Application of reagent for detecting anti-ap2a1 autoantibody in assisting diagnosis of nervous system autoimmune disease, kit

Abstract

The application discloses a kind of reagent for detecting anti-AP2A1 autoantibody in the application of assisting in diagnosing nervous system autoimmune disease, kit, belong to protein diagnostic reagent preparation technical field. Including: the application of reagent for detecting anti-AP2A1 autoantibody in the preparation of product for assisting in diagnosing nervous system autoimmune disease. The application discloses that anti-AP2A1 autoantibody is closely related to nervous system autoimmune disease for the first time, can distinguish nervous system autoimmune disease and other autoimmune diseases, especially distinguish autoimmune encephalitis and other autoimmune diseases, realizes the auxiliary diagnosis of nervous system autoimmune disease, and improves the accuracy of diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of protein diagnostic reagent preparation technology, specifically relating to the application of reagents for detecting anti-AP2A1 autoantibodies in the auxiliary diagnosis of autoimmune diseases of the nervous system, and kits. Background Technology

[0002] Neurological autoimmune diseases constitute a significant class of conditions in neurology, affecting the central nervous system, peripheral nervous system, and neuromuscular junctions. Their pathological mechanism primarily involves autoimmune factors and antibodies attacking the nervous system. Neurological autoimmune diseases are relatively rare, exhibiting the complexity of immune disorders and the high lethality and disabling potential of neurological diseases, thus attracting considerable attention from clinicians and researchers. While progress has been made in identifying autoantibody-mediated immune mechanisms in a large proportion of patients with neurological disorders, specific "neurological" antibodies remain undetectable in some patient samples, and a number of cases with unknown etiologies remain undiagnosed even after extensive evaluation of infectious causes. Further elucidation of the immune mechanisms in seronegative patients holds promise for opening new treatment options for these patients. Therefore, the identification of neoantigens that bind to autoantibodies is necessary to improve diagnostic and therapeutic outcomes.

[0003] AP2A1 (Adaptor Related Protein Complex 2 Subunit Alpha 1) is a core component of the AP-2 adaptor complex, a key component in clathrin-mediated endocytosis. As its α1 subunit, it plays an indispensable role in vesicle formation, membrane receptor recognition, and signal transduction coordination. The AP-2 complex is a heterotetramer composed of four subunits: α, β2, μ2, and σ2. The α1 subunit encoded by AP2A1 not only constitutes the structural backbone of the complex but also directly participates in regulating its conformational activation state and membrane binding capacity. The structural characteristics of AP2A1 determine its dual function in the AP-2 complex: on the one hand, assisting in the assembly and bending of clathrin cages; on the other hand, maintaining the integrity of the entire complex. As the α1 subunit of the AP-2 adaptor complex in clathrin-mediated endocytosis, AP2A1 is widely expressed in various tissues and cell types. Early studies have confirmed that AP2A1 is basally expressed in cells including the brain, liver, kidney, lung, and various epithelial and mesenchymal-derived cells, suggesting its universal importance in maintaining basic cell membrane transport functions. AP2A1 is not only the structural core of the AP-2 complex, but also a multifunctional hub integrating lipid signaling, cargo recognition, clathrin recruitment and conformational regulation. Its functional integrity is crucial for maintaining cellular endocytic homeostasis.

[0004] Autoantibodies are antibodies produced by the body against its own tissues or cellular components, playing a crucial role in the development and progression of various diseases, especially autoimmune diseases, tumors, and neurodegenerative diseases. Accurate detection and in-depth research of autoantibodies are irreplaceable for early disease diagnosis, disease monitoring, treatment planning, and elucidation of pathogenesis.

[0005] Currently, there are no reports on the correlation between anti-AP2A1 autoantibodies and neurological autoimmune diseases, and there is also a lack of technical solutions for using this antibody to diagnose related diseases. Summary of the Invention

[0006] The purpose of this invention is to provide a reagent for detecting anti-AP2A1 autoantibodies in the preparation of products for the auxiliary diagnosis of neurological autoimmune diseases. By detecting anti-AP2A1 autoantibodies, the accuracy of diagnosis of neurological autoimmune diseases can be improved, and specific auxiliary diagnosis of such diseases can be achieved. To achieve the above objectives, the present invention employs the following technical solution: This invention discloses the application of reagents for detecting anti-AP2A1 autoantibodies in the preparation of products for the auxiliary diagnosis of autoimmune diseases of the nervous system.

[0007] Preferably, in this invention, the reagent for detecting anti-AP2A1 autoantibodies preferably includes one or more of AP2A1 protein, cells expressing AP2A1 protein, vectors expressing AP2A1 protein, and tissues containing AP2A1 protein, and more preferably AP2A1 protein.

[0008] More preferably, the AP2A1 protein of the present invention refers to an immunogenic polypeptide capable of binding to AP2A1 autoantibodies. The polypeptide is preferably a polymer of ≥2 amino acids, more preferably a polymer of 2-70 amino acids, and even more preferably a polymer of 2, 3, 4, 5, 6, 7, 8, 10, 12, 20, 30, 40, 50, 60, or 70 amino acids. The polypeptide of the present invention preferably contains one or more epitopes derived from the AP2A1 protein.

[0009] More preferably, the polypeptide of the present invention further includes a fusion protein incorporating AP2A1 and other amino acids, wherein the amino acids are preferably linked to the N-terminus or C-terminus and have the function of promoting the purification, immobilization, precipitation or identification of the polypeptide or protein. The amino acids can constitute tags known in the art, such as His tags, thioredoxin, maltose-binding protein, glutathione S-transferase, flag tags, myc tags or strep tags.

[0010] In this invention, there is no particular limitation on the source of the polypeptide; recombinant polypeptides and / or purified polypeptides may be used. The AP2A1 protein in this invention is preferably an immobilized protein, and more preferably immobilized on a solid support.

[0011] Preferably, the amino acid sequence of the AP2A1 protein includes any one of a) to c): a) The amino acid sequence shown in SEQ ID NO.1; b) The amino acid sequence shown in SEQ ID NO.1, after modification or mutation, can recognize the amino acid sequence of anti-AP2A1 autoantibody; c) The amino acid sequence is ≥70% and <100% identical to the amino acid sequence in a) or b), and is capable of recognizing the amino acid sequence of anti-AP2A1 autoantibody.

[0012] More preferably, in b), the amino acid sequence shown in SEQ ID NO.1 is modified or mutated to recognize the amino acid sequence of the anti-AP2A1 autoantibody as shown in SEQ ID NO.3.

[0013] More preferably, in this invention, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1 preferably includes any one of I) to III): I) the nucleotide sequence shown in SEQ ID NO.2; II) a nucleotide sequence that, after mutation of the nucleotide sequence shown in SEQ ID NO.2, can encode an amino acid sequence that recognizes an anti-AP2A1 autoantibody; III) a nucleotide sequence that has ≥70% and <100% identity with the nucleotide sequence in I) or II) and can encode an amino acid sequence that recognizes an anti-AP2A1 autoantibody.

[0014] The sequence represented by SEQ ID NO.1 is as follows: MPAVSKGDGMRGLAVFISDIRNCKSKEAEIKRINKELANIRSKFKGDKALDGYSKKKYVCKLLFIFLLGHDIDFGHMEAVNLLSSNKYTEKQIGYLFISVLVNSNSELIRLINNAIKNDLASRNPTFMCLALHCIANVGSREMGEAFAADIPRILVAGDSMDSVKQSAALCLLRLYKASPDLVPMGEWTARVVHLLNDQHMGVVTAAVSLITCLCKKNPDDFKTCVSLAVSRLSRIVSSASTDLQDYTYYFVPAPWLSVKLLRLLQCYPPPEDAAVKGRLVECLETVLNKAQEPPKSKKVQHSNAKNAILFETISLIIHYDSEPNLLVRACNQLGQFLQHRETNLRYLALESMCTLASSEFSHEAVKTHIDTVINALKTERDVSVRQRAADLLYAMCDRSNAKQIVSEMLRYLETADYAIREEIVLKVAILAEKYAVDYSWYVDTILNLIRIAGDYVSEEVWYRVLQIVTNRDDVQGYAAKTVFEALQAPACHENMVKVGGYILGEFGNLIAGDPRSSPPVQFSLLHSKFHLCSVATRALLLSTYIKFINLFPETKATIQGVLRAGSQLRNADVELQQRAVEYLTLSSVASTDVLATVLEEMPPFPERESSILAKLKRKKGPGAGSALDDGRRDPSSNDINGGMEPTPSTVSTPSPSADLLGLRAAPPPAAPPASAGAGNLLVDVFDGPAAQPSLGPTPEEAFLSPGPEDIGPPIPEADELLNKFVCKNNGVLFENQLLQIGVKSEFRQNLGRMYLFYGNKTSVQFQNFSPTVVHPGDLQTQLAVQTKRVAAQVDGGAQVQQVLNIECLRDFLTPPLLSVRFRYGGAPQALTLKLPVTINKFFQPTEMAAQDFFQRWKQLSLPQQEAQKIFKANHPMDAEVTKAKLLGFGSALLDNVDPNPENFVGAGIIQTKALQVGCLLRLEPNAQAQMYRLTLRTSKEPVSRHLCELLAQQF.

[0015] The sequence set forth in SEQ ID NO.2 is as follows:

[0016] The sequence set forth in SEQ ID NO.3 is as follows: MPAVSKGDGMRGLAVFISDIRNCKSKEAEIKRINKELANIRSKFKGDKALDGYSKKKYVCKLLFIFLLGHDIDFGHMEAVNLLSSNKYTEKQIGYLFISVLVNSNSELIRLINNAIKNDLASRNPTFMCLALHCIANVGSREMGEAFAADIPRILVAGDSMDSVKQSAALCLLRLYKASPDLVPMGEWTARVVHLLNDQHMGVVTAAVSLITCLCKKNPDDFKTCVSLAVSRLSRIVSSASTDLQDYTYYFVPAPWLSVKLLRLLQCYPPPEDAAVKGRLVECLETVLNKAQEPPKSKKVQHSNAKNAILFETISLIIHYDSEPNLLVRACNQLGQFLQHRETNLRYLALESMCTLASSEFSHEAVKTHIDTVINALKTERDVSVRQRAADLLYAMCDRSNAKQIVSEMLRYLETADYAIREEIVLKVAILAEKYAVDYSWYVDTILNLIRIAGDYVSEEVWYRVLQIVTNRDDVQGYAAKTVFEALQAPACHENMVKVGGYILGEFGNLIAGDPRSSPPVQFSLLHSKFHLCSVATRALLLSTYIKFINLFPETKATIQGVLRAGSQLRNADVELQQRAVEYLTLSSVASTDVLATVLEEMPPFPERESSILAKLGNLLVDVFDGPAAQPSLGPTPEEAFLSPGPEDIGPPIPEADELLNKFVCKNNGVLFENQLLQIGVKSEFRQNLGRMYLFYGNKTSVQFQNFSPTVVHPGDLQTQLAVQTKRVAAQVDGGAQVQQVLNIECLRDFLTPPLLSVRFRYGGAPQALTLKLPVTINKFFQPTEMAAQDFFQRWKQLSLPQQEAQKIFKANHPMDAEVTKAKLLGFGSALLDNVDPNPENFVGAGIIQTKALQVGCLLRLEPNAQAQMYRLTLRTSKEPVSRHLCELLAQQF.

[0017] Preferably, in this invention, the cells preferably include prokaryotic cells or eukaryotic cells; the prokaryotic cells preferably include Escherichia coli cells; the eukaryotic cells preferably include immortalized human cells, insect cells, or yeast. In specific implementations of this invention, selectable cells may include HEK293 cells, HeLa cells, CHO, Pichia pastoris, Saccharomyces cerevisiae, sf9, BL21, or Rosetta, etc.

[0018] Preferably, the vectors described in this invention include vectors from the pTriEx vector family, pcDNA3 family, pET series, or pBac series. The tissues described in this invention are preferably brain tissue, and more preferably brain tissue from humans, rats, primates, mice, goats, horses, sheep, or cattle.

[0019] This invention screens serum samples from subjects exhibiting clinical symptoms related to neurological autoimmune diseases for antibodies. Primary neurons and rat brain tissue sections were incubated with patient serum and healthy human serum. By amplifying the signal with a fluorescent secondary antibody, an unknown fluorescent signal was found in patient serum after incubation with primary neurons and rat brain tissue sections, compared to healthy human serum. A biodetector chip was used to screen for this unknown fluorescent signal, identifying it as the fluorescence signal generated by autoantibodies in the patient serum recognizing the AP2A1 antigen. Furthermore, the authenticity of the target antigen AP2A1 protein was verified by co-staining commercially available antibodies with patient serum on neurons and AP2A1-overexpressing cells, as well as by serum neutralization experiments. More serum samples from patients with or without neurological diseases and healthy individuals were collected as control samples and screened using a cytokine assay (CBA) to verify the specificity of the target antibody.

[0020] In this invention, the symptoms of the autoimmune disease of the nervous system preferably include one or more of the following: memory dysfunction, epileptic seizures, mental and behavioral abnormalities, impaired consciousness, and speech disorders.

[0021] Preferably, the neurological autoimmune disease described in this invention is autoimmune encephalitis; the patient with symptoms of neurological autoimmune disease is preferably a patient to whom a physician highly suspects autoimmune encephalitis, and the serum sample tests for autoimmune encephalitis-related autoantibodies (NMDAR, AMPA1, AMPA2, LGI1, CASPR2, GABABR, DPPX, IgLON5, GlyRα1, GABAARα1, GABAARβ3, GABAARγ2) are all negative.

[0022] In this invention, the diagnosis is an auxiliary diagnosis. Anti-AP2A1 autoantibodies are closely related to autoimmune diseases of the nervous system and can distinguish between autoimmune diseases of the nervous system and other autoimmune diseases, especially autoimmune encephalitis and other autoimmune diseases. In the field of autoimmune encephalitis detection, the diagnostic criteria for autoimmune encephalitis usually include four aspects: clinical manifestations, auxiliary examinations, confirmatory tests, and exclusion of other causes. Among them, when the patient's clinical manifestations and auxiliary examinations are consistent with autoimmune encephalitis and other causes are excluded, the diagnosis is antibody-negative autoimmune encephalitis. When the patient's clinical manifestations and auxiliary examinations are consistent with autoimmune encephalitis and other causes are excluded, and the autoimmune encephalitis-related marker (anti-neuronal cell antibody) is positive, the diagnosis is confirmed autoimmune encephalitis.

[0023] This invention uses anti-AP2A1 autoantibodies as biomarkers for the diagnosis of autoimmune encephalitis. Anti-AP2A1 autoantibodies are closely related to autoimmune diseases of the nervous system, especially autoimmune encephalitis, and can be used as an auxiliary diagnostic tool for autoimmune encephalitis. By detecting anti-AP2A1 autoantibodies, doctors can combine the results with the patient's clinical manifestations, auxiliary examinations, and disease biomarker detection results to determine whether the patient has the autoimmune disease of the nervous system described in this invention or to rule out the possibility of another autoimmune disease of the nervous system. This helps doctors select a more promising treatment plan or drug for the patient.

[0024] The present invention also provides a kit for the auxiliary diagnosis of autoimmune diseases of the nervous system, including reagents for detecting anti-AP2A1 autoantibodies.

[0025] As one implementation method, the kit described in this invention can be a kit based on enzyme-linked immunosorbent assay (ELISA) or a kit based on Western blotting. Preferably, the kit also includes one or more of labeled antibodies, reaction buffers, and sample diluents. This invention does not impose strict requirements on the specific composition of the labeled antibodies, buffers, and sample diluents; conventional selections are acceptable.

[0026] This invention establishes a kit for the auxiliary diagnosis of autoimmune diseases of the nervous system, using reagents for detecting anti-AP2A1 autoantibodies as the main component. This kit can qualitatively or quantitatively analyze anti-AP2A1 autoantibodies, thereby diagnosing autoimmune diseases of the nervous system, and is particularly useful for auxiliary diagnosis. When using the kit for detection, there are no strict requirements on the detection method used; any method well-known in the art can be used, such as CBA, TBA, ELISA, immunogold immunoassay, Western blotting, immunospotting, chemiluminescence, radioimmunoassay, liquid chromatography-array, lateral chromatography, or flow cytometry.

[0027] Compared with the prior art, the present invention has the following beneficial effects: This invention, by comparing the serum of patients with symptoms of neurological autoimmune diseases with that of healthy individuals, yielded the following results: Compared to healthy serum, the serum of patients with symptoms of neurological autoimmune diseases contained anti-AP2A1 autoantibodies. Extensive experimental verification confirmed that anti-AP2A1 autoantibodies can serve as a biomarker for the auxiliary diagnosis of neurological autoimmune diseases, particularly as an anti-neuronal cell antibody for the auxiliary diagnosis of these diseases. Detection of anti-AP2A1 autoantibodies can facilitate the auxiliary diagnosis of neurological autoimmune diseases, especially autoimmune encephalitis.

[0028] This invention also provides a kit for the auxiliary diagnosis of autoimmune diseases of the nervous system, including reagents for detecting anti-AP2A1 autoantibodies. This invention is the first to establish a kit for the auxiliary diagnosis of autoimmune diseases of the nervous system using reagents for detecting anti-AP2A1 autoantibodies as the main component. Using this kit, anti-AP2A1 autoantibodies can be qualitatively or quantitatively analyzed; the operation is simple, enabling the auxiliary diagnosis of autoimmune diseases of the nervous system, especially autoimmune encephalitis. Attached Figure Description

[0029] Figure 1 Image showing the staining results of patient serum and healthy human serum on primary neurons; Figure 2 The staining results show the co-localization of patient serum and neuronal marker MAP2 antibody on primary neurons. Figure 3 The image shows the staining results of the patient's serum on primary rat brain tissue; Figure 4 The image shows the co-staining results of patient serum and NCDN antibody on primary rat brain tissue; Figure 5 A CBA result image showing a positive signal generated when the patient's serum reacted with the same antigen AP2A1 on the biodetector chip; Figure 6 The staining results of AP2A1 antibody on rat primary neurons to verify the signal detected by patient serum are shown in the figure. Figure 7 The staining results of AP2A1 antibody on AP2A1-overexpressing cell smears to verify the signal detected in patient serum; Figure 8 Western blot (WB) results for AP2A1 antibody, patient serum, and healthy human serum; Figure 9The staining results of the serum neutralization experiment were obtained by verifying the signals detected in the patient's serum on AP2A1-overexpressing cell smears, rat primary neuron cell smears, and rat brain tissue sections. Figure 10 The staining results are shown in the serum of some patients who are positive for anti-AP2A1 antibodies, the serum of disease controls, and the serum of healthy individuals. Figure 11 This image shows the staining results of a portion of serum on a cell slide overexpressing the recombinant AP2A1 deletion mutant. Detailed Implementation

[0030] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0031] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0032] The present invention will now be described in further detail with reference to the accompanying drawings: The technical terms involved in this invention are defined as follows: The term "diagnosis" described in this invention refers to the process of making a clear determination of a patient's health status and disease-related attributes. It covers situations including: determining whether an individual has a certain disease; identifying the specific type and subtype of the disease suffered by an individual who has already contracted the disease; determining the severity, stage, or grade of the disease; tracing the cause or trigger of the disease; differentiating multiple suspected diseases with overlapping symptoms; further confirming the disease after screening asymptomatic individuals for abnormalities; adjusting the original diagnosis according to changes in the condition during disease treatment or follow-up; or a combination of the above.

[0033] The term "assisted diagnosis" as described in this invention refers to the process of providing objective evidence, supplementary information, or decision-making reference for "diagnosis" through specific methods, technologies, or test results (including but not limited to laboratory testing reagents, imaging techniques, pathological staining techniques, molecular detection methods, etc.). It cannot independently complete a full diagnosis and needs to be combined with core clinical information such as patient medical history and physical examination. The scope includes: in the disease screening stage, initially identifying high-risk individuals who may have the disease; in the disease diagnosis stage, providing key evidence to support "whether the patient has the disease" and "what disease the patient has"; in the disease differential diagnosis stage, excluding or narrowing the range of suspected diseases; in the disease classification and staging stage, assisting in clarifying the specific attributes of the disease; in the disease follow-up or disease monitoring stage, assisting in assessing disease activity, treatment response, or recurrence; in the prognostic assessment stage, assisting in predicting disease outcome or recovery; or a combination of the above. The core objective of "assisted diagnosis" is to reduce diagnostic uncertainty, improve diagnostic accuracy, shorten the diagnostic cycle, and provide reliable support for diagnostic decisions.

[0034] The term "identity" percentage described in this invention refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when two sequences are optimally aligned. The amino acid sequence identity percentage alignment can be performed using various methods within the art, such as software well-known in the field, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA.

[0035] To further illustrate the present invention, the application of the anti-AP2A1 autoantibody provided by the present invention in the preparation of products for diagnosing autoimmune diseases of the nervous system is described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0036] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments: The patient serum described in this invention was donated by the hospital with the consent of the individual. The serum from healthy individuals came from the serum of healthy individuals undergoing physical examinations donated by the hospital's physical examination center with the consent of the individual undergoing the physical examinations.

[0037] Example 1: Identification and Confirmation of Novel Autoantibodies Step 1: Sample Collection Collected data from January 1, 2018 to December 31, 2020, for 12 tests related to autoimmune encephalitis (NMDAR, AMPA1, AMP A2, LGI1, CASPR2, GABA). B R, DPPX, IgLON5, GlyR, GABA A Rα1, GABA ARγ2, GABA A Serum samples from patients who tested negative for Rβ3 and all 12 autoantibody tests were further screened, with the following inclusion criteria: (1) Symptoms of an autoimmune disease of the nervous system, including one or more of the following: memory impairment, seizures, mental and behavioral abnormalities, impaired consciousness, speech disorders, cognitive impairment, motor disorders, brainstem dysfunction, and autonomic dysfunction; (2) At least one inflammatory condition, such as cerebrospinal fluid leukocytosis (>5×10⁻⁶). 6 / L), elevated oligoclonal bands or IgG index in cerebrospinal fluid, MRI showing encephalitis (high signal on brain MRI limited to one or both medial temporal lobes, or involving gray matter, white matter, or both in multifocal areas), brain biopsy showing lymphocytic infiltration; (3) Detection of autoantibodies (NMDAR, AMPA1, AMPA2, LGI1, CASPR2, GABA) associated with autoimmune encephalitis using cell-based immunofluorescence assay (CBA). B R, DPPX, IgLON5, GlyR, GABA A Rα1, GABA A Rγ2, GABA A All Rβ3 were negative; (4) Age ≥ 14 years; (5) Exclude infectious diseases (including viral encephalitis, neurosyphilis, and nervous system infections caused by bacteria, fungi and parasites), metabolic and toxic encephalopathy (including hepatic encephalopathy and pulmonary encephalopathy, toxic encephalopathy caused by antibiotics, chemotherapy drugs or immunosuppressants, radiation encephalopathy), hereditary diseases (mitochondrial encephalopathy, methylmalonic acidemia, adrenoleukodystrophy), and neurodegenerative diseases (Lewy body dementia, frontotemporal dementia, multiple system atrophy and hereditary cerebellar degeneration).

[0038] (6) Expanded antibody profile detection negative: The CBA method was used to detect more reported neurological-related autoantibodies, including paraneoplastic syndrome-related autoantibodies (Hu, Ri, YO, Ma1, Ma2, CV2 / CRMP5, Amphiphysin, PKCγ). Detection of Zic4, SOX1, Tr(DNER), Titin, Recoverin, GAD65; detection of central nervous system demyelination-related autoantibodies (AQP4, MOG, MBP, GFAP, AQP1, Flotillin1 / 2); and 60 other reported nervous system-related autoantibodies (mGluR5, D2R, Neurexin-3α, KCTD16, GAD67, KCNA4 (Kv1.4), KLHL11, AK5, TRIB2, GluR3, Gephyrin, CaVα2δ (CACNA2D1), TPO, TGM2, TGM6, MUNC18-1, mGluR1, GABAARAP, Drebrin, AGO, NAE, PDE10A, ADAM2). 2. The results of tests for the following 80 autoantibodies were all negative: ADAM23, ROCK2, mGluR3, mGluR4, CACNB1, VAMP2, CRMP2, CACNA1A, Homer3, ATP1A3, ARHGAP26, ITPR1, septin-5, NCDN, GRID2, AP3B2, mGluR2, GRIK2 (GluR6 / GluK2), Rab6A, Rab6B, CA8 (CARPVIII), PDE10A, PLP1, NF155, NF186, CNTN1, CNTN2, CASPR1, Gliomedin, Agrin, SYN1, AChR, LRP4, Musk, MAG, neurobeachin, and VGLUT2.

[0039] The methods for detecting 80 known neurological-related autoantibodies in patient serum are as follows: (1) Construction of recombinant vectors: The gene sequences encoding the above 80 proteins were found on NCBI and sent to the sequencing company. They were ligated into pCDNA3.1 to obtain 80 recombinant vectors. After the sequences were confirmed to be correct, they were extracted for later use. (2) Transfection of target genes: 293T cells were cultured in 81 culture dishes with 6cm×6cm spreaders at the bottom. When the cell density reached 60%~80%, the recombinant vectors of 80 corresponding genes and the empty vector pCDNA3.1 were transfected into the 293T cells using PEI transfection reagent and then labeled. (3) Cell crawling sheet fixation: After transfection, culture for 24-48h, wash twice with PBS, fix with acetone for 5min, wash twice with PBS, and after drying, cut the crawling sheet into 2.5mm×2.5mm size, attach 81 kinds of small pieces to a glass slide, and prepare a biodetection chip for later use. (4) Immunofluorescence staining: Patient serum was diluted with PBST at a volume ratio of 1:10 and incubated onto a biodetector chip at room temperature for 1 hour, followed by washing with PBST three times for 5 minutes each time; FITC-labeled goat anti-human IgG antibody diluted 1:200 was added, incubated at room temperature for 30 minutes, followed by washing with PBST three times for 5 minutes each time; the results were observed under a fluorescence microscope. No positive signals were observed in the above tests in all serum samples from the included patients (i.e., all 80 known autoantibodies were negative).

[0040] Patients who did not meet the inclusion criteria were excluded, resulting in data from 373 patients, randomly assigned to numbers H1-H373. Serum samples were then randomly collected from 20 healthy individuals, assigned to numbers J1-J20.

[0041] Step 2: Screening for fluorescence signals in primary neurons from patient serum (1) Isolation of primary rat neurons: Rats were anesthetized with 10% chloral hydrate at a dose of 3 mL / kg body weight. After anesthesia, the rat brain tissue was disinfected by soaking in 75% alcohol and removed in a biosafety cabinet. The brain tissue was placed in pre-cooled DMEM containing 1% BSA and cut into a paste. 10 mL of papain was added and digested at 37°C for 30 minutes. After digestion, the cells were transferred to a new centrifuge tube, resuspended in 10 mL of DMEM containing 1% BSA, centrifuged at 400g and 4°C for 5 minutes, and the supernatant was discarded. The precipitate was resuspended in 5 mL of DMEM containing 1% BSA, centrifuged at 200g and 4°C for 5 minutes, and the supernatant was discarded again. The cells were resuspended in 20 mL of DMEM medium and seeded at an appropriate density in a culture dish containing cell spreaders to obtain primary rat brain neurons.

[0042] (2) Fixation of cell crawling sheets: Take the cultured rat primary neuron cell crawling sheets, fix them with 4% paraformaldehyde (FPA) at room temperature for 10 minutes, wash twice with PBS, add 1.25M glycine to terminate the fixation for 10 minutes, wash twice with PBS to obtain rat primary neuron cell crawling sheets.

[0043] (3) Serum incubation: Serum from 373 patients and 20 healthy individuals was diluted with PBST at a volume ratio of 1:10 and incubated onto rat primary neuronal cell smears. The cells were incubated at room temperature for 1 hour, washed three times with PBST for 5 minutes each time. Then, FITC-labeled goat anti-human IgG antibody diluted 1:200 was added, and the cells were incubated at room temperature for 30 minutes, washed three times with PBST for 5 minutes each time. DAPI was used to stain the cell nuclei at room temperature for 10 minutes, followed by three washes with PBST for 5 minutes each time. Serum-incubated cell smears were obtained, and the results were observed and photographed under a fluorescence microscope. The results showed that 152 of the 373 patients' serum samples showed positive signals on rat primary neuronal cells, while 221 patients' serum samples and 20 healthy individuals' samples did not show positive signals. This indicates that the serum from 152 patients contained novel autoantibodies that bind to antigens in rat primary neuronal cells, unlike previously reported autoantibodies that can recognize neurons. Some staining results are shown below. Figure 1 As shown.

[0044] (4) Antibody colocalization The neuronal cell-specific cytoskeleton marker MAP2 antibody was diluted 1:500 (v / v) using PBST and incubated with patient serum-incubated cell smears for antibody co-staining. The cells were incubated overnight at 4°C, washed three times with PBST for 5 minutes each time. Then, Alexa Fluor 594-labeled goat anti-mouse IgG antibody (1:200 dilution) was added, and the cells were incubated at room temperature for 30 minutes, washed three times with PBST for 5 minutes each time. The resulting antibody-co-stained cell smears were observed and photographed under a fluorescence microscope. The results showed that the signal stained by the patient serum overlapped with the signal of the MAP2 antibody on primary neurons, indicating that the antigen recognized by the antibody in the patient serum is expressed on neurons. Some staining results are shown below. Figure 2 As shown.

[0045] Step 3: Screening for fluorescence signals in rat brain tissue from patient serum (1) Preparation of frozen sections of rat brain tissue: Adult rats were anesthetized, and after the limbs were stiffened, the abdominal cavity was opened to expose the apex of the heart. PBS was perfused from the left apex of the heart. The brain tissue was removed and fixed with methanol for 10-30 min. It was then transferred to 30 wt.% sucrose solution and dehydrated at 4°C until the tissue block settled. After OCT embedding, it was sectioned by a cryostat at -20°C to obtain frozen sections of rat brain tissue.

[0046] (2) Serum incubation: The serum of 152 patients and 5 healthy individuals who showed positive signals on primary rat neurons were diluted with PBST at a volume ratio of 1:10 and incubated on frozen sections of rat brain tissue at room temperature for 1 hour. The sections were washed with PBST 3 times for 5 minutes each time. FITC-labeled goat anti-human IgG antibody diluted 1:200 was added and incubated at room temperature for 30 minutes. The sections were washed with PBST 3 times for 5 minutes each time. The results were observed under a fluorescence microscope and photographed.

[0047] The results showed that among the 152 patients with positive signals on primary rat neurons, 6 patients' serum also showed positive signals in the cerebellar molecular layer, granular layer, Purkinje cell layer, and white matter in frozen sections of rat brain tissue, as well as in the hippocampus and midbrain regions. One healthy human sample showed no positive signal. This indicates that autoantibodies specifically binding to rat brain tissue proteins were indeed present in the serum of these 6 patients. Some staining results are shown below. Figure 3 As shown.

[0048] (4) Antibody colocalization Antibody against the neuronal cell-specific protein NCDN was diluted 1:500 (v / v) with PBST and incubated with patient serum-incubated tissue sections for antibody co-staining. The incubation was performed overnight at 4°C, followed by three washes with PBST for 5 minutes each. Then, Alexa Fluor 594-labeled goat anti-mouse IgG antibody (1:200 dilution) was added, and the sections were incubated at room temperature for 30 minutes, followed by three washes with PBST for 5 minutes each. The resulting co-stained tissue sections were observed and photographed under a fluorescence microscope. The results showed that the staining signal from the patient serum overlapped with the signal from the NCDN antibody in rat brain tissue sections. This suggests that the antigen recognized by the abnormal autoantibody in the patient is expressed on neurons in brain tissue. Some staining results are shown below. Figure 4 As shown.

[0049] Step 4: Screening of target antigens To further explore whether the serum of the 152 patients who had been excluded from having known neurological-related autoantibodies (all 80 antibodies detected by the CBA method were negative) and who showed positive reactions on primary neurons contained novel autoantibodies that could specifically recognize neurons, 100 proteins with relatively high expression levels in the human brain were identified from the human protein atlas (https: / / www.proteinatlas.org / ). A biodetector chip was prepared, and the immunofluorescence assay was used to detect whether the patient serum could generate an immune response after incubation with the biodetector chip. The specific steps are as follows: (1) Preparation of biodetection chip: The gene sequences encoding each protein were found from NCBI and sent to a sequencing company to synthesize the genes into pCDNA3.1. The constructed recombinant vector was sequenced and then extracted for later use. The above recombinant vector was transfected into 293T cells grown on 6cm×6cm slides using PEI transfection reagent. After 24-48h of transfection, the cells were washed, fixed and dried. The cell slides were cut into 2.5mm×2.5mm pieces and attached to a glass slide in sequence to prepare a biodetection chip for later use.

[0050] (2) Immunofluorescence staining: Serum from 152 patients and 20 healthy individuals was diluted with PBST at a volume ratio of 1:10 and incubated onto a biodetector chip. The chips were incubated at room temperature for 1 hour, followed by washing with PBST three times for 5 minutes each time. FITC-labeled goat anti-human IgG antibody diluted 1:200 was added, and the chips were incubated at room temperature for 30 minutes, followed by washing with PBST three times for 5 minutes each time. The results were observed under a fluorescence microscope and photographed. The results are as follows: Figure 5 As shown.

[0051] The results showed that the serum of 7 out of 152 patients (numbered H47, H53, H95, H118, H172, H276, and H315) showed a significantly stronger positive signal than that of healthy serum for the same antigen on the biodetection chip. Further investigation revealed that the target antigen recognized by the serum of these 7 patients was AP2A1 protein (adaptor-related protein complex 2-subunit alpha 1, accession number: NM_130787.3), with the amino acid sequence shown in SEQ ID NO.1 and the nucleotide sequence encoding this protein shown in SEQ ID NO.2. In addition, the serum of 10 patients showed significantly stronger positive signals than those of healthy individuals for seven different antigens (the serum of patients H14 and H35 recognized antigen 1; the serum of patients H28 and H76 recognized antigen 2; the serum of patients H58 and H81 recognized antigen 3; and the serum of patients H24, H69, H153, and H215 recognized antigens 4, 5, 6, and 7, respectively). The serum of the other 135 patients and the serum of 20 healthy individuals did not produce signals with any of the antigens on the biodetection chip.

[0052] Step 5: Organize clinical information from patient samples The clinical information of the above 7 patients who were positive for anti-AP2A1 autoantibodies was compiled and is shown in Table 1.

[0053] Table 1. Clinical information of patients H47, H53, H95, H118, H172, H276, and H315

[0054] Conclusion: In this study, immunofluorescence staining of primary rat neurons and staining of rat brain tissue sections were performed. The results showed that specific positive fluorescent signals were detected in the serum of the tested patients in both primary rat neurons and rat brain tissue sections, while no specific fluorescent expression was observed in the serum of healthy controls. This suggests the presence of unknown autoantibodies targeting neurons in the serum of the patients. Target antigen screening and identification confirmed the presence of a novel anti-AP2A1 autoantibody in the serum of 7 enrolled patients, with the AP2A1 protein as its target antigen.

[0055] Example 2: Validation of AP2A1 antibody in patient serum 1. Verification of serum staining signals on primary rat neurons using commercial AP2A1 antibody. Preparation of rat primary neuronal cell crawling slides: Following step 2 of Example 1, rat primary neuronal cell crawling slides were obtained. Serum from seven patients (numbered H47, H53, H95, H118, H172, H276, and H315) was incubated and co-stained with a commercially available AP2A1 antibody. The slides were observed and photographed under a fluorescence microscope. Results showed that the staining signals from the serum of the seven patients (numbered H47, H53, H95, H118, H172, H276, and H315) on the rat primary neurons overlapped with the staining signals from the commercially available AP2A1 antibody. This indicates the presence of autoantibodies in the serum of the seven patients (numbered H47, H53, H95, H118, H172, H276, and H315) that can recognize the AP2A1 protein, a target antigen on primary neurons. Some staining results are shown below. Figure 6 As shown.

[0056] 2. Verification of staining signal of serum on overexpressing cell smears using commercial AP2A1 antibody. Following the procedure in step 1(6) of Example 1, AP2A1 overexpressing cell smears and serum samples from 7 patients (numbered H47, H53, H95, H118, H172, H276, and H315) were prepared and co-stained with commercial AP2A1 antibody. The samples were observed and photographed under a fluorescence microscope. The results showed that the commercial antibody showed a positive signal on the AP2A1 overexpressing cell smears, indicating successful overexpression of AP2A1 protein on 293T cells. The staining signals from the serum samples of the 7 patients (numbered H47, H53, H95, H118, H172, H276, and H315) overlapped with those from the commercial AP2A1 antibody on the AP2A1 overexpressing cell smears, indicating the presence of autoantibodies in the serum samples of the 7 patients (numbered H47, H53, H95, H118, H172, H276, and H315) that can recognize the target antigen AP2A1 protein on the AP2A1 overexpressing cell smears. Some results are shown below. Figure 7 As shown.

[0057] 3. Verification of Western blot signal in AP2A1 overexpressed protein using commercial AP2A1 antibody (1) Preparation of protein samples: Overexpression of AP2A1 protein: When the cultured 293T cells reached a density of 30%–40%, the recombinant vector from step 4 of Example 1 was transfected using PEI transfection reagent. AP2A1- pCDNA3.1 and empty pCDNA3.1 were transfected into 293T cells and labeled. After 24-48 hours, one dish of 293T cells overexpressing AP2A1 was collected, the supernatant was discarded, and the cells were scraped into a 1.5mL centrifuge tube using a cell scraper. The cells were centrifuged at 800rpm at room temperature to remove the supernatant, and 200μL of PBS was added. The cells were then sonicated (10% power, 3s disruption, 6s pause, 1 minute total) to obtain AP2A1 overexpressing protein. One dish of cells transfected with empty pCDNA3.1 was used to prepare pCDNA3.1 control protein, using the same conditions and methods as the AP2A1 overexpressing protein preparation.

[0058] Rat brain tissue protein: 50 mg of whole rat brain tissue and cerebellum tissue were minced and transferred to a mortar. Liquid nitrogen was added and the mixture was cryogenically ground until a fine powder was obtained. 500 μL of RIPA lysis buffer (composed of 150 mM NaCl, 1 mM EDTA, 100 mM Tris-HCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, and 5% glycerol, pH 7.5) was added to each sample. A 1× protease inhibitor was added, and the mixture was sonicated (10% power, 3 s lysis, 6 s pause, 1 min total sonication). After sonication, the mixture was lysed on ice for 30 min, with shaking every 5 min to obtain whole rat brain tissue protein and cerebellum tissue protein. Five dishes of primary neurons were collected, 200 μL of PBS was added, and the mixture was sonicated (under the same conditions as tissue protein) to obtain primary neuronal protein. All samples were collected and centrifuged at 15000 rpm for 30 min. The supernatant was used to determine the protein concentration using the BCA method.

[0059] (2) Sample loading and transfer: 20 μg each of AP2A1 overexpression protein, pCDNA3.1 control protein, rat whole brain tissue protein, rat cerebellum tissue protein and primary neuron protein were loaded and prepared in two groups. After electrophoresis, the transfer was performed using a wet transfer method at 300 mA for 90 min. 5% skim milk powder was used for blocking at room temperature for 1 h. (3) Antibody incubation and color development: Commercial AP2A1 antibody diluted 1:2000 with TBST, serum from 3 patients (numbered H53, H95, H118) and serum from 3 healthy individuals (numbered J1, J2, J3) diluted 1:200 as primary antibodies, and incubated overnight at 4°C; the next day, washed 3 times with TBST for 5 min each time; added HRP-labeled goat anti-rabbit / human secondary antibody, and incubated at room temperature for 1 h; washed 3 times with TBST for 5 min each time; added After chemiluminescence staining and imaging, the results showed that the commercially available AP2A1 antibody and the serum of three patients all exhibited specific bands (approximately 110 KD) on the immunoblots of AP2A1 overexpressing protein, rat whole brain tissue protein, rat cerebellar tissue protein, and primary neuron protein. However, no signal was observed on the pCDNA3.1 control protein lane. This indicates the presence of autoantibodies capable of recognizing the target antigen AP2A1 protein in the sera of patients H53, H95, and H118. Some results are shown below. Figure 8 As shown.

[0060] 4. Neutralization test to verify the signal detected in the patient's serum (1) Preparation of neutralizing protein: Refer to step 3 of Example 2 to prepare overexpressed AP2A1 protein as neutralizing protein and prepare empty vector pCDNA3.1 protein as control protein.

[0061] (2) Neutralization experiment to verify the signal detected in the patient's serum on the AP2A1 overexpressing cell smear. Refer to step 1 (6) of Example 1 to prepare AP2A1 overexpressing cell smears. Two portions of commercial AP2A1 antibody diluted 1:200 were prepared using PBST. Two portions of patient H47 serum diluted 1:10 were also prepared using PBST. Each portion was 100 μL. 20 μL of AP2A1 neutralizing protein and 20 μL of control protein were added to each portion and incubated at room temperature for 30 minutes. Two portions of patient H53 serum, two portions of patient H95 serum, two portions of patient H118 serum, two portions of patient H172 serum, two portions of patient H276 serum, and two portions of patient H315 serum were prepared in the same way and incubated to the prepared AP2A1 overexpressing cell smears. The cells were incubated at room temperature for 1 hour and washed 3 times with PBST for 5 minutes each time. FITC-labeled goat anti-rabbit IgG antibody diluted 1:200 was added and incubated at room temperature for 30 minutes. The cells were washed 3 times with PBST for 5 minutes each time. The cells were observed under a fluorescence microscope and photographed.

[0062] (3) Neutralization experiment to verify the signal detected in the patient's serum on the primary neuronal cell slice. Following the procedure in step 2 of Example 1, primary neuronal cell crawling slides were prepared. Following the procedure in step 4 (2) of Example 2, the neutralizing protein group and control protein group of the serum of 7 patients (numbered H47, H53, H95, H118, H172, H276, H315) were incubated into AP2A1 overexpressing cell crawling slides, observed under a fluorescence microscope, and photographed.

[0063] (4) Neutralization experiment to verify the signal detected in rat brain tissue sections by patient serum. Following the procedure in Example 1, frozen sections of rat brain tissue were prepared. Following the procedure in step 4(2) of Example 2, the neutralizing protein group and control protein group of serum from 7 patients (numbered H47, H53, H95, H118, H172, H276, H315) were incubated onto rat brain tissue sections, observed under a fluorescence microscope, and photographed.

[0064] The results showed that the signals from commercially available AP2A1 antibody and the serum of seven patients (numbered H47, H53, H95, H118, H172, H276, and H315) were weakened or disappeared after being blocked by neutralizing proteins on AP2A1-overexpressing cell slices, primary neuronal cell slices, and rat brain tissue sections. However, the control protein did not block these signals. This indicates that the signals appearing in the serum of the seven patients (numbered H47, H53, H95, H118, H172, H276, and H315) on the aforementioned AP2A1-overexpressing cell slices, primary neuronal cell slices, and rat brain tissue sections are signals specifically recognizing the AP2A1 antigen. Some results are shown below. Figure 9 As shown.

[0065] Conclusion: This embodiment employed a protocol involving co-labeling and staining of patient serum with commercially available AP2A1 antibodies, as well as a serum neutralization assay, to validate the assays in primary neurons, AP2A1-overexpressing cell smears, and Western blotting (WB). Results showed that the positive signals detected in these experimental systems were generated by the specific binding of autoantibodies in patient serum to the target antigen AP2A1. This confirmed that all seven tested patients' serum contained anti-AP2A1 autoantibodies targeting AP2A1, and that the target antigen of these antibodies was the AP2A1 protein expressed in brain tissue. In summary, this demonstrates that AP2A1 autoantibodies are generated by the body's immune system in response to the AP2A1 protein in neural tissue, thus establishing a specific binding relationship between anti-AP2A1 autoantibodies and the AP2A1 target antigen.

[0066] Example 3: Verification of the clinical specificity and detection rate of anti-AP2A1 autoantibodies using cell-based immunofluorescence assay Step 1: Sample Collection To verify the clinical specificity and detection rate of anti-AP2A1 antibodies, samples submitted between January 1, 2021 and December 31, 2025 were collected. Patients who did not meet the inclusion criteria (inclusion criteria: (1) age ≥ 14 years; (2) discharge diagnosis information) were excluded, resulting in data from 900 patients, including 783 serum samples and 117 cerebrospinal fluid samples. Anti-AP2A1 autoantibodies in all collected samples were detected using the CBA method. Following step (6) of Example 1, overexpressing AP2A1 cell crawling slides and empty pCDNA3.1 control cell crawling slides were prepared. Using these slides, 21 positive AP2A1 autoantibody samples were screened out. All positive samples were serum. Some results are shown below. Figure 10 As shown.

[0067] Step 2: Sample Statistical Analysis (1) Patients were classified and numbered according to their diagnosis at discharge. Among them, 445 patients were antibody-negative or diagnosed with autoimmune encephalitis, including 292 diagnosed autoimmune encephalitis cases and 153 antibody-negative autoimmune encephalitis cases; 346 patients had other diagnosed neurological immune diseases, including paraneoplastic syndromes, myasthenia gravis, multiple sclerosis, neuromyelitis optica spectrum disorders, Guillain-Barré syndrome, and immune-related peripheral neuropathy; and 109 patients had non-neurological autoimmune diseases, including infectious diseases (including viral encephalitis, meningitis, and neurological infections caused by bacteria, fungi, and parasites), metabolic and toxic encephalopathy (including hepatic encephalopathy and pulmonary encephalopathy, toxic encephalopathy caused by antibiotics, chemotherapy drugs, or immunosuppressants, and radiation encephalopathy), tumors (gliomatosis, primary central nervous system lymphoma, and multiple metastatic cancers), and patients without neurological symptoms. Serum samples were then randomly collected from 100 healthy individuals and numbered J21-J120. Detailed patient classifications and numbers are shown in Table 2.

[0068] Table 2. Patient Classification and Numbering Table for the Validation Set

[0069] The results showed that 21 cases of positive anti-AP2A1 antibodies were detected in 900 samples; among them, 10 cases of positive anti-AP2A1 antibodies were detected in 292 cases of autoimmune encephalitis patients who were negative for antibodies, with a detection rate of 3.42% (4.15% in serum samples and 0.00% in cerebrospinal fluid samples); among them, 5 cases of positive anti-AP2A1 antibodies were detected in 153 confirmed autoimmune encephalitis patients, with a detection rate of 3.27% (3.91% in serum samples and 0.00% in cerebrospinal fluid samples). These 5 patients were positive for anti-NMDAR antibody, anti-LGI1 antibody, anti-CASPR2 antibody, anti-NMDAR antibody, and anti-DPPX antibody, respectively. In addition to autoimmune encephalitis, 34 cases of positive anti-AP2A1 antibodies were detected in 34 other cases. Five out of six patients with other neurological immune-related diseases tested positive, with a detection rate of 1.45% (1.63% in serum samples and 0.00% in cerebrospinal fluid samples). These five patients were diagnosed with paraneoplastic syndrome (1 case), myasthenia gravis (1 case), neuromyelitis optica spectrum disorder (1 case), Guillain-Barré syndrome (1 case), and immune-related peripheral neuropathy (1 case). One case (patients without neurological symptoms) was detected in one of the 109 patients with other non-neurological immune-related diseases (as controls), with a detection rate of 0.92% (0.93% in serum samples and 0.00% in cerebrospinal fluid samples). No positive results were found in any of the 100 healthy controls, with a detection rate of 0.00%.

[0070] Conclusion: The above results indicate that anti-AP2A1 autoantibodies were mainly detected in patients with autoimmune encephalitis but not in healthy subjects, suggesting that this autoantibody has auxiliary diagnostic value for autoimmune encephalitis and can serve as a potential diagnostic biomarker for this type of disease.

[0071] (2) The 7 patients with positive anti-AP2A1 antibodies collected in the previous clinical trials (numbered H47, H53, H95, H118, H172, H276, H315) and the 21 newly added patients with positive anti-AP2A1 antibodies (numbered PAE21, PAE34, PAE60, PAE71, PAE149, PAE158, PAE165, PAE181, PAE224, PAE232, AE49, AE57, AE61, AE109, AE120, PNS5, MG33, CNS32, GBS19, IRPN8, Oth18) were integrated into a total of 28 patients. All patients were verified by CBA to be positive for the target antibody and cases with other neurological infections, tumors and metabolic diseases were excluded. Clinical symptoms were classified into 9 categories (memory impairment, epileptic seizures, mental and behavioral abnormalities, consciousness disorders, speech disorders, cognitive impairment, motor disorders, brainstem dysfunction, and autonomic dysfunction). To avoid double counting, if a patient presented with both "incoherent speech and hallucinations," the patient was only classified as "mental and behavioral abnormalities." The symptom distribution data of 28 patients who were positive for anti-AP2A1 antibodies were statistically analyzed, and the results are shown in Table 3.

[0072] Table 328 Symptom Distribution of Anti-AP2A1 Antibody Positive Patients

[0073] Among them, memory dysfunction, epileptic seizures, mental and behavioral abnormalities, consciousness disorders, and speech disorders are the most frequent symptoms associated with positive anti-AP2A1 antibodies.

[0074] Conclusion: Autoantibodies binding to human AP2A1 antigen were present in the serum of these 28 patients. When patients exhibit one or more of the following neurological symptoms, such as memory impairment, seizures, abnormal mental behavior, impaired consciousness, and speech disorders, and AP2A1 autoantibodies are detected in the patient's sample, the patient is highly likely to have autoimmune encephalitis.

[0075] Step 3: Sensitivity and specificity analysis of anti-AP2A1 antibody in the auxiliary diagnosis of autoimmune encephalitis: (1) First, based on the detection results of step 2, a systematic evaluation was conducted on the sensitivity and specificity of anti-AP2A1 antibody detected by the CBA method in the auxiliary diagnosis of autoimmune diseases of the nervous system, especially autoimmune encephalitis. The correspondence between the detection results and the clinical diagnosis is shown in Table 4.

[0076] Table 4. Correspondence between test results and clinical diagnosis

[0077] Based on Table 4, the sensitivity and specificity of anti-AP2A1 antibodies in the auxiliary diagnosis of autoimmune encephalitis were calculated, and the results are as follows: Sensitivity (positive concordance rate) = 3.37%, 95% CI: 2.0% 5.6%; Specificity (negative concordance rate) = 98.68%, 95% CI: 97.1% 99.4%.

[0078] Based on the above verification results, this invention provides a novel antigen that binds to autoantibodies for the auxiliary diagnosis of autoimmune diseases of the nervous system, especially autoimmune encephalitis; anti-AP2A1 autoantibodies can be detected in patients with autoimmune diseases of the nervous system with neurological symptoms, indicating that the antibody has an auxiliary role in the diagnosis of autoimmune diseases of the nervous system, especially in the auxiliary diagnosis of autoimmune encephalitis.

[0079] Example 4: Detection of AP2A1 autoantibodies in patient serum using AP2A1 mutants In this embodiment, a mutant of the human AP2A1 gene was selected, specifically one that lacks amino acids 617-678 of the AP2A1 protein, with the following sequence: KRKKGPGAGSALDDGRRDPSSNDINGGMEPTPSTVSTPSPSADLLGLRAAPPPAAPPASAGA.

[0080] The mutant of the AP2A1 protein, lacking amino acids 617-678 (amino acid sequence as shown in SEQ ID NO. 3), was subjected to vector construction, preparation of cell slices overexpressing the recombinant AP2A1 deletion mutant, and analysis of the 28 cases (numbered H47, H53, H95, H118, H172, H276, H315, PAE21, PAE34, PAE60, PAE71, PAE149, PAE158, PAE165, PAE181, PAE224, PAE232, AE49, AE57, AE61, AE109, AE120, PNS5) according to the steps described in Example 1. Serum samples from patients positive for anti-AP2A1 antibodies (MG33, CNS32, GBS19, IRPN8, Oth18) and serum samples from 5 healthy individuals (numbered J21, J22, J23, J24, and J25) were tested. The results showed that serum from all 28 patients positive for anti-AP2A1 antibodies produced positive signals on cell smears overexpressing the recombinant AP2A1 deletion mutant, while no positive signals were produced in the 5 healthy individuals. Therefore, the AP2A1 deletion mutant can still recognize anti-AP2A1 antibodies in patient serum. Some results are shown below. Figure 11 As shown.

[0081] In summary, this invention discloses for the first time that anti-AP2A1 autoantibodies are closely related to neurological autoimmune diseases and can differentiate between neurological autoimmune diseases and other autoimmune diseases, especially autoimmune encephalitis and other autoimmune diseases. Detection of anti-AP2A1 autoantibodies can facilitate the auxiliary diagnosis of neurological autoimmune diseases, providing a new diagnostic biomarker for clinical practice. This invention also provides a kit containing this reagent, capable of qualitative or quantitative analysis of anti-AP2A1 autoantibodies, with simple operation, helping doctors select appropriate treatment plans for patients.

[0082] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. Application of reagents for detecting anti-AP2A1 autoantibodies in the preparation of products for the auxiliary diagnosis of autoimmune diseases of the nervous system.

2. The application according to claim 1, characterized in that, The reagent for detecting anti-AP2A1 autoantibodies includes one or more of the following: AP2A1 protein, cells expressing AP2A1 protein, vectors expressing AP2A1 protein, and tissues containing AP2A1 protein.

3. The application according to claim 2, characterized in that, The amino acid sequence of the AP2A1 protein includes: a) The amino acid sequence shown in SEQ ID NO.1; b) The amino acid sequence shown in SEQ ID NO.1, after modification or mutation, can recognize the amino acid sequence of anti-AP2A1 autoantibody; c) The amino acid sequence is ≥70% and <100% identical to the amino acid sequence in a) or b), and is capable of recognizing the amino acid sequence of anti-AP2A1 autoantibody.

4. The application according to claim 3, characterized in that, In b), after modifying or mutating the amino acid sequence shown in SEQ ID NO.1, the amino acid sequence that can recognize the anti-AP2A1 autoantibody is shown in SEQ ID NO.

3.

5. The application according to claim 3, characterized in that, The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1 includes any one of (I) to (III): I) The nucleotide sequence shown in SEQ ID NO.2; Ⅱ) A nucleotide sequence that, after mutation of the nucleotide sequence shown in SEQ ID NO.2, encodes an amino acid sequence that recognizes the anti-AP2A1 autoantibody; Ⅲ) A nucleotide sequence that is ≥70% and <100% identical to the nucleotide sequence in Ⅰ) or Ⅱ) and that encodes an amino acid sequence that recognizes an anti-AP2A1 autoantibody.

6. The application according to claim 1, characterized in that, The neurological autoimmune disease is autoimmune encephalitis, and the diagnosis is an auxiliary diagnosis.

7. A reagent kit for the auxiliary diagnosis of autoimmune diseases of the nervous system, characterized in that, This includes reagents for detecting anti-AP2A1 autoantibodies.

8. The kit for assisting in the diagnosis of autoimmune diseases of the nervous system according to claim 7, characterized in that, Reagents for detecting anti-AP2A1 autoantibodies include one or more of the following: AP2A1 protein, cells expressing AP2A1 protein, vectors expressing AP2A1 protein, and tissues containing AP2A1 protein; The amino acid sequence of the AP2A1 protein includes: a) The amino acid sequence shown in SEQ ID NO.1; b) The amino acid sequence shown in SEQ ID NO.1, after modification or mutation, can recognize the amino acid sequence of anti-AP2A1 autoantibody; c) The amino acid sequence is ≥70% and <100% identical to the amino acid sequence in a) or b), and is capable of recognizing the amino acid sequence of anti-AP2A1 autoantibody.

9. The kit for assisting in the diagnosis of autoimmune diseases of the nervous system according to claim 7, characterized in that, The kit uses enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence of cells, immunogold immunoassay, immunospot assay, chemiluminescence, radioimmunoassay, liquid microarray assay, lateral chromatography, or flow cytometry for detection.

10. The kit for assisting in the diagnosis of autoimmune diseases of the nervous system according to claim 7, characterized in that, The kit also includes one or more of labeled antibodies, reaction buffers, and sample diluents.

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