CN117007819A

CN117007819A - Kit for auxiliary diagnosis of Alzheimer's disease

Info

Publication number: CN117007819A
Application number: CN202311023715.4A
Authority: CN
Inventors: 王培昌; 王小灵; 官志超; 安源
Original assignee: Suzhou Yuce Biotechnology Co ltd; Xuanwu Hospital
Current assignee: Suzhou Yuce Biotechnology Co ltd; Xuanwu Hospital
Priority date: 2023-08-15
Filing date: 2023-08-15
Publication date: 2023-11-07

Abstract

The application relates to a kit for auxiliary diagnosis of Alzheimer's Disease (AD), and application of a detection reagent for synaptic vesicle protein SV2A content in a biological sample in preparation of a kit for auxiliary diagnosis of AD. The application firstly finds the application of a detection reagent for the content of synaptic vesicle protein SV2A in biological samples in preparing a kit for assisting in diagnosing AD, and the AD is assisted by detecting the content of synaptic vesicle protein SV2A in serum and the like. The kit disclosed by the application has the advantages of simple components, low cost, high accuracy and good application prospect.

Description

Kit for auxiliary diagnosis of Alzheimer's disease

Technical Field

The application relates to the field of immunological diagnosis, in particular to a kit for assisting in diagnosing whether a subject suffers from Alzheimer's disease or not, and application of a detection reagent for SV2A content in a biological sample in preparing a kit for assisting in diagnosing AD.

Background

Alzheimer's disease (Alzheimer disease, hereinafter sometimes abbreviated as AD) is a chronic neurodegenerative disorder of unknown cause whose pathological changes are mainly manifested by senile plaques caused by the deposition of betA-Amyloid, neurofibrillary tangles caused by abnormal phosphorylation of Tau protein, loss of neurons and synapses, and clinical manifestations of progressive cognitive decline and non-cognitive neuropsychiatric symptoms. The main diagnostic means of AD at present are neuropsychological scales, imaging examinations, biomarker detection, etc. The neuropsychological scale evaluates the cognitive function of the subject, and the simple mental state scale, the teryle cognitive evaluation scale, the Changguchun dementia scale and the like are mainly adopted in the prior art, but the test result is greatly floated due to different education degrees and individual understanding abilities of the subject; the neuroimaging detection mainly comprises Magnetic Resonance Imaging (MRI), positron Emission (PET), computer Tomography (CT) and the like, which are strong evidence of AD diagnosis, and the CT can directly display the brain atrophy phenomenon of an AD patient, but has limited value due to poor resolution of soft tissues, and the MRI and the PET are expensive and are not suitable for crowd screening.

Accurate and early diagnosis of AD facilitates early medical intervention by patients, thereby providing the possibility to delay the onset or progression of AD. Both cerebrospinal fluid aβ42/aβ40 (or brain ridge aβ2) and aβpet are associated with brain aβ pathology, and are considered the earliest biomarkers for AD. Recently, with attention paid to AD, more and more biomarkers for AD are reported, and the following is briefly introduced in connection with patent documents 1 to 7.

Patent document 1 finds 7 mirnas which have significant correlation with the P-tau/aβ ratio by detecting the miRNA level in the blood of an AD patient, establishes a calculation model for predicting the P-tau/aβ ratio of cerebrospinal fluid of the AD patient by the 7 mirnas, verifies the diagnostic efficacy of the model in two independent populations, and verifies that a model consisting of 7 mirnas in the blood can accurately predict the ratio of P-tau/aβ42 in cerebrospinal fluid.

Patent document 2 provides a Kit for early diagnosis of alzheimer's disease, containing epidermal growth factor, growth-regulated alpha protein/C-X-C motif chemokine, macrophage-derived chemokine/C-C motif chemokine 22, monocyte chemokine 1/C-C motif chemokine 2, monocyte chemokine 2/C-C motif chemokine 8, monocyte chemokine 4/C-C motif chemokine 13, thymus and activation-regulated chemokine/C-C 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/C-C motif chemokine 27 and gamma-tubulin complex component 2 biomarker.

Patent document 3 discloses a group of biomarkers for early diagnosis of alzheimer's disease, including brain-derived nerve growth factor, insulin-like growth factor-1, tumor growth factor β1, vascular endothelial growth factor, interleukin 18 and monocyte chemotactic protein-1. Determining the level of the biomarker in the biological sample of the individual by detecting at least four of the specific biomarkers, and determining an increase or decrease in the level of the biomarker as compared to a reference level of the biomarker, thereby utilizing the biomarker to indicate Alzheimer's disease.

Patent document 4 discloses a group of biomarkers for aiding diagnosis of alzheimer's disease in a subject or determining the risk of developing alzheimer's disease in a subject, including cholic acid, chenodeoxycholic acid, allocholic acid, indole-3-lactic acid and tryptophan, and by detecting the content of the biomarkers in plasma, early evaluation of alzheimer's disease can be performed.

Patent document 5 discloses a group of markers for Alzheimer's disease detection, specifically, a combination of any 3 or more proteins selected from the group consisting of alpha-1-chymotrypsin, alpha-1-antitrypsin, alpha albumin, apolipoprotein A-1, apolipoprotein B-100, and the like.

Patent document 6 discloses an exosome protein for diagnosing alzheimer's disease, specifically a combination of six agents of Ig-like domain containing protein, complement C1q subunit C, complement component C9, platelet glycoprotein ibβ chain, RAS inhibitor protein 1 and disintegrin and metalloprotease domain 10, and its use.

Patent document 7 discloses protein markers for evaluating alzheimer's disease, which predict AD risk by integrating 12 or 19 plasma proteins (such as CD164, CETN2, GAMT, GSAP, hK14, LGMN, NELL1, PRDX1, PRKCQ, TMSB10, VAMP5, VPS37A, and the like).

The above patent documents are all combined with various markers to assist in diagnosing AD, and the expression level of the various markers needs to be measured, which will obviously increase the preparation cost and the process complexity of the kit, increase the burden of patients, and require a kit for assisting in diagnosing AD for a single marker on the basis of ensuring the diagnosis accuracy.

Synaptic vesicle protein2 (synaptic vesicle protein, SV 2) is a class of glycoproteins having 12 transmembrane structures, present in all synaptic and endocrine vesicles. SV2A is the most widely distributed subtype, which is almost present in various neurons and is widely distributed in the cerebral cortex, and is closely related to neurotransmitter release, endocrine-vesicle exocytosis, maintenance of synaptic vesicle homeostasis, and the like. In recent years, the mechanism of action of SV2A protein in epileptic related cognitive dysfunction has been studied extensively (see non-patent document 1), its association with AD is also being studied (see non-patent document 2), and the interaction with key causative agents in AD has also been reported (see non-patent document 3). However, current studies on SV2A protein in AD have been mainly to quantify synaptic damage or loss in AD clinical trials by PET imaging scanning of SV2A protein in 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 GAPDH, CTSD, NRGN, MBP, GFAP, tau, phosphorylated Tau, or SV2A, etc., using antibodies against at least two different markers on the surface of exosomes, although SV2A proteins are broadly mentioned, the use of a single SV2A protein in a kit for aiding in the diagnosis of AD is not mentioned. In addition, although it is mentioned in this document that several tens of markers including phosphorylated Tau, αβ -42, tau protein, SV2A protein are associated with neurological disorders, as is known from PET imaging, the association of these proteins with neurological disorders such as AD is known in the art and does not mean that the person skilled in the art is able to think of using only SV2A protein to aid in the diagnosis of AD, nor is it studied at all for SV2A protein in all examples. Moreover, this document uses vesicles as an analyte, and the vesicles are required to be enriched from a sample, and such a treatment requires expensive reagents and is time-consuming, which increases the treatment burden. Thus, in summary, the use of a detection reagent for the content of SV2A protein in a biological sample in the preparation of a kit for aiding in the diagnosis of AD has not been reported, nor has a kit for aiding in the diagnosis of AD based on a single SV2A protein been reported.

Prior art literature

Patent document 1: CN112980941a;

patent document 2: CN106885909B;

patent document 3: CN106062563a;

patent document 4: CN110333310a;

patent document 5: CN106018827a;

patent document 6: CN114150057a;

patent document 7: CN115461474a;

patent document 8: WO2018218090A1;

non-patent document 1: synaptic Vesicle Glycoprotein 2A Ligands in the Treatment of Epilepsy and Beyond,Wolfgang Loscher et al CNS Drugs 2016, 30:1055-1077;

non-patent document 2: the Synaptic Vesicle Glycoprotein 2 Structure, function, and Disease Relevance, kristen A. Stout et al, ACS chem. Neurosci. 2019, 10:3927-3938;

non-patent document 3: the Synaptic Vesicle Protein 2A Interacts With Key Pathogenic Factors in Alzheimer's Disease: implications for Treatment, yanian Kong et al, front, cell dev, biol 2021,9:1-13;

non-patent document 4: the clinical promise of biomarkers of synapse damage or loss in Alzheimer's disease, martI Colom-Cadena et al, alzheimer's Research & Therapy 2020, 12:21, a step of;

non-patent document 5: in vivo measurement of widespread synaptic loss in Alzheimer's disease with SV A PET, adam P.Mecca et al, alzheimer's device 2020, 16:974-982.

Disclosure of Invention

Problems to be solved by the application

As described above, in the prior art, there are many studies on AD markers, and many studies on markers that can be sampled from non-cerebrospinal fluid such as blood, but most of them involve a combination of multiple proteins or RNAs, which results in a complex diagnostic system, and a large number of kit components, which greatly increases the cost and limits the practical clinical use. There is no report of applying a single marker to AD diagnosis (such as detecting taurine or cholic acid in the feces of AD patients) in the prior art, but the accuracy is poor and is less than 50%.

In view of the above-described state of the art, it is an object of the present application to provide a kit for assisting in diagnosing AD by detecting the content (concentration) of only one biomarker on the basis of ensuring the accuracy of diagnosis. It is another object of the present application to provide the use of a reagent for detecting the content of synaptic vesicle protein SV2A (hereinafter sometimes simply referred to as SV2A protein) in a biological sample for preparing a kit for aiding diagnosis of AD.

Means for solving the problems

The applicant conducted intensive studies on markers for aiding diagnosis of AD, and as a result, unexpectedly found for the first time that the amount of SV2A protein in peripheral blood (serum, etc.) and cerebrospinal fluid of AD patients is significantly low compared to normal persons, and that the content of SV2A protein is inversely related to the severity of AD. Based on this finding, the inventors believe that SV2A protein, which has been conventionally used as a target for epilepsy, is highly suitable as a marker for AD-assisted diagnosis, and in particular, by measuring the SV2A protein content in a sample after sampling from serum or the like, it is possible to assist in distinguishing AD patients from non-AD patients with better diagnostic accuracy, and compared with detection of local reduction in synaptic density in neurodegenerative diseases (including AD) by PET imaging technique and imaging of SV2A protein in the brain using a tracer (SV 2A-PET), the technique of the present application greatly simplifies the diagnostic procedure, reduces the diagnostic cost, and has good application prospects. It should be noted that although the prior art has studied that the synaptic density of an AD patient is different from that of a normal person by using SV2A-PET imaging technology, according to the technical knowledge in the art, it cannot be expected by those skilled in the art, and even by experts in the art, whether the SV2A protein content of each part of the AD patient is significantly different from that of the normal person, which requires a great deal of research, analysis and creative labor to know. After the applicant fully searches the prior art, the applicant finds that no relevant technology for specifically detecting the SV2A protein content exists at present, and the research on the difference of the SV2A protein content in blood or cerebrospinal fluid of AD patients and normal people is more impossible.

One technical scheme of the application is as follows,

use of a detection reagent for SV2A protein content in a biological sample for the preparation of a kit for aiding in the diagnosis of AD in a subject.

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

Preferably, the biological sample is serum or cerebrospinal fluid, further preferably, the biological sample is serum. In addition, none of the samples described above were used directly without enrichment or extraction of vesicles (including exosomes, microparticles/microvesicles, apoptotic bodies, tumor vesicles, and other various EV subpopulations).

Preferably, the detection reagent detects the concentration of SV2A protein in the biological sample by a single molecule immunoassay technique featuring counting, a chemiluminescent immunoassay technique, a radioimmunoassay technique, or a flow fluorescent technique. Further preferably, the detection reagent detects the concentration of SV2A protein in the biological sample by a single molecule immunoassay technique featuring counting. Still further preferably, the single molecule immunodetection 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 for enhancing fluorescent signals in situ, comprise fluorescent probes and carriers, and have an average particle size of 180-350 nm. Further preferably, the support is silica, polystyrene, or polyacrylamide, preferably polyacrylamide or silica. Preferably, the fluorescent probe is a fluorescein luminescent material, a rhodamine luminescent material, an aggregation-induced emission material or a quantum dot luminescent material.

Preferably, the reagent comprises a capture antibody and a detection antibody against the SV2A protein, which bind to the first and second sites of the SV2A protein, respectively, and do not comprise antibodies against other proteins. The capture antibody is bound to magnetic beads, and in addition, the binding of the capture antibody to the first site of the SV2A protein is performed at a pH ranging from 8 to 10.

Preferably, the reagent 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, tris, cellulose salt, cellulose derivative, sodium azide, or glycerol. The agent is not a PET tracer.

Another aspect of the application relates to a kit for aiding in the diagnosis of AD comprising an antibody directed against the SV2A protein. The kit does not contain antibodies to other proteins, and the kit is not an ELISA kit.

Preferably, the antibodies to the SV2A protein are a capture antibody and a detection antibody, respectively, which bind to different sites of the SV2A protein. The capture antibodies are bound to magnetic beads and the detection antibodies are bound to the in situ signal enhancing particles described above.

Preferably, the kit is used to assist in distinguishing between subjects suffering from AD and normal subjects.

The above-described kits do not contain reagents for enrichment or capture of vesicles (vesicles) including exosomes, microparticles/microvesicles, apoptotic bodies, tumor vesicles, and other various EV sub-populations.

Preferably, the kit further comprises in-situ signal enhancement particles and magnetic beads, wherein the in-situ signal enhancement particles are nanoparticles for enhancing fluorescent signals in situ, comprise fluorescent probes and carriers, and have an average particle size of 180-350 nm.

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

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, tris, cellulose salt, cellulose derivative, sodium azide, or glycerol.

Effects of the application

According to the present application, it is possible to provide a kit for assisting diagnosis of AD by detecting only the content (concentration) of one biomarker on the basis of ensuring the accuracy of diagnosis.

Drawings

FIG. 1 is a comparison of SV2A protein content in serum (serum) in AD patients (DAT) and cognitive normal populations (HC).

FIG. 2 is a graph of a correlation analysis of serum SV2A protein with cognitive function scores.

FIG. 3 is a subject operating profile (ROC) based on a predictive model of AD for the SV2A protein level of cerebrospinal fluid of AD patients.

FIG. 4 is a comparison of SV2A protein content in cerebrospinal fluid (CSF) in AD patients (DAT) and cognitive normal populations (HC).

FIG. 5 is a graph of a correlation analysis of SV2A protein in cerebrospinal fluid with cognitive function score.

FIG. 6 is a subject operating profile (ROC) based on a predictive model of AD for the level of SV2A protein in cerebrospinal fluid of AD patients.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more apparent, the technical solutions in the embodiments of the present application will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

As used herein, the term "Alzheimer's Disease (AD)" is a common neurodegenerative disease of the elderly, characterized by cognitive dysfunction as a major clinical feature. For diagnosis of AD, methods such as Magnetic Resonance Imaging (MRI), positron Emission Tomography (PET), and biomarker diagnosis can be used.

As used herein, the term "marker" refers to a biochemical marker that can mark alterations in system, organ, tissue, cell and subcellular structure or function, or alterations that may be suffered from, having a very broad range of uses. Biomarkers can be used for disease diagnosis, for judging disease stage or for evaluating the safety and effectiveness of new drugs or new therapies in a target population.

As used herein, a kit refers to a cartridge for containing chemical reagents for detecting chemical components, drug residues, virus species, etc., and in this specification refers specifically to a cartridge for detecting the content of SV2A protein in a sample, which contains a plurality of reagents. The kit further includes a kit instruction, a reaction cup, a waste cup, and the like, and a detailed description thereof is omitted.

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

(1) The first scheme of the application: use of detection reagent for SV2A protein content in biological sample in preparation of kit for auxiliary diagnosis of AD

The first aspect of the application relates to the use of a reagent for detecting the content of SV2A protein in a biological sample for the preparation of a kit for aiding in the diagnosis of AD in a subject. The biological sample may be serum, plasma, whole blood (e.g., peripheral blood), cerebrospinal fluid, etc., and is preferably serum or cerebrospinal fluid from the viewpoint of diagnostic accuracy, and more preferably serum from the viewpoint of ease of sampling. In addition, none of the samples described above were used directly without enrichment or extraction of vesicles (including exosomes, microparticles/microvesicles, apoptotic bodies, tumor vesicles, and other various EV subpopulations). The applicant has also studied the expression level of SV2A in the muscle, liver, etc. of AD patients, and found that the SV2A protein content at these sites is not significantly different from that of normal persons, probably because the expression level of SV2A protein in blood, cerebrospinal fluid, hippocampal tissue, cortex, skeletal muscle, etc. is different.

The detection reagent detects the concentration of SV2A protein in a biological sample by various immunoassay techniques, such as a single molecule immunoassay technique featuring counting, a chemiluminescent immunoassay technique, a radioimmunoassay technique, or a flow fluorescent technique, as long as the concentration of SV2A protein in a relatively low content in the biological sample can be accurately determined. In the prior art, the immunodetection technology for SV2A protein is not perfect, which is probably due to the fact that the SV2A protein content in blood, cerebrospinal fluid and the like is low and is not easy to detect. Further preferably, the detection reagent detects the concentration of SV2A protein in the biological sample by a single-molecule immunoassay technique featuring counting, in view of improving sensitivity. Single Molecule Detection (SMD) is a method that is built on the basis of analysis of single molecules, which has been of great interest in life sciences research to date. In recent years, the method has been applied to cell imaging, research of protein interactions, and quantitative detection of proteins and nucleic acids. In the above mentioned applications, the quantitative detection is achieved by counting target molecules one after the other, which means of quantitative detection represent the final limit of detection. In the SMD quantification method, the molecules that can generate signals are counted, so that the method is more preferable because the method has higher visibility and digitality, and ensures higher reproducibility while obtaining extremely high sensitivity.

More specifically, the single-molecule immunodetection technique characterized by counting is characterized by using in-situ signal enhancement particles and magnetic beads, and detecting the concentration of SV2A protein based on a double antibody sandwich method, wherein the in-situ signal enhancement particles are nanoparticles for enhancing fluorescent signals in situ, comprise fluorescent probes and carriers, and have an average particle diameter of 180-350 nm. Further preferably, the carrier is silica, polystyrene, or polyacrylamide, preferably polyacrylamide or silica. Preferably, the fluorescent probe is a fluorescein luminescent material, a rhodamine luminescent material, an aggregation-induced emission material or a quantum dot luminescent material. The in-situ signal enhancement particles are preferably fluorescent particles formed by coating fluorescent dye molecules (such as fluorescein) with silicon dioxide, fluorescent particles formed by coating fluorescent dye molecules (such as fluorescein) with polyacrylamide, fluorescent particles formed by coating quantum dots with polystyrene, and the like.

Preferably, the detection reagent comprises a capture antibody and a detection antibody for the SV2A protein, wherein the capture antibody and the detection antibody are respectively combined with a first site and a second site of the SV2A protein, and the capture antibody is combined with magnetic beads. And the above detection reagent does not contain antibodies against other proteins. In addition, the binding of the capture antibody to the first site of the SV2A protein is performed at a pH in the range of 8-10.

The surface of the magnetic beads is modified with reactive functional groups capable of covalent coupling with antibodies, such as one or more of hydroxyl, carboxyl, amino, succinimidyl, sulfonyl (e.g., tosyl) and derivatives thereof.

As described above, the antibodies against SV2A protein in the present application may be a capture antibody and a detection antibody that bind to different sites of SV2A protein. The capture antibodies are classified according to antibody specificity, and may be one or both of polyclonal antibodies and monoclonal antibodies. The capture antibody is classified according to source, and can be one or more of murine antibody, rabbit antibody, sheep antibody and alpaca antibody. The detection antibodies are classified according to the specific characteristics of the antibodies, and can be one or two of polyclonal antibodies and monoclonal antibodies. The detection antibody is classified according to sources and can be one or more of a murine antibody, a rabbit antibody, a sheep antibody and an alpaca antibody.

Examples of monoclonal and polyclonal antibodies derived from mammals include: antibodies produced in animal blood, antibodies produced by hybridomas, and antibodies produced by a host transfected with an expression vector containing an antibody gene obtained by genetic engineering techniques, antibodies produced in large quantities in CHO cell factories using the gene of the optimal antibody, or human antibodies produced directly using an anti-gene mouse producing a human antibody, and the like. Monoclonal and polyclonal antibodies can be produced by methods known to those skilled in the art, and commercially available products can be purchased.

(2) The second scheme of the application: a kit for aiding in the diagnosis of AD comprising an antibody directed against SV2A protein.

A second aspect of the application relates to a kit product for aiding in the diagnosis of AD. The use of the kit is limited to the specific use of the kit for the diagnosis of AD, and the limitation also determines that the kit of the present embodiment does not contain a component that is clearly unsuitable for the diagnosis of AD, for example, a conventional diagnosis component for epilepsy, and determines that the instructions attached thereto are instructions matching the diagnosis of AD.

As described above, the antibodies against the SV2A protein may be a capture antibody and a detection antibody, which bind to different sites of the SV2A protein, respectively, in this case based on the double antibody sandwich method. It may also be a capture antibody that binds only to the SV2A protein. In addition, the kit does not include antibodies to other proteins.

As used herein, "antibody" refers to monoclonal antibodies, monospecific antibodies (e.g., either monoclonal antibodies or antibodies produced by methods other than normal germ cells), multispecific antibodies, humanized antibodies (fully or partially humanized antibodies), animal antibodies (e.g., without limitation, birds (e.g., ducks or geese)), sharks, whales and mammals, including non-primates (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, etc.), or non-human primates (e.g., monkeys, chimpanzees, etc.), recombinant antibodies, chimeric antibodies, single chain Fv ("scFv"), single domain antibodies, fab fragments, F (ab') 2 fragments, etc., preferably monoclonal/polyclonal murine/rabbit antibodies.

The kit can further comprise in-situ signal enhancement particles and magnetic beads, wherein the in-situ signal enhancement particles are nanoparticles for enhancing fluorescent signals in situ, comprise fluorescent probes and carriers, and have an average particle size of 180-350 nm. The kit does not contain reagents for enrichment or extraction of vesicles (including exosomes, microparticles/microvesicles, apoptotic bodies, tumor vesicles, and other various EV sub-populations) from biological samples.

The kit may further comprise 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, tris, cellulose salt, cellulose derivative, sodium azide, or glycerol. Specifically, 10 mM-40 mM PBS, 0.05 mL-0.30 mL of nonionic surfactant, 0.4 g-1.0 g of inorganic alkali metal salt, 0.2 g-0.8 g of animal serum albumin, 0.03 mL-0.06 mL of biological preservative, and sterile distilled water are contained in each 100mL of buffer solution. The nonionic surfactant is Tween-20, tween-40, tween-60, tween-80 or a mixture of at least two of them, preferably Tween-20, and the nonionic surfactant is 0.08 mL-0.20 mL per 100mL of the buffer. Preferably, the inorganic alkali metal salt is sodium chloride or potassium chloride, preferably sodium chloride, and the inorganic alkali metal salt is 0.5g to 0.8g per 100mL of the buffer solution. Preferably, the animal serum albumin is bovine serum albumin, and the mass of the animal serum albumin is 0.4g to 0.6g in each 100mL of the buffer solution. Preferably, the biological preservative is proclin-300, and the volume of the biological preservative is 0.04-0.06 mL in every 100mL of buffer. Preferably, the buffer further comprises 2g to 10g of trehalose, preferably 4g to 6g of trehalose per 100mL of the buffer.

The kit may be a kit based on a single molecule immunoassay technique featuring counting.

Example 1

The present application will be described in further detail with reference to examples, but the present application is not limited thereto.

With respect to statistical analysis

The continuous variable between two independent samples was compared using either the t-test or the Mann-Whitney U test. Diagnostic accuracy was assessed using a subject work characteristic curve (ROC) analysis. The area under the curve (AUC) and representative optimal sensitivity and specificity were used to evaluate the performance of the model. All assays were double-tailed assays, and p <0.05 was considered statistically significant. All analyses were performed using SPSS24 version (IBM, armonk, NY, USA). Data were presented using GraphPad Prism 9 software (San Diego, CA, USA).

Example 1: detection of SV2A protein levels in serum of AD patients and cognitive normal control group

The data for this example were from 108 cognitive normal control groups and 164 AD patients, all recruited at the university of capital medical university Xuan Wu hospital; following recruitment, demographic characteristics of AD groups and cognitive normal subjects, including age, gender, education age, montreal cognitive assessment (MoCA), simple mental state examination (MMSE) scores, are recorded as shown in table 1 below.

Patient inclusion criteria were as follows: (1) A cognitively impaired patient in first hospitalization and not receiving systemic medical treatment; (2) The patient completed a cognitive scale test, including MMSE and MOCA, and brain atrophy was confirmed by imaging (CT or MRI); (3) diagnosis of AD is based on criteria published by NIA-AA in the United states. The patient exclusion criteria were as follows: (1) Excluding patients suffering from traumatic brain disorders and other mental disorders; (2) Excluding patients suffering from systemic diseases such as tumors, hematological disorders, hypertension, hyperglycemia; (3) patients who were refused to participate in the study were excluded. Inclusion criteria for participants in the cognitive normal control group were as follows: (1) The participants were first hospitalized patients who did not receive systemic treatment; (2) imaging findings without cognitive impairment such as hippocampal atrophy; (3) At least two neurologists demonstrated that they did not have a clinical manifestation of cognitive impairment. All procedures and procedures were in accordance with the protocol approved by the ethics committee of human research activities at the university of capital medical science Xuan Wu hospital, which has obtained written informed consent from all participants or guardians.

Serum collection mode: after 12 hours fasted, blood samples were drawn from enrolled individuals by venipuncture in the morning and stored in the pigtail along with the clot activator. Serum was extracted from the blood samples by centrifugation at 2000 Xg for 10 minutes. Serum was then stored in polypropylene tubes at-80 ℃ until the next test, avoiding repeated freezing and thawing. Enrichment and extraction of vesicles are not performed.

Serum SV2A protein was detected using human SV2A kit (self-grinding, not commercially available) and a single-molecule immunodetector (AST-Sc-Ulti, commercially available) from Yu-test biotechnology Co., ltd. In Suzhou, the concentration (expressed in pg/mL) was calculated from a standard curve based on a single-molecule immunodetection technique featuring counting (specific steps can be referred to patent CN111771126B by the inventors). Specifically, the human SV2A kit contains a capture antibody and a detection antibody (both monoclonal antibodies that are self-developed) against SV2A protein, carboxyl-activated magnetic beads (purchased from Merck), fluorescent particles (particle size 250nm, homemade) of silica-coated fluorescein isothiocyanate, buffers (homemade, see above for components), calibrator, quality control, and instructions related to AD diagnosis. The kit does not contain reagents for enrichment or extraction of vesicles (including exosomes, microparticles/microvesicles, apoptotic bodies, tumor vesicles, and other various EV sub-populations) from biological samples.

Table 1, cognitive normal control and AD group demographic results

	Cognitive normal group (N=108)	AD group (N=164)
			Age (years+ -SD)	65.59±7.89	67.20±8.63
Sex (Male,%)	51，47%	77，47%
			Education years (years + -SD)	10.25±2.35	9.30±3.26
MoCA scoring	29.13±2.41	12.52±6.32
			MMSE scoring	28.35±1.41	17.35±6.61
SV2A level (pg/mL)	4956.07	2081.87

The average level of SV2A protein (2081.87 pg/ml) in the serum of AD patient group was found to be significantly lower than the average level of SV2A protein (4956.07 pg/ml) in the serum of cognitive normal group (as shown in FIG. 1). Meanwhile, correlation analysis found that SV2A protein levels in serum had a positive correlation with MoCA scores (as shown in fig. 2, where the correlation coefficient r=0.14, p=0.0052, i.e., p < 0.05) and also had a positive correlation with MMSE scores (as shown in fig. 2, where the correlation coefficient r=0.133, p=0.0081, i.e., p < 0.05). This suggests: reduced levels of SVA2A protein in serum are associated with reduced cognitive function; AD is a disease characterized by cognitive impairment as the primary clinical feature, and therefore the above results indicate: the SV2A protein in serum can be used as a protein marker for clinical diagnosis of Alzheimer's disease.

To evaluate the diagnostic accuracy, the area under the curve (see fig. 3) was analyzed using the subject operating characteristic curve (ROC), and as can be seen from fig. 3, the AUC was about 0.70, and the accuracy was excellent.

Example 2: detection of SV2A protein levels in cerebrospinal fluid of AD patients and cognitive Normal control groups

The data for this example were from 35 cognitive normal control groups and 46 AD patients, all enrolled in the university of capital medical university Xuan Wu hospital; following recruitment, demographic characteristics, including age, gender, education, moCA, MMSE scale scores, were recorded for AD groups and cognitive normal subjects, as shown in table 2 below. All procedures and procedures were in accordance with the protocol approved by the ethics committee of human research activities at the university of capital medical science Xuan Wu hospital, which has obtained written informed consent from all participants or guardians. And collecting a human cerebrospinal fluid sample. The cerebrospinal fluid collection steps are as follows: when lumbar puncture is performed, the subject is placed at the left side position; selecting an L3-L5 intervertebral disc space as a puncture site; CSF was collected with a 20 gauge atraumatic needle and centrifuged at 2000×g for 10 min at room temperature. The cerebrospinal fluid was then stored in polypropylene tubes at-80 ℃ until the next test, avoiding repeated freezing and thawing.

Cerebrospinal fluid SV2A protein detection was also performed using a human SV2A kit (self-grinding, not commercially available, supra) and a single molecule immunoassay instrument (AST-Sc-Ulti, commercially available) produced by the Souzhou biosciences, inc., the concentration (expressed in pg/mL) was calculated from a standard curve based on a single molecule immunoassay technique characterized by counting.

Table 2, cognitive normal control and AD group demographic results

	Cognitive normal group (N=35)	AD group (N=46)
			Age (year)	62.23±8.43	61.80±7.37
Sex (Male duty cycle)	17（48%）	21（46%）
			Education years (years)	9.35±2.48	9.85±4.16
MoCA scoring	28.24±3.15	12.30±6.84
			MMSE scoring	29.05±2.67	17.04±7.17
SV2A level (pg/mL)	6197.46	3522.20

The average level of SV2A protein (3522.20 pg/ml) in the cerebrospinal fluid of AD patient groups was found to be significantly lower than the average level of SV2A protein (6197.46 pg/ml) in the cerebrospinal fluid of cognitive normal groups as shown in FIG. 4. Meanwhile, correlation analysis shows that the SV2A protein level in cerebrospinal fluid has positive correlation with MoCA score (as shown in fig. 5, where the correlation coefficient r=0.20, p=0.0315, i.e. p < 0.05) and also has positive correlation with MMSE score (as shown in fig. 5, where the correlation coefficient r=0.20, p= 0.0309, i.e. p < 0.05). This suggests: reduced levels of SVA2A protein in cerebrospinal fluid are associated with reduced cognitive function; AD is a disease characterized by cognitive impairment as the primary clinical feature, and therefore the above results indicate: SV2A protein in cerebrospinal fluid can be used as a protein marker for clinical diagnosis of Alzheimer's disease.

To evaluate the diagnostic accuracy, the area under the curve (see fig. 6) was analyzed using the subject operating characteristic curve (ROC), and as can be seen from fig. 6, the AUC was about 0.8, and the accuracy was excellent.

Claims

1. Use of a detection reagent for synaptic vesicle protein SV2A content in a biological sample in the preparation of a kit for aiding in the diagnosis of whether a subject is suffering from alzheimer's disease.

2. The use of claim 1, wherein the biological sample is serum, cerebrospinal fluid, whole blood, or plasma, and the subject is a human.

3. The use according to claim 1 or 2, wherein the biological sample is serum or cerebrospinal fluid.

4. The use according to claim 1 or 2, wherein the detection reagent detects the concentration of synaptic vesicle protein SV2A in the biological sample by a single molecule immunoassay featuring counting, a chemiluminescent immunoassay, a radioimmunoassay or a flow fluorescent technique.

5. The use according to claim 4, wherein the detection reagent detects the concentration of synaptic vesicle protein SV2A in the biological sample by a single molecule immunoassay characterized by counting.

6. Kit for aiding in the diagnosis of alzheimer's disease, characterized in that it comprises antibodies against the synaptic vesicle protein SV 2A.

7. The kit of claim 6, wherein the antibodies to the synaptic vesicle protein SV2A are a capture antibody and a detection antibody that bind to different sites of the synaptic vesicle protein SV2A, respectively.

8. The kit of claim 6 or 7, wherein the kit is used to aid in distinguishing between subjects suffering from alzheimer's disease and normal subjects.

9. The kit of claim 6 or 7, further comprising in-situ signal enhancing particles and magnetic beads, wherein the in-situ signal enhancing particles are nanoparticles for enhancing fluorescent signals in situ, comprise fluorescent probes and carriers, and have an average particle diameter of 180-350 nm.