Kit for assisting diagnosis of Alzheimer's disease

Abstract

The present application relates to a kit for assisting the diagnosis of Alzheimer's disease (AD), and the use of a detection reagent for the content of synaptic vesicle protein SV2A in a biological sample in the preparation of a kit for assisting the diagnosis of AD. The present application first discovered the use of a detection reagent for the content of synaptic vesicle protein SV2A in a biological sample in the preparation of a kit for assisting the diagnosis of AD, and assists in the diagnosis of AD by detecting the content of synaptic vesicle protein SV2A in serum, etc. The kit of the present application has simple components, low cost, high accuracy, and good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of immunological diagnosis, and in particular to a kit for assisting in diagnosing whether a subject suffers from Alzheimer's disease, and use of a detection reagent for SV2A content in a biological sample in preparing a kit for assisting in diagnosing AD. Background Art

[0002] Alzheimer's disease (hereinafter referred to as AD) is a chronic neurodegenerative disease of unknown cause. Its pathological changes are mainly manifested by the deposition of β-amyloid protein leading to senile plaques, abnormal phosphorylation of Tau protein leading to neurofibrillary tangles, and loss of neurons and synapses. The clinical manifestations are progressive cognitive impairment and non-cognitive neuropsychiatric symptoms. At present, the main diagnostic methods for AD are neuropsychological scales, imaging examinations, and biomarker detection. Neuropsychological scales evaluate the cognitive function of subjects. Currently, the main scales used are the Mini-Mental State Examination, the Trier Cognitive Assessment Scale, and the Hasegawa Dementia Rating Scale. However, due to the different educational levels and personal understanding abilities of the subjects, the test results fluctuate greatly. Neuroimaging tests mainly include magnetic resonance imaging (MRI), positron emission tomography (PET), and computed tomography (CT), which are also strong evidence for the diagnosis of AD. CT can directly show brain atrophy in AD patients, but its value is limited due to poor soft tissue resolution. MRI and PET are expensive and not suitable for population screening.

[0003] Accurate and early diagnosis of AD is conducive to early medical intervention for patients, thus providing the possibility of delaying the onset or progression of AD. Both cerebrospinal fluid Aβ42 / Aβ40 (or cerebrospinal Aβ2) and AβPET are related to Aβ pathology in the brain and are considered to be the earliest biomarkers of AD. Recently, with the increasing attention paid to AD, more and more biomarkers for AD have been reported. The following is a brief introduction in conjunction with patent documents 1 to 7.

[0004] Patent document 1 detected the miRNA levels in the blood of AD patients and discovered 7 miRNAs that were significantly correlated with the p-tau / Aβ ratio. A calculation model for predicting the p-tau / Aβ ratio in the cerebrospinal fluid of AD patients was established using these 7 miRNAs. The diagnostic efficacy of the model was verified in two independent populations, confirming that the model composed of 7 miRNAs in the blood can accurately predict the P-tau / Aβ42 ratio in the cerebrospinal fluid.

[0005] Patent document 2 provides a kit for early diagnosis of Alzheimer's disease, which contains epidermal growth factor, growth-regulated alpha protein / CXC motif chemokine, macrophage-derived chemokine / CC motif chemokine 22, monocyte chemoattractant protein 1 / CC motif chemokine 2, monocyte chemoattractant protein 2 / CC motif chemokine 8, monocyte chemoattractant protein 4 / CC motif chemokine 13, thymus and activation-regulated chemokine / CC motif chemokine 17, stem cell factor / Kit ligand, TNF-related apoptosis-inducing ligand / tumor necrosis factor ligand superfamily member 10, skin T cell attracting chemokine / CC motif chemokine 27 and γ-tubulin complex component 2 biomarkers.

[0006] Patent document 3 discloses a group of biomarkers for early diagnosis of Alzheimer's disease, including brain-derived neurotrophic factor, insulin-like growth factor-1, tumor growth factor β1, vascular endothelial growth factor, interleukin 18, and monocyte chemoattractant protein-1. By detecting at least four specific biomarkers, the levels of biomarkers in an individual's biological sample are determined, and compared with the reference levels of the biomarkers, the increase or decrease of the biomarker levels is determined, thereby using biomarkers to indicate Alzheimer's disease.

[0007] Patent document 4 discloses a group of biomarkers for assisting in the diagnosis of Alzheimer's disease in a subject or determining the risk of developing Alzheimer's disease in a subject, including bile acid, chenodeoxycholic acid, allocholic acid, indole-3-lactic acid and tryptophan. By detecting the content of biomarkers in plasma, early assessment of Alzheimer's disease can be performed.

[0008] Patent document 5 discloses a group of markers for detecting Alzheimer's disease, specifically a combination of any three or more proteins selected from α-1-chymotrypsin, α-1-antitrypsin, α-albumin, apolipoprotein A-1, apolipoprotein B-100, etc.

[0009] Patent document 6 discloses exosome proteins for diagnosing Alzheimer's disease and their uses. The exosome proteins are specifically a combination of six reagents: Ig-like domain-containing protein, complement C1q subunit C, complement component C9, platelet glycoprotein Ib β chain, RAS inhibitory protein 1, and disintegrin and metalloproteinase domain 10.

[0010] Patent document 7 discloses a protein marker for assessing Alzheimer's disease, which predicts AD risk by integrating 12 or 19 plasma proteins (such as CD164, CETN2, GAMT, GSAP, hK14, LGMN, NELL1, PRDX1, PRKCQ, TMSB10, VAMP5 and VPS37A, etc.).

[0011] The above patent documents all combine multiple markers to assist in the diagnosis of AD. It is necessary to measure the expression levels of multiple markers, which will inevitably significantly increase the preparation cost and process complexity of the kit and increase the burden on patients. There is a need for a kit for assisting in the diagnosis of AD that targets a single marker while ensuring diagnostic accuracy.

[0012] Synaptic vesicle protein 2 (SV2) is a glycoprotein with 12 transmembrane structures, which is present in all synaptic vesicles and endocrine vesicles. SV2A is the most widely distributed isoform, which is present in almost all neurons and widely distributed in the cerebral cortex. It is closely related to the release of neurotransmitters, endocrine vesicle exocytosis, and the maintenance of synaptic vesicle homeostasis. In recent years, the mechanism of action of SV2A protein in epilepsy-related cognitive dysfunction has been widely studied (see non-patent document 1), and its association with AD is also being studied (see non-patent document 2), and its interaction with key pathogenic factors in AD has also been reported (see non-patent document 3). However, the current research on SV2A protein in AD is mainly to quantify synaptic damage or loss in AD clinical trials by performing PET imaging scans of SV2A protein in the brains of AD patients (see non-patent documents 4 and 5). Patent document 8 relates to methods, compositions and kits for detecting and quantifying biomarkers on vesicles, and mentions a kit for diagnosing or prognosing a neurological disorder in a subject, comprising (1) a capture reagent for CD171, CD63, CD81, SNAP25, EAAT1 or OMG; and (2) a detection reagent that selectively binds to GAPDH, CTSD, NRGN, MBP, GFAP, Tau, phosphorylated Tau, or SV2A, etc., which is detected using antibodies against at least two different markers on the surface of exosomes. Although SV2A protein is generally mentioned, there is no mention of the use of a single SV2A protein in a kit for assisting the diagnosis of AD. In addition, although the document generally mentions that dozens of markers including phosphorylated Tau, αβ-42, Tau protein, and SV2A protein are associated with neurological disorders, as known from PET imaging, it is known in the art that these proteins are associated with neurological disorders such as AD, and it does not mean that a person skilled in the art can think of using only SV2A protein to assist in the diagnosis of AD, and none of the embodiments thereof have studied SV2A protein at all. Moreover, the document uses vesicles as the analysis object, which needs to be enriched from the sample before use. Such processing requires expensive reagents and is time-consuming, which will increase the processing burden. Therefore, in summary, there has been no report on the use of a detection reagent for the content of SV2A protein in a biological sample in the preparation of a kit for assisting the diagnosis of AD, nor has a kit for assisting the diagnosis of AD based on a single SV2A protein been reported.

[0013] Prior art literature

[0014] Patent document 1: CN112980941A;

[0015] Patent document 2: CN106885909B;

[0016] Patent document 3: CN106062563A;

[0017] Patent document 4: CN110333310A;

[0018] Patent document 5: CN106018827A;

[0019] Patent document 6: CN114150057A;

[0020] Patent document 7: CN115461474A;

[0021] Patent document 8: WO2018218090A1;

[0022] Non-patent literature 1: Synaptic Vesicle Glycoprotein 2A Ligands in the Treatment of Epilepsy and Beyond, Wolfgang Loscher et al., CNS Drugs 2016, 30: 1055-1077;

[0023] Non-patent literature 2: The Synaptic Vesicle Glycoprotein 2: Structure, Function, and Disease Relevance, Kristen A. Stout et al., ACS Chem. Neurosci. 2019, 10: 3927-3938;

[0024] Non-patent document 3: The Synaptic Vesicle Protein 2A Interacts With KeyPathogenic Factors in Alzheimer's Disease: Implications for Treatment, YanyanKong et al., Front. Cell Dev. Biol. 2021, 9: 1-13;

[0025] Non-patent document 4: The clinical promise of biomarkers of synapse damage or loss in Alzheimer's disease, Martí Colom-Cadena et al., Alzheimer's Research&Therapy 2020, 12: 21;

[0026] Non-patent document 5: In vivo measurement of widespread synaptic loss in Alzheimer's disease with SV2A PET, Adam P. Mecca et al., Alzheimer's Dement 2020, 16: 974-982. Summary of the invention

[0027] Problems to be solved by the invention

[0028] As mentioned above, there are many studies on AD markers in the prior art, and there are also many studies on markers that can be sampled from non-cerebrospinal fluids such as blood, but most of them involve a combination of multiple proteins or RNAs, which leads to a complex diagnostic system and a variety of test kit components, greatly increasing the cost and limiting its actual clinical use. There are also reports in the prior art on the application of single markers in AD diagnosis (such as detecting taurine or bile acid in the feces of AD patients), but its accuracy is poor, less than 50%.

[0029] In view of the above-mentioned state of the prior art, the purpose of the present application is to provide a kit for assisting in the diagnosis of AD by only detecting the content (concentration) of one biomarker while ensuring diagnostic accuracy. Another purpose of the present application is to provide a detection reagent for the content of synaptic vesicle protein SV2A (hereinafter, sometimes referred to as SV2A protein) in a biological sample for use in the preparation of a kit for assisting in the diagnosis of AD.

[0030] Means for solving problems

[0031] The applicant has conducted in-depth research on markers for assisting the diagnosis of AD, and unexpectedly found for the first time that the amount of SV2A protein in the peripheral blood (serum, etc.) and cerebrospinal fluid of AD patients is significantly lower than that of normal people, and the content of SV2A protein is negatively correlated with the severity of AD. Based on this discovery, the inventor is convinced that the SV2A protein, which was previously commonly used as a target for epilepsy, is very suitable as a marker for assisting the diagnosis of AD, especially by measuring the content of SV2A protein in the sample after sampling from serum, etc., which can assist in distinguishing AD patients from non-AD patients with better diagnostic accuracy. Compared with the use of PET imaging technology and using tracers to image SV2A protein in the brain (SV2A-PET) to detect localized reductions in synaptic density in neurodegenerative diseases (including AD), the technology of the present application greatly simplifies the diagnostic process, reduces diagnostic costs, and has good application prospects. It should be noted that although the SV2A-PET imaging technology is used in the prior art to study the difference in synaptic density between AD patients and normal people, based on the technical knowledge in this field, the technicians in this field and even the experts in this field cannot predict whether there is a significant difference in the SV2A protein content in various parts of AD patients and normal people, which requires a lot of research and analysis and creative labor to know. After a thorough search of the prior art, the applicant found that there is currently no relevant technology for targeted detection of SV2A protein content, let alone studying the difference in SV2A protein content in the blood or cerebrospinal fluid between AD patients and normal people.

[0032] A technical solution of this application is as follows:

[0033] Use of a detection reagent for SV2A protein content in a biological sample in the preparation of a kit for assisting in diagnosing whether a subject suffers from AD.

[0034] Preferably, the biological sample is whole blood (eg peripheral blood), plasma, serum or cerebrospinal fluid, and the subject is a mammal or a human, more preferably a human.

[0035] Preferably, the biological sample is serum or cerebrospinal fluid, and more preferably, the biological sample is serum. In addition, the above samples are used directly without enrichment or extraction of vesicles (including exosomes, microparticles / microvesicles, apoptotic bodies, tumor vesicles and other EV subpopulations).

[0036] Preferably, the detection reagent detects the concentration of SV2A protein in a biological sample by a single molecule immunoassay technique characterized by counting, a chemiluminescent immunoassay technique, a radioimmunoassay technique, or a flow fluorescence technique. Further preferably, the detection reagent detects the concentration of SV2A protein in a biological sample by a single molecule immunoassay technique characterized by counting. Further preferably, the single molecule immunoassay technique characterized by counting is characterized in that in situ signal enhancement particles and magnetic beads are used, and the concentration of SV2A protein is detected based on a double antibody sandwich method, wherein the in situ signal enhancement particles are nanoparticles that enhance the fluorescence signal in situ, comprising a fluorescent probe and a carrier, and having an average particle size of 180 to 350 nm. Further preferably, the carrier is silica, polystyrene, or polyacrylamide, preferably polyacrylamide or silica. Preferably, the fluorescent probe is a fluorescein-based luminescent material, a rhodamine-based luminescent material, an aggregation-induced luminescent material, or a quantum dot-based luminescent material.

[0037] Preferably, the reagent comprises a capture antibody and a detection antibody against the SV2A protein, wherein the capture antibody and the detection antibody bind to the first site and the second site of the SV2A protein, respectively, and does not comprise antibodies against other proteins. The capture antibody binds to magnetic beads, and the binding of the capture antibody to the first site of the SV2A protein is carried out in the range of pH 8 to 10.

[0038] Preferably, the reagent comprises a buffer, more preferably, the pH of the buffer is 7.0 to 7.6, comprising PBS, animal serum albumin, nonionic surfactant, inorganic alkali metal salt, biological preservative, and sterile distilled water, and does not contain Tris base, urea, trishydroxymethylaminomethane, cellulose salt, cellulose derivative, sodium azide or glycerol. The reagent is not a PET tracer.

[0039] Another technical solution of the present application relates to a kit for assisting in the diagnosis of AD, which comprises an antibody against the SV2A protein. The kit does not comprise antibodies against other proteins, and the kit is not an ELISA kit.

[0040] Preferably, the antibody against SV2A protein is a capture antibody and a detection antibody that bind to different sites of SV2A protein, respectively. The capture antibody binds to magnetic beads, and the detection antibody binds to the aforementioned in situ signal enhancement particles.

[0041] Preferably, the kit is used to assist in distinguishing subjects suffering from AD from normal subjects.

[0042] The above kits do not contain reagents for enrichment or capture of vesicles (including exosomes, microparticles / microvesicles, apoptotic bodies, tumor vesicles, and other various EV subsets).

[0043] Preferably, the kit further comprises in situ signal enhancing particles and magnetic beads. The in situ signal enhancing particles are nanoparticles that enhance the fluorescent signal in situ, comprise fluorescent probes and carriers, and have an average particle size of 180-350 nm.

[0044] Preferably, the fluorescent probe is a fluorescein-based luminescent material, a rhodamine-based luminescent material, an aggregation-induced emission material, or a quantum dot-based luminescent material.

[0045] Preferably, the kit comprises a buffer. Further preferably, the buffer has a pH of 7.0 to 7.6, comprises PBS, animal serum albumin, a nonionic surfactant, an inorganic alkali metal salt, a biological preservative, and sterile distilled water, and does not contain Tris base, urea, trishydroxymethylaminomethane, cellulose salts, cellulose derivatives, sodium azide or glycerol.

[0046] Effects of the Invention

[0047] According to the present invention, a kit for assisting in the diagnosis of AD can be provided by detecting only the content (concentration) of one biomarker while ensuring diagnostic accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 To compare the SV2A protein content in serum between AD patients (DAT) and cognitively normal people (HC).

[0049] Figure 2 This is a correlation analysis chart between serum SV2A protein and cognitive function score.

[0050] Figure 3 Figure 2 Receiver operating characteristic (ROC) curve of the AD prediction model based on the cerebrospinal fluid SV2A protein level of AD patients.

[0051] Figure 4 To compare the SV2A protein content in cerebrospinal fluid (CSF) between AD patients (DAT) and cognitively normal people (HC).

[0052] Figure 5 This is a correlation analysis chart between cerebrospinal fluid SV2A protein and cognitive function score.

[0053] Figure 6 Figure 2 Receiver operating characteristic (ROC) curve of the AD prediction model based on the cerebrospinal fluid SV2A protein level of AD patients. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] As used herein, the term "Alzheimer's disease (AD)" is a common neurodegenerative disease in the elderly, with cognitive dysfunction as the main clinical feature. AD can be diagnosed using methods such as magnetic resonance imaging (MRI), positron emission tomography (PET) and biomarker diagnosis.

[0056] As used herein, the term "marker" refers to a biochemical index that can mark changes or possible changes in the structure or function of a system, organ, tissue, cell, or subcellular structure, and has a very wide range of uses. Biomarkers can be used to diagnose diseases, determine disease stages, or evaluate the safety and effectiveness of new drugs or new therapies in target populations.

[0057] As used herein, a test kit refers to a box for containing chemical reagents for detecting chemical components, drug residues, virus types, etc., and in this specification, it specifically refers to a box containing multiple reagents for detecting the content of SV2A protein in a sample. In addition, the test kit also includes a matching instruction manual, reaction cup, waste cup, etc., which are omitted here for detailed description.

[0058] As used herein, the term "subject" includes, but is not limited to, various animals, particularly mammals and humans.

[0059] (1) The first scheme of the present application: Use of a detection reagent for the content of SV2A protein in a biological sample in the preparation of a kit for auxiliary diagnosis of AD

[0060] The first scheme of the present application relates to the use of a reagent for detecting the content of SV2A protein in a biological sample in the preparation of a kit, which is used to assist in the diagnosis of whether a subject suffers from AD. The biological sample can be serum, plasma, whole blood (such as peripheral blood, etc.), cerebrospinal fluid, etc. From the perspective of diagnostic accuracy, serum and cerebrospinal fluid are preferred, and serum is further preferred from the perspective of ease of sampling. In addition, the above samples are not enriched or extracted from vesicles (including exosomes, microparticles / microvesicles, apoptotic bodies, tumor vesicles, and various other EV subpopulations) and are used directly. The applicant has also studied the expression of SV2A in the muscles, liver, etc. of AD patients and found that the SV2A protein content in these parts is not significantly different from that of normal people. This may be because the expression of SV2A protein in blood, cerebrospinal fluid, hippocampal tissue, cortex, skeletal muscle, etc. is different.

[0061] The detection reagent detects the concentration of SV2A protein in the biological sample by various immunoassay techniques, such as single molecule immunoassay technique characterized by counting, chemiluminescent immunoassay technique, radioimmunoassay technique, or flow fluorescence technique, as long as the concentration of SV2A protein at a lower content in the biological sample can be accurately determined. In the prior art, the immunoassay technique for SV2A protein is not perfect, which may be due to the low content of SV2A protein in blood, cerebrospinal fluid, etc., which is not easy to detect. Further preferably, from the aspect of improving sensitivity, the detection reagent detects the concentration of SV2A protein in the biological sample by a single molecule immunoassay technique characterized by counting. Single molecule detection (SMD) is a method based on the analysis of a single molecule, and so far, it has received much attention in the research of life sciences. In recent years, this method has been applied to cell imaging, protein interaction research, and quantitative detection of proteins and nucleic acids. In the above-mentioned applications, quantitative detection is achieved by counting the target molecules one by one, and this quantitative detection method represents the ultimate limit of detection. In the SMD quantitative method, counting the molecules that can generate signals is more visual and digital, and it achieves extremely high sensitivity while ensuring high reproducibility, so it is more preferred.

[0062] More specifically, the above-mentioned single-molecule immunoassay technology characterized by counting is characterized in that in-situ signal enhancement particles and magnetic beads are used, and the concentration of SV2A protein is detected based on the double antibody sandwich method, wherein the in-situ signal enhancement particles are nanoparticles that enhance the fluorescence signal in situ, comprising a fluorescent probe and a carrier, and the average particle size is 180~350nm. Further preferably, the above-mentioned carrier is silica, polystyrene, or polyacrylamide, preferably polyacrylamide or silica. Preferably, the fluorescent probe is a fluorescein-based luminescent material, a rhodamine-based luminescent material, an aggregation-induced luminescent material, or a quantum dot-based luminescent material. The above-mentioned in-situ signal enhancement particles are preferably fluorescent particles formed by encapsulating fluorescent dye molecules (such as fluorescein) with silica, fluorescent particles formed by encapsulating fluorescent dye molecules (such as fluorescein) with polyacrylamide, fluorescent particles formed by encapsulating quantum dots with polystyrene, etc.

[0063] Preferably, the detection reagent comprises a capture antibody and a detection antibody against the SV2A protein, wherein the capture antibody and the detection antibody bind to the first site and the second site of the SV2A protein, respectively, and the capture antibody binds to the magnetic beads. The detection reagent does not comprise antibodies against other proteins. In addition, the binding of the capture antibody to the first site of the SV2A protein is carried out in the range of pH 8 to 10.

[0064] The surface of the magnetic beads is modified with active functional groups that can be covalently coupled with antibodies, such as one or more of hydroxyl, carboxyl, amino, succinimidyl ester, sulfonyl (such as toluenesulfonyl) and their derivatives.

[0065] As described above, the antibodies against SV2A protein in the present invention can be capture antibodies and detection antibodies that bind to different sites of SV2A protein. The above-mentioned capture antibodies are classified according to the antibody specificity characteristics, and can be one or both of polyclonal antibodies and monoclonal antibodies. The above-mentioned capture antibodies are classified according to the source, and can be one or more of mouse antibodies, rabbit antibodies, sheep antibodies, and alpaca antibodies. The detection antibodies are classified according to the antibody specificity characteristics, and can be one or both of polyclonal antibodies and monoclonal antibodies. The detection antibodies are classified according to the source, and can be one or more of mouse antibodies, rabbit antibodies, sheep antibodies, and alpaca antibodies.

[0066] Examples of monoclonal antibodies and polyclonal antibodies derived from mammals include antibodies produced in animal blood, antibodies produced by hybridomas, antibodies produced by hosts transfected with expression vectors (the expression vectors contain antibody genes obtained by genetic engineering techniques), antibodies mass-produced in CHO cell factories using genes of optimal antibodies, or human antibodies directly produced using anti-gene mice that produce human antibodies, etc. Monoclonal antibodies and polyclonal antibodies can be produced using methods known to those skilled in the art, or commercial products can be purchased.

[0067] (2) The second solution of the present application: a kit for assisting the diagnosis of AD, comprising an antibody against the SV2A protein.

[0068] The second solution of the present application relates to a kit product, which is used for auxiliary diagnosis of AD. It should be noted that, through the limitation of use, it is specifically limited to auxiliary diagnosis of AD. This limitation also determines that the kit of this solution does not contain components that are obviously not suitable for auxiliary diagnosis of AD, such as the previous diagnostic components for epilepsy, and also determines that the accompanying instructions are instructions matching AD diagnosis.

[0069] As mentioned above, the antibody against SV2A protein can be a capture antibody and a detection antibody that bind to different sites of SV2A protein, respectively, based on a double antibody sandwich method. It can also be only a capture antibody that binds to SV2A protein. In addition, the kit does not contain antibodies against other proteins.

[0070] The term "antibody" as used in this specification refers to a monoclonal antibody, a monospecific antibody (for example, a monoclonal antibody or an antibody produced by methods other than ordinary germ cell production), a multispecific antibody, a human antibody, a humanized antibody (a fully or partially humanized antibody), an animal antibody (for example, but not limited to, birds (such as ducks or geese), sharks, whales, and mammals, including non-primates (such as cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, etc.), or non-human primates (such as monkeys, chimpanzees, etc.), a recombinant antibody, Chimeric antibodies, single-chain Fv ("scFv"), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, etc., preferably monoclonal / polyclonal mouse / rabbit antibodies. In addition, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing analyte binding sites. Immunoglobulin molecules can be protein molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass, preferably IgG.

[0071] Examples of monoclonal antibodies and polyclonal antibodies derived from mammals include antibodies produced in animal blood, antibodies produced by hybridomas, antibodies produced by hosts transfected with expression vectors (the expression vectors contain antibody genes obtained by genetic engineering techniques), antibodies mass-produced in CHO cell factories using genes of optimal antibodies, or human antibodies directly produced using anti-gene mice that produce human antibodies, etc. Monoclonal antibodies and polyclonal antibodies can be produced using methods known to those skilled in the art, or commercial products can be purchased.

[0072] The kit may further include in situ signal enhancement particles and magnetic beads, wherein the in situ signal enhancement particles are nanoparticles that enhance the fluorescence signal in situ, include fluorescent probes and carriers, and have an average particle size of 180-350 nm. The kit does not include reagents for enriching or extracting vesicles (including exosomes, microparticles / microvesicles, apoptotic bodies, tumor vesicles, and various other EV subpopulations) from biological samples.

[0073] The kit may further include a buffer. Further preferably, the pH of the buffer is 7.0 to 7.6, comprising PBS, animal serum albumin, nonionic surfactant, inorganic alkali metal salt, biological preservative, and sterile distilled water, and does not contain Tris base, urea, trishydroxymethylaminomethane, cellulose salt, cellulose derivative, sodium azide or glycerol. Specifically, each 100 mL of buffer contains 10 mM to 40 mM PBS, 0.05 mL to 0.30 mL of nonionic surfactant, 0.4 g to 1.0 g of inorganic alkali metal salt, 0.2 g to 0.8 g of animal serum albumin, 0.03 mL to 0.06 mL of biological preservative, and sterile distilled water. The aforementioned nonionic surfactant is Tween-20, Tween-40, Tween-60, Tween-80 or a mixture of at least two of them, preferably Tween-20, and in each 100 mL of the aforementioned buffer, the aforementioned nonionic surfactant is 0.08 mL to 0.20 mL. Preferably, the aforementioned inorganic alkali metal salt is sodium chloride or potassium chloride, preferably sodium chloride, and the aforementioned inorganic alkali metal salt is 0.5g to 0.8g per 100mL of the aforementioned buffer. Preferably, the aforementioned animal serum albumin is bovine serum albumin, and its mass is 0.4g to 0.6g per 100mL of the buffer. Preferably, the aforementioned biological preservative is proclin-300, and its volume is 0.04mL to 0.06mL per 100mL of buffer. Preferably, each 100mL of the aforementioned buffer further comprises 2g to 10g of trehalose, preferably 4g to 6g of trehalose.

[0074] The kit may be a kit based on a single molecule immunoassay technique characterized by counting. Example 1

[0075] Hereinafter, the present application will be described in further detail with reference to Examples, but the present application is not limited thereto.

[0076] About Statistical Analysis

[0077] Continuous variables between two independent samples were compared using t-test or Mann-Whitney U test. Receiver operating characteristic curve (ROC) analysis was used to evaluate diagnostic accuracy. Area under the curve (AUC) and representative optimal sensitivity and specificity were used to evaluate the performance of the model. All tests were two-tailed tests, and p < 0.05 was considered statistically significant. All analyses were performed using SPSS 24 version (IBM, Armonk, NY, USA). GraphPad Prism 9 software (San Diego, CA, USA) was used to present data.

[0078] Example 1: Detection of SV2A protein levels in serum of AD patients and cognitively normal control group

[0079] The data of this embodiment are from 108 cognitively normal control groups and 164 AD patients, all of whom were recruited from Xuanwu Hospital of Capital Medical University; after recruitment, the demographic characteristics of the AD group and cognitively normal subjects were recorded, including age, gender, years of education, Montreal Cognitive Assessment (MoCA), and Mini-Mental State Examination (MMSE) scores, as shown in Table 1 below.

[0080] The inclusion criteria for patients were as follows: (1) patients with cognitive impairment who were hospitalized for the first time and had not received systemic medical treatment; (2) patients completed cognitive scale tests, including MMSE and MOCA, and brain atrophy was confirmed by imaging (CT or MRI); (3) the diagnosis of AD was based on the criteria published by the NIA-AA of the United States. The exclusion criteria for patients were as follows: (1) patients with traumatic brain disorder and other psychiatric disorders were excluded; (2) patients with systemic diseases such as tumors, blood diseases, hypertension, and hyperglycemia were excluded; (3) patients who refused to participate in this study were excluded. The inclusion criteria for participants in the cognitive normal control group were as follows: (1) participants were patients who were hospitalized for the first time and had not received systemic treatment; (2) there were no imaging findings of cognitive impairment such as hippocampal atrophy; (3) at least two neurologists confirmed that they had no clinical manifestations of cognitive impairment. All operations and procedures were in accordance with the protocol approved by the Human Research Behavior Ethics Committee of Xuanwu Hospital of Capital Medical University, and the study obtained written informed consent from all participants or guardians.

[0081] Serum Collection Method: Blood samples were drawn from enrolled individuals in the morning by venipuncture after a 12-hour fast and stored in serum tubes with a clot activator. Serum was extracted from blood samples by centrifugation at 2000 × g for 10 minutes. Serum was then stored at -80°C in polypropylene tubes until the next test to avoid repeated freezing and thawing. No enrichment or extraction of vesicles was performed.

[0082] Serum SV2A protein detection uses the human SV2A kit (self-developed, not commercially available) and single-molecule immunoassay instrument (AST-Sc-Ulti, commercially available) produced by Suzhou Yuce Biotechnology Co., Ltd., based on the single-molecule immunoassay technology characterized by counting (the specific steps can refer to the applicant's authorized patent CN111771126B), and its concentration (expressed in pg / mL) is calculated by the standard curve. Specifically, the human SV2A kit contains capture antibodies and detection antibodies for SV2A protein (both self-developed monoclonal antibodies), carboxyl-activated magnetic beads (purchased from Merck), fluorescent particles made of silica-encapsulated fluorescein isothiocyanate (particle size of 250nm, homemade), buffer (self-made, ingredients can be found in the previous text), calibrators, quality control products and instructions related to AD diagnosis. The kit does not contain reagents for enriching or extracting vesicles (including exosomes, microparticles / microvesicles, apoptotic bodies, tumor vesicles and various other EV subpopulations) from biological samples.

[0083] Table 1. Demographic characteristics of the control group and the AD group

[0084] Normal cognitive group (N=108) AD group (N=164) Age (years ± SD) 65.59±7.89 67.20±8.63 Gender (Male, %) 51，47% 77，47% Years of education (years ± SD) 10.25±2.35 9.30±3.26 MoCA Scoring 29.13±2.41 12.52±6.32 MMSE score 28.35±1.41 17.35±6.61 SV2A level (pg / mL) 4956.07 2081.87

[0085] The above kit detected that the average level of SV2A protein in the serum of the AD patient group (2081.87 pg / ml) was significantly lower than that in the normal cognitive group (4956.07 pg / ml) (e.g. Figure 1 At the same time, correlation analysis found that the SV2A protein level in serum was positively correlated with the MoCA score (as shown in Figure 2 As shown, the correlation coefficient is r=0.14, p=0.0052, i.e. p<0.05), and it is also positively correlated with the MMSE score (such as Figure 2 As shown, the correlation coefficient is r=0.133, p=0.0081, i.e. p<0.05). This suggests that the decrease in the level of SVA2A protein in serum is related to the decrease in cognitive function; and AD is a disease with cognitive impairment as the main clinical feature. Therefore, the above results indicate that the SV2A protein in serum can be used as a protein marker for the clinical diagnosis of Alzheimer's disease.

[0086] To evaluate the diagnostic accuracy, receiver operating characteristic (ROC) curve analysis was performed and the area under the curve was determined (see Figure 3 ),Depend on Figure 3 It can be seen that the AUC is about 0.70, which is excellent in accuracy.

[0087] Example 2: Detection of SV2A protein levels in cerebrospinal fluid of AD patients and cognitively normal control group

[0088] The data of this embodiment are from 35 cognitively normal controls and 46 AD patients, all of whom were recruited in Xuanwu Hospital of Capital Medical University; after recruitment, the demographic characteristics of the AD group and cognitively normal subjects were recorded, including age, gender, education, MoCA, and MMSE scale scores, as shown in Table 2 below. All operations and processes were in accordance with the protocol approved by the Human Research Behavior Ethics Committee of Xuanwu Hospital of Capital Medical University, and the study has obtained written informed consent from all participants or guardians. Collect human cerebrospinal fluid samples. The steps for collecting cerebrospinal fluid are as follows: when performing a lumbar puncture, place the subject in a left position; select the L3-L5 intervertebral disc space as the puncture site; collect CSF with a 20-gauge non-destructive needle and centrifuge at 2000×g for 10 minutes at room temperature. The cerebrospinal fluid was then stored in a polypropylene tube at -80°C until the next test to avoid repeated freezing and thawing.

[0089] The detection of SV2A protein in cerebrospinal fluid was also performed using the human SV2A kit (self-developed, not commercially available, same as above) and single-molecule immunoassay detector (AST-Sc-Ulti, commercially available) produced by Suzhou Yuce Biotechnology Co., Ltd., based on the single-molecule immunoassay technology characterized by counting, and its concentration (expressed in pg / mL) was calculated using a standard curve.

[0090] Table 2. Demographic characteristics of the control group and the AD group

[0091] Normal cognitive group (N=35) AD group (N=46) Age (years) 62.23±8.43 61.80±7.37 Gender (male percentage) 17（48%） 21（46%） Years of education (years) 9.35±2.48 9.85±4.16 MoCA Scoring 28.24±3.15 12.30±6.84 MMSE score 29.05±2.67 17.04±7.17 SV2A level (pg / mL) 6197.46 3522.20

[0092] The above kit detected that the average level of SV2A protein in the cerebrospinal fluid of the AD patient group (3522.20 pg / ml) was significantly lower than the average level of SV2A protein in the cerebrospinal fluid of the cognitively normal group (6197.46 pg / ml) (e.g. Figure 4 At the same time, correlation analysis found that the SV2A protein level in cerebrospinal fluid was positively correlated with the MoCA score (as shown in Figure 5 As shown, the correlation coefficient is r=0.20, p=0.0315, that is, p<0.05, and it is also positively correlated with the MMSE score (such as Figure 5As shown, the correlation coefficient is r=0.20, p=0.0309, i.e. p<0.05). This suggests that the decrease in the level of SVA2A protein in cerebrospinal fluid is related to the decrease in cognitive function; AD is a disease with cognitive impairment as the main clinical feature. Therefore, the above results show that SV2A protein in cerebrospinal fluid can be used as a protein marker for the clinical diagnosis of Alzheimer's disease.

[0093] To evaluate the diagnostic accuracy, receiver operating characteristic (ROC) curve analysis was performed and the area under the curve was determined (see Figure 6 ),Depend on Figure 6 It can be seen that the AUC is about 0.8, which is excellent in accuracy.

Claims

1. Use of a detection reagent for the content of synaptic vesicle protein SV2A in a biological sample in the preparation of a kit, characterized in that: The kit is used to assist in diagnosing whether a subject suffers from Alzheimer's disease. Wherein, the biological sample is serum or cerebrospinal fluid, the subject is a human, and the detection reagent detects the concentration of synaptic vesicle protein SV2A in the biological sample by a single-molecule immunoassay technique characterized by counting.

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