WO2024237108A1 - Anti-aplp1 monoclonal antibody and use thereof - Google Patents

Abstract

The present invention pertains to: an anti-APLP1 monoclonal antibody of which the epitope is the amino acid sequence from no. 232 to no. 238 in SEQ ID NO. 1; and an anti-APLP1 monoclonal antibody in which CDR1, CDR2, and CDR3 in a light chain respectively have the amino acid sequences described in SEQ ID NO. 5, 7, and 9, and CDR1, CDR2, and CDR3 in a heavy chain respectively have the amino acid sequences described in SEQ ID NO. 12, 14, and 16. The present invention also pertains to: a method for recovering extracellular vesicles derived from brain neurons by using the anti-APLP1 monoclonal antibody of the present invention; methods for detecting a neuropsychiatric disease biomarker and for recovering neuron-derived components, the methods each comprising recovering extracellular vesicles by said method; and a test reagent and a test kit which are for use in recovering extracellular vesicles and which include the anti-APLP1 monoclonal antibody of the present invention. The present invention provides: an anti-APLP1 monoclonal antibody having a higher specificity with respect to NDE; a method for recovering extracellular vesicles by using said antibody; a method for detecting a neuropsychiatric disease biomarker using the recovered extracellular vesicles; a test drug; and a test kit.

Description

Anti-APLP1 monoclonal antibody and its use

The present invention relates to an anti-APLP1 monoclonal antibody and uses thereof.
CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Japanese Patent Application No. 2023-79084, filed May 12, 2023, the entire disclosure of which is expressly incorporated herein by reference.

　Measures to combat dementia are a major issue in modern times, and methods to diagnose dementia using biomarkers are being developed. However, these involve imaging diagnostics such as PET or cerebrospinal fluid diagnostics, and problems have been raised in terms of cost and invasiveness. Therefore, there is hope for the development of biomarkers that use peripheral blood, which are less invasive and less expensive.

Exosomes are released from various cells into body fluids such as blood. Therefore, neuron-derived extracellular vesicles (NDEs) are present in the blood. NDEs are surrounded by a lipid bilayer, which protects the contents from proteolytic and nucleolytic enzymes and preserves the environment of brain cells. In addition, since the membrane of NDEs reflects that of brain neurons, neurospecific proteins exist on the NDE membrane. One of these is APLP1 (amyloid beta precursor like protein 1). A schematic diagram of an NDE presenting APLP1 on the NDE membrane is shown in Figure 1.

Patent document 1: WO2021/132352

Non-patent document 1: Alzheimer's & Dementia 15 (2019) 1071-1080
The entire disclosures of Patent Document 1 and Non-Patent Document 1 are each expressly incorporated herein by reference.

Patent Document 1 discloses a method for recovering extracellular vesicles using an anti-APLP1 antibody, a method for detecting neuropsychiatric disorders using the recovered extracellular vesicles, a test agent, a test kit, and the like. The anti-APLP1 antibody used in the invention described in Patent Document 1 is a commercially available antibody and is a polyclonal antibody.

Non-Patent Document 1 describes a method for recovering NDE using an anti-NCAM antibody (mouse anti-human neural cell adhesion molecule (NCAM)). The anti-NCAM antibody is an antibody that shows reactivity with a completely different target than the anti-APLP1 antibody.

The present invention aims to provide an anti-APLP1 monoclonal antibody, which is a new antibody with higher specificity for NDE, and to provide a method for recovering extracellular vesicles using this anti-APLP1 monoclonal antibody, a method for detecting biomarkers for neuropsychiatric disorders using the recovered extracellular vesicles, a test agent, a test kit, etc.

The inventors focused on the integral membrane protein of NDE and prepared multiple monoclonal antibodies that recognize specific sites of this protein as epitopes. From among these, they discovered an anti-APLP1 monoclonal antibody that specifically binds to NDE, and completed an invention relating to a method for recovering NDE using this anti-APLP1 monoclonal antibody, a method for detecting biomarkers for neuropsychiatric disorders using recovered NDE, and a test agent and test kit.

The present invention is as follows.
[1]
An anti-APLP1 monoclonal antibody having as its epitope the amino acid sequence from positions 232 to 238 of the amino acid sequence set forth in SEQ ID NO:1 in the sequence listing.
[2]
An anti-APLP1 monoclonal antibody, wherein the light chain CDR1, CDR2 and CDR3 have the amino acid sequences set forth in SEQ ID NOs: 5, 7 and 9, respectively, and the heavy chain CDR1, CDR2 and CDR3 have the amino acid sequences set forth in SEQ ID NOs: 12, 14 and 16, respectively.
[3]
The anti-APLP1 monoclonal antibody according to [2], wherein FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences of SEQ ID NOs: 4, 6, 8 and 10, respectively, and FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences of SEQ ID NOs: 11, 13, 15 and 17, respectively.
[4]
A step of mixing a sample containing an anti-APLP1 antibody and extracellular vesicles to form a complex of the anti-APLP1 antibody and the extracellular vesicles, and a step of recovering the complex of the anti-APLP1 antibody and the extracellular vesicles.
Including,
The anti-APLP1 antibody is an anti-APLP1 monoclonal antibody according to any one of [1] to [3].
A method for recovering extracellular vesicles derived from brain neurons.
[5]
The method according to [4], wherein the sample containing extracellular vesicles is plasma.
[6]
A method for detecting a biomarker for a psychiatric and neurological disorder, comprising the steps of recovering extracellular vesicles by the method according to [4] or [5], and obtaining measurements of polypeptides and/or polynucleotides that serve as biomarkers for the psychiatric and neurological disorder from the recovered extracellular vesicles.
[7]
The detection method according to [6], further comprising a step of comparing the acquired measurement value with a corresponding reference value and determining whether the measurement value is within or outside the reference range.
[8]
The detection method according to [6] or [7], wherein the biomarker is tau protein or phosphorylated tau protein.
[9]
A method for recovering components derived from brain and nerve cells, comprising the steps of recovering extracellular vesicles by the method described in [4] or [5], and recovering at least one biological molecule selected from the group consisting of sugars, lipids, polypeptides, and polynucleotides from the recovered extracellular vesicles.
[10]
A test reagent used for recovering extracellular vesicles, comprising the anti-APLP1 monoclonal antibody according to any one of [1] to [3].
[11]
The test reagent described in [10], which is used to carry out the extracellular vesicle recovery method described in [4] or [5].
[12]
The test reagent according to [10], which is used for carrying out the method for detecting a biomarker for a psychiatric and neurological disorder according to any one of [6] to [8].
[13]
The test reagent described in [10] is used to carry out the method for recovering components derived from brain nerve cells described in [9].
[14]
A test kit comprising the test reagent according to [10].

The present invention can provide an anti-APLP1 monoclonal antibody that specifically binds to NDE. Furthermore, the present invention can provide a method for recovering NDE and a method for detecting biomarkers for neuropsychiatric disorders using the recovered NDE.

FIG. 1 shows a schematic diagram of an NDE presenting APLP1 on the NDE membrane. Figure 2 shows the human APLP1 amino acid sequence (NCBI accession No. NP_031493.2) (SEQ ID NO: 1). FIG. 3 shows the results of analysis using a flow cytometer in Example 1-1. FIG. 4 shows the results of ELISA analysis of the reactivity of the antibody obtained in Example 1-1 to the APLP1 peptide. FIG. 5 shows the results of immunostaining in Example 1-1. FIG. 6 shows the results of Western blotting in Example 1-1. FIG. 7 shows the results of cell staining in Example 1-2. FIG. 8 shows the results of WB analysis of the extract from 293FT cells in which APLP1 was forcibly expressed in Example 1-3(1). FIG. 9 shows the results of signal detection by cell staining in Example 1-3(2). FIG. 10-1 shows the results of Western blotting in Example 2. FIG. 10-2 shows the comparative results of Western blotting in Example 2, using the specific monoclonal antibody #11 of the present invention or a commercially available polyclonal antibody. FIG. 11 shows the results of Western blotting in Example 3. FIG. 12 shows a scatter plot showing the correlation between the total amount of tau in plasma brain neuron-derived extracellular vesicles (NDE) and the total amount of tau in cerebrospinal fluid (CSF) in Example 4. FIG. 13 shows a scatter plot showing the correlation between the total amount of tau in plasma brain neuron-derived extracellular vesicles (NDE) in Example 4, phosphorylated tau (pTau181) in cerebrospinal fluid (CSF) (left figure), and the Aβ42/Aβ40 ratio in cerebrospinal fluid (CSF) (right figure).

1. Anti-APLP1 Monoclonal Antibody The first aspect of the anti-APLP1 monoclonal antibody of the present invention is an anti-APLP1 monoclonal antibody having an epitope of the amino acid sequence of 232 to 238 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing. The anti-APLP1 monoclonal antibody having an epitope of the amino acid sequence of 232 to 238 of the amino acid sequence described in SEQ ID NO: 1 is an antibody that recognizes a peptide having an amino acid sequence of 232 to 238 of the amino acid sequence described in SEQ ID NO: 1 as an antigen. The recognition of a peptide having an amino acid sequence of 232 to 238 of the amino acid sequence described in SEQ ID NO: 1 as an antigen can be determined by the antibody showing reactivity with a peptide having an amino acid sequence of 232 to 238. The reactivity of the antibody with the peptide can be tested by standard methods, such as flow cytometry (FACS), enzyme-linked immunosorbent assay (ELISA), western blot, fluorescence microanalysis (FMAT), surface plasmon resonance (BIAcore), immunostaining, immunoprecipitation, and the like.

APLP1 (amyloid beta precursor like protein 1) is an integral membrane protein of brain neuronal-derived extracellular vesicles (NDEs) and a member of the amyloid precursor protein gene family. APLP1 protein is a membrane-bound glycoprotein expressed in the brain that is cleaved by secretases similar to the cleavage of the amyloid beta A4 precursor protein. This cleavage releases intracellular cytoplasmic fragments that may act as transcriptional activators.

Brain nerve cells may include, for example, neurons and glial cells. Glial cells may include astrocytes, oligodendrocytes, and microglial cells. Extracellular vesicles are particles with a size of tens to thousands of nanometers that are covered with a membrane mainly composed of phospholipids and are released from cells.

Human APLP1 is registered, for example, under Gene ID: 333. The NCBI Reference Sequence is, for example, NM_001024807.3. In the present invention, the human APLP1 peptide has the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing.

The first embodiment of the anti-APLP1 monoclonal antibody of the present invention is an anti-APLP1 monoclonal antibody having, as an epitope, the amino acid sequence from positions 232 to 238 of the amino acid sequence set forth in SEQ ID NO:1 in the sequence listing. As shown in the examples, the amino acid sequence from positions 232 to 238 of the amino acid sequence set forth in SEQ ID NO:1 in the sequence listing is the minimum amino acid sequence that serves as an epitope.

A second embodiment of the anti-APLP1 monoclonal antibody of the present invention is an anti-APLP1 monoclonal antibody in which CDR1, CDR2, and CDR3 of the light chain have the amino acid sequences of SEQ ID NOs: 5, 7, and 9, respectively, and CDR1, CDR2, and CDR3 of the heavy chain have the amino acid sequences of SEQ ID NOs: 12, 14, and 16, respectively. In the second embodiment of the anti-APLP1 monoclonal antibody of the present invention, preferably, FR1, FR2, FR3, and FR4 of the light chain have the amino acid sequences of SEQ ID NOs: 4, 6, 8, and 10, respectively, and FR1, FR2, FR3, and FR4 of the heavy chain have the amino acid sequences of SEQ ID NOs: 11, 13, 15, and 17, respectively.

The amino acid sequences represented by the sequence numbers of the light and heavy chains are shown in Tables 1 and 2 below.

The anti-APLP1 monoclonal antibody of the present invention is not particularly limited in terms of immunoglobulin class and subclass, and may be, for example, IgG. Furthermore, the anti-APLP1 monoclonal antibody may be a fragment (e.g., Fab, F(ab'), F(ab) ₂ , etc.), a chimeric antibody, scFv, etc.

Method for Producing Anti-APLP1 Monoclonal Antibody of the Present Invention The anti-APLP1 monoclonal antibody of the present invention can be produced using the methods described in, for example, Kurosawa et al., “Rapid production of antigen-specific monoclonal antibodies from a variety of animals” BMC Biology 2021, 10:80 (Non-Patent Document 2), Kurosawa et al., “Novel method for the high-throughput production site-specific monoclonal antibodies” SCIENTIFIC REPORT 6:25174 DOI:10.1038 srep25174 (Non-Patent Document 3) and WO2011/027808 (Patent Document 2) and WO2012/133572 (Patent Document 3). The entire disclosures of Patent Documents 2 and 3, and Non-Patent Documents 2 and 3, respectively, are expressly incorporated herein by reference.

　Monoclonal antibodies with specific light and heavy chain amino acid sequences, as well as anti-APLP1 monoclonal antibodies with specific light chain amino acid sequences CD1, CD2, CD3, FR1, FR2, FR3, and FR4, and heavy chain CD1, CD2, CD3, FR1, FR2, FR3, and FR4, can be produced by known methods, including those described in Non-Patent Documents 2 and 3, and Patent Documents 2 and 3, but are not intended to be limited thereto.

　A monoclonal antibody that uses an integral membrane protein of extracellular vesicles derived from brain nerve cells as an epitope is produced by immunizing an animal with an amino acid fragment (target antigen) that is to be used as an epitope, collecting lymph or lymphatic tissue from the immunized animal, collecting cells from the lymph or lymphatic tissue, and collecting specific plasma cells that are reactive with the target antigen from the collected cells. cDNA is synthesized from the collected target antigen-specific plasma cells, and the cDNA is used as a template to amplify the γ-chain and κ-chain variable region genes. The amplified variable regions are incorporated into an expression vector to obtain the variable region genes. The obtained γ-chain and κ-chain variable region genes are used to create heavy and light chain immunoglobulin genes, which are then introduced into, for example, 293FT cells to create a recombinant antibody. If necessary, an antibody with high reactivity to the target antigen can be selected from the multiple recombinant antibodies created. A specific method is described in Example 1.

2. Method for recovering extracellular vesicles This embodiment relates to a method for recovering extracellular vesicles derived from brain nerve cells. The method for recovering extracellular vesicles includes a step of mixing an anti-APLP1 monoclonal antibody (hereinafter, sometimes referred to as anti-APLP1 antibody) with a sample containing extracellular vesicles, and a step of recovering a complex of the anti-APLP1 antibody and the extracellular vesicles. By mixing the anti-APLP1 antibody with a sample containing extracellular vesicles, the anti-APLP1 antibody and the extracellular vesicles can form a complex.

Extracellular vesicles include exosomes, microvesicles, apoptotic bodies, etc. In many cases, biomolecules are present in extracellular vesicles. For example, exosomes or microvesicles contain at least one biomolecule selected from the group consisting of polypeptides and polypeptides (RNA such as mRNA, miRNA, non-coding RNA, and DNA). For example, apoptotic bodies contain at least one type selected from the group consisting of fragmented nuclei and organelles. Extracellular vesicles preferably contain at least one biomolecule selected from the group consisting of polypeptides and polypeptides. More preferably, extracellular vesicles contain at least one biomolecule selected from the group consisting of polypeptides and polypeptides. Here, a polypeptide refers to a compound in which multiple amino acids are bound by peptide bonds, and includes proteins with relatively large molecular weights and peptides with relatively small molecular weights.

The sample contains extracellular vesicles roughly purified from a specimen containing extracellular vesicles. The sample may be a dispersion of extracellular vesicles, or a pellet of extracellular vesicles.

Methods for crudely purifying extracellular vesicles from a specimen are known, and extracellular vesicles can be crudely purified, for example, by size exclusion chromatography, ultracentrifugation, affinity purification, polymer precipitation, and combinations thereof. Known methods can be used as these crude purification methods. Size exclusion chromatography is not limited as long as it can fractionate extracellular vesicles by size. For example, size exclusion chromatography can be performed using an extracellular vesicle extraction kit qEV (Izon Science). Ultracentrifugation can be performed, for example, by ultracentrifugation at 100,000 g to 150,000 g for about 2 to 3 hours to obtain extracellular vesicles. Preferably, the specimen is diluted with PBS, HEPES buffer, cell culture medium, or the like, as necessary, so that the specific gravity is about 1.000 to 1.010 when ultracentrifuging. Affinity purification methods may include, for example, phosphatidylserine affinity purification methods, CD63 affinity purification methods, anion affinity purification methods, etc. Polymer precipitation methods are methods for precipitating extracellular vesicles using polyethers such as polyethylene glycol, polypropylene glycol, and polytetramethylene glycol. It is preferable to roughly purify extracellular vesicles using a method that can obtain extracellular vesicles of about 70 nm to 1000 nm.

The sample is collected from an animal, preferably a mammalian living body such as a human, mouse, rat, rabbit, dog, cat, cow, pig, or horse, and is not limited as long as it contains extracellular vesicles. For example, the sample includes whole blood, serum, plasma, lymph, urine, ascites, pleural fluid, cerebrospinal fluid, interstitial fluid, tears, nasal secretion, saliva, etc.

The anti-APLP1 antibody is the anti-APLP1 monoclonal antibody of the present invention. However, the antibody may be a complex bound to a carrier. Examples of the carrier include known carriers used in immunoprecipitation. For example, the carrier is magnetic beads, agarose beads, cellulose beads, a microplate, a tube, etc.

The mixing of the anti-APLP1 antibody with the sample containing extracellular vesicles is not limited as long as the anti-APLP1 antibody is mixed with the APLP1 present in the extracellular vesicles under conditions that allow the antibody to bind to APLP1 present in the extracellular vesicles. Examples of the buffer include Tris-HCl buffer, phosphate buffer, HEPES buffer, maleic acid buffer, CHAPS buffer, etc., with a pH of about 6 to about 9. It is preferable to add the same amount of sodium chloride as physiological saline to these buffers. In addition, bovine serum albumin, skim milk, etc. may be added as a blocking reagent. The reaction temperature is about 4°C to 37°C. In addition, it is preferable to carry out the reaction between the antibody and the extracellular vesicles while stirring. The reaction time depends on the reaction temperature, but is about 4 to 48 hours when the reaction temperature is about 4°C, and about 0.5 to 4 hours when the reaction temperature is about 37°C.

The above reaction causes the anti-APLP1 antibody to bind to the extracellular vesicles, forming a complex of the anti-APLP1 antibody and the extracellular vesicles (also called the "anti-APLP1 antibody-extracellular vesicle complex").

The anti-APLP1 antibody-extracellular vesicle complex can be collected by known methods. When the anti-APLP1 antibody is bound to a carrier in advance, the collection method can be selected according to the properties of the carrier. For example, when the carrier is magnetic beads, the anti-APLP1 antibody-extracellular vesicle complex can be adsorbed to a magnet and collected. When the carrier is non-magnetic beads such as agarose beads or cellulose beads, the anti-APLP1 antibody-extracellular vesicle complex can be collected by centrifugation.

When the anti-APLP1 antibody is not bound to a carrier in advance, the anti-APLP1 antibody-extracellular vesicle complex can be recovered using a substance that has affinity for the antibody used, such as a secondary antibody that binds to the anti-APLP1 antibody, or a carrier bound to protein A or protein G. The method for recovering the carrier and the anti-APLP1 antibody-extracellular vesicle complex bound to the carrier is the same as when the anti-APLP1 antibody is bound to a carrier in advance.

The process for recovering the anti-APLP1 antibody-extracellular vesicle complex may include a washing step of the anti-APLP1 antibody-extracellular vesicle complex by performing B (bound)/F (free) separation to remove extracellular vesicles that have not reacted with the anti-APLP1 antibody, and anti-APLP1 antibody that has not reacted with the extracellular vesicles.

APLP1 is a protein that is specifically expressed in brain neurons, so the above recovery method can recover extracellular vesicles derived from brain neurons. The anti-APLP1 monoclonal antibody of the present invention has high specific reactivity to human APLP1 peptide, so it can specifically recover extracellular vesicles that have APLP1 peptide as a membrane protein.

As one embodiment of the method for recovering extracellular vesicles,
(1) Separating total extracellular vesicles from plasma by size exclusion chromatography;
(2) Immunoprecipitation of the total extracellular vesicle fraction using anti-APLP1 monoclonal antibody;
(3) treating the obtained fraction with acid to separate extracellular vesicles;
Examples of the method for separating extracellular vesicles from plasma include the following. In addition, extracellular vesicles can be separated by peptide treatment instead of acid treatment.

3. Method for detecting biomarkers for psychiatric and neurological disorders In this embodiment, biomarkers for psychiatric and neurological disorders are detected using the extracellular vesicles collected in 2 above.

The method for detecting a biomarker for a psychiatric disorder includes obtaining a measurement value of a polypeptide and/or polynucleotide of the biomarker for a psychiatric disorder from the extracellular vesicles collected in 2 above. The method for detecting a biomarker for a psychiatric disorder may further include a step of comparing the measurement value with a corresponding reference value and determining whether the measurement value is within or outside the reference range. If the measurement value is outside the reference range, it can be determined that the subject from whom the sample was collected is suffering from a psychiatric disorder. Alternatively, if the measurement value is within the reference range, it can be determined that the patient from whom the sample was collected is not suffering from a psychiatric disorder.

Furthermore, the severity of the neuropsychiatric disorder may be determined by determining the extent to which the measured values deviate from the baseline values.

Psychiatric and neurological disorders may include psychiatric disorders and neurological disorders. Neurological disorders may include, for example, neurodegenerative disorders, neurological dysfunction after brain and spinal cord trauma, brain tumors, brain and spinal cord disorders associated with infections, and multiple sclerosis. Neurodegenerative disorders may include, for example, dementia, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, multiple system atrophy, and triplet repeat disease. Dementia may include, for example, Alzheimer's disease, senile dementia, Lewy body disease, frontotemporal dementia, vascular dementia, alcoholic dementia, and corticobasal degeneration. Brain and spinal cord disorders associated with infections may include, for example, meningitis, brain abscess, Crotzfeldt-Jakob disease, and AIDS dementia. Brain tumors may include, for example, astrocytoma.

Mental illnesses may include, for example, schizophrenia, depression, and bipolar disorder.

For example, tau protein, particularly phosphorylated tau protein, contained in extracellular vesicles can be a biomarker for Alzheimer's disease. If 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.

Biomarkers reported for each disease include α-synuclein for Parkinson's disease and dementia with Lewy bodies; TAR DNA-binding protein (TDP-43) for amyotrophic lateral sclerosis and frontotemporal dementia; abnormal prion protein for Creutzfeldt-Jakob disease; and Neurofilament Light Chain for neurological dysfunction after brain and spinal cord trauma, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, multiple system atrophy, triplet repeat disease, Alzheimer's disease, disease with Lewy bodies, frontotemporal dementia, vascular dementia, and corticobasal degeneration. Other examples include insulin-like growth factor (IGF-1) and brain-derived neurotrophic factor (BDNF) for depression; microRNAs hsa-miR-34a and hsa-miR-432 for schizophrenia; and IGF-1 and BDNF for bipolar disorder.

The biomarkers for neuropsychiatric disorders contained in extracellular vesicles can be detected as polypeptides or polynucleotides. The polynucleotides may be RNA or DNA, and the RNA may include not only mRNA but also microRNA, ncRNA, etc. The polypeptides and/or polynucleotides of the biomarkers for neuropsychiatric disorders may include full-length ones as well as fragments.

Methods for detecting biomarkers for psychiatric and neurological disorders as polypeptides include known methods such as Western blotting and Enzyme-Linked Immunosorbent Assay (ELISA). Methods for detecting biomarkers for psychiatric and neurological disorders as RNA include known methods such as RT-PCR (including quantitative RT-PCR), microarrays, and RNA-Seq. Methods for detecting biomarkers for psychiatric and neurological disorders as DNA include known methods such as PCR (including quantitative PCR), microarrays, and sequencing.

When biomarkers for psychiatric and neurological disorders are detected as polypeptides using Western blotting, ELISA, etc., the extracellular vesicles are dissolved in a specified dissolution buffer as a pretreatment. The sample dissolved in the dissolution buffer is used as the test sample.

The primary antibody for detecting a biomarker for a psychiatric and neurological disorder by Western blotting, ELISA, etc. is not limited as long as it can detect the biomarker for a psychiatric and neurological disorder.

When biomarkers for psychiatric and neurological disorders are detected as RNA using RT-PCR, microarrays, RNA-Seq, etc., RNA is extracted from extracellular vesicles as a pretreatment. If necessary, the extracted RNA may be used as a template for reverse transcription to synthesize complementary DNA (cDNA). RNA or cDNA can be used to detect the biomarkers.

The primers used in RT-PCR (which may include a probe in the case of quantitative RT-PCR) can be commercially available. Commercially available microarrays can also be used.

RNA-Seq can obtain the number of reads of biomarker mRNA for neuropsychiatric disorders using a next-generation sequencer (e.g., manufactured by Illumina).

When biomarkers for psychiatric and neurological disorders are detected as DNA using PCR, microarrays, sequencing, etc., DNA is extracted from extracellular vesicles as a pretreatment. If necessary, an amplification reaction may be performed using the extracted DNA as a template. The DNA extracted from extracellular vesicles or the amplified DNA can be used to detect the biomarkers.

　Commercially available primers can be used for PCR (which may include a probe in the case of quantitative PCR). Commercially available microarrays can also be used.

Sequencing can be performed using a next-generation sequencer (e.g., manufactured by Illumina) to obtain the number of DNA reads associated with biomarkers for neuropsychiatric disorders.

When a biomarker for a neuropsychiatric disorder is detected by Western blotting, ELISA, RT-PCR, PCR, RNA-Seq, sequencing, microarray, or the like, if the biomarker for a neuropsychiatric disorder is detected in extracellular vesicles, it may be determined that "the biomarker for a neuropsychiatric disorder has been detected" or that "the expression of the biomarker for a neuropsychiatric disorder is positive." Alternatively, by comparing the amount of the polypeptide or the amount of the polynucleotide of the biomarker for a neuropsychiatric disorder derived from the extracellular vesicles of a subject with the amount of the polypeptide or the polynucleotide of the biomarker for a neuropsychiatric disorder derived from the extracellular vesicles of a healthy individual, it may be determined that "the biomarker for a neuropsychiatric disorder has been detected" or that "the expression of the biomarker for a neuropsychiatric disorder is positive." If the amount of the polypeptide or the amount of the polynucleotide of the biomarker for a neuropsychiatric disorder in the test sample derived from the subject is higher than the amount of the polypeptide or the polynucleotide of the biomarker for a neuropsychiatric disorder in the extracellular vesicles derived from the healthy individual, it may be determined that "the biomarker for a neuropsychiatric disorder has been detected" or that "the expression of the biomarker for a neuropsychiatric disorder is positive." In addition, when the amount of the polypeptide of the biomarker for a neuropsychiatric disease or the amount of the polynucleotide of the biomarker for a neuropsychiatric disease in the extracellular vesicles derived from the subject is comparable to the amount of the polypeptide of the biomarker for a neuropsychiatric disease or the amount of the polynucleotide of the biomarker for a neuropsychiatric disease in the extracellular vesicles derived from a healthy individual, it may be determined that "the biomarker for a neuropsychiatric disease is not detected" or that "the expression of the biomarker for a neuropsychiatric disease is negative." Here, "showing a high value" can be exemplified by 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 higher. "Similar" can be exemplified by about 0.8 times to less than 1.2 times. In addition, before comparing the amount of the polypeptide of the biomarker for a neuropsychiatric disease or the amount of the polynucleotide of the biomarker for a neuropsychiatric disease, the amount of extracellular vesicles purified from each specimen may be normalized by the amount of the polypeptide of CD9, CD63, CD81, etc., which are markers of extracellular vesicles. The number of extracellular vesicles may be normalized based on the number of particles measured by the Nanoparticle Tracking Analysis method or the like. The amount of polypeptide may be expressed as mass or concentration, or may be expressed as the luminescence intensity of the substrate. The amount of polynucleotide may be the copy number or read number of the polynucleotide, or may be expressed as fluorescence intensity or the like.

In another embodiment, a reference value for the amount of a biomarker polypeptide or RNA for a neuropsychiatric disorder may be determined in advance, and when the amount of a biomarker polypeptide or RNA for a neuropsychiatric disorder in extracellular vesicles derived from a subject is outside the reference range, it may be determined that a "biomarker for a neuropsychiatric disorder has been detected" or that the expression of a biomarker for a neuropsychiatric disorder is positive. In addition, when the amount of a biomarker polypeptide or RNA for a neuropsychiatric disorder derived from extracellular vesicles derived from a subject is within the reference range, it may be determined that a "biomarker for a neuropsychiatric disorder has not been detected" or that the expression of a biomarker for a neuropsychiatric disorder is negative. The reference value is not limited as long as it is a value that can determine whether the amount of a polypeptide for a biomarker for a neuropsychiatric disorder or the amount of a polynucleotide for a biomarker for a neuropsychiatric disorder has been detected or that the expression is positive, and can be determined by a known method. The value that can determine whether the amount of a polypeptide of a biomarker for a neuropsychiatric disorder or the amount of a polynucleotide of a biomarker for a neuropsychiatric disorder is detected or whether expression is positive can also be determined by a receiver operating characteristic curve (ROC) curve, discriminant analysis method, mode method, Kittler method, 3σ method, p-tile method, etc. Examples of reference values include sensitivity, specificity, negative predictive value, positive predictive value, first quartile, etc.

One embodiment of the method for detecting biomarkers for psychiatric and neurological disorders is a method for measuring the total amount of tau in extracellular vesicles, which includes measuring the total amount of tau in extracellular vesicles obtained by the method for isolating extracellular vesicles from plasma using an ultrasensitive digital ELISA.

4. Method for recovering components derived from brain nerve cells In this embodiment, components derived from brain nerve cells are recovered using the extracellular vesicles recovered in 2 above.

The method for recovering components derived from brain nerve cells includes a step of recovering at least one biomolecule selected from the group consisting of sugars, lipids, polypeptides, and polynucleotides from the extracellular vesicles recovered in 1 above.

The sugars may include monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The sugars may also be bound to lipids, proteins, or the like. The sugars can be recovered from the extracellular vesicles by known methods using hydrazide-oxyamine-bearing polymers, lectins, and the like.

Lipids may include fatty acids, eicosanoids, triacylglycerols, wax esters, phospholipids, sphingolipids, isoprenoids, lipoproteins, etc. Lipids can be extracted from extracellular vesicles using the Folch method, a lipid extraction kit (Cell Biolabs, Inc.), etc.

Polypeptides and polynucleotides may include the biomarkers described in 2. above, as well as components other than the biomarkers. The explanation of the biomarkers is incorporated herein by reference.

Polypeptides and polynucleotides can be extracted according to known methods. Commercially available extraction kits may also be used.

5. Test Reagent In this section, a test reagent for recovering extracellular vesicles will be described. The test reagent contains the anti-APLP1 monoclonal antibody of the present invention. The description of the anti-APLP1 monoclonal antibody is incorporated herein by reference in the above 1.

The test reagent may contain one or more types of anti-APLP1 monoclonal antibodies.

The anti-APLP1 monoclonal antibody contained in the test reagent may be in a dry state or may be dissolved in a buffer such as phosphate-buffered saline. Furthermore, the test reagent may contain at least one of the following: a stabilizer such as β-mercaptoethanol or DTT; a protective agent such as albumin; a surfactant such as polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (10) octylphenyl ether; or a preservative such as sodium azide.

The anti-APLP1 monoclonal antibody may be labeled with an enzyme or a fluorescent dye. The antibody that binds to adipophilin may be immobilized on a microplate, magnetic beads, etc.

6. Test Kit In this section, a kit for recovering extracellular vesicles containing the test reagent described in 5 above will be described.

The test kit may be provided as a test kit including a test reagent and a package insert that describes how to use the reagent or that lists the URL of a web page that describes how to use the reagent. In addition, when the anti-APLP1 antibody is not bound to a carrier or solid phase, the test kit may include secondary antibody-bound beads, protein A beads, protein G beads, secondary antibody-immobilized microplates, protein A-immobilized microplates, protein G-immobilized microplates, secondary antibody-immobilized tubes, etc., for binding the anti-APLP1 antibody to a carrier or solid phase.

Furthermore, the test kit may contain a size exclusion chromatography column, an affinity purification column, a polyether such as polyethylene glycol, etc., for crude purification of extracellular vesicles.

Furthermore, the sample may contain antibodies, probes, primers, etc. for detecting biomarkers for neuropsychiatric disorders present in the extracellular vesicles.

The present invention will be described in more detail below based on examples. However, the examples are merely illustrative of the present invention, and the present invention is not intended to be limited to the examples.

Example 1-1
(Generation of human APLP1-specific monoclonal antibodies)
(A) To prepare a human APLP1-specific monoclonal antibody, iliac lymph nodes were collected from guinea pigs immunized with human APLP1 peptide (GTAVGDPSTRSWPPGSRVEG) (SEQ ID NO: 1) and a cell suspension was prepared from the lymph nodes. The cells ( ^1x107 cells) were fixed in 2% paraformaldehyde in PBS at 4°C for 10 minutes (fixation).

Cells were collected by centrifugation, and 250 μl of staining solution (PBST containing anti-guinea pig IgG-Dylight650, APLP1 peptide-streptavidin Dylight488, streptavidin Dylight550, and 200 units of RNaseOUT) was added to the cells, and the cells were left on ice for 15 minutes for staining (cell membrane lysis and reaction of the antigen to be labeled).

3 ml of PBS solution containing 0.2 μg/ml DAPI was added to this solution to stain the cell nuclei.

These cells were analyzed using a flow cytometer to separate APLP1 peptide-specific plasma cells. Single cell populations were identified by setting the R1 gate (FSC vs SSC) and R2 gate (DAPI). Autofluorescent cells and non-specifically stained cells were further removed from the single cell population using the FL2 channel (R3 gate). Among these cells, cells that were strongly anti-mouse IgG positive, strongly APLP1 peptide positive, and streptavidin Dylight550 negative were identified as APLP1 peptide-specific plasma cells (R4 gate). The results are shown in Figure 3.

(C) Single cell sorting was performed on the cells in the R4 gate (cell isolation). After that, cDNA was synthesized from the cells separated by the cell sorter according to the method of Kurosawa et al. (Non-Patent Documents 2 and 3), and the rat γ-chain and κ-chain variable region genes were amplified using this as a template by 5'-RACE PCR. The amplified variable regions were incorporated into a linearized expression vector to create guinea pig γ-chain and κ-chain immunoglobulin expression units. The results of agarose electrophoresis of the amplified DNA are shown.

From the obtained variable region genes, full-length heavy and light chain immunoglobulin genes were created according to the method of Kurosawa et al. (Non-Patent Documents 2 and 3), and these were introduced into 293FT cells to produce recombinant antibodies. The reactivity of the obtained antibodies against the APLP1 peptide was analyzed by ELISA. Among these, antibody #11 showed the highest reactivity against the APLP1 peptide. The results are shown in Figure 4.

Using the obtained APLP1 peptide-positive antibody clone, immunostaining was performed on SHSY-5Y cells, a human neuroblastoma that expresses endogenous APLP1, and on APLP1 gene knockout SHSY-5Y cells. #11 showed dot-like localization in the cytoplasm of SHSY-5Y cells, but no signal was observed in the knockout cells. The results are shown in Figure 5.

　The extract from HEK293 cells in which the human APLP1 gene was forcibly expressed was separated by SDS electrophoresis, and Western blotting was performed using #11 as the primary antibody. In HEK293 cells in which the human APLP1 gene was forcibly expressed, a single band was observed at 75 kDa, which corresponds to the molecular weight of APLP1, but no signal was observed in the negative control wild-type HEK293 cells. The results are shown in Figure 6.

The amino acid sequence of human APLP1 (SEQ ID NO: 1) is shown in Figure 2, the #11 light chain amino acid sequence (SEQ ID NO: 2) is shown in Table 3, and the #11 heavy chain amino acid sequence (SEQ ID NO: 3) is shown in Table 4.

Example 1-2
(Identification of the minimal epitope for human APLP1-specific monoclonal antibodies)
Fusion proteins in which the APLP1-derived peptide sequence (RSWPPGSRVE (SEQ ID NO: 20), RSWPPGSRV (SEQ ID NO: 21), WPPGSRVE (SEQ ID NO: 22), PPGSRVE (SEQ ID NO: 23), PGSRVE (SEQ ID NO: 24)) was added to the C-terminus of GFP were expressed in HEK293 cells by gene transfer. After 24 hours of culture, the cells were fixed with 4% formalin/PBS, membranes were solubilized with 0.1% TritonX-100/PBS, and then reacted with #11 (0.1 μg/mL PBS) for 1 hour. Signals were detected by staining the cells with an anti-guinea pig antibody (Dylight 650). The results are shown in Figure 7.

Results: When the peptide RSWPPGSRVE was added to GFP, GFP-positive cells were positive for #11, but when the peptide RSWPPGSRV was added to GFP, GFP-positive cells were not positive for #11. This indicates that E is required for the C-terminal epitope recognized by #11.

Next, when the peptide PPGSRVE was added to GFP, GFP-positive cells were positive for #11, but when the peptide PGSRVE was added to GFP, GFP-positive cells were not positive for #11. These results demonstrated that the minimal epitope recognized by #11 is PPGSRVE.

Examples 1-3
(1) APLP1 cDNA was introduced into HEK293 cells, and after 48 hours of culture, the cells were treated with 0.1% TritonX-100/PBS to obtain a cell extract. Proteins (equivalent to 10 μg) in the cell extract were separated using SDS polyacrylamide and transferred to a nitrocellulose membrane, after which APLP1 was detected using #11. The results are shown in Figure 8. A band corresponding to the molecular weight of APLP1 of 75 kDa was detected.

(2) SHSY-5Y cells were fixed in 4% formalin/PBS, membranes were solubilized in 0.1% TritonX-100/PBS, and then reacted with #11 (0.1 μg/mL PBS) for 12 hours. Signals were detected by cell staining with anti-guinea pig antibody (Dylight 650). The results are shown in Figure 9.

Example 2: Recovery of brain neuron-derived extracellular vesicles from plasma using human APLP1-specific monoclonal antibody #11 (1) Sample Blood was collected from healthy adults using EDTA-2K tubes, and 20 mL of plasma was separated. The plasma was stored at -80°C until testing. When used, the plasma was thawed and centrifuged at 2,500×g and 4°C for 10 minutes, and the supernatant was collected.

(2) Crude purification of plasma extracellular vesicles Using qEV10 (Izon Science), 20 mL of size exclusion chromatography (SEC) fraction was collected from 10 mL of plasma according to the kit protocol. The above procedure was repeated twice, and 40 mL of SEC fraction was collected. The SEC fraction was concentrated to 3,000 μL using an Amicon Ultra-15 100 kDa MWCO.

(3) Preparation of beads for immunoprecipitation Human APLP1-specific monoclonal antibody #11 prepared in Example 1 was biotinylated using EZ-Link NHS-PEG4-Biotin No-Weigh Format (Thermo Fisher Scientific) according to the protocol included with the kit, and excess biotin was removed and purified using Zeba Spin Desalting Columns 40K MWCO 0.5mL (Thermo Fisher Scientific) according to the protocol included with the kit.

20 μL of Dynabeads® MyOne Streptavidin C1 (Thermo Fisher Scientific) and 10 μg of biotinylated human APLP1-specific monoclonal antibody #11 were mixed in Dulbecco's PBS containing 0.01% Tween-20 to produce magnetic beads bound to human APLP1-specific monoclonal antibody #11.

Furthermore, the antibody-bound magnetic beads were added to 800 μL of blocking buffer containing 2% (w/v) ECL ^™ Prime Blocking Reagent, and incubated overnight at 4° C. to block the surface.

(4) Immunoprecipitation of brain neuron-derived extracellular vesicles 1,444 μL of concentrated SEC fraction was added to the immunoprecipitation beads prepared in (3) above, and incubated at 4°C for 6 hours with inversion mixing. The tube was spun down, attached to a magnetic stand, waited for 1 minute, and the supernatant was removed. Next, to wash the magnetic beads, 800 μL of D-PBS(-) was added to the tube and mixed by inversion. The tube was spun down, attached to a magnetic stand, waited for 1 minute, and the supernatant was removed. The tube was spun down again, attached to a magnetic stand, waited for 1 minute, and the supernatant was completely removed.

17.4μL of 0.1M glycine-HCl (pH 2.8) was added to the tube containing the remaining magnetic beads, which was then vortexed and incubated at 4°C for 10 minutes. The tube was then spun down, placed on a magnetic stand and left for 1 minute. The supernatant was then collected and 2.6μL of 1M Tris-HCl (pH 8.0) was added to adjust the pH before preparation for analysis.

(5) Western Blot
The supernatant collected in the previous step was mixed with a denaturing buffer and heated to prepare a sample for application. The sample was loaded onto a 5-15% Tris-glycine SDS gel and electrophoresed at 200 V for 55 minutes. After electrophoresis, the SDS gel was electrophoresed at 15 V for 30 minutes in Towbin Buffer (containing 5% methanol) under semi-dry conditions, and the gel was transferred to a PVDF membrane.

Blocking was performed at room temperature for 5 minutes using Blocking One (Nacalai Tesque, Inc.). Primary antibodies were diluted with 5% Blocking One dissolved in TBS-T and incubated overnight at 4°C. The antibodies used were anti-APLP1 antibody (R&D Systems, AF3129), anti-CD81 antibody (Cosmo Bio, SHI-EXO-M03), and anti-CD9 antibody (Cosmo Bio, SHI-EXO-M01). Anti-APLP1 antibody and anti-CD81 antibody were diluted 2,000-fold, and anti-CD9 antibody was diluted 1,000-fold. After incubation with the primary antibodies, the cells were washed six times with TBS-T for 5 minutes each.

The secondary antibody was diluted with 5% Blocking One dissolved in TBS-T and incubated 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. Six washes were performed using TBS-T for 5 minutes each.

1,400μL of ImmunoStar (registered trademark) LD A+B solution was added to the membrane and allowed to react at room temperature for 5 minutes. HRP was induced to emit light using a MyECL Imager (Thermo Scientific) and the signal was imaged.

(6) Results The results of Western Blot are shown in FIG. 10-1.

In addition to the target APLP1, EV proteins CD81 and CD9 were detected by immunoprecipitation using plasma. This demonstrated that APLP1-positive EVs, which are extracellular vesicles derived from brain neurons, can be isolated by using specific monoclonal antibody #11.

Furthermore, in immunoprecipitation using plasma, the detection of APLP1 and the EV proteins CD9 and CD63 was compared using the specific monoclonal antibody #11 of the present invention or a commercially available polyclonal antibody (Human APLP-1 Antibody (R&D Systems, #AF3129)). The results of Western Blot are shown in Figure 10-2. As a result, APLP1, CD9, and CD63 could be detected using specific monoclonal antibody #11. In contrast, when a commercially available polyclonal antibody was used, APLP1 could not be detected, and it was difficult to detect CD9 and CD63.

Example 3 Recovery of extracellular vesicles from culture supernatant of neurons differentiated from iPS cells (1) Preparation of beads for immunoprecipitation Human APLP1-specific monoclonal antibody #11 prepared in Example 1 was biotinylated using EZ-Link NHS-PEG4-Biotin No-Weigh Format (Thermo Fisher Scientific) according to the protocol included in the kit, and excess biotin was removed and purified using Zeba Spin Desalting Columns 40K MWCO 0.5mL (Thermo Fisher Scientific) according to the protocol included in the kit.

20 μL of Dynabeads® MyOne Streptavidin C1 (Thermo Fisher Scientific) and 10 μg of biotinylated human APLP1-specific monoclonal antibody #11 were mixed in Dulbecco's PBS containing 0.01% Tween-20 to produce magnetic beads bound to human APLP1-specific monoclonal antibody #11.

Furthermore, the antibody-bound magnetic beads were added to 800 μL of blocking buffer containing 2% (w/v) ECL ^™ Prime Blocking Reagent, and incubated overnight at 4° C. to block the surface.

(2) Preparation of extracellular vesicles (EVs) in culture supernatant Culture supernatant of neural cells differentiated from iPS cells was centrifuged at 2,500g for 10 minutes, and 80 mL of the supernatant was concentrated to 20 mL using an Amicon Ultra-15 100kDa MWCO. Using qEV10 (Izon Science), 20 mL of size exclusion chromatography (SEC) fraction was collected from 10 mL of concentrated culture supernatant according to the kit protocol. The above was carried out twice in total, and 40 mL of SEC fraction was collected. The SEC fraction was concentrated to 1,300 μL using an Amicon Ultra-15 100kDa MWCO.

(3) Immunoprecipitation and Western Blot
300 μL of concentrated SEC fraction was added to the immunoprecipitation beads and incubated for 8 hours at 4°C with inversion mixing. The tube was spun down and then attached to a magnetic stand, waited for 1 minute, and the supernatant was removed. Next, to wash the magnetic beads, 800 μL of D-PBS(-) was added to the tube and mixed by inversion. The tube was spun down and then attached to a magnetic stand, waited for 1 minute, and the supernatant was removed. The tube was spun down again, attached to a magnetic stand, waited for 1 minute, and the supernatant was completely removed.

64μL of 1x RIPA buffer (without SDS) (Nacalai Tesque, Inc., 08714-04) prepared according to the kit instructions was added to the tube containing the beads, vortexed, and incubated at 4℃ for 10 minutes. After centrifugation, the tube was attached to a magnetic stand and left to stand for 1 minute to collect the supernatant, which was then mixed with denaturing buffer and heated to prepare the sample for application. The sample was loaded onto a 5-15% Tris-glycine SDS gel and electrophoresed at 200V for 55 minutes. After electrophoresis, the SDS gel was electrophoresed under semi-dry conditions in Towbin Buffer (containing 5% methanol) at 15V for 30 minutes, and the gel was transferred to a PVDF membrane.

Blocking was performed at room temperature for 5 minutes using Blocking One (Nacalai Tesque, Inc.). APLP1 C-terminus antibody (Calbiochem 171615) diluted 10,000 times in 5% Blocking One dissolved in TBS-T, CD63 antibody (Santa Cruz sc-5275) diluted 2,000 times, and CD81 antibody (Santa Cruz sc-23962) diluted 2,000 times were added and incubated overnight at 4°C with shaking. After incubation, the membrane was washed six times for 5 minutes with 0.1% TBS-T.

The secondary antibody was diluted with 5% Blocking One dissolved in TBS-T and incubated 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. Six washes were performed using TBS-T for 5 minutes each.

800 μL each of ImmunoStar® LD (Wako) solution A and B were mixed, the membrane was immersed in the solution and left to stand for 5 minutes. Signals were detected (10-300 seconds) using a MY ECL Imager (Thermo Fisher Scientific).

(4) Results The results of Western Blot are shown in FIG.

Lane 1 is 1μg of iPS neuron lysate, lane 2 is EV before immunoprecipitation, lane 3 is input, lane 4 is the supernatant from immunoprecipitating EV, and lane 5 is antibody-coupled beads from immunoprecipitating EV. 1/40 of the total was electrophoresed for input and supernatant.

APLP1, EV marker proteins (CD63, Flotillin-1), and neural-derived proteins (L1CAM, SNAP25) were detected with antibody beads. This demonstrated that extracellular vesicles can be recovered by using human APLP1-specific monoclonal antibody #11.

Example 4
Comparison of total tau amount in CSF and NDE, comparison of total tau amount in NDE with phosphorylated tau (pTau181) and Aβ42/Aβ40 in CSF (1) Samples Blood was collected from patients using EDTA-2K tubes, and 10 mL of plasma was separated. The plasma was stored at -80°C until testing. When used, the plasma was thawed and centrifuged at 10,000×g at room temperature for 5 minutes, and the supernatant was collected. 100 μL of D-PBS(-) was added to 400 μL of plasma.

(2) Crude purification of plasma extracellular vesicles Using qEV original (Izon Science), 1,500 μL of size exclusion chromatography (SEC) fraction was collected from 500 μL of D-PBS(-)-added plasma according to the protocol attached to the kit.

(3) Preparation of beads for immunoprecipitation Human APLP1-specific monoclonal antibody #11 prepared in Example 1 was biotinylated using EZ-Link NHS-PEG4-Biotin No-Weigh Format (Thermo Fisher Scientific) according to the protocol included with the kit, and excess biotin was removed and purified using Zeba Spin Desalting Columns 40K MWCO 0.5mL (Thermo Fisher Scientific) according to the protocol included with the kit.

10 μL of Dynabeads (registered trademark) MyOne Streptavidin C1 (Thermo Fisher Scientific) and 4 μg of biotinylated human APLP1-specific monoclonal antibody #11 were mixed in D-PBS(-) containing 0.01% Tween-20 to prepare magnetic beads bound to specific monoclonal antibody #11.

Furthermore, the antibody-bound magnetic beads were added to 800 μL of blocking buffer containing 2% (w/v) ECL ^™ Prime Blocking Reagent, and incubated overnight at 4° C. to block the surface.

(4) Immunoprecipitation of brain neuron-derived extracellular vesicles 1,444 μL of concentrated SEC fraction was added to the immunoprecipitation beads prepared in (3) above, and incubated at 4°C for 6 hours with inversion mixing. The tube was spun down, attached to a magnetic stand, waited for 1 minute, and the supernatant was removed. Next, to wash the magnetic beads, 800 μL of D-PBS(-) was added to the tube and mixed by inversion. The tube was spun down, attached to a magnetic stand, waited for 1 minute, and the supernatant was removed. The tube was spun down again, attached to a magnetic stand, waited for 1 minute, and the supernatant was completely removed.

105μL of 0.1M glycine-HCl (pH 2.8) was added to the tube containing the remaining magnetic beads, which was then vortexed and incubated at 4°C for 10 minutes. The tube was then spun down, placed on a magnetic stand and left for 1 minute. The supernatant was then collected and 15.8μL of 1M Tris-HCl (pH 8.0) was added to adjust the pH before preparation for analysis.

(5) Simoa Tau 2.0 assay
The supernatant collected in the previous step was dispensed into a dedicated 96-well plate, and the Tau concentration was measured using the Simoa HD-1 Analyzer instrument and the Simoa Tau 2.0 Advantage Kit according to the kit instructions. This resulted in the determination of the total amount of Tau contained in extracellular vesicles isolated from plasma using human APLP1-specific monoclonal antibody #11.

Cerebrospinal fluid was collected by lumbar puncture on the same day as plasma was collected in (1). The cerebrospinal fluid was centrifuged at 430xg for 5 minutes at room temperature, aliquoted, and stored at -80°C. The amounts of total tau, phosphorylated tau (pTau181), Aβ42, and Aβ40 were measured by sandwich ELISA.

(6) Results Figure 12 shows a scatter plot showing the correlation between the measured total tau amount in plasma brain neuron-derived extracellular vesicles and the total tau in cerebrospinal fluid, and Figure 13 shows a scatter plot showing the correlation between the measured total tau amount in plasma brain neuron-derived extracellular vesicles and phosphorylated tau (pTau181) (left figure) and the Aβ42/Aβ40 ratio (right figure).

The total amount of tau contained in the extracellular vesicles isolated by immunoprecipitation using human APLP1-specific monoclonal antibody #11 and plasma correlated well with the total tau, phosphorylated (pTau181), and Aβ42/Aβ40 ratio contained in the cerebrospinal fluid. This demonstrated that APLP1-positive EVs, which are extracellular vesicles derived from brain neurons, could be isolated and reflect the amount of biomarkers in the cerebrospinal fluid. Furthermore, it was found that the total amount of tau contained in the extracellular vesicles isolated from plasma using human APLP1-specific monoclonal antibody #11 could be a biomarker for neuropsychiatric disorders, and that the total amount of tau detected by this method could be used to detect neuropsychiatric disorders using plasma, which is easy to collect, as a sample instead of cerebrospinal fluid, which is difficult to collect.

The present invention is useful in fields related to biomarkers for neuropsychiatric disorders.

SEQ ID NO: 1: Human APLP1 amino acid sequence (NCBI accession no. NP_031493.2)
SEQ ID NO:2: #11 light chain amino acid sequence SEQ ID NO:3: #11 heavy chain amino acid sequence SEQ ID NO:4: #11 light chain FR1
SEQ ID NO:5: #11 Light chain CDR1
SEQ ID NO:6: #11 Light chain FR2
SEQ ID NO:7: #11 Light chain CDR2
SEQ ID NO: 8: #11 Light chain FR3
SEQ ID NO: 9: #11 Light chain CDR3
SEQ ID NO: 10: #11 light chain FR4
SEQ ID NO: 11: #11 heavy chain FR1
SEQ ID NO: 12: #11 heavy chain CDR1
SEQ ID NO: 13: #11 heavy chain FR2
SEQ ID NO: 14: #11 heavy chain CDR2
SEQ ID NO: 15: #11 heavy chain FR3
SEQ ID NO: 16: #11 heavy chain CDR3
SEQ ID NO: 17: #11 heavy chain FR4
SEQ ID NO:18: #11 light chain signal sequence SEQ ID NO:19: #11 heavy chain signal sequence SEQ ID NO:20: Peptide sequence for identifying minimal epitope SEQ ID NO:21: Peptide sequence for identifying minimal epitope SEQ ID NO:22: Peptide sequence for identifying minimal epitope SEQ ID NO:23: Peptide sequence for identifying minimal epitope SEQ ID NO:24: Peptide sequence for identifying minimal epitope

Claims (14)

An anti-APLP1 monoclonal antibody that has as its epitope the amino acid sequence from 232 to 238 in the amino acid sequence set forth in SEQ ID NO:1 in the sequence listing. An anti-APLP1 monoclonal antibody in which the light chain CDR1, CDR2, and CDR3 have the amino acid sequences set forth in SEQ ID NOs: 5, 7, and 9, respectively, and the heavy chain CDR1, CDR2, and CDR3 have the amino acid sequences set forth in SEQ ID NOs: 12, 14, and 16, respectively. An anti-APLP1 monoclonal antibody according to claim 2, in which FR1, FR2, FR3 and FR4 of the light chain have the amino acid sequences set forth in SEQ ID NOs: 4, 6, 8 and 10, respectively, and FR1, FR2, FR3 and FR4 of the heavy chain have the amino acid sequences set forth in SEQ ID NOs: 11, 13, 15 and 17, respectively. A step of mixing a sample containing an anti-APLP1 antibody and extracellular vesicles to form a complex of the anti-APLP1 antibody and the extracellular vesicles, and a step of recovering the complex of the anti-APLP1 antibody and the extracellular vesicles.
Including,
The anti-APLP1 antibody is an anti-APLP1 monoclonal antibody according to claim 1 or 2.
A method for recovering extracellular vesicles derived from brain neurons. The method of claim 4, wherein the sample containing extracellular vesicles is plasma. A method for detecting a biomarker for a neuropsychiatric disorder, comprising the steps of recovering extracellular vesicles by the method according to claim 4, and obtaining measurements of polypeptides and/or polynucleotides that serve as biomarkers for a neuropsychiatric disorder from the recovered extracellular vesicles. The detection method of claim 6, further comprising the step of comparing the obtained measurement value with a corresponding reference value and determining whether the measurement value is within or outside the reference range. The detection method according to claim 6, wherein the biomarker is tau protein or phosphorylated tau protein. A method for recovering components derived from brain nerve cells, comprising the steps of recovering extracellular vesicles by the method of claim 4, and recovering at least one biomolecule selected from the group consisting of sugars, lipids, polypeptides, and polynucleotides from the recovered extracellular vesicles. A test reagent used for recovering extracellular vesicles, comprising the anti-APLP1 monoclonal antibody according to claim 1 or 2. The test reagent according to claim 10, which is used to carry out the method for recovering extracellular vesicles according to claim 4. The test reagent according to claim 10, which is used to carry out the method for detecting a biomarker for a neuropsychiatric disorder according to claim 6. The test reagent according to claim 10, which is used to carry out the method for recovering components derived from brain nerve cells according to claim 9. A test kit comprising the test reagent according to claim 10.