Application of reagent for detecting SLC17A7 autoantibody in diagnosis of nervous system diseases

Abstract

The invention discloses application of a reagent for detecting an SLC17A7 autoantibody in diagnosis of nervous system diseases. By comparing different fluorescence modes of autoimmune antibodies related to IgG of healthy people in mouse brain tissue slices of patients with nervous system diseases, it is found that fluorescence signals only exist in the patients with nervous system diseases and do not exist in the healthy people, and it is prompted that the patients possibly have the autoimmune antibodies. According to the invention, IgG specific protein related to nervous system diseases is sought through a co-immunoprecipitation technology, a specific SLC17A7 autoantibody is found through cell immunofluorescence (CBA) detection, verification is carried out in cerebrospinal fluid, and finally it is determined that the SLC17A7 autoantibody can be used as a specific marker for diagnosis of nervous system autoimmune diseases. The SLC17A7 is taken as a detection antigen, detection of the expression of the SLC17A7 autoantibody can be applied to a kit for detecting the SLC17A7 autoantibody, the kit can be applied to detection of nervous system diseases, markers for identifying the nervous system diseases are enriched, and the detection accuracy of nervous system related diseases is improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of reagents for detecting SLC17A7 autoantibodies in the diagnosis of neurological diseases. Background Technology

[0002] Autoimmune neurology has become a research hotspot in the field of neuroimmunology in recent years. In the past, the identification and diagnosis of autoimmune neurological diseases in clinical practice were limited, and effective treatments were scarce. However, with the continuous deepening of research and the optimization of antibody detection technology, the number of pathogenic antibody biomarkers for autoimmune neurological diseases has increased rapidly, such as anti-NMDAR antibodies, anti-LGI1 antibodies, anti-AMPAR antibodies, and anti-GAD65 antibodies. These novel autoimmune antibodies have further improved our understanding of autoimmune neurological diseases. Simultaneously, these discoveries have also promoted research into the pathological mechanisms of these diseases, revealing the immunological basis of many diseases and providing a theoretical basis for the development of new therapies.

[0003] After an autoimmune neurological disease is clinically considered, neuroantibody testing helps to clarify the diagnosis and provide information for prognosis and treatment. In most cases, testing readily available biological samples such as serum and cerebrospinal fluid for autoimmune antibodies not only aids in early diagnosis but also allows for the selection of appropriate immunosuppressive therapy based on the pathogenic mechanism of antibody markers, thereby improving the targeting and effectiveness of treatment. The presence of autoimmune antibodies makes early diagnosis and treatment possible. With the discovery of more clinically validated novel autoimmune antibodies, an increasing number of neurological diseases can be diagnosed using immunological methods, significantly improving the level of clinical diagnosis and treatment.

[0004] The SLC17A7 gene (Solute carrier family 17member 7) encodes vesicular glutamate transporter 1, a member of solute carrier family 17. SLC17A7 is a multifunctional membrane transporter, possibly with a 12-span membrane structure, primarily located on synaptic vesicle membranes. Its main function is to transport glutamate from the cytoplasm to synaptic vesicles at the presynaptic nerve endings of excitatory neurons, thus participating in excitatory neurotransmission. It is also responsible for the transport of phosphate, proton, chloride, and sodium ions. SLC17A7 is primarily expressed in the nervous system, especially in regions rich in glutamatergic neurons. In the central nervous system (CNS), it is mainly expressed on the synaptic vesicle membranes of glutamatergic neurons, particularly widely distributed in the cortex, hippocampus, olfactory cortex, brainstem, and cerebellar cortex. In peripheral tissues, it is mainly expressed in the eye, especially the retina, and in pacemaker cells of the sinoatrial node of the heart. SLC17A7 is also expressed in some tumor cells, such as glioblastoma. Further research into SLC17A7 has revealed a potential link between it and autoimmune neurological diseases. Recent studies show that SLC17A7 is expressed not only in neurons but also in certain astrocyte subsets. A specific type of glutamatergic astrocyte (expressing SLC17A7) can release glutamate through vesicles, participating in the regulation of neuronal excitability. Research has demonstrated that this specific glutamatergic astrocyte, by releasing glutamate, can enhance synaptic plasticity, strengthen memory formation, and protect against epileptic seizures. One of the core pathological mechanisms of autoimmune neurological diseases is neuroinflammation. In autoimmune neurological diseases such as multiple sclerosis, neuroinflammation and activation of immune cells infiltrate the central nervous system, leading to glial cell dysfunction. This dysfunction may affect the normal function of glutamatergic glial cells, causing abnormal activation or inhibition, thereby interfering with neuronal excitability and synaptic transmission, and exacerbating nerve damage. Furthermore, the activation of glutamatergic glial cells may further promote the release of inflammatory factors, exacerbating neuroinflammation. Furthermore, some reports have mentioned the association between SLC17A7 and other neurological diseases, such as epilepsy, Parkinson's disease, Alzheimer's disease, autism spectrum disorders, depression, anxiety disorders, neurodevelopmental disorders, and neurodegenerative diseases. Although the role of SLC17A7 in autoimmune neurological diseases is not fully understood, and there are no reports of SLC17A7 autoantibodies, its crucial role in glutamatergic neurotransmission and glial cell function makes it a hot research topic. Future research will further elucidate its specific role in disease pathogenesis and explore its potential as a therapeutic target, contributing to a better understanding of the physiological and pathological mechanisms of the central nervous system. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned problems and provide a reagent for detecting SLC17A7 autoantibodies in the diagnosis of neurological diseases. This invention utilizes SLC17A7 autoantibodies as a biomarker for the discovery and application of these markers in the detection of neurological diseases. By comparing different fluorescence patterns of IgG-related autoantibodies in mouse brain tissue sections from patients with neurological diseases and healthy individuals, this invention identifies fluorescence signals present only in patients with neurological diseases and not in healthy individuals, suggesting the possible presence of autoimmune antibodies in these patients. Through immunoprecipitation, specific IgG proteins associated with neurological diseases are sought. Cellular immunofluorescence (CBA) is used to detect specific SLC17A7 autoantibodies, which are then validated in cerebrospinal fluid. Ultimately, it is determined that SLC17A7 autoantibodies can serve as a specific biomarker for the diagnosis of neurological autoimmune diseases. Using SLC17A7 as the detection antigen, the detection of SLC17A7 autoantibody expression can be applied to kits for detecting SLC17A7 autoantibodies, which can be used to detect neurological diseases, enriching the biomarkers for identifying neurological diseases and improving the accuracy of detecting related diseases.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: the application of reagents for detecting SLC17A7 autoantibodies in the preparation of products for diagnosing neurological diseases.

[0007] Preferably, the reagent for detecting SLC17A7 autoantibodies includes one or more of the following: cells expressing SLC17A7 protein, tissues expressing SLC17A7 protein, and lysates containing SLC17A7 protein.

[0008] Preferably, the SLC17A7 protein includes one or more of a) to d):

[0009] a) SLC17A7 protein in cell lysis products;

[0010] b) SLC17A7 protein in the brain or brain nerve tissue;

[0011] c) The SLC17A7 protein whose amino acid sequence is encoded by the nucleotide sequence shown in SEQ ID NO.1;

[0012] d) The SLC17A7 protein encoded by the amino acid sequence shown in SEQ ID NO.2.

[0013] Preferably, the symptoms of the neurological disorder include epileptic seizures.

[0014] Preferably, the test sample for the neurological disease is an individual biological sample, including serum or cerebrospinal fluid.

[0015] Preferably, the reagent includes one for detecting SLC17A7 autoantibodies.

[0016] Preferably, the reagent for detecting SLC17A7 autoantibodies includes one or more of cells expressing SLC17A7 protein, tissues expressing SLC17A7 protein, and lysates containing SLC17A7 protein.

[0017] Preferably, the SLC17A7 protein includes one or more of a) to d):

[0018] a) SLC17A7 protein in cell lysis products;

[0019] b) SLC17A7 protein in the brain or brain nerve tissue;

[0020] c) The SLC17A7 protein whose amino acid sequence is encoded by the nucleotide sequence shown in SEQ ID NO.1;

[0021] d) The SLC17A7 protein encoded by the amino acid sequence shown in SEQ ID NO.2.

[0022] The specific sequences of SEQ ID NO.1 and SEQ ID NO.2 of this invention are as follows:

[0023] SEQ ID NO.1:

[0024]

[0025] SEQ ID NO.2:

[0026] MEFRQEEFRKLAGRALGKLHRLLEKRQEGAETLELSADGRPVTTQTRDPPVVDCTCFGLPRRYIIAIMSGLGFCISFGIRCNLGVAIVSMVNNSTTHRGGHVVVQKAQFSWDPETVGLIHGSFFWGYIVTQIPGGFICQK FAANRVFGFAIVATSTLNMLIPSAARVHYGCVIFVRILQGLVEGVTYPACHGIWSKWAPPLERSRLATTAFCGSYAGAVVAMPLAGVLVQYSGWSSSVFYVYGSFGIFWYLFWLLVSYESPALHPSISEEERKYIEDAIGE SAKLMNPLTKFSTPWRRFFTSMPVYAIIVANFCRSWTFYLLLISQPAYFEEVFGFEISKVGLVSALPHLVMTIIVPIGGQIADFLRSRRIMSTTNVRKLMNCGGFGMEATLLLVVGYSHSKGVAISFLVLAVGFSGFAIS GFNVNHLDIAPRYASILMGISNGVGTLSGMVCPIIVGAMTKHKTREEWQYVFLIASLVHYGGVIFYGVFASGEKQPWAEPEEMSEEKCGFVGHDQLAGSDDSEMEDEAEPPGAPPAPPPSYGATHSTFQPPRPPPPVRDY

[0027] Compared with existing technologies, the beneficial effects of this solution are:

[0028] Detecting the presence or absence of SLC17A7 autoantibodies can specifically identify patients with neurological disorders, particularly epilepsy. This not only aids in early diagnosis but also allows for the provision of appropriate immunosuppressive therapy, thereby improving the targetedness and effectiveness of treatment. As the experimental results mentioned in the document show, SLC17A7 autoantibodies are positive in the serum of patients, while they are negative in the serum of healthy individuals, indicating that SLC17A7 autoantibodies can serve as a biomarker to distinguish between patients and healthy individuals.

[0029] The discovery of SLC17A7 autoantibodies enriches the existing database of biomarkers for neurological diseases, providing clinicians with more diagnostic tools. This not only helps improve diagnostic capabilities but may also drive the development of personalized treatment strategies for specific neurological disorders. For example, the document mentions that the detection of SLC17A7 autoantibodies can be used in kits for detecting SLC17A7 antibodies and can also be applied to the detection of neurological diseases, thereby enriching the database of biomarkers for neurological diseases. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the immunofluorescence staining effect of serum on mouse brain tissue in an embodiment of the present invention (green: immunofluorescence reaction of patient serum with C57 mouse brain tissue sections. red: immunofluorescence reaction of commercial anti-SCL17A7 antibody with C57 mouse brain tissue sections. blue: DAPI cell nuclear staining);

[0031] Figure 2 This is a schematic diagram of the immunofluorescence staining effect of serum on mouse brain tissue in an embodiment of the present invention (green: immunofluorescence reaction of patient serum with C57 mouse brain tissue sections. red: immunofluorescence reaction of commercial anti-SCL17A7 antibody with C57 mouse brain tissue sections. blue: DAPI cell nuclear staining);

[0032] Figure 3 This is a gel silver staining result of immunoprecipitation of patient serum and healthy control serum in mouse brain tissue lysate in an embodiment of the present invention;

[0033] Figure 4 This is a schematic diagram of the co-localization of patient serum and SLC17A7 autoantibody staining on mouse brain tissue in an embodiment of the present invention;

[0034] Figure 5 This is a schematic diagram illustrating the immunofluorescence staining effect of patient cerebrospinal fluid on a smear of 293T cells overexpressing SLC17A7 in an embodiment of the present invention.

[0035] Figure 6 This is a schematic diagram of the immunofluorescence staining effect of patient serum on 293T cells overexpressing SLC17A7 in an embodiment of the present invention. Detailed Implementation

[0036] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0037] This invention demonstrates the presence of autoantibodies against the SLC17A7 protein in neurological diseases. These SLC17A7 autoantibodies can be used as biomarkers in the detection of neurological diseases and can be incorporated into kits for detecting SLC17A7 antibodies.

[0038] The symptoms of the neurological diseases described in this invention include one or more manifestations of limb convulsions, abnormal muscle tone, loss of consciousness, cognitive impairment, involuntary movements, and autonomic dysfunction; the neurological diseases include epilepsy. The samples used to detect the neurological diseases described in this invention are serum or cerebrospinal fluid.

[0039] The SLC17A7 autoantibody described in this invention is closely related to neurological diseases, and provides a basis for the diagnosis of neurological diseases in patients.

[0040] In this invention, the reagent for detecting SLC17A7 autoantibodies includes one or more of cells expressing SLC17A7 protein, tissues expressing SLC17A7 protein, and lysates containing SLC17A7 protein.

[0041] The SLC17A7 protein includes any one of a) to d): a) SLC17A7 protein in cell lysis products; b) SLC17A7 protein in brain or brain nerve tissue; c) SLC17A7 protein whose amino acid sequence is encoded by the nucleotide sequence shown in SEQ ID NO.1; d) SLC17A7 protein encoded by the amino acid sequence shown in SEQ ID NO.2.

[0042] The specific sequences of SEQ ID NO.1 and SEQ ID NO.2 of this invention are as follows:

[0043] SEQ ID NO.1:

[0044]

[0045] SEQ ID NO.2:

[0046] MEFRQEEFRKLAGRALGKLHRLLEKRQEGAETLELSADGRPVTTQTRDPPVVDCTCFGLPRRYIIAIMSGLGFCISFGIRCNLGVAIVSMVNNSTTHRGGHVVVQKAQFSWDPETVGLIHGSFFWGYIVTQIPGGFICQK FAANRVFGFAIVATSTLNMLIPSAARVHYGCVIFVRILQGLVEGVTYPACHGIWSKWAPPLERSRLATTAFCGSYAGAVVAMPLAGVLVQYSGWSSSVFYVYGSFGIFWYLFWLLVSYESPALHPSISEEERKYIEDAIGE SAKLMNPLTKFSTPWRRFFTSMPVYAIIVANFCRSWTFYLLLISQPAYFEEVFGFEISKVGLVSALPHLVMTIIVPIGGQIADFLRSRRIMSTTNVRKLMNCGGFGMEATLLLVVGYSHSKGVAISFLVLAVGFSGFAIS GFNVNHLDIAPRYASILMGISNGVGTLSGMVCPIIVGAMTKHKTREEWQYVFLIASLVHYGGVIFYGVFASGEKQPWAEPEEMSEEKCGFVGHDQLAGSDDSEMEDEAEPPGAPPAPPPSYGATHSTFQPPRPPPPVRDY

[0047] In this invention, the tissue expressing SLC17A7 protein is preferably mammalian tissue, which preferably includes human or mouse, and the tissue is preferably brain tissue; the cell expressing SLC17A7 protein is preferably 293T cell expressing SLC17A7 protein; the lysate containing SLC17A7 protein preferably includes mammalian tissue, which is preferably mouse, and the tissue is preferably brain tissue.

[0048] In this invention, the solid-phase support preferably includes a glass slide, magnetic beads, chromatographic packing material, or a poly-L-lysine-coated culture plate; the labeled antibody preferably includes horseradish peroxidase-labeled antibody, alkaline phosphatase-labeled antibody, biotin-labeled antibody, FITC-labeled antibody, or Alexa Fluor dye. The detection methods for the SLC17A7 autoantibody of this invention include indirect immunofluorescence (including cell substrate-based immunofluorescence and tissue substrate-based immunofluorescence) and immunoprecipitation. Preferred instruments for observing the results include a fluorescence microscope, an ELISA reader, a chemiluminescence analyzer, and a film viewing lamp.

[0049] To further illustrate the present invention, the application of SLC17A7 autoantibody in the detection of neurological diseases is described in detail below with reference to the accompanying drawings and embodiments. The drawings and embodiments are for illustrative purposes only and should not be construed as limiting the scope of protection of the present invention.

[0050] In all embodiments of this invention, patient samples were obtained with the patients' consent. Patient information is as follows:

[0051] Patient 1: A 38-year-old male presented to the hospital with a history of unexplained paroxysmal loss of consciousness, accompanied by limb convulsions, frothing at the mouth, limb numbness, vocalizations, and leftward deviation of the mouth. He had a history of right upper limb fracture surgery. Brain MRI and oxygenation level-dependent brain imaging (BOLD) revealed activation areas in the bilateral frontal lobes, cingulate gyrus, bilateral occipital lobes, and left temporal lobe, with more pronounced activation in the left superior frontal gyrus. Multimodal epilepsy imaging showed a smaller volume in the left hippocampus and amygdala compared to the contralateral side. TBA screening showed positive cerebellar cells. The clinical diagnosis was refractory epilepsy.

[0052] Patient 2: A 33-year-old female presented to the hospital with a history of paroxysmal altered consciousness accompanied by immobility over the past four years without any apparent cause. She had a history of right frontal head trauma. Cranial MRI revealed bilateral frontoparietal arachnoid cysts; SWI and MRV scans of the brain showed no significant abnormalities. Electroencephalography (EEG) showed increased slow waves, with numerous scattered medium-to-high sharp waves and sharp-slow waves observed in the left anterior, middle, and posterior temporal regions and the sphenoid bone during awake periods. Blood and cerebrospinal fluid autoimmune tests for brain paraneoplastic and demyelinating antibodies were negative. The clinical diagnosis was focal epilepsy with perceptual disturbances.

[0053] Patient 3: A 37-year-old male presented to the hospital with symptoms including intermittent facial twitching, drooling, drooling, upward slant of the mouth, and head tilting during sleep at night for the past 16 years, accompanied by recent memory decline. He had previously undergone epilepsy surgery and had a 3-month history of elevated blood pressure. A cranial MRI revealed a continuous abnormal signal pattern in the deep left temporal lobe, basal ganglia, periventricular region, and frontal lobe; cerebellar atrophy; and a large cisterna magna. A cranial MRA scan showed no significant abnormalities. The clinical diagnosis was epilepsy.

[0054] Patient 4: Male, 30 years old, presented to the hospital with a history of paroxysmal generalized contractions, limb convulsions and weakness, accompanied by trismus, drooling, difficulty swallowing, and tongue biting, occurring during meals over the past 5 months. He has a history of hyperlipidemia and hyperuricemia, and his mother has a history of epilepsy. A cranial MRI revealed a right maxillary sinus cyst. An abnormal video EEG was also observed. The clinical diagnosis was epilepsy.

[0055] Example 1: Immunofluorescence (TBA) staining based on mouse brain tissue

[0056] Preparation of mouse brain tissue sections:

[0057] Mouse brain tissue was fixed with 4% paraformaldehyde and then dehydrated in sucrose solution. The cryostat was turned on, and the chamber temperature and the sectioning head temperature were both set to -20℃. The section thickness was set to 20μm. The dehydrated mouse brain tissue was taken out and embedded with OCT tissue embedding medium. After the tissue was completely frozen, it was sectioned. After sectioning to the target brain region, the slides were collected.

[0058] Sealing process:

[0059] Three mouse brain tissue sections were selected and rinsed with PBS containing 0.2% Triton X-100 for 10 minutes, followed by rinsing twice with PBS for 10 minutes each time. The sections were then blocked at room temperature for 1 hour using blocking buffer (4 mL PBS solution, 500 μL goat serum stock solution, 500 μL 2% Triton X-100 solution, 0.1 g BSA).

[0060] Incubation of primary antibody:

[0061] The three mouse brain tissue sections prepared in step 1 were incubated with blocking buffer diluted to 1:100 using serum from patients 1, 2, 3, and 4, as well as serum from healthy individuals. The sections were incubated at 4°C for 14 hours, and then washed three times with PBS for 10 minutes each time to obtain mouse brain tissue sections incubated with the primary antibody.

[0062] Incubation of secondary antibodies:

[0063] Tissue sections were incubated with Alexa Fluor 488-labeled goat anti-human IgG (purchased from Invitrogen) at a blocking solution diluted to 1:1000 and incubated at room temperature in the dark for 1 hour. The sections were then washed three times with PBS for 10 minutes each time and mounted with mounting medium containing DAPI.

[0064] Detection of immunofluorescence signals:

[0065] Observe and photograph under a fluorescence microscope. Results are as follows: Figure 1-2As shown, the patient's serum exhibited a distinct green fluorescent signal in mouse brain tissue, indicating the presence of autoimmune antibodies in the serum. The blue fluorescent signal represents the cell nucleus, and the colocalization of the fluorescent signal suggests that the autoimmune antibodies target the cytoplasm or cell membrane antigens of brain cells.

[0066] Example 2: Immunoprecipitation of mouse brain tissue

[0067] Dissociated tissue:

[0068] Mouse brain tissue was collected and washed twice with PBS solution on ice. Then, lysis buffer (500 μL GPCR extraction buffer, 10 μL PMSF solution, 500 μL RIPA solution) was added. The tissue was minced using ophthalmic scissors and sonicated (lysis conditions: 10% power, 30 seconds of lysis, 30 seconds of pause, 5 minutes of sonication). The lysate was then lysed on ice for 30 minutes. The obtained lysate was centrifuged at 13,000 rpm for 20 minutes at 4°C and the supernatant was collected into a new centrifuge tube.

[0069] Preparation of antigen-antibody complexes:

[0070] 1 μL of serum from each patient was diluted to 1:200 with RIPA, and 100 μL of the brain tissue lysis buffer obtained in step 1 was added. The mixture was incubated at 4°C for 40 minutes by rotation. Subsequently, 50 μL of protein A / G magnetic beads (purchased from MCE and used according to instructions), balanced with non-denaturing lysis buffer and RIPA, were incubated overnight at 4°C by rotation to capture antigen-antibody complexes. The next day, the magnetic beads were washed three times each with RIPA and PBST, and then eluted using elution buffer (20 μL of pH 2.6 glycine-HCl buffer and 10 μL of pH 7.4 1M Tris-HCl) on a magnetic rack. 1×SDS-PAGE loading buffer was added to the eluted supernatant, and the sample was heated at 100°C to obtain the patient's antigen-antibody complex. The preparation method for antigen-antibody complexes in healthy individuals was the same as that for patients.

[0071] Mass spectrometry detection:

[0072] The prepared antigen-antibody complex was subjected to SDS-PAGE gel electrophoresis and then silver stained. The results are as follows: Figure 2 As shown, no specific bands were observed in the patient's serum compared to healthy serum. Therefore, the entire gel was sent to a mass spectrometry company (Zhongke Xinsheng) for sequencing identification. The mass spectrometry results showed that, compared with healthy individuals, the patient's gel lanes contained SLC17A7 protein, indicating the presence of SLC17A7 autoantibodies in the patient's serum.

[0073] Example 3: Co-localization of TBA serum antibodies based on mouse brain tissue

[0074] Mouse brain tissue sections prepared in step 1 of Example 1 were incubated with anti-SLC17A7 antibody diluted 1:200 (purchased from ProteinTech), followed by incubation with Alexa Fluor 647-labeled fluorescent secondary antibody diluted 1:1000. The sections were observed and photographed under a fluorescence microscope, following the same procedures as in Example 1. The results were compared with those of the immunofluorescence-stained tissue sections from Example 1. Figure 3-4 As shown, the red fluorescent signal represents the SLC17A7 protein in the tissue, and its location is highly consistent with the green fluorescent signals of patient 1 serum, patient 2 serum, patient 3 serum and patient 4 serum.

[0075] Example 4: Detection of SLC17A7 autoantibodies using immunofluorescence (CBA) staining based on 293T cells

[0076] Construction of 293T cells overexpressing SLC17A7:

[0077] The SLC17A7 gene sequence (NM_020309.4) was obtained from NCB I, as shown in SEQ ID NO.1. The recombinant expression vector N1-mCherry-SLC17A7 was synthesized by General Biotech using N1-mCherry as the vector. The nucleotide sequence of the recombinant expression vector is shown in SEQ ID NO.3. After confirming the sequence was correct, the constructed recombinant vector was extracted for later use. The recombinant expression vector was transfected into 293T cells using Lipo2000 transfection reagent (purchased from Biosharp, transfection was performed according to the instructions). The cells were cultured in poly-L-lysine-coated culture plates.

[0078] The specific sequence of SEQ ID NO.3 of this invention is as follows:

[0079] SEQ ID NO.3:

[0080]

[0081] 2. Preparation of cell slides:

[0082] The 293T cells overexpressing SLC17A7 constructed in step 1 were cultured to a density of 80%–90%, and washed twice with PBS for 5 minutes each time at room temperature. They were then fixed with 4% paraformaldehyde for 10 minutes at room temperature, followed by washing three times with PBS for 5 minutes each time. The cells were then rinsed with PBS containing 0.2% triton X100 for 8 minutes. Finally, the cells were blocked with 10% goat serum at 37°C for 30 minutes to obtain blocked cell slides.

[0083] 3. Incubation of primary antibody:

[0084] Take the cell slides blocked in step 2 and incubate them separately with serum from TBA-positive patients and healthy individuals diluted 1:10 with 10% goat serum at a constant temperature of 37°C for 1 hour. Then wash three times with PBS for 10 minutes each time. This yields the cell slides incubated with the primary antibody.

[0085] 4. Incubation of secondary antibodies:

[0086] Cell slides incubated with Alexa Fluor 488-labeled goat anti-human IgG diluted 1:1000 with 10% goat serum were incubated at room temperature in the dark for 1 hour. The slides were then washed three times with PBS for 10 minutes each time.

[0087] 5. Detect fluorescence signal:

[0088] Observe and photograph under a fluorescence microscope. Results are as follows: Figure 5 , Figure 6 As shown, red fluorescent signals indicate cells overexpressing SLC17A7, green fluorescent signals indicate positive reactions in serum / cerebrospinal fluid, and co-localization of red and green fluorescent signals suggests the presence of SLC17A7 autoantibodies in the patient's serum / cerebrospinal fluid.

[0089] Based on the results of the above embodiments, it can be inferred that SLC17A7 autoantibody can be used as a biomarker for detecting neurological diseases and applied to kits for detecting SLC17A7 autoantibody. Screening for neurological diseases can be achieved by detecting SLC17A7 autoantibody.

[0090] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. Application of reagents for detecting SLC17A7 autoantibodies in the preparation of products for diagnosing neurological diseases.

2. The application as described in claim 1, characterized in that: The reagent for detecting SLC17A7 autoantibodies includes one or more of the following: cells expressing SLC17A7 protein, tissues expressing SLC17A7 protein, and lysates containing SLC17A7 protein.

3. The application as described in claim 2, characterized in that: The SLC17A7 protein includes one or more of a) to d): a) SLC17A7 protein in cell lysis products; b) SLC17A7 protein in the brain or brain nerve tissue; c) The SLC17A7 protein whose amino acid sequence is encoded by the nucleotide sequence shown in SEQ ID NO.1; d) The SLC17A7 protein encoded by the amino acid sequence shown in SEQ ID NO.

2.

4. The application as described in claim 1, characterized in that: The symptoms of the neurological disorders include one or more of the following: limb twitching, abnormal muscle tone, loss of consciousness, cognitive impairment, involuntary movements, and autonomic dysfunction.

5. The application as described in claim 1, characterized in that: The test samples for the neurological diseases are individual biological samples, including serum or cerebrospinal fluid.

6. A reagent kit for diagnosing neurological diseases, characterized in that, This includes reagents for detecting SLC17A7 autoantibodies.

7. The kit as described in claim 6, characterized in that: The reagent for detecting SLC17A7 autoantibodies includes one or more of the following: cells expressing SLC17A7 protein, tissues expressing SLC17A7 protein, and lysates containing SLC17A7 protein.

8. The kit according to claim 7, characterized in that: The SLC17A7 protein includes one or more of a) to d): a) SLC17A7 protein in cell lysis products; b) SLC17A7 protein in the brain or brain nerve tissue; c) The SLC17A7 protein whose amino acid sequence is encoded by the nucleotide sequence shown in SEQ ID NO.1; d) The SLC17A7 protein encoded by the amino acid sequence shown in SEQ ID NO.2.

Images