluR1、CACNA2D1、RyR1、PLP1、PDE10A、ADAM22、ROCK2、mGluR2、Rab6A、Rab6B、Tg、LRP4、MuSK、MAG、NF155、NF186、CNTN1、CNTN2、CASPR1、mGluR1、PCA2、agrin、MAP1B、NAE), searching gene sequences encoding the 60 proteins from NCBI and sending to a sequencing company for gene synthesis to obtain recombinant vectors of the corresponding genes, then transfecting the recombinant vectors into 293T cells to obtain recombinant cells, preparing a biological detection chip material for over-expressing target antigens, detecting whether serum/cerebrospinal fluid of a patient can generate immune reaction after incubation with the biological detection chip by an immunofluorescence method, and searching whether the serum/cerebrospinal fluid of the patient contains specific autoantibodies of the series of genes or not, wherein the specific steps are as follows:

(1) Recombinant vector construction: connecting the 60 genes to pCDNA3.1 through a molecular cloning method by a PCR or artificial synthesis method to obtain 60 recombinant vectors, and carrying out large extraction on the constructed recombinant vectors for later use after the sequencing is correct;

(2) Transfection of the target Gene: 293T cells with 6cm multiplied by 6cm climbing slices are paved at the bottom of a culture dish in a 5% CO ₂ cell incubator at 37 ℃ by using 10% FBS-DMEM high-sugar culture, when the cell density reaches 30% -40%, 60 recombinant vectors of corresponding genes and empty pCDNA3.1 are respectively transfected into the 293T cells by using PEI transfection reagent (manufacturer: thermo, product number: BMS 1003) and marked;

(3) Cell climbing sheet fixation: washing 48h of cells after transfection with PBS for 2 times, adding acetone for fixing for 5min, washing with PBS for 2 times, drying at 45 ℃ for 30min, cutting the climbing slices into 2.5mm multiplied by 2.5mm, and pasting 61 cell climbing slices with the size of 2.5mm multiplied by 2.5mm on a glass slide to prepare a biological detection chip for screening target antigens for later use;

(4) Immunofluorescent staining: patient 1 serum and control serum were each prepared according to 1:10, incubating the diluted solution on a biological detection chip at room temperature for 1h, and washing the solution with PBST for 3 times, each time for 5min; use 1:200 diluted FITC-labeled goat anti-human secondary antibodies are incubated for 30min at room temperature, and PBST is washed 3 times for 5min each time; the fluorescent microscope shows that the signals of the serum of the patient 1 and 60 biological detection chips are not obviously stronger than the color reaction of the control serum, but the staining result of the serum of the patient 1 on the hippocampus part of the neuron is obvious according to the staining result of the brain tissue section of the rat, so that the serum of the patient 1 is suspected to possibly contain new autoantibodies which can identify the neuron cells and are different from the prior reports.

Example 2

Co-immunoprecipitation of primary cell lysates and validation and identification of antigens of interest

1. Taking primary neuron cells of a 6-dish rat, discarding the supernatant, washing with PBS for 2 times, fixing 0.4wt.% paraformaldehyde for 10min, and washing with 1×HEPES for 3 times; patient 1 serum and normal control serum were each 1: diluting with 1000, filtering with 0.22 μm filter membrane, adding into immobilized primary neuron cells, and incubating at room temperature for 2 hr; 15. Mu.L of ProteinA/G immunoprecipitation beads (manufacturer: selleck) were added to a 2mL tube, equilibrated washed 3 times, and blocked with 300. Mu.L of 4% BSA for 2h; placing the incubated cells on ice, discarding the supernatant, washing with PBS for 2 times, adding 500 μl of lysis solution (containing 150mM NaCl,1mM EDTA,100mM Tris-HCl,0.5% sodium deoxycholate, 1% Triton X-100 and 0.1% SDS, pH 7.5) with water as solvent, collecting cells, adding protease inhibitor with final concentration of 1×for lysis for 30min, shaking at intervals of 15000rpm for 30min, collecting supernatant, and measuring concentration; adding the collected supernatant into treated ProteinA/G immunoprecipitation magnetic beads, and incubating at 4 ℃ overnight in a rotary manner; washing the incubated magnetic beads with the lysate for 4 times, eluting with 80 μL of 2×loading buffer, and collecting eluate; adding 5 XSDS-PAGE loading buffer into the eluent, adding DTT with the final concentration of 0.01M, heating at 100 ℃ for 10min, adding iodoacetamide with the final concentration of 2wt.% into the eluent, and standing at room temperature for 30min to obtain immunoprecipitation complex of a patient 1 serum sample and a normal control serum sample; after SDS-PAGE electrophoresis of the prepared immunocomplexes, staining was performed using a silver staining kit (manufacturer: thermo), and the results showed that a protein of about 100 KD-130 KD was detected in immunoprecipitates captured with patient 1 serum in rat neurons, which was not present in controls prepared in normal control serum by a similar method (FIG. 3).

2. Immunoblot validation

(1) Electrophoresis: performing SDS-PAGE on the immune complex sample obtained in the step 1;

(2) Transferring: after electrophoresis, transferring film by wet method, the film transferring condition is 200mA,90min;

(3) Closing: sealing for 1h at room temperature by using 5% skimmed milk powder;

(4) Serum incubation: patient 1 serum and normal control serum were each 1:100 dilution and incubation at room temperature for 2h;

(5) Washing: TBST is washed for 3 times, each time for 5min;

(6) Secondary antibody incubation: adding HRP-labeled goat anti-human IgG secondary antibody (manufacturer: jackson), and incubating for 1h at room temperature;

(7) Washing: TBST is washed for 3 times, each time for 5min;

(8) Color development: the results showed that the presence of a band reactive with autoantibodies in patient 1 serum in the immunoprecipitate captured in primary neuronal cells with patient 1 serum, the band of the protein of interest was between 100KD and 130KD, whereas the immunoprecipitate was not reactive with normal control serum between 100KD and 130KD (fig. 4), demonstrating the presence of autoantibodies reactive with neuronal proteins in patient 1 serum.

3. Identification of antigens of interest by mass spectrometry

The strip of the gel cutting recovery step 1 (arrow position in FIG. 3) at the position of 100 KD-130 KD is sent to the norstanding organism for mass spectrometry analysis, and the mass spectrometry result shows that the cut gel strip contains IDE protein (insulin degrading enzyme, accession number: NM_ 004969.4).

Example 3

Commercial antibody in rat brain tissue frozen section immunofluorescence experiment to verify the expression of target antigen

1. Preparation of protein samples

Rat brain tissue was cut up at 50mg each, transferred to a mortar, frozen and ground with liquid nitrogen to a fine powder, and each group of samples was added with 500. Mu.L of RIPA lysate (consisting of 150mM NaCl,1mM EDTA,100mM Tris-HCl,0.1%SDS,0.5% sodium deoxycholate, 1% Triton X-100 and 5% glycerol, pH 7.5) and with 1X final concentration of protease inhibitor, sonicated (disruption conditions: 10% power, disruption for 3s, disruption for 6s, total sonication for 1 min). Cracking on ice for 30min after ultrasonic treatment, and vibrating once at intervals of 5 min; collecting 293T cells over-expressing IDE in 1 dish and empty pCDNA3.1 cells in 1 dish, respectively adding 200 μl PBS, ultrasonic crushing (under the same condition as the tissue protein), centrifuging at 15000rpm for 30min after collecting all samples, and measuring protein concentration of supernatant by BCA method;

2. Sample loading and transfer film

Respectively taking 40 mug of rat whole brain tissue protein, over-expressed IDE protein and pCDNA3.1 protein for loading, and carrying out wet transfer under the condition of 300mA and 90min after electrophoresis; sealing 5% of skimmed milk powder for 1h at room temperature;

3. Antibody incubation and color development

Use 1:1000 dilutions of IDE commercial antibody (manufacturer: abcam cat# ab 32216) or internal reference GAPDH antibody (manufacturer: jackson) were incubated on the membrane-transferred PVDF membrane overnight at 4 ℃; the next day, TBST is washed 3 times for 5min each time; adding HRP-labeled goat anti-rabbit secondary antibody (manufacturer: jackson), and incubating for 1h at room temperature; TBST is washed for 3 times, each time for 5min; the ECL chemiluminescent solution was added for chromogenic photographing, and the result shows that the expression of IDE protein exists in both over-expressed IDE cells and rat brain tissues (FIG. 5).

Example 4

Serum neutralization experiments verify patient serum detected signals

1. Construction of recombinant vectors

The IDE gene (SEQ ID NO. 3) is connected to the pCDNA3.1 by a PCR or artificial synthesis method, the insertion site is XbaI/NotI, the recombinant vector pCDNA3.1-IDE is obtained, and the constructed recombinant vector is greatly extracted for later use after the sequencing is correct.

2. Preparation of neutralizing serum

(1) Transfection of the target Gene: 293F suspension cells were cultured in 20mL of a total of 2 flasks using CD05 medium (manufacturer: olprimex) in a 100mL flask and placed in a 5% CO ₂ cell culture shaker at 37 ℃. When the cell density reaches 3X 10 ⁶/mL, using a PEI transfection reagent (manufacturer: thermo, product number: BMS 1003) to respectively transfect the pCDNA3.1-IDE recombinant vector and the empty pCDNA3.1 into 293F cells, marking, and centrifuging and changing the liquid the next day;

(2) Cell fixation: respectively centrifuging two bottles of suspension cells grown for 96 hours after transfection, washing with PBS for 2 times, re-suspending with 5mL of PBS, adding absolute ethanol for fixation for 10min, centrifuging to remove ethanol, washing with PBS for 2 times, placing in a 2mL EP tube, and adding 1mLPBS for re-suspending;

(3) Serum neutralization: and respectively adding 12 mu L of patient 1 serum into the two tubes of fixed cells, incubating overnight at 4 ℃, centrifuging the cells on the next day, and labeling the centrifugal supernatant of the cells which are subjected to over-expression IDE suspension as neutralizing serum and labeling the centrifugal supernatant of the cells which are subjected to empty pCDNA3.1 suspension as control serum.

3. Immunofluorescence assay

Reference example 1, an over-expressed IDE cell slide and an empty pcdna3.1 control cell slide were prepared for use; incubating the over-expressed IDE cell slide and the pCDNA3.1 control cell slide with the neutralizing serum and the control serum respectively for 1h at room temperature, and washing with PBST for 3 times, each time for 5min; adding diluted FITC-labeled goat anti-human secondary antibody (manufacturer: jackson cat# 109-095-170), incubating at room temperature for 30min, and washing with PBST 3 times each for 5min; microscopic observations showed that on the over-expressed IDE cell slide, the neutralizing serum signal was reduced, while the control serum signal was still evident (fig. 6), indicating that the signal of patient 1 serum neutralized by the over-expressed IDE suspension cells was a signal specifically recognizing IDE antigen.

Western Blot experiments

Protein samples were prepared according to reference example 3, and subjected to electrophoresis, transfer and blocking steps, using the neutralized serum and control serum, respectively, incubated on the transferred PVDF membrane, overnight at 4 ℃; the next day, TBST is washed 3 times for 5min each time; adding HRP-labeled goat anti-human secondary antibody (manufacturer: jackson), and incubating for 1h at room temperature; TBST is washed for 3 times, each time for 5min; the result of chromogenic photographing by adding ECL chemiluminescent liquid shows that the band signal of the neutralizing serum is weakened at 100-130KD in the lane over-expressing IDE protein, while the band signal of the control serum is still obvious at the place (figure 7), which shows that the signal of neutralizing the serum of the patient 1 by the over-expressed IDE suspension cells is a signal for specifically recognizing IDE antigen (the incubation mode of the internal reference GAPDH antibody is the same as in example 3).

Example 5

Recovery of serum autoantibodies to verify patient serum detection signals

1. Preparation of recovered serum autoantibodies

Two tubes of cells after centrifugation and collection of the neutralized serum in step 2 of example 4 were resuspended in PBS and washed repeatedly for 4 times, 5min each time; after washing, adding 500 mu LpH =3 of 0.1M glycine eluent into each tube, eluting for 15min by a rotary table at room temperature, centrifugally collecting eluent after eluting, adding 10 mu L of 1M Tris into the eluent to neutralize until the pH of the eluent is 7.0-8.0, adding 1/10 volume of PBS to obtain 2 parts of 500 mu L eluting samples, wherein one part is experimental eluting antibody after the combination of patient 1 serum and over-expressed IDE suspension cells; the other fraction was control eluted antibody after binding of patient 1 serum to control pcdna3.1 suspension cells.

2. Immunofluorescence assay

Incubating the over-expression IDE cell slide and the empty pCDNA3.1 control cell slide by using an experimental elution antibody and a control elution antibody respectively, incubating for 1h at room temperature, and washing by PBST for 3 times, wherein each time is 5min; use 1:200 diluted FITC-labeled goat anti-human IgG, incubated at room temperature for 30min, and washed with PBST 3 times for 5min each; the results of the observation under the fluorescence microscope show that the experimental elution antibody combined with the over-expression IDE suspension cells has obvious positive signals on the over-expression IDE cell slide, while the control elution antibody does not (figure 8), which shows that the experimental elution antibody combined with the over-expression IDE suspension cells can specifically recognize the IDE antigen on the over-expression IDE cell slide.

Western Blot experiments

Preparing a protein sample according to reference example 3, performing electrophoresis, membrane transfer and sealing, and respectively incubating the protein sample and the reference eluted antibody on the membrane-transferred PVDF membrane at 4 ℃ overnight; the next day, TBST is washed 3 times for 5min each time; adding HRP-labeled goat anti-human secondary antibody (manufacturer: jackson), and incubating for 1h at room temperature; TBST is washed for 3 times, each time for 5min; the results of the chromogenic photographing with ECL chemiluminescent solution added show that the experimental eluting antibody has obvious band signals at the positions of 100-130KD of the over-expressed IDE proteome lane and the rat brain tissue protein lane, while the band signals of the control eluting antibody are not obvious (figure 9), which shows that the experimental eluting antibody combined with the over-expressed IDE suspension cells can specifically recognize IDE antigens in the over-expressed cells and the rat brain tissue (the incubation mode of the internal reference GAPDH antibody is the same as in example 3).

Example 6

Detection rate of anti-IDE autoantibody in suspected nervous system autoimmune disease sample

1. Immunofluorescent staining

(1) Serum incubation: serum from patient 1 was diluted 10-fold with PBST, and the over-expressed IDE cell slide and empty pcdna3.1 control cell slide were incubated separately for 1h at room temperature; PBST is washed for 3 times, each time for 5min; adding FITC marked goat anti-human IgG secondary antibody, and incubating for 1h at room temperature; PBST is washed for 3 times, each time for 5min;

(2) Co-staining of antibodies: commercial IDE antibody 1 using PBST: after 100 dilution, continuing to incubate the cell slide expressing IDE in the previous step and the cell slide of the empty pCDNA3.1 control cell slide, and incubating for 1h at room temperature; PBST is washed for 3 times, each time for 5min; adding AlexaFluor594 marked goat anti-rabbit IgG, and incubating for 1h at room temperature; PBST is washed for 3 times, each time for 5min; microscopic observations showed that the staining signal of patient 1 serum on the over-expressed IDE cell slide overlapped with the staining signal of the commercial IDE antibody on the over-expressed IDE cell slide (fig. 10), indicating that the antibody in patient 1 serum specifically recognized the IDE protein on the over-expressed IDE cell slide.

2. 3890 Patient samples (3328 of them, 562 of cerebrospinal fluid) with symptoms of neurological diseases were taken, and these patients exhibited symptoms of: suspected encephalitis, paraneoplastic syndrome, myasthenia gravis, neuromyelitis optica and peripheral neuropathy, and the detection of the autoantibodies of the nervous system, which are reported by the prior art, are all negative results. The cell slide over-expressing IDE and the empty pCDNA3.1 control cell slide were used to perform detection, and 44 positive samples of anti-IDE autoantibodies (42 cases of positive patients with serum positive, 2 cases of positive cerebrospinal fluid, 1:1 brain ridge titer, and corresponding positive serum, 1:10 titer) were screened out, the total detection rate of anti-IDE autoantibodies was 1.13% (1.26% of serum samples and 0.3% of cerebrospinal fluid samples), the staining results of some patients were shown in FIG. 11, and the clinical symptoms of some patients were shown in Table 1. 44 patients who were diagnosed by the clinician and detected anti-IDE antibodies had autoimmune encephalitis, 2 of which had complicated central demyelinating diseases. The invention provides a new antigen combined with an autoantibody to be detected for realizing the diagnosis of autoimmune encephalitis, and the anti-IDE autoantibody can be detected in patients suffering from nervous system symptoms, which indicates that the autoantibody has an auxiliary effect on the diagnosis of nervous system autoimmune diseases.

TABLE 1 partial patient clinical information

Example 7

Cell-based immunofluorescence method for verifying specificity of anti-IDE autoantibody

57 Cases (wherein case number of anti-NMDAR ⁺, case number of anti-GABABR ⁺, case number of anti-CASPR2 ⁺, case number of anti-LGI1 ⁺, case number of anti-AMPAR ⁺, case number of anti-Hu ⁺, case number of anti-Ri ⁺, case number of anti-Yo ⁺, case number of anti-CV2 ⁺, case number of anti-Ma2 ⁺, case number of anti-amphiphysin ⁺ 4, case number of anti-GAD65 ⁺, case number of anti-AQP4 ⁺, case number of anti-MBP ⁺, case number of anti-MOG ⁺) were selected, immunofluorescence detection was performed using the over-expressed cell slide, and the selected case number of 57-neural autoimmune disease samples and 40 healthy control samples were not similar to the cell slide samples screened out in example number of example 6.

Example 8

Detection of anti-IDE autoantibodies in patient serum by IDE mutant

1 Mutant of human IDE gene, namely the mutant (SEQ ID NO. 2) deleted from amino acids 572-599 of IDE protein, was selected, vector construction was performed, cell climbing sheets over-expressing the recombinant IDE deletion mutant were prepared and serum of the patient was examined according to the procedure described in example 2, and the result showed that the IDE deletion mutant still recognized anti-IDE antibodies in the serum of the patient.

According to the above embodiments, it can be seen that the anti-IDE autoantibody can be used as a biomarker for detecting and/or diagnosing autoimmune diseases of the nervous system, and diagnosis and screening of autoimmune diseases of the nervous system, in particular, auxiliary diagnosis of autoimmune diseases of the nervous system can be achieved by detecting the anti-IDE autoantibody.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (10)

1. The use of a reagent for detecting anti-IDE autoantibodies in the manufacture of a product for detecting and/or diagnosing autoimmune diseases of the nervous system.

2. The use of claim 1, wherein the agent for detecting anti-IDE autoantibodies comprises one or more of IDE protein, IDE protein-expressing cells, IDE protein-expressing tissues, and IDE protein lysate.

3. The use of claim 2, wherein the amino acid sequence of the IDE protein comprises any one of a) to c):

a) An amino acid sequence shown in SEQ ID NO. 1;

b) 10% -80% of the amino acid sequence shown in SEQ ID NO.1, and can recognize the amino acid sequence of the anti-IDE autoantibody;

c) The amino acid sequence of a) or b) is modified or mutated and the amino acid sequence of an anti-IDE autoantibody can be recognized.

4. The use according to claim 3, wherein the modification comprises one or more of glycosylation modification, phosphorylation modification, acetylation modification, methylation modification and ubiquitination modification.

5. The use according to claim 3, wherein the nucleotide sequence encoding the amino acid sequence of the IDE protein comprises any one of i) to iii):

i) the nucleotide sequence shown in SEQ ID NO. 3;

II) 10% -80% of the nucleotide sequence shown in SEQ ID NO.3, and the coded amino acid sequence can recognize an anti-IDE autoantibody;

The encoded amino acid sequence recognizes an anti-IDE autoantibody after modification or mutation of the nucleotide sequence of III) I) or II).

6. The use according to claim 1, wherein the symptoms of the neurological autoimmune disease comprise one or more of headache, fever, muscle soreness, vomiting, insomnia, snoring, abnormal gait, decreased memory, dysphagia, mental abnormalities, seizures, cognitive dysfunction, language disorders, disturbance of consciousness and movement disorders.

7. The use according to claim 6, wherein the neurological autoimmune disease is autoimmune encephalitis.

8. A kit for detecting and/or diagnosing an autoimmune disease of the nervous system, comprising a reagent for detecting an anti-IDE autoantibody and a labeled antibody.

9. The kit of claim 8, wherein the reagent for detecting anti-IDE autoantibodies comprises one or more of IDE protein, IDE protein-expressing cells, IDE protein-expressing tissue, and IDE protein-containing tissue.

10. The kit of claim 9, wherein the amino acid sequence of the IDE protein comprises any one of a) to c):

a) An amino acid sequence shown in SEQ ID NO. 1;

b) 10% -80% of the amino acid sequence shown in SEQ ID NO.1, and can recognize the amino acid sequence of the anti-IDE autoantibody;

c) The amino acid sequence of a) or b) is modified or mutated and the amino acid sequence of an anti-IDE autoantibody can be recognized.