Methods of collecting extracellular vesicles derived from cells of the nervous system

Abstract

The invention solves the problem of providing a method for collecting extracellular vesicles derived from nervous system cells with improved efficiency. The problem is solved by a method for collecting extracellular vesicles derived from nervous system cells, the method comprising the step of mixing an anti-APLP1 antibody with a sample containing extracellular vesicles to form anti-APLP1 antibody-extracellular vesicle complexes and the step of collecting the anti-APLP1 antibody-extracellular vesicle complexes.

Description

Technical Field

[0001] This specification discloses methods for collecting extracellular vesicles derived from nervous system cells, methods for detecting neuropsychiatric disorders, methods for collecting components derived from nervous system cells, reagents for collecting extracellular vesicles, and kits for detecting extracellular vesicles containing the reagents. Background Technology

[0002] Patent Document 1 discloses biomarkers for Alzheimer's disease and other neurodegenerative diseases, as well as methods for diagnosis and prognosis. Patent Document 2 discloses the detection of biomarkers in vesicles (e.g., exosomes) isolated from biological samples for the diagnosis and prognosis of Alzheimer's disease and other neurodegenerative diseases. Patent Document 3 discloses methods for quantifying exosome subsets and methods for the diagnosis and prognosis of neurodegenerative diseases (e.g., Alzheimer's disease). Patent Document 4 discloses how to use exosomes and exosome biomarkers in methods for the diagnosis and prognosis of neurological diseases, immune diseases, placental diseases, cancer, blood diseases, kidney diseases, gastrointestinal diseases, liver diseases, and musculoskeletal diseases.

[0003] Reference List

[0004] Patent documents

[0005] Patent Document 1: WO2015 / 061634

[0006] Patent Document 2: WO2017 / 193115

[0007] Patent Document 3: WO2018 / 094120

[0008] Patent document 4: WO2019 / 144056. Summary of the Invention

[0009] Technical issues

[0010] Patent documents 2-4 disclose the use of exosomes in the diagnosis and prognosis of neurodegenerative diseases. However, for example, NCAM or CD171, used for collecting exosomes as described in Patent Document 2, are not proteins specific to nervous system cells. To efficiently collect extracellular vesicles derived from nervous system cells, proteins highly specific to other nerve cells are required.

[0011] The present invention solves the following problem: providing a method for collecting extracellular vesicles derived from nervous system cells with improved efficiency.

[0012] Solution to the problem

[0013] As a result of diligent research, the inventors discovered that extracellular vesicles derived from nervous system cells can be effectively collected by immunoprecipitation targeting APLP1 present in extracellular vesicles.

[0014] The present invention includes, for example, the following aspects as embodiments.

[0015] Item 1. A method for collecting extracellular vesicles derived from nervous system cells, comprising the steps of mixing an anti-APLP1 antibody with a sample containing extracellular vesicles to form a complex of the anti-APLP1 antibody and extracellular vesicles, and collecting the complex of the anti-APLP1 antibody and extracellular vesicles.

[0016] Item 2, the method for collecting extracellular vesicles according to Item 1, wherein the sample containing extracellular vesicles comprises extracellular vesicles crudely purified from the sample.

[0017] Item 3. The method for collecting extracellular vesicles according to Item 1 or 2, wherein the crude purification is performed by size exclusion chromatography, ultracentrifugation, affinity purification, polymer precipitation, or a combination thereof.

[0018] Item 4. A method for detecting neuropsychiatric disorders, comprising the steps of obtaining measurements of polypeptides and / or polynucleotides as biomarkers of neuropsychiatric disorders from extracellular vesicles collected by the method according to any one of claims 1 to 3, and comparing the measurements with corresponding reference values ​​to determine whether the measurements are within or outside the reference range.

[0019] Item 5. The method for detecting neuropsychiatric disorders according to Item 4, wherein the neuropsychiatric disorders are selected from neurodegenerative disorders, post-traumatic brain and spinal cord dysfunction, brain tumors, infection-related brain and spinal cord disorders, multiple sclerosis, schizophrenia, and bipolar disorder.

[0020] Item 6. The method for detecting neuropsychiatric disorders according to Item 5, wherein the neurodegenerative disease is selected from dementia, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, multiple system atrophy, and triadic repeat sequence disease.

[0021] Item 7. A method for collecting components derived from nervous system cells, comprising the step of collecting at least one biomolecule selected from sugars, lipids, polypeptides and polynucleotides from extracellular vesicles collected by the method according to any one of items 1 to 3.

[0022] Item 8-1, Detection reagent for collecting extracellular vesicles, which contains anti-APLP1 antibody.

[0023] Item 8-2, the detection reagent according to Item 8-1, wherein the extracellular vesicles are derived from nervous system cells.

[0024] Item 8-3, a detection reagent comprising an anti-APLP1 antibody, wherein the detection reagent is used to implement the method for collecting extracellular vesicles according to any one of items 1 to 3, the method for detecting neuropsychiatric disorders according to any one of items 4 to 6, or the method for collecting components derived from nervous system cells according to item 7.

[0025] Item 9-1, a detection kit comprising a detection reagent containing an anti-APLP1 antibody for collecting extracellular vesicles.

[0026] Item 9-2, the detection kit according to Item 9-1, wherein the extracellular vesicles are derived from nervous system cells.

[0027] Item 9-3, a detection kit comprising a detection reagent containing an anti-APLP1 antibody, wherein the detection kit is used to implement a method for collecting extracellular vesicles according to any one of items 1 to 3, a method for detecting neuropsychiatric disorders according to any one of items 4 to 6, or a method for collecting components derived from nervous system cells according to item 7.

[0028] Beneficial effects of the invention

[0029] According to the present invention, an improved and efficient method for collecting extracellular vesicles derived from nervous system cells can be provided. Attached Figure Description

[0030] Figure 1 The results of Western blot analysis of extracellular vesicles collected from plasma are shown.

[0031] Figure 2 The results of Western blot analysis of extracellular vesicles collected from the culture supernatant of nerve cells differentiated from iPS cells are shown. Detailed Implementation

[0032] 1. Methods for collecting extracellular vesicles

[0033] This embodiment relates to a method for collecting extracellular vesicles derived from nervous system cells. The method for collecting extracellular vesicles includes the steps of mixing an anti-APLP1 antibody with a sample containing extracellular vesicles and collecting the complex of the anti-APLP1 antibody and the extracellular vesicles. In the method for collecting extracellular vesicles, the complex of the anti-APLP1 antibody and the extracellular vesicles can be formed by mixing the anti-APLP1 antibody with the sample containing extracellular vesicles.

[0034] Extracellular vesicles are particles ranging in size from tens to thousands of nm in size, covered by a membrane primarily composed of phospholipids released from cells. Extracellular vesicles include exosomes, microvesicles, apoptotic bodies, etc. Biomolecules are typically present in extracellular vesicles. For example, exosomes or microvesicles contain at least one biomolecule selected from polypeptides and polynucleotides (RNAs such as mRNA, miRNA, and non-coding RNA and DNA). For example, apoptotic bodies contain at least one biomolecule selected from the group consisting of fragmented cell nuclei and organelles. Extracellular vesicles preferably contain at least one biomolecule selected from polypeptides and polynucleotides. More preferably, extracellular vesicles contain at least one biomolecule selected from polypeptides and polynucleotides. In this document, a polypeptide refers to a compound composed of multiple amino acids linked by peptide bonds, and includes proteins with relatively large molecular weights and peptides with relatively small molecular weights.

[0035] Amyloid β precursor-like protein 1 (APLP1): APLP1 is a member of the amyloid precursor protein gene family. Expressed in the brain, APLP1 is a membrane-bound glycoprotein cleaved by secretases, similar to the amyloid βA4 precursor protein. Intracellular cytoplasmic fragments that can act as transcription activators are released through this cleavage. Human APLP1 has been registered, for example, with gene ID: 333. The NCBI reference sequence is, for example, NM_001024807.3. Two variants of human APLP1 have been reported, but in this embodiment, the type of variant is not limited.

[0036] The nervous system can include nerve cells and glial cells. Glial cells can include astrocytes, oligodendrocytes, and microglia.

[0037] The sample comprises extracellular vesicles that have been roughly purified from a sample containing extracellular vesicles. The sample may be a dispersion of extracellular vesicles or microspheres of extracellular vesicles.

[0038] Methods for the crude purification of extracellular vesicles from a sample are known. For example, extracellular vesicles can be crudely purified by size exclusion chromatography, ultracentrifugation, affinity purification, polymer precipitation, or a combination thereof. These crude purification methods can utilize known techniques. Size exclusion chromatography is not limited as long as the extracellular vesicles can be fractionated by size. For example, size exclusion chromatography can be performed using an extracellular vesicle isolation kit such as qEV (Izon Science). In the case of ultracentrifugation, extracellular vesicles can be obtained, for example, by ultracentrifugation at 100,000 g to 150,000 g for approximately 2 to 3 hours. Preferably, as needed, the sample is diluted with PBS, HEPES buffer, cell culture medium, etc., during ultracentrifugation to a specific gravity of approximately 1.000 to 1.010. Affinity purification can include, for example, phosphatidylserine affinity purification, CD63 affinity purification, anion affinity purification, etc. Polymer precipitation is a method of precipitating extracellular vesicles using polyethers such as polyethylene glycol, polypropylene glycol, or polybutylene glycol. The crude purification of extracellular vesicles is preferably carried out by a method that can obtain extracellular vesicles with a size of about 70 nm to 1000 nm.

[0039] The sample is taken from a living animal, preferably a mammal such as a human, mouse, rat, rabbit, dog, cat, cow, pig, or horse, as long as it contains extracellular vesicles. For example, the sample includes whole blood, serum, plasma, lymph, urine, ascites, pleural effusion, cerebrospinal fluid, interstitial fluid, tears, nasal mucus, saliva, etc.

[0040] Anti-APLP1 antibodies are not limited as long as they can bind to at least a portion of the APLP1 protein. Furthermore, anti-APLP1 antibodies can be of a single type or a mixture of multiple types. As an "antibody," any of the following can be used: polyclonal antibodies, monoclonal antibodies, and their fragments (e.g., Fab, F(ab'), F(ab)2, etc.). There are no particular restrictions on the type and subclass of immunoglobulins. In addition, antibodies can be selected from antibody libraries, such as chimeric antibodies, scFv, etc. For example, R&DSYSTEMS AF3129 can be used as an anti-APLP1 antibody.

[0041] Antibodies do not need to be purified; they can be antiserum containing antibodies, ascites, immunoglobulin fractions isolated from these, etc.

[0042] In addition, antibodies can bind to carriers. Examples of carriers include known carriers used for immunoprecipitation. For example, carriers are magnetic beads, agarose beads, cellulose beads, microplates, tubes, etc.

[0043] Mixing anti-APLP1 antibodies and samples containing extracellular vesicles is not limited as long as the anti-APLP1 antibody can bind to APLP1 present in the extracellular vesicles. Examples include Tris-HCl buffer, phosphate buffer, HEPES buffer, maleic acid buffer, and CHAPS buffer with a pH of approximately 6 to 9. It is preferable to add the same amount of sodium chloride as the saline solution to these buffers. Additionally, bovine serum albumin, skim milk, etc., can be added as blocking reagents. The reaction temperature is approximately 4°C to 37°C. Furthermore, the reaction between the antibody and the extracellular vesicles is preferably carried out with stirring. The reaction time depends on the reaction temperature, but is approximately 4 to 48 hours at approximately 4°C and approximately 0.5 to 4 hours at approximately 37°C.

[0044] Through the above reaction, the anti-APLP1 antibody binds to extracellular vesicles to form an anti-APLP1 antibody-extracellular vesicle complex (also known as the "anti-APLP1 antibody-extracellular vesicle complex").

[0045] Anti-APLP1 antibody-extracellular vesicle complexes can be collected using known methods. When the anti-APLP1 antibody is pre-bound to a carrier, the collection method can be selected based on the properties of the carrier. For example, when the carrier is a magnetic bead, the anti-APLP1 antibody-extracellular vesicle complex can be adsorbed onto a magnet and collected. When the carrier is a non-magnetic bead such as agarose beads or cellulose beads, the anti-APLP1 antibody-extracellular vesicle complex can be collected by centrifugation.

[0046] When the anti-APLP1 antibody is not pre-bound to a carrier, a carrier bound to a substance with affinity for the antibody used, such as a secondary antibody bound to the anti-APLP1 antibody, protein A, or protein G, can be used to collect the anti-APLP1 antibody-extracellular vesicle complex. The method for collecting the carrier and the carrier-bound anti-APLP1 antibody-extracellular vesicle complex is the same as for anti-APLP1 antibodies pre-bound to a carrier.

[0047] The process of collecting anti-APLP1 antibody-extracellular vesicle complexes may include a step of washing the anti-APLP1 antibody-extracellular vesicle complexes to perform B (bound) / F (free) separation, wherein extracellular vesicles that have not reacted with anti-APLP1 and anti-APLP1 antibodies that have not reacted with extracellular vesicles may be appropriately removed.

[0048] Since APLP1 is a protein specifically expressed in nervous system cells, extracellular vesicles derived from nervous system cells can be collected using the methods described above.

[0049] 2. Methods for detecting neuropsychiatric disorders

[0050] In this embodiment, the extracellular vesicles collected in step 1 above are used to detect neuropsychiatric disorders.

[0051] Methods for detecting neuropsychiatric disorders include obtaining measurements of peptides and / or polynucleotides that are biomarkers of neuropsychiatric disorders from extracellular vesicles collected in section 1 above, and comparing these measurements with corresponding reference values ​​to determine whether the measurements are within or outside the reference range. When the measurements are outside the reference range, it can be determined that the subject from whom the sample was collected has a neuropsychiatric disorder. Alternatively, if the measurements are within the reference range, it can be determined that the subject from whom the sample was collected does not have a neuropsychiatric disorder.

[0052] In addition, the severity of neuropsychiatric disorders can be determined by assessing the degree of separation between measured and reference values.

[0053] Neuropsychiatric disorders can include mental disorders and neurological diseases. Neurological diseases can include neurodegenerative diseases, post-traumatic brain and spinal cord dysfunction, brain tumors, infection-related brain and spinal cord diseases, multiple sclerosis, etc. Neurodegenerative diseases can include dementia, Parkinson's disease, amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy, multiple system atrophy, triadic repetition disorder, etc. Dementia can include Alzheimer's disease, senile dementia, Lewy body disease, frontotemporal dementia, vascular dementia, alcohol-related dementia, and corticobasal degeneration. Infection-related brain and spinal cord diseases can include meningitis, brain abscess, Creutzfeldt-Jakob disease (CJD), and AIDS-related dementia. Brain tumors can include astrocytomas.

[0054] Mental disorders can include schizophrenia, depression, bipolar disorder, etc.

[0055] For example, Tau protein, particularly phosphorylated Tau protein, found in extracellular vesicles, is a biomarker for Alzheimer's disease. When the measured value of Tau protein or phosphorylated Tau protein in a sample taken from a subject is higher than the reference value, it can be determined that the subject has Alzheimer's disease.

[0056] Biomarkers reported for each disease may include α-synuclein for Parkinson's disease and Lewy body dementia; TAR DNA-binding protein (TDP-43) for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia; Creutzfeldt-Jakob disease; and aberrant prion proteins of neurofilament light chains for post-traumatic neurological dysfunction, multiple sclerosis, ALS, progressive supranuclear palsy, multiple system atrophy, triadic repeat sequence disease, Alzheimer's disease, Lewy body disease, frontotemporal dementia, vascular dementia, and corticobasal degeneration. Additionally, biomarkers may include insulin-like growth factor (IGF-1) and brain-derived neurotrophic factor (BDNF) for depression; microRNA hsa-miR-34a and microRNA hsa-miR-432 for schizophrenia; and IGF-1 and BDNF for bipolar disorder.

[0057] Biomarkers for neuropsychiatric disorders are detected in the form of peptides or polynucleotides contained in extracellular vesicles. Polynucleotides can be RNA or DNA; in addition to mRNA, RNA can include microRNA, ncRNA, etc. Peptides and / or polynucleotides for neuropsychiatric disorders can include fragments of them as well as their full length.

[0058] Methods for detecting peptide biomarkers for neuropsychiatric disorders can include known methods such as Western blotting and enzyme-linked immunosorbent assay (ELISA). Methods for detecting RNA biomarkers for neuropsychiatric disorders can include known methods such as RT-PCR (including quantitative RT-PCR), microarrays, and RNA-Seq. Methods for detecting DNA biomarkers for neuropsychiatric disorders can include known methods such as PCR (including quantitative PCR), microarrays, and sequencing.

[0059] When detecting peptide biomarkers for neuropsychiatric disorders using methods such as Western blotting and ELISA, extracellular vesicles are lysed with a predetermined lysis buffer as a pretreatment. Samples lysed with the lysis buffer are then used as test samples.

[0060] There are no restrictions on primary antibodies used to detect biomarkers of neuropsychiatric disorders by Western blotting, ELISA, etc., as long as they can detect the biomarkers of neuropsychiatric disorders.

[0061] When detecting RNA-based biomarkers for neuropsychiatric disorders using methods such as RT-PCR, microarrays, and RNA-Seq, RNA is extracted from extracellular vesicles as a pretreatment. Furthermore, if desired, the extracted RNA can be used as a reverse transcription template to synthesize complementary DNA (cDNA). RNA or cDNA can then be used to detect the biomarkers.

[0062] Commercially available primers can be used as primers for RT-PCR (and probes in the case of quantitative RT-PCR). Alternatively, commercially available microarrays can also be used.

[0063] For RNA-Seq, next-generation sequencers (e.g., those manufactured by Illumina) can be used to obtain mRNA readings for biomarkers of neuropsychiatric disorders.

[0064] When detecting DNA-based biomarkers for neuropsychiatric disorders using methods such as PCR, microarrays, and sequencing, DNA is extracted from extracellular vesicles as a pretreatment. Additionally, the extracted DNA can be used as a template for amplification reactions if desired. Both the extracted DNA and the amplified DNA can be used to detect the biomarkers.

[0065] Commercially available primers can be used as primers for PCR (probes may be included in the case of quantitative PCR). Alternatively, commercially available microarrays can also be used.

[0066] For sequencing, next-generation sequencers (e.g., those manufactured by Illumina) can be used to obtain readings of DNA associated with biomarkers for neuropsychiatric disorders.

[0067] When detecting biomarkers for neuropsychiatric disorders using methods such as Western blotting, ELISA, RT-PCR, PCR, RNA-Seq, sequencing, and microarrays, if the biomarkers for neuropsychiatric disorders are detected in extracellular vesicles, it can be determined that "a biomarker for neuropsychiatric disorders has been detected" or "the expression of the biomarker for neuropsychiatric disorders is positive." Alternatively, when comparing the amount of peptides targeting neuropsychiatric disorders derived from extracellular vesicles of a subject with the amount of peptides targeting neuropsychiatric disorders derived from extracellular vesicles of a healthy individual, or when comparing the amount of polynucleotides targeting neuropsychiatric disorders derived from extracellular vesicles of a subject with the amount of polynucleotides targeting neuropsychiatric disorders derived from extracellular vesicles of a healthy individual, if the amount of peptides or polynucleotides targeting neuropsychiatric disorders in the test sample taken from the subject is higher than the amount of peptides or polynucleotides targeting neuropsychiatric disorders in the extracellular vesicles of a healthy individual, it can be determined as "a biomarker for neuropsychiatric disorders has been detected" or "the expression of the biomarker for neuropsychiatric disorders is positive". Furthermore, if the amount of peptides or polynucleotides of biomarkers for neuropsychiatric disorders in extracellular vesicles taken from subjects is the same as that in healthy individuals, it can be determined that "no biomarker for neuropsychiatric disorders was detected" or "the expression of the biomarker for neuropsychiatric disorders is negative." In this text, the expression "a higher value" can be exemplified as a value that is 1.2 times or more, preferably 1.5 times or more, more preferably 2 times or more, and even more preferably 5 times or more. The expression "the same level" can be exemplified as a value that is approximately 0.8 times to less than 1.2 times. Furthermore, before comparing the amount of peptides or polynucleotides of biomarkers for neuropsychiatric disorders in subjects and healthy individuals, the number of extracellular vesicles purified from each sample can be standardized by the amount of peptides such as CD9, CD63, and CD81, which are markers for extracellular vesicles. Furthermore, the number of extracellular vesicles can be standardized by measuring the number of particles using methods such as nanoparticle tracking analysis. The amount of peptides can be expressed as mass or concentration, or as the luminescence intensity of the substrate. The amount of polynucleotides can be expressed as the copy number or read count of the polynucleotide, or as fluorescence intensity.

[0068] As another implementation, a reference value for the amount of peptide or RNA of a biomarker for neuropsychiatric disorders is predetermined. If the amount of peptide or RNA of a biomarker for neuropsychiatric disorders derived from the subject's extracellular vesicles is outside the reference range, it can be determined that "a biomarker for neuropsychiatric disorders has been detected" or "the expression of a biomarker for neuropsychiatric disorders is positive." Furthermore, if the amount of peptide or RNA of a biomarker for neuropsychiatric disorders derived from the subject's extracellular vesicles is within the reference range, it can be determined that "a biomarker for neuropsychiatric disorders has not been detected" or "the expression of a biomarker for neuropsychiatric disorders is negative." The reference value is not limited as long as it can determine whether the amount of peptide, polynucleotide, or expression of a biomarker for neuropsychiatric disorders is detected or positive. And it can be determined by known methods. The amount of peptide or polynucleotide of a biomarker for neuropsychiatric disorders, or the expression of a biomarker for neuropsychiatric disorders, can be determined using ROC (receiver operating characteristic) curves, discriminant analysis, modal analysis, Kittler's method, 3σ method, p-tile method, etc. Additionally, as reference values, sensitivity, specificity, negative predictive value, positive predictive value, and first quartile can be listed.

[0069] 3. Methods for collecting components derived from nerve cells

[0070] In this embodiment, the extracellular vesicles collected in step 1 above are used to collect components derived from nervous system cells.

[0071] The method for collecting components derived from nervous system cells includes the step of collecting at least one biomolecule selected from sugars, lipids, peptides and polynucleotides from extracellular vesicles collected in step 1 above.

[0072] Sugars can include monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Furthermore, sugars can bind to lipids, proteins, and other substances. Sugars can be collected from extracellular vesicles using known methods such as polymers loaded with hydrazides / oxamines, exogenous lectins, etc.

[0073] Lipids can include fatty acids, arachidic acids, triacylglycerols, wax esters, phospholipids, sphingolipids, isoprene kinases, lipoproteins, etc. Lipids can be extracted from extracellular vesicles using methods such as the Folch method and lipid extraction kits (Cell Biolabs, Inc.).

[0074] Polypeptides and polynucleotides may also contain the biomarkers described in section 2 above and other components not described above. The descriptions of the biomarkers are incorporated herein by reference.

[0075] The steps for collecting peptides and polynucleotides can be performed using known methods. Alternatively, commercially available extraction kits can be used.

[0076] 4. Test reagents

[0077] This section describes a detection reagent for collecting extracellular vesicles. The detection reagent contains at least an anti-APLP1 antibody. The description of the anti-APLP1 antibody is incorporated herein by reference to the description in section 1 above.

[0078] The test reagent may contain one or more anti-APLP1 antibodies.

[0079] The anti-APLP1 antibody contained in the assay reagent may be in a dry state or may be dissolved in a buffer, such as phosphate-buffered saline. Further, the assay reagent may include at least one of the following: stabilizers such as β-mercaptoethanol and DTT; protectants such as albumin; surfactants such as polyoxyethylene (20) sorbitol monolaurate and polyoxyethylene (10) octylphenyl ether; and preservatives such as sodium azide.

[0080] Anti-APLP1 antibodies can be labeled with enzymes or fluorescent dyes. Lipophilic antibodies can be immobilized on microplates, magnetic beads, etc.

[0081] 5. Test kit

[0082] In this section, a kit containing the detection reagents for collecting extracellular vesicles described in section 4 above will be described.

[0083] Test kits may be available in the form of a packaging insert that includes a description of the test reagent and how to use it, or a URL for a webpage describing how to use the reagent. Additionally, when the anti-APLP1 antibody is not bound to a carrier or solid, the kit may include secondary antibody conjugation beads, protein A beads, protein G beads, secondary antibody immobilization microplates, protein A immobilization microplates, protein G immobilization microplates, secondary antibody immobilization tubes, etc., for binding the anti-APLP1 antibody to a carrier or solid.

[0084] Furthermore, the kit may include columns for size exclusion chromatography, columns for affinity purification, and polyethers such as polyethylene glycol for the crude purification of extracellular vesicles.

[0085] In addition, it can contain antibodies, probes, primers, etc., for detecting biomarkers of neuropsychiatric disorders present in extracellular vesicles.

[0086] Example

[0087] In the following description, this embodiment will be described in more detail with reference to the embodiments, but the invention is not to be construed as being limited to these embodiments.

[0088] 1. Example 1: Extracellular vesicles derived from nervous system cells were collected from plasma using anti-APLP1 antibody.

[0089] (I) Sample

[0090] Blood was collected from the patient using EDTA-2K tubes, and 20 mL of plasma was separated. The plasma was stored at -80°C until testing. When needed, the plasma was thawed, centrifuged at 2500g, 4°C for 10 minutes, and the supernatant was collected.

[0091] (ii) Crude purification of extracellular vesicles in plasma

[0092] According to the accompanying protocol, 20 mL of size exclusion chromatography (SEC) fraction was collected from 10 mL of plasma using qEV10 (Meiwafosis Group Ltd). This procedure was performed twice in total, collecting 40 mL of SEC fraction. The SEC fraction was then concentrated to 600 μL using an Amicon Ultra-15 100 kDa MWCO.

[0093] (iii) Preparation of beads for immunoprecipitation

[0094] Couple 5 mg according to the regimen provided with the kit. Antibody Coupling Kit (ThermoFisher Scientific) M-270Epoxy and 10 μg of anti-APLP1 antibody (R&D SYSTEMSAF3129) were used to prepare magnetic beads incorporating the anti-APLP1 antibody.

[0095] (iv) Immunoprecipitation of extracellular vesicles derived from nervous system cells

[0096] Add 100 μL of the immunoprecipitation beads prepared in (iii) above to 600 μL of concentrated SEC fraction in a tube and incubate at 4 °C for 4 hours, inverting and mixing. After rotating the tube, attach it to a magnetic rack, wait 1 minute, and then remove the supernatant. Subsequently, to wash the magnetic beads, add 800 μL of D-PBS(-) to the tube and mix by inverting. After rotating the tube, attach it to a magnetic rack, wait 1 minute, and then remove the supernatant. Rotate the tube again, attach it to a magnetic rack, wait 1 minute, and then completely remove the supernatant.

[0097] Add 17 μL of 2×Laemmli Buffer to the test tube containing the magnetic beads, vortex, and incubate at 97°C for 5 minutes. After rotating the tube, attach it to a magnetic rack, wait 1 minute, collect the supernatant, and use it for analysis.

[0098] (5) Western blotting method

[0099] The supernatant collected in the previous step was mixed with denaturation buffer and then heated to prepare the sample for application. The sample was loaded into a 5%–15% Tris-glycine SDS gel and run at 200V for 55 minutes. The SDS gel after electrophoresis was run for 30 minutes at 15V semi-dry conditions using Towbin buffer (containing 5% methanol) to perform the transfer from the gel to the PVDF membrane.

[0100] Blocking was performed for 1 hour at room temperature using 2% ECL Prime blocking agent dissolved in TBS-T. The primary antibody was diluted with 2% ECL Prime blocking agent dissolved in TBS-T and reacted overnight at 4°C. The antibodies used were anti-APLP1 antibody (Calbiochem, 171615), anti-CD81 antibody (Santa Cruz, sc-23962), and anti-Tau antibody (Merck Millipore, 5778801), all diluted 1000-fold before use. After reaction with the primary antibody, the antibody was washed 6 times within 5 minutes using TBS-T.

[0101] The secondary antibody was diluted with 2% ECL Prime blocking agent dissolved in TBS-T and reacted at room temperature for 1 hour. The antibodies used were HRP-labeled anti-mouse antibody (Promega, W4021) and HRP-labeled anti-rabbit antibody (Promega, W4011), all diluted 10,000 times before use. The secondary antibody was washed 6 times with TBS-T for 5 minutes each.

[0102] Add 1,400 μL to the membrane The LD A+B solution mixture was reacted at room temperature for 5 minutes. The HRP signal was emitted and imaged using a myECL Imager (Thermo Scientific).

[0103] (6) Results

[0104] The results of Western blot analysis are as follows: Figure 1 As shown.

[0105] In addition to targeting APLP1, CD81, an extracellular vesicle (EV) protein, and Tau, a neuroprotein, were detected using plasma immunoprecipitation. This revealed that APLP1-positive EVs containing neuroproteins could be isolated, allowing for the collection of extracellular vesicles derived from nervous system cells.

[0106] 2. Example 2: Extracellular vesicles from the culture supernatant of nerve cells differentiated from iPS cells

[0107] (i) Preparation of beads for immunoprecipitation

[0108] Couple 5 mg according to the protocol provided with the kit. Antibody Coupling Kit (ThermoFisher Scientific) M-270Epoxy and 10 μg of anti-APLP1 antibody (R&D SYSTEMSAF3129) were used to prepare magnetic beads incorporating the anti-APLP1 antibody.

[0109] (ii) Preparation of extracellular vesicles (EVs) in culture supernatant

[0110] The culture supernatant of nerve cells differentiated from iPS cells was centrifuged at 2,500g for 10 minutes, and 12 mL of the supernatant was transferred to four ultracentrifuge tubes. After ultracentrifugation at 120,000g for 2.5 hours, the supernatant was removed and microspheres containing extracellular vesicles were collected. 12 mL of PBS was added to each microsphere, pipetteted, and the microspheres were again ultracentrifuged at 120,000g for 2.5 hours to collect the microspheres. 25 μL of PBS was added to each microsphere, pipetteted, and transferred to 1.5 mL test tubes.

[0111] (iii) Immunoprecipitation and Western blotting

[0112] 100 μL conjugated with APLP1 antibody Place the mixture in a screw-cap tube. Add 20 μL of ultracentrifuged EV, 80 μL × 10 Complete (Roche) buffer, and 600 μL of PBS. Incubate the mixture at 4 °C for 4 hours, rotating and mixing simultaneously. Unscrew the tube, attach it to a magnetic holder, and let it stand for 1 minute. Transfer the supernatant to another tube, add 800 μL of PBS to the beads, and vortex the tube. Unscrew the tube, attach it to a magnetic holder, and let it stand for 1 minute. After removing the supernatant, add 12 μL × 2 of Western blotting sample buffer to the tube containing the magnetic beads, vortex, and heat the mixture at 97 °C for 5 minutes. After centrifugation, attach the tube to a magnetic holder and let it stand for 1 minute. Electrophoresis 10 μL onto a 5%–20% or 15% SDS-PAGE gel. In another tube, take 20 μL of the immunoprecipitated supernatant, add 4 μL × 6 of sample buffer, vortex, and heat at 97 °C for 5 minutes. After centrifugation, 20 μL was electrophoresed on a 5%–20% or 15% SDS-PAGE gel at 120 V for 100 minutes.

[0113] The electrophoretically deposited gel was transferred onto a PVDF membrane via wet transfer (400 mA, 1 hour). The membrane was then blocked with 2% ECL Prime blocking reagent for 2 hours.

[0114] Subsequently, the membrane was added with APLP1 C-terminal antibody (Calbiochem 171615) diluted 10,000-fold with 2% ECL Prime blocking agent, L1CAM antibody (Santa Cruz sc-53386) diluted 1,000-fold, CD63 antibody (Santa Cruz sc-5275) diluted 2,000-fold, Flotillin-1 antibody (BD Transduction 610820) diluted 1,000-fold, and SNAP25 antibody (abcam ab5666) diluted 1,000-fold, and incubated overnight at 4°C with shaking. After incubation, the membrane was washed 6 times over 5 minutes with 0.1% TBS-T.

[0115] Membranes reacted with L1CAM, CD63, and Flotillin-1, respectively, were incubated with anti-mouse IgG-HRP (Promega W402B) diluted 10,000-fold with 2% ECL Prime blocking agent as secondary antibodies, and incubated at room temperature with shaking for 1 hour. Membranes reacted with APLP1 and SNAP25, respectively, were incubated with anti-rabbit IgG-HRP (Promega W401B) diluted 10,000-fold with 2% ECL Prime blocking agent as secondary antibodies, and incubated at room temperature with shaking for 1 hour. After incubation, the membranes were washed 6 times over 5 minutes with 0.1% TBS-T.

[0116] Solution A and solution B of LD (Wako) were mixed in 800 μL amounts each, the membrane was impregnated and allowed to stand for 5 minutes. The signal was detected (10 to 300 seconds) using a myECL Imager (Thermo Fisher Scientific).

[0117] (iv) Results

[0118] The results of Western blot analysis are as follows: Figure 2 As shown.

[0119] Lane 1 contained 1 μg of iPS neuron lysate, lane 2 contained EVs before immunoprecipitation, lane 3 contained the input, lane 4 contained the supernatant after EV immunoprecipitation, and lane 5 contained antibody-conjugated beads after EV immunoprecipitation. The input and supernatant volumes for electrophoresis were one-fortieth of the total volume.

[0120] APLP1, EV-labeled proteins (CD63, Flotillin-1), and neural-derived proteins (L1CAM, SNAP25) were detected in the antibody beads. This indicates that extracellular vesicles can be collected using anti-APLP1 antibody.

Claims

1. A method of collecting extracellular vesicles originating from cells of the nervous system, comprising a step of mixing an anti-APLP1 antibody and a sample containing extracellular vesicles to form a complex of the anti-APLP1 antibody and the extracellular vesicles, and a step of collecting the complex of the anti-APLP1 antibody and the extracellular vesicles.

2. The method of collecting extracellular vesicles according to claim 1, wherein, The sample containing extracellular vesicles comprises the extracellular vesicles coarsely purified from a test sample.

3. The method of collecting extracellular vesicles according to claim 2, wherein, The coarsely purification is performed by size exclusion chromatography, ultracentrifugation, affinity purification, polymer precipitation or a combination thereof.

4. Use of an anti-APLP1 antibody for the manufacture of a detection reagent for use in a method of detecting a neuropsychiatric disorder, the method comprising: a step of obtaining a measure of a polypeptide and / or a polynucleotide as a biomarker of a neuropsychiatric disorder from the extracellular vesicles collected according to the method of any one of claims 1 to 3, and a step of comparing the measure to a corresponding reference value to determine whether the measure is within or outside a reference range.

5. Use according to claim 4, wherein, The neuropsychiatric disorder is selected from the group consisting of a neurodegenerative disease, a post-cerebral-spinal trauma neurological dysfunction, a brain tumor, an infection-related cerebral-spinal disease, multiple sclerosis, schizophrenia, depression and bipolar disorder.

6. Use according to claim 5, wherein, The neurodegenerative disease is selected from the group consisting of dementia, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, multiple system atrophy and triplet repeat disorders.

7. A method of collecting components originating from cells of the nervous system, comprising a step of collecting at least one biomolecule selected from the group consisting of a sugar, a lipid, a polypeptide and a polynucleotide from the extracellular vesicles collected according to the method of any one of claims 1 to 3.

8. Use of an anti-APLP1 antibody for the manufacture of a detection reagent for use in a method of collecting extracellular vesicles.

9. Use according to claim 8, wherein, The extracellular vesicles originate from cells of the nervous system.

10. Use according to claim 8 or 9, wherein, The detection reagent can be used for implementing the method of collecting extracellular vesicles according to any one of claims 1 to 3, or implementing the method of collecting components originating from cells of the nervous system according to claim 7.

11. Use of a detection reagent comprising an anti-APLP1 antibody for the manufacture of a kit for use in a method of collecting extracellular vesicles.

12. Use according to claim 11, wherein, The extracellular vesicles originate from cells of the nervous system.

13. Use of a detection reagent comprising an anti-APLP1 antibody for the manufacture of a kit for use in the method of collecting extracellular vesicles according to any one of claims 1 to 3, or the method of collecting components originating from cells of the nervous system according to claim 7.

14. Use of a detection reagent comprising an anti-APLP1 antibody for the manufacture of a kit for use in a method of detecting a neuropsychiatric disorder, the method comprising: a step of obtaining a measure of a polypeptide and / or a polynucleotide as a biomarker of a neuropsychiatric disorder from the extracellular vesicles collected according to the method of any one of claims 1 to 3, and a step of comparing the measure to a corresponding reference value to determine whether the measure is within or outside a reference range. comparing the measurement value with a corresponding reference value to determine whether the measurement value is within a reference range or outside the reference range.

15. Use according to claim 14, wherein, The neuropsychiatric disorder is selected from neurodegenerative diseases, post-encephalocerebrospinal trauma, brain tumors, infection-related encephalomyelopathy, multiple sclerosis, schizophrenia, depression and bipolar disorder.

16. The use according to claim 15, wherein, The neuropsychiatric disorder is selected from dementia, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, multiple system atrophy and triplet repeat disorders.

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