WO2022131208A1 - Method for detecting membrane structural body having lipid bilayer - Google Patents

Method for detecting membrane structural body having lipid bilayer Download PDF

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Publication number
WO2022131208A1
WO2022131208A1 PCT/JP2021/045817 JP2021045817W WO2022131208A1 WO 2022131208 A1 WO2022131208 A1 WO 2022131208A1 JP 2021045817 W JP2021045817 W JP 2021045817W WO 2022131208 A1 WO2022131208 A1 WO 2022131208A1
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Prior art keywords
membrane structure
sample
lipid bilayer
virus
test strip
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PCT/JP2021/045817
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French (fr)
Japanese (ja)
Inventor
憲祐 齊藤
逸明 楊
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株式会社堀場製作所
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Priority to JP2022569983A priority Critical patent/JPWO2022131208A1/ja
Publication of WO2022131208A1 publication Critical patent/WO2022131208A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a method for detecting a membrane structure having a lipid bilayer, a kit for performing the method, a test strip, and the like.
  • Patent Document 1 a method that requires an expensive dedicated device such as MACS for detection
  • Patent Document 2 Only what is needed (Patent Document 2) is known.
  • the present inventors have investigated in detail a conventional method for detecting a membrane structure having a lipid bilayer such as a virus, a bacterium, or an exosome.
  • a method of labeling a membrane structure having a lipid bilayer with a labeling reagent such as a fluorescent dye.
  • a labeling reagent such as a fluorescent dye.
  • the completed detection method will be further studied, and by detecting a protein specifically expressed in a disease in a membrane structure having a lipid bilayer by the detection method of the present invention, the presence or absence of disease, etc. It was also found that the present invention could be further completed.
  • a method for detecting a membrane structure having a lipid bilayer (1) A step of labeling a membrane structure having a lipid bilayer present in a sample, A method comprising (2) a step of separating a fraction containing a membrane structure having a predetermined lipid bilayer, and (3) a step of detecting a membrane structure having the predetermined lipid bilayer in the fraction. ..
  • a kit for detecting a membrane structure having a lipid bilayer is A reagent container containing a labeling reagent for labeling the membrane structure present in the sample, and It has a test strip for detecting the membrane structure from a sample containing the membrane structure labeled with the labeling reagent.
  • the test strip A sample receiving unit that receives a sample containing the labeled membrane structure, and a sample receiving unit.
  • a filter unit that selectively passes the labeled membrane structure in the sample received by the sample receiving unit, and a filter unit.
  • the kit comprising a detection unit for detecting the labeled membrane structure that has passed through the filter unit based on the label.
  • a test strip for detecting a membrane structure having a lipid bilayer is A sample receiving unit that receives a sample containing the membrane structure, A conjugate section for labeling the membrane structure in the sample received by the sample receiving section, and a conjugate section. A filter unit that selectively passes the membrane structure labeled by the conjugate unit, and a filter unit.
  • the test strip comprising a detection unit for detecting the labeled membrane structure that has passed through the filter unit based on the label.
  • the present invention it is possible to provide a method for detecting a membrane structure having a lipid bilayer. Further, according to the present invention, it is possible to provide a method for determining a disease or the like based on a protein or the like expressed in a membrane structure having a lipid bilayer.
  • the method of the present invention is highly accurate and simple because it does not require an expensive dedicated device such as MACS for detection and it is not necessary to perform a plurality of reactions or processes before detection.
  • a membrane structure having a lipid bilayer can be detected easily and with high accuracy, a membrane structure having a lipid bilayer expressing a disease-specific protein can be efficiently detected, and therefore reliable. It is also possible to determine a highly sexual disease.
  • FIG. 1 is a schematic diagram illustrating the configuration of the kit and the test strip of the present invention.
  • the reagent container included in the kit is shown in a cross-sectional view and the test strip is shown in a perspective view for easy understanding (the same applies to FIG. 2 (a)).
  • shading and hatching are appropriately added to each part in order to distinguish the areas (the same applies to FIGS. 2 (a) and 2 (b)).
  • FIG. 2 is a schematic diagram showing an example of a preferable configuration of the kit and the test strip of the present invention.
  • FIGS. 1 is a schematic diagram illustrating the configuration of the kit and the test strip of the present invention.
  • the detection method of the present invention is as follows: A method for detecting a membrane structure having a lipid bilayer. (1) A step of labeling a membrane structure having a lipid bilayer present in a sample, A method comprising (2) a step of separating a fraction containing a membrane structure having a predetermined lipid bilayer, and (3) a step of detecting a membrane structure having the predetermined lipid bilayer in the fraction. I will provide a.
  • the detection method of the present invention is not particularly limited as long as it can detect a membrane structure having a lipid bilayer (membrane), but may be used, for example, in a test strip for lateral flow or the like.
  • a test strip may be configured to be used with a reagent container containing a labeling reagent (a test strip having no conjugate portion), as in the kit of the present invention described later, and may be described later.
  • all the elements necessary for carrying out the detection method may be arranged in one test strip.
  • a test strip suitable for carrying out the detection method of the present invention is, for example, a membrane structure having a lipid bilayer to be detected by the method of the present invention contained in a sample (hereinafter, “test membrane structure”). It detects (sometimes referred to as “body”) and quantifies the concentration, and is a sample dropping member (i) a sample pad (sample receiving part), in the direction in which the sample flows, and optionally (ii) a filter. (Iii) Expanded membrane (membrane), which has a test line where a capture substance having a specific affinity for the test membrane structure to be detected is immobilized, and the expanded membrane is optionally a lipid bilayer.
  • the test strip may also optionally have (ii) a conjugate section in front of the filter section for labeling a membrane structure having a lipid bilayer in the (ii') sample.
  • the width of the test strip suitable for carrying out the detection method of the present invention is not particularly limited as long as a membrane structure having a desired lipid bilayer can be detected, and is, for example, 1.5 mm to 5.0 mm.
  • the sample pad is a member that holds a sample (sample) containing a dropped analysis object for a certain period of time.
  • Examples of the sample pad include those made of cellulose, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, cotton cloth and the like, and more specifically, for example, Cellulose Absorbent Pad (manufactured by Pall) and the like. Can be mentioned.
  • the sample pad may be used after the sample pad is infiltrated with a buffer solution and dried.
  • the volume of the sample dropped on the sample pad is, for example, 25 ⁇ l to 100 ⁇ l.
  • the conjugate portion is a portion for labeling the membrane structure having a lipid bilayer in the sample, and contains a reagent capable of labeling the membrane structure.
  • the reagent may be contained in the conjugate portion in any state as long as it can label the membrane structure having a lipid bilayer in the sample.
  • the reagent is dissolved in an organic solvent such as DMSO. After that, it may be contained in a state where it is infiltrated into the conjugate portion and dried.
  • conjugate portion examples include those made of cellulose, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, cotton cloth, etc., and more specifically, for example, Glass Fiber Pad (manufactured by Millipore). And so on.
  • the conjugate portion may be integrated with the sample pad (sample receiving portion) or may be independent of each other.
  • the sample pad and the conjugate portion may be provided on a developing membrane (membrane) described later.
  • the developing film has a developing portion, and the developing portion may be the whole or a part of the developing film.
  • the shape of the test line provided with the detection portion on the developing membrane and the control line optionally provided is not particularly limited as long as the trapping substance is immobilized, and is, for example, line-shaped, circular, or circular. Examples include strips. Of the shapes, it is preferable that the width is a line shape of 0.5 mm to 2.0 mm.
  • the amount of the capture substance to be immobilized is not particularly limited as long as a membrane structure having a desired lipid bilayer can be detected. For example, when the capture substance is an antibody, the amount of antibody per 1 cm of line is 0.01. It is ⁇ 20 ⁇ g.
  • test line and the control line are not particularly limited as long as a substance (eg, a membrane structure having a lipid bilayer) desired to be detected in the sample can be detected, and for example, the development direction of the sample. It may be provided perpendicular to, parallel to, or the like. More specifically, when they are provided in parallel, the test line and the control line may be arranged in parallel on the left and right so that the distance from the position where the sample is dropped is the same as the test line and the control line (Fig.). See 240a and 240b (test line / control line) in 2 (a) and 340a and 340b (test line / control line) in Figure 2 (b)).
  • a substance eg, a membrane structure having a lipid bilayer
  • the water absorption pad is a member that absorbs the sample that has moved through the developing membrane due to the capillary phenomenon and constantly generates a constant flow.
  • Examples of the material of the water absorption pad include cellulose, non-woven fabric, cotton cloth, cellulose acetate and the like, and a fibrous cellulose material is preferable, and more specifically, for example, Cellulose Fiber Sample Pad (manufactured by Millipore) and the like. Be done.
  • the backing sheet has adhesiveness for immobilizing four members: (i) sample pad (sample receiving part), (ii) filter, (iii) developing membrane (membrane), and optionally (iv) absorption pad. It is a sheet, and examples of the sheet include a backing sheet AR9020 (manufactured by Adhesives Research).
  • the step (1) of the detection method of the present invention is a step of contacting a membrane structure having a lipid bilayer existing in a sample with a labeling reagent such as a fluorescent dye to label the membrane structure.
  • the step is not particularly limited as long as the membrane structure having the lipid bilayer present in the sample can be brought into contact with a labeling reagent such as a fluorescent dye to label the membrane structure, but on a test strip or the like. It may be done in, or in another place (eg, in a tube, etc.). If it is on a test strip, it may be performed, for example, in a conjugate section containing a labeling reagent as described later on the test strip.
  • the sample used in the detection method of the present invention is preferably a biological sample that is liquid, and examples of the biological sample include samples collected from animals or cells. If necessary, intracellular components (eg, exosomes, etc.) may be extracted or isolated by disrupting, lysing, or the like. Further, the sample may be a sample known to contain a membrane structure having a lipid bilayer (test membrane structure) to be detected by the method of the present invention, or may include a test membrane structure. It may be an unknown sample. Further, the sample may be appropriately diluted with a solvent (water, physiological saline, buffer solution, organic solvent, etc.) by a method known per se, or using various columns (eg, silica monolith, etc.). It may be purified or concentrated.
  • a solvent water, physiological saline, buffer solution, organic solvent, etc.
  • animal-derived samples include blood, serum, plasma, saliva, urine, tears, sweat, milk, nasal juice, semen, pleural effusion, gastrointestinal secretions, cerebrospinal fluid, intertissue fluid, lymph fluid and the like.
  • blood, serum or plasma Preferably blood, serum or plasma.
  • serum or plasma can be prepared by collecting blood from a test animal according to a conventional method and separating the humoral component, and cerebrospinal fluid can be prepared. It can be collected by a known means such as spinal puncture.
  • test membrane structure examples include enveloped viruses, bacteria, cells, exosomes (diameter 40 to 100 nm), microvesicles (diameter 100 to 1000 nm), apoptotic bodies (diameter 1000 to 5000 nm), and the like. ..
  • the enveloped virus may be a DNA virus or an RNA virus.
  • the DNA virus include a herpesvirus family (Family Herpesviridae) virus, a Poxviridae virus, a hepadnaviridae virus, and the like.
  • RNA viruses include Flavivirus family (Family Flaviviridae) virus, Togavirus family (Family Togaviridae) virus, Coronavirus family (Family Coronaviridae) virus, Hepatitis D virus (Hepatitis D virus), and Orthomixovirus family (Family). Orthomyxoviridae virus, Family Paramyxoviridae virus, Family Rhabdoviridae virus, Family Bunyaviridae virus, Family Filoviridae virus, Family Retroviridae virus, etc. Can be mentioned.
  • herpesvirus family virus examples include Varisellovirus virus (eg, Varicella Zoster virus, etc.) and Simplexvirus virus (eg, simple herpesvirus type 2 (eg, simple herpesvirus type 2). Herpes simplex virus 2), etc.), Roseolovirus virus (eg, human Herpesvirus 6, 7), etc.).
  • poxvirus family viruses include Genus Orthopoxvirus virus (eg, Variola virus, Cowpox virus, Vaccinia virus, etc.) and Parapox virus (Genus). Parapoxvirus) virus (eg, Orf virus, etc.) and the like.
  • hepadonavirus family virus examples include orthohepadnavirus (genus Orthohepadnavirus) virus (eg, Hepatitis B virus), Abihepadnavirus (Genus Avihepadnavirus) virus (eg, duck B). Hepatitis virus (Duck hepatitis B virus), etc.), Ross's goose hepatitis B virus, etc. can be mentioned.
  • orthohepadnavirus genus Orthohepadnavirus
  • Hepatitis B virus eg, Hepatitis B virus
  • Abihepadnavirus Gene Avihepadnavirus
  • Hepatitis virus Duck hepatitis B virus
  • Ross's goose hepatitis B virus etc.
  • Flaviviridae viruses include, for example, Hepacivirus virus (eg, Hepatitis C virus, etc.), Flaviviridae virus (eg, Japanese encephalitis virus). , Zikavirus, etc.).
  • Examples of the Togavirus family virus include a Genus Rubivirus virus (eg, Rubella virus, etc.) and the like.
  • Examples of the coronavirus family virus include beta coronavirus (Genus Betacoronavirus) virus (eg, SARS coronavirus (Severe acute respiratory syndrome coronavirus: SARS-CoV), SARS coronavirus 2 (SARS-CoV-2)), and MERS. Corona virus (Middle East respiratory syndrome corona virus, etc.) and the like can be mentioned.
  • Examples of the orthomixovirus family virus include influenza A virus (Influenzavirus A), influenza B virus (Influenzavirus B), influenza C virus (Influenzavirus C), and the like.
  • Examples of the paramyxovirus family virus include a genus Morbillivirus virus (eg, Measles morbillivirus), a Genus Orthopneumovirus virus (eg, RS virus (Respiratory syncytial virus)). Etc.) etc.
  • Examples of the rhabdoviridae virus include Lyssavirus virus (eg, Rabies virus, etc.) and the like.
  • Bunyaviridae virus examples include Nairovirus (Genus Nairovirus) virus (eg, Crimean-Congo hemorrhagic fever virus) and the like.
  • examples of the phyllovirus family virus include Ebolavirus (Genus Ebolavirus) virus (eg, Bundibugyoebolavirus), Reston ebolavirus, Sudanebolavirus, and Thai Forest Ebolavirus. (Tai Forest ebolavirus), Zaire ebolavirus, etc.), Genus Marburgvirus virus (eg, Marburgmarburgvirus, etc.) and the like.
  • Ebolavirus Genus Ebolavirus
  • Bundibugyoebolavirus Reston ebolavirus
  • Sudanebolavirus Sudanebolavirus
  • Thai Forest Ebolavirus Tritial Forest ebolavirus
  • Genus Marburgvirus virus eg, Marburgmarburgvirus, etc.
  • retrovirus family virus examples include Lentivirus (Genus Lentivirus) virus (e
  • Bacteria include, for example, Pseudomonas (Pyogenic bacteria, etc.), Escherichia coli (E. coli, etc.), Klebsiella (Pneumococcus, Klebsiella, Oxytoca, etc.), Hemophilus (Influenza, Parainfluenza, etc.), Bordetella (Pertussis).
  • mycoplasma examples include M. gallisepticum, M. genitalium, M. hominis, M. hyopneumoniae, M. laboratorium, M. mycoides, M. ovipneumoniae, M. pneumonia, Staphylococcus aureus and the like.
  • the cell as the test membrane structure may be a prokaryotic cell or a eukaryotic cell, and may be appropriately selected depending on the intended purpose.
  • yeast, insect cells, animal cells and the like are used.
  • animal cells are particularly preferable.
  • animal cells include cells of mammals (eg, mice, rats, hamsters, guinea pigs, dogs, monkeys, orangutans, chimpanzees, humans, etc.).
  • Examples of cell types include nerve cells, oligodendrocytes, erythrocytes, mononuclear cells (eg, lymphocytes (NK cells, B cells, T cells, monospheres, dendritic cells, etc.)), granulocytes (eg, favorable).
  • epithelial cells eg, retinal pigment epithelial cells, etc.
  • endothelial cells eg, vascular endothelial cells, hepatic sinus endothelial cells, etc.
  • muscle cells eg, fibroblasts Differentiation of (eg, skin cells, etc.),
  • Reagents that can label membrane structures with lipid bilayers include fluorescent dyes or visible dyes.
  • fluorescent dyes include divalent manganese, lead, antimony, cerium, trivalent cerium, trivalent chromium, divalent or trivalent iron, trivalent or tetravalent titanium, and copper.
  • Silver divalent sumalium, divalent or trivalent europium, trivalent terbium, trivalent dysprosium, trivalent holmium, trivalent erbium, uranyl compound, lutetium compound, tin compound, thulium compound, bismus compound, Examples thereof include tungonic acid compound, molybdic acid compound, sulfur, vanadium compound, lanthanum compound, praseodymium compound, neodymium compound, promethium compound, gadrinium compound, thulium compound, ytterbium compound, lutetium compound, organic fluorescent dye compound, organic visible dye compound and the like. ..
  • organic fluorescent dye compound and the organic visible dye compound examples include DY630, DY631, DY633, DY635, DY636, DY650, DY651 (above, manufactured by Dyomics GmbH), Oyster643, Oyster656 (above, manufactured by Denovo Biolabels) 5-carboxy.
  • BODIPY FL BODIPYTM / 503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665 (above, made by Invitrogen), FM3-25, FM2 -10, FM1-43, FM1-84, FM4-64, FM5-95 (above, manufactured by Biotium) methoxycoumarin, eodin, NBD, pyrene, Cy5, Cy5.5, Cy7 and the like.
  • the step (2) of the detection method of the present invention is a step of separating a fraction containing a membrane structure having a predetermined lipid bilayer, and is not particularly limited as long as the fraction can be separated.
  • the separation is performed by a capillary phenomenon, and a sample containing the membrane structure labeled in step (1) is optionally passed through a filter (eg, blood cell separation filter, etc.) to remove unnecessary contaminants, and then the separation is performed.
  • a filter eg, blood cell separation filter, etc.
  • each substance is separated by the mass of the substance contained in the sample, the adsorbing force on the membrane, or the electric charge.
  • the degree of separation is such that the binding between the capture molecule (eg, antibody, etc.) present on the test line in step (3) described later and the membrane structure having the lipid bilayer desired to be detected is not inhibited.
  • the degree of separation does not matter.
  • the developing membrane (membrane) used in the step (2) of the present invention has a bond between a capture molecule (eg, an antibody, etc.) existing on the test line and a membrane structure having a lipid bilayer that is desired to be detected. It is not particularly limited as long as it can be deployed to the extent that it is not hindered. Specifically, for example, polymethylmethyl methacrylate (PMMA), polycarbonate, polypropylene, polyethylene, polymethylpentene, polystyrene, polytetrafluoroethylene, ABS resin, polydimethylsiloxane, nitrocellulose, silicon resin, and high of them.
  • PMMA polymethylmethyl methacrylate
  • PMMA polycarbonate
  • polypropylene polyethylene
  • polymethylpentene polystyrene
  • polytetrafluoroethylene polydimethylsiloxane
  • ABS resin polydimethylsiloxane
  • Examples include copolymers or composites containing molecular compounds, silica (silica monolith), quartz glass, Pyrex (registered trademark) glass, soda glass, borate glass, silicate glass, borosilicate glass, ceramics and their composites. Be done. Further, a capture molecule that specifically binds to a membrane structure desired to be detected is immobilized on the developed membrane.
  • the membrane structure in the fraction containing the membrane structure having a predetermined lipid bilayer layer for which detection desired to be fractionated by step (2) is desired is used as a developing membrane. It is not particularly limited as long as it can be captured and detected by the immobilized capture molecule.
  • the capture is referred to as an antibody or a fragment thereof (hereinafter referred to as "antibody or the like” that can specifically bind to a surface antigen (eg, a membrane protein or the like) of a membrane structure having a predetermined lipid bilayer that is desired to be detected. It may be done using an aptamer or the like.
  • the surface antigens of the envelope virus include, for example, hepatitis B virus antigens such as HBV surface (HBsAg) and core (HBcAg) antigens, and influenza virus antigens such as hemagglutinin antigen (HA) and neurominidase antigen (NA).
  • hepatitis B virus antigens such as HBV surface (HBsAg) and core (HBcAg) antigens
  • influenza virus antigens such as hemagglutinin antigen (HA) and neurominidase antigen (NA).
  • Ebola virus antigen such as glycoprotein (GP)
  • HIV antigen such as HIV envelope protein gp41, gp120
  • SARS-Cov-2 such as spike (S), envelope (E), membrane (M) and nucleocapsid (N) protein
  • S serum
  • E envelope
  • M membrane
  • N nucleocapsid
  • cell surface antigens examples include CD3 (T cells), CD19 (B cells), NK1.1 (NK cells), CD11c (dendritic cells), CD11b (monocytes), Fc ⁇ R1 (basophils), and Gr1. (Granulocytes) and the like.
  • examples of surface antigens of apoptotic bodies include Annexin V and the like.
  • examples of surface antigens of microvesicles include selectin, integrin, floritrin, CD40 and the like.
  • examples of surface antigens of exosomes include tetraspanin, cell adhesion molecule, antigen presentation-related protein, cytokine receptor and the like.
  • proteins known to be abundant in the membrane of exosomes in the body fluids of cancer patients can be mentioned.
  • examples of tetraspanin include CD9, CD37, CD63, CD81 and the like.
  • Examples of cell adhesion molecules include integrin, CEA family member, ICAM-1 (intercellular adhesion molecule-1), CD31 and the like.
  • examples of the antigen-presenting-related protein include MHC I, MHC II and the like.
  • examples of the cytokine receptor include EGFRvIII and the like.
  • proteins known to be abundant in the membrane of exosomes in the body fluids of cancer patients include CD9, CD24, CD91, CD147, caveolin-1, glypican-1, and EpCAM (Epithelial cell adhesion molecule). Can be mentioned.
  • glycoproteins CD9, CD37, CD63, integrin, CEA family members ICAM-1, CD31, MHCI, MHCII, EGFRvIII, CD24, CD147, glypican-1, EpCAM are preferable, and the CEA family Members are more preferred.
  • CEA family members include CEA, CEACAM1, CEACAM3, CEACAM4, CEACAM6, CEACAM7, CEACAM8, and the like, preferably CEA, CEACAM1, and CEACAM6.
  • the capture substance used in the detection method of the present invention and having a specific affinity for the test membrane structure to be detected is not particularly limited as long as it has a specific affinity for the test membrane structure.
  • the trapping material is immobilized on the test line by a chemical bond or a physical bond.
  • the capture substance that recognizes the lipid bilayer is not particularly limited as long as it has a specific affinity for the lipid bilayer.
  • the trapping material is immobilized on the control line by a chemical bond or a physical bond.
  • the capture substance having a specific affinity for the test membrane structure immobilized on the test line and the capture substance recognizing the lipid bilayer immobilized on the control line may be the same. It may be different, but it is preferable that it is different.
  • the capture substance include amino acids, nucleic acids, polypeptides, polynucleotides, lipids, phospholipids, carbohydrates, polysaccharides, low molecular weight compounds, inorganic substances, and fusions thereof.
  • Factors, receptors, receptor fragments, hormones and the like can be mentioned.
  • the above antibody can be obtained as a polyclonal or monoclonal antibody by using known means.
  • a commercially available product may be used.
  • the origin of the antibody used in the present invention is not particularly limited, but is preferably derived from mammals.
  • the monoclonal antibody derived from a mammal may be either one produced by a hybridoma or one produced by a host transformed with an expression vector containing an antibody gene by a genetic engineering technique.
  • An antibody-producing hybridoma can be produced by a method known per se, for example, using a protein contained in the membrane of a test membrane structure or a part thereof as an antigen, and immunizing the antigen according to a conventional immunization method. It can be produced by fusing the obtained immune cells with known parent cells by a conventional cell fusion method and screening monoclonal antibody-producing cells by a conventional screening method.
  • the antibody fragment is not particularly limited as long as it contains the same complementarity determining regions, and is a fusion of a light chain fragment and a light chain fragment via a linker or the like (eg, scFv (single chain).
  • scFv single chain
  • Antibodies etc.
  • Specific antibodies or fragments thereof include, for example, fragments such as Fab, Fab', F (ab') 2, and genetically engineered conjugated molecules such as scFv, scFv-Fc, minibody, and diabody.
  • Or derivatives thereof modified with a molecule having a protein stabilizing action such as polyethylene glycol (PEG).
  • the test line and the method for detecting or measuring fluorescence (emission) in the test line are not particularly limited, but are equipped with a general optical fiber type fluorescence detector, a microplate reader, a fluorescence microscope, and a camera (eg, CCD camera, etc.). It can be detected or measured by using a detector (eg, Celvin, etc.). Further, as in the method of the ELISA method, a calibration curve is prepared using a sample (standard product) having a known concentration of the target test membrane structure, and the calibration curve is used to prepare the test membrane structure in the sample. It is also possible to quantify the concentration of.
  • the determination method of the present invention is as follows: It is a method for determining (detecting) a disease in a subject. (1) A step of labeling a membrane structure having a lipid bilayer present in a sample derived from the subject. A method comprising (2) a step of separating a fraction containing a membrane structure having a predetermined lipid bilayer, and (3) a step of detecting a membrane structure having the predetermined lipid bilayer in the fraction. I will provide a.
  • the determination method of the present invention is a method for determining whether or not a subject has a virus or bacterial infection or a disease such as cancer (detection of a virus or bacterial infection in the subject or a disease such as cancer). Is. The determination is made by detecting the surface antigen of the membrane structure having a lipid bilayer such as a virus, a bacterium, an exosome, etc. as described in the above "1. Detection method of the present invention". For example, in the case of a virus or a bacterium, it can be determined that the subject (subject) is infected with (or may be infected with) the virus or the bacterium by detecting the presence of the virus or the bacterium.
  • specific surface antigens eg, EGFRIII (collagenoma), Del-1 (breast cancer), etc.
  • Fibronectin (breast cancer), CD147 (colon cancer), GPC-1 (pancreatic cancer), EphA2 (pancreatic cancer), EGFR, EpCAM, MUC1, WNT2, GPC1 (pancreatic cancer), CD91 (lung cancer), CD24, EpCAM (ovarian cancer), CD24, EpCAM, CA-125 (ovarian cancer), Survivin (prostatic cancer), PD-L1 (pharyngeal cancer), TACSTD2 (bladder cancer), PD-L1 (melanoma), CD63, Caveolin-1 (melanoma), etc.) can be expressed (significantly).
  • the specific disease is determined (affected by a specific disease) by detecting both the surface antigen of the exosome itself (eg, CD9, etc.) and the surface antigen of the exosome caused by the specific disease. , Or it may be determined that it may be affected).
  • the determination method of the present invention is performed on a test strip for lateral flow
  • the surface antigen of the exosome itself eg, CD9, etc.
  • the surface antigen of the exosome caused by a specific disease is detected. Judgment may be made by detecting on the test line.
  • the amount of the membrane structure having a predetermined lipid double layer derived from the subject (subject) detected in the step (3) of the method of the present invention and the amount of the membrane structure having a predetermined lipid double layer of a healthy person may be determined that the patient has or may have a specific disease (eg, cancer, etc.). More specifically, for example, if the amount of co-positive exosomes of CD9 and CD147 derived from a subject (subject) is (significantly) higher than that of a healthy subject, the subject has colorectal cancer. It can be determined (or may be affected). The comparison may be performed using, for example, the fluorescence intensity, the cutoff value obtained by a method known per se, or the like.
  • a specific disease eg, cancer, etc.
  • the determination method of the present invention may assist a definitive diagnosis by a doctor or the like, and therefore, the determination method can be said to be a method for assisting a determination (diagnosis).
  • the determination method of the present invention the contents described in the above "1. Detection method of the present invention” shall be appropriately incorporated as necessary.
  • the kit is for carrying out the detection method of the present invention, and includes a reagent container 100 and a test strip 200.
  • the reagent container 100 contains the labeling reagent 110.
  • the labeling reagent 110 is a reagent for labeling the above-mentioned membrane structure present in the sample S1.
  • the test strip 200 has at least a sample receiving unit 210, a filter unit 220, and a detecting unit 240, which are arranged on the support layer 200b in the example of FIG. 1 (a).
  • the overall laminated structure of the test strip 200, the length of each part, the overall length, and the like, conventionally known test strips can be referred to.
  • each part of the kit and the usage method thereof are as follows. First, as shown by the thick arrow in FIG. 1 (a), when the sample S1 is injected into the reagent container 100, the membrane structure in the sample S1 is labeled with the labeling reagent 110.
  • labeling reagent 110 the sample containing the labeled membrane structure
  • sample S2 the sample containing the labeled membrane structure
  • sample S2 the sample containing the labeled membrane structure
  • sample S2 simply “sample S2”.
  • FIG. 1 (a) for the sake of brevity, an example in which the sample S1 is injected into the reagent container 100 is shown, but another mixing container such as a well is used to label the sample S1 with the sample S1. It may be mixed with the reagent 110.
  • the sample receiving section 210 is a portion that receives the labeled sample S2 and is configured to temporarily hold a sufficient amount of the supplied sample S2 and release it into the test strip.
  • the structure of the sample receiving portion 210 itself can refer to the structure of the sample receiving portion in a conventionally known test strip, and is made of, for example, cellulose, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, cotton cloth or the like.
  • Cellulose Absorbent Pad manufactured by Pall
  • the like are exemplified.
  • the sample S2 received by the sample receiving unit 210 travels through the test strip 200, passes through the filter unit 220, and reaches the detection unit 240, as shown by the thick broken line arrow in FIG. 1 (a).
  • the driving force (propulsive force or suction force) for the sample S2 to travel through the test strip 200 is not particularly limited, and for example, gravity and capillarity are exemplified, and a combination thereof may be used.
  • the expansion unit 230 is provided between the filter unit 220 and the detection unit 240.
  • the developing portion 230 may be the whole or a part of the developing membrane, and has a capture molecule (eg, an antibody, etc.) present on the test line and a lipid bilayer layer desired to be detected. It is a part provided for development to the extent that the bond with the membrane structure is not hindered.
  • the sample S2 is configured to pass through the detection unit 240.
  • the filter unit 220 is a portion that selectively passes the labeled membrane structure in the sample S2 received by the sample receiving unit 210.
  • the presence of the filter unit 220 makes it possible to remove impurities contained in the sample S2, and facilitates separation in the developing unit 230.
  • the configuration of the filter unit 220 is not particularly limited as long as impurities can be removed, and conventionally known filter configurations can be referred to. Further, the sample receiving unit 210 and the filter unit 220 may exist individually or as a unit.
  • the detection unit 240 is a portion configured to detect the labeled membrane structure in the sample S2 based on the label.
  • the configuration of the detection unit 240 is as described in detail in "1. Detection method of the present invention", and has a test line and optionally a control line.
  • test strip 300 is for carrying out the detection method of the present invention, and has a sample receiving unit 310, a conjugate unit 350, a filter unit 320, and a detection unit 340. In the example of FIG. 1 (b), these are arranged on the support layer 300b.
  • the difference between the test strip 300 and the test strip 200 of the kit shown in FIG. 1 (a) is that the test strip 300 has a conjugate portion 350. Since the conjugate portion 350 is provided, the user can carry out the detection method of the present invention using only the test strip 300.
  • the sample receiving unit 310, the filter unit 320, the expanding unit 330, the detecting unit 340, and the support layer 300b included in the test strip 300 are the sample receiving unit 210, the filter unit 220, and the expanding unit 230, respectively. This is the same as the detection unit 240 and the support layer 200b.
  • the overall laminated structure of the test strip 200, the length of each part, the overall length, and the like, conventionally known test strips can be referred to.
  • the sample S1 is supplied (for example, dropped) to the sample receiving portion 310 of the test strip 300 using a pipette or the like.
  • the sample S1 received by the sample receiving section 310 travels through the test strip 300 and passes through the conjugate section 350, as shown by the thick dashed arrow in FIG. 1 (b).
  • the conjugate portion 350 is configured to label the membrane structure in sample S1.
  • sample S1 passes through the conjugate section 350, it becomes a sample containing the labeled membrane structure (ie, sample S2).
  • the configuration in which the sample S2 passes through the filter unit 320 and reaches the detection unit 340 is the same as the test strip of the above-mentioned kit.
  • test strip Apply anti-CD9 mouse monoclonal antibody to the control line and anti-CD147 mouse monoclonal antibody to the test line at 1 ⁇ l / cm using an antibody coater on the membrane (HiFlow Plus HF120 (Milipore)). .. Regarding the control line and the test line, the test line and the control line are arranged in parallel on the left and right so that the distance from the position where the sample solution is dropped is the same as the test line and the control line (Fig. 2 (a)). And (b)).
  • the backing sheet is cut to a width of 5 mm using a cutting device, and then placed in a housing case to form a test strip.
  • the fluorescence intensity due to FM4-64 in the test line is compared with the fluorescence intensity due to FM4-64 in the test line in the serum derived from a healthy subject detected by the same method. From the results of the comparison, it can be seen that the prepared test strips are sufficiently usable.
  • the method of the present invention for detecting a membrane structure having a lipid bilayer can be used to detect a desired membrane structure having a lipid bilayer with high accuracy and convenience.
  • the disease determination method of the present invention can efficiently detect a membrane structure having a lipid bilayer expressing a disease-specific protein, and can be used for highly reliable disease determination.

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Abstract

The present invention provides: a method for detecting a membrane structural body having a lipid bilayer; a kit for conducting the method; a test strip; and the like.　The method for detecting a membrane structural body having a lipid bilayer according to the present invention comprises: (1) a step for labeling a membrane structural body having a lipid bilayer present in a sample; (2) a step for separating a fraction containing a membrane structural body having a predetermined lipid bilayer; and (3) a step for detecting the membrane structural body having the predetermined lipid bilayer in the fraction.

Description

脂質二重層を有する膜構造体の検出方法Method for detecting a membrane structure having a lipid bilayer
　本発明は、脂質二重層を有する膜構造体の検出方法、該方法を行うためのキット、及びテストストリップ等に関する。 The present invention relates to a method for detecting a membrane structure having a lipid bilayer, a kit for performing the method, a test strip, and the like.
　近年の医学の目覚ましい進歩により、以前は治療等を行うことができなかった数々の疾患も、治療可能なものになりつつある。このような状況の中、特に望まれているのは、疾患の早期発見である。疾患の早期発見は、治療効果を最大限にもたらすにとどまらず、我が国が抱える超高齢化社会における医療費の削減という問題に対する解決の一助となる。 Due to the remarkable progress in medicine in recent years, many diseases that could not be treated before are becoming treatable. In such a situation, what is particularly desired is early detection of the disease. Early detection of diseases not only maximizes the therapeutic effect, but also helps solve the problem of reducing medical costs in Japan's super-aging society.
　疾患の早期発見に関しては、例えば、ウイルスや細胞の表面上に存在する特定のタンパク質等を標的として、それらを検出することにより、ウイルスや細菌感染の有無の判断を行う試みが為されている。また、最近、細胞外小胞であるエクソソームやマイクロベシクルの細胞表面に、疾患特異的なタンパク質が発現している可能性が徐々に分かってきており、該疾患特異的なタンパク質の検出という点についても研究が行われている。 Regarding early detection of diseases, for example, attempts have been made to determine the presence or absence of virus or bacterial infection by targeting specific proteins existing on the surface of viruses or cells and detecting them. Recently, it is gradually becoming clear that disease-specific proteins may be expressed on the cell surface of extracellular vesicles such as exosomes and microvesicles, and the detection of the disease-specific proteins has been revealed. Is also being researched.
　しかしながら、例えば、エクソソームの検出に関しては、研究が為されているものの、検出にMACS等の高価な専用の装置を必要とする方法（特許文献1）や、検出までに複数の反応や処理を行う必要のあるもの（特許文献2）等が知られているにとどまる。 However, for example, although research has been conducted on the detection of exosomes, a method that requires an expensive dedicated device such as MACS for detection (Patent Document 1), and a plurality of reactions and treatments are performed before detection. Only what is needed (Patent Document 2) is known.
特開2018-179957号公報Japanese Unexamined Patent Publication No. 2018-179957 国際公開第2020/027185号International Publication No. 2020/0271 185
　本発明の課題は、脂質二重層を有する膜構造体を検出する方法、該方法を行うためのキット、及びテストストリップを提供することにある。また、本発明の課題は、脂質二重層を有する膜構造体に発現するタンパク質等に基づく、疾患の判定方法等を提供することである。 An object of the present invention is to provide a method for detecting a membrane structure having a lipid bilayer, a kit for performing the method, and a test strip. Another object of the present invention is to provide a method for determining a disease or the like based on a protein or the like expressed in a membrane structure having a lipid bilayer.
　本発明者らは、従来のウイルスや細菌、あるいはエクソソーム等の脂質二重層を有する膜構造体の検出方法について詳細に検討したところ、高価な専用の装置を必要とせず、検出までに複数の反応や処理を行う必要のない効率的な方法として、最初に脂質二重層を有する膜構造体を蛍光色素等の標識試薬で標識する工程を行う方法を着想した。該着想に基づいて、さらに鋭意検討を行ったところ、上記工程を最初に行なうことで、検出を所望する脂質二重層を有する膜構造体を、高精度且つ簡便に検出し得ることを見出し、本発明を完成するに至った。また、完成した検出方法について、一層検討を進めて、脂質二重層を有する膜構造体において、疾患特異的に発現するタンパク質を、本発明の検出方法で検出することで、疾患の罹患の有無等を判定し得ることも見出し、さらなる本発明を完成するに至った。 The present inventors have investigated in detail a conventional method for detecting a membrane structure having a lipid bilayer such as a virus, a bacterium, or an exosome. As an efficient method that does not require treatment, we first conceived a method of labeling a membrane structure having a lipid bilayer with a labeling reagent such as a fluorescent dye. As a result of further diligent studies based on the idea, it was found that a membrane structure having a lipid bilayer layer desired to be detected can be detected easily and with high accuracy by performing the above step first. The invention was completed. In addition, the completed detection method will be further studied, and by detecting a protein specifically expressed in a disease in a membrane structure having a lipid bilayer by the detection method of the present invention, the presence or absence of disease, etc. It was also found that the present invention could be further completed.
　即ち、本発明は以下に関する。
［1］脂質二重層を有する膜構造体を検出する方法であって、
（1）サンプル中に存在する脂質二重層を有する膜構造体を標識する工程、
（2）所定の脂質二重層を有する膜構造体を含む画分を分離する工程、及び
（3）前記画分中の前記所定の脂質二重層を有する膜構造体を検出する工程
を含む、方法。
［2］脂質二重層を有する膜構造体を検出するキットであって、当該キットは、
　サンプル中に存在する前記膜構造体を標識するための標識試薬が収容された試薬容器と、
　前記標識試薬によって標識された膜構造体を含んだサンプルから、該膜構造体を検出するテストストリップとを有し、
　前記テストストリップは、
　　前記標識された膜構造体を含んだサンプルを受入れるサンプル受入れ部と、
　　前記サンプル受入れ部で受け入れたサンプル中の前記標識された膜構造体を選択的に通過させるフィルター部と、
　　前記フィルター部を通過した前記標識された膜構造体を、前記標識に基づいて検出する検出部と
を有するものである、前記キット。
［3］脂質二重層を有する膜構造体を検出するテストストリップであって、当該テストストリップは、
　前記膜構造体を含んだサンプルを受入れるサンプル受入れ部と、
　前記サンプル受入れ部で受け入れたサンプル中の前記膜構造体を標識するためのコンジュゲート部と、
　前記コンジュゲート部によって標識された膜構造体を選択的に通過させるフィルター部と、
　前記フィルター部を通過した前記標識された膜構造体を、前記標識に基づいて検出する検出部と
を有する、前記テストストリップ。 That is, the present invention relates to the following.
[1] A method for detecting a membrane structure having a lipid bilayer.
(1) A step of labeling a membrane structure having a lipid bilayer present in a sample,
A method comprising (2) a step of separating a fraction containing a membrane structure having a predetermined lipid bilayer, and (3) a step of detecting a membrane structure having the predetermined lipid bilayer in the fraction. ..
[2] A kit for detecting a membrane structure having a lipid bilayer, and the kit is
A reagent container containing a labeling reagent for labeling the membrane structure present in the sample, and
It has a test strip for detecting the membrane structure from a sample containing the membrane structure labeled with the labeling reagent.
The test strip
A sample receiving unit that receives a sample containing the labeled membrane structure, and a sample receiving unit.
A filter unit that selectively passes the labeled membrane structure in the sample received by the sample receiving unit, and a filter unit.
The kit comprising a detection unit for detecting the labeled membrane structure that has passed through the filter unit based on the label.
[3] A test strip for detecting a membrane structure having a lipid bilayer, and the test strip is
A sample receiving unit that receives a sample containing the membrane structure,
A conjugate section for labeling the membrane structure in the sample received by the sample receiving section, and a conjugate section.
A filter unit that selectively passes the membrane structure labeled by the conjugate unit, and a filter unit.
The test strip comprising a detection unit for detecting the labeled membrane structure that has passed through the filter unit based on the label.
　本発明によれば、脂質二重層を有する膜構造体を検出する方法等を提供することができる。また、本発明によれば、脂質二重層を有する膜構造体に発現するタンパク質等に基づく、疾患の判定方法等を提供することができる。本発明の方法は、検出にMACS等の高価な専用の装置を必要とせず、検出までに複数の反応や処理を行う必要もないため、高精度且つ簡便である。また、高精度且つ簡便に脂質二重層を有する膜構造体を検出し得るため、疾患特異的なタンパク質を発現する脂質二重層を有する膜構造体を効率的に検出することができ、そのため、信頼性の高い疾患の判定も行い得る。 According to the present invention, it is possible to provide a method for detecting a membrane structure having a lipid bilayer. Further, according to the present invention, it is possible to provide a method for determining a disease or the like based on a protein or the like expressed in a membrane structure having a lipid bilayer. The method of the present invention is highly accurate and simple because it does not require an expensive dedicated device such as MACS for detection and it is not necessary to perform a plurality of reactions or processes before detection. In addition, since a membrane structure having a lipid bilayer can be detected easily and with high accuracy, a membrane structure having a lipid bilayer expressing a disease-specific protein can be efficiently detected, and therefore reliable. It is also possible to determine a highly sexual disease.
図1は、本発明のキットおよびテストストリップの構成を説明する模式図である。図1（a）では、分かりやすく説明するために、キットに含まれる試薬容器を断面図で示し、テストストリップを斜視図で示している（図2（a）も同様である）。図1（a）、（b）における、テストストリップの図では、領域を区別するために、各部に網掛とハッチングを適宜に加えている（図2（a）、（b）も同様である）。FIG. 1 is a schematic diagram illustrating the configuration of the kit and the test strip of the present invention. In FIG. 1 (a), the reagent container included in the kit is shown in a cross-sectional view and the test strip is shown in a perspective view for easy understanding (the same applies to FIG. 2 (a)). In the test strip diagram in FIGS. 1 (a) and 1 (b), shading and hatching are appropriately added to each part in order to distinguish the areas (the same applies to FIGS. 2 (a) and 2 (b)). .. 図2は、本発明のキットおよびテストストリップの好ましい構成の一例を示す模式図である。図2（a）、（b）では、分かりやすく説明するために、テストストリップ上に滴下された試料液が、２方向（２つの破線矢印）に分かれてテストストリップ上を長手方向に進行するかのように表現しているが、試料液の進行の実体は図1と同様である。FIG. 2 is a schematic diagram showing an example of a preferable configuration of the kit and the test strip of the present invention. In FIGS. 2 (a) and 2 (b), for the sake of easy understanding, whether the sample liquid dropped on the test strip is divided into two directions (two dashed arrows) and travels in the longitudinal direction on the test strip. However, the substance of the progress of the sample solution is the same as in Fig. 1.
1．本発明の検出方法
　本発明は、以下：
　脂質二重層を有する膜構造体を検出する方法であって、
（1）サンプル中に存在する脂質二重層を有する膜構造体を標識する工程、
（2）所定の脂質二重層を有する膜構造体を含む画分を分離する工程、及び
（3）前記画分中の前記所定の脂質二重層を有する膜構造体を検出する工程
を含む、方法
を提供する。 1. 1. The detection method of the present invention The present invention is as follows:
A method for detecting a membrane structure having a lipid bilayer.
(1) A step of labeling a membrane structure having a lipid bilayer present in a sample,
A method comprising (2) a step of separating a fraction containing a membrane structure having a predetermined lipid bilayer, and (3) a step of detecting a membrane structure having the predetermined lipid bilayer in the fraction. I will provide a.
　本発明の検出方法は、脂質二重層（膜）を有する膜構造体を検出することができる限り特に限定されないが、例えば、ラテラルフロー用等のテストストリップにおいて為されてもよい。そのようなテストストリップは、後述する本発明のキットのように、標識試薬を収容した試薬容器と共に用いるように構成されたもの（コンジュゲート部を持たないテストストリップ）でもよいし、また、後述する本発明のテストストリップのように、１つのテストストリップ中に、当該検出方法を実施するために必要な全ての要素が配置されたものであってもよい。 The detection method of the present invention is not particularly limited as long as it can detect a membrane structure having a lipid bilayer (membrane), but may be used, for example, in a test strip for lateral flow or the like. Such a test strip may be configured to be used with a reagent container containing a labeling reagent (a test strip having no conjugate portion), as in the kit of the present invention described later, and may be described later. As in the test strip of the present invention, all the elements necessary for carrying out the detection method may be arranged in one test strip.
　具体的には、本発明の検出方法の実施に適したテストストリップは、例えば、サンプルに含まれる本発明の方法により検出の対象となる脂質二重層を有する膜構造体（以下、「被験膜構造体」と称することがある）を検出して濃度を定量するものであって、サンプル滴下部材である（i）サンプルパッド（サンプル受入れ部）、サンプルが流れる方向に、任意により、（ii）フィルター、検出対象である被験膜構造体に特異的親和性のある捕捉物質が固相化された部位であるテストラインを有する（iii）展開膜（メンブレン）、該展開膜は、任意により脂質二重層を認識する捕捉物質が固相化された部位であるコントロールラインを有してもよく、任意により、更にその下流に毛細管現象により浸潤してきたサンプルを吸収する（iv）吸収パッドの4種の部材が（v）バッキングシートを介して直列に連結されたような構成を有する。
　本願明細書中において、（ii）フィルターと（iii）展開膜（メンブレン）を合わせてフィルター部と称してもよい。また、該テストストリップは、任意により、（ii）フィルター部の前に、（ii'）サンプル中の脂質二重層を有する膜構造体を標識するためのコンジュゲート部を有してもよい。 Specifically, a test strip suitable for carrying out the detection method of the present invention is, for example, a membrane structure having a lipid bilayer to be detected by the method of the present invention contained in a sample (hereinafter, “test membrane structure”). It detects (sometimes referred to as "body") and quantifies the concentration, and is a sample dropping member (i) a sample pad (sample receiving part), in the direction in which the sample flows, and optionally (ii) a filter. (Iii) Expanded membrane (membrane), which has a test line where a capture substance having a specific affinity for the test membrane structure to be detected is immobilized, and the expanded membrane is optionally a lipid bilayer. It may have a control line where the capture substance that recognizes the substance is immobilized, and optionally further downstream of the control line that absorbs the sample infiltrated by the capillary phenomenon (iv) Four kinds of members of the absorption pad. Has a configuration in which (v) are connected in series via a backing sheet.
In the specification of the present application, (ii) a filter and (iii) a developing film (membrane) may be collectively referred to as a filter unit. The test strip may also optionally have (ii) a conjugate section in front of the filter section for labeling a membrane structure having a lipid bilayer in the (ii') sample.
　本発明の検出方法の実施に適したテストストリップの幅は、所望する脂質二重層を有する膜構造体を検出し得る限り、特に限定されないが、例えば、1.5 mm～5.0 mmである。
　サンプルパッド（サンプル受入れ部）は、滴下された分析対象物を含む試料（サンプル）を一定時間保持する部材である。サンプルパッドとしては、例えば、セルロース、ガラス繊維、ポリウレタン、ポリアセテート、酢酸セルロース、ナイロン、綿布等を素材としたものが挙げられ、より詳細には、例えば、Cellulose Absorbent Pad（Pall社製）等が挙げられる。また、滴下された試料によりpHが異なることを鑑み、サンプルパッドに緩衝液を浸潤させて乾燥させた後に、そのサンプルパッドを使用してもよい。サンプルパッドに滴下するサンプルの体積は、例えば、25μl～100μlである。
　コンジュゲート部は、サンプル中の脂質二重層を有する膜構造体を標識するための部分であり、膜構造体を標識し得る試薬を含む。該試薬は、サンプル中の脂質二重層を有する膜構造体を標識することができる限り、如何なる状態でコンジュゲート部に含まれていてもよいが、例えば、試薬を、DMSO等の有機溶媒に溶解した後、コンジュゲート部に浸潤させて乾燥させた状態で含まれていてもよい。コンジュゲート部の材質は、例えば、セルロース、ガラス繊維、ポリウレタン、ポリアセテート、酢酸セルロース、ナイロン、綿布等を素材としたものが挙げられ、より詳細には、例えば、Glass Fiber Pad（Millipore社製）等が挙げられる。コンジュゲート部は、サンプルパッド（サンプル受入れ部）と一体となっていてもよく、互いに独立していてもよい。 The width of the test strip suitable for carrying out the detection method of the present invention is not particularly limited as long as a membrane structure having a desired lipid bilayer can be detected, and is, for example, 1.5 mm to 5.0 mm.
The sample pad (sample receiving unit) is a member that holds a sample (sample) containing a dropped analysis object for a certain period of time. Examples of the sample pad include those made of cellulose, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, cotton cloth and the like, and more specifically, for example, Cellulose Absorbent Pad (manufactured by Pall) and the like. Can be mentioned. Further, considering that the pH differs depending on the dropped sample, the sample pad may be used after the sample pad is infiltrated with a buffer solution and dried. The volume of the sample dropped on the sample pad is, for example, 25 μl to 100 μl.
The conjugate portion is a portion for labeling the membrane structure having a lipid bilayer in the sample, and contains a reagent capable of labeling the membrane structure. The reagent may be contained in the conjugate portion in any state as long as it can label the membrane structure having a lipid bilayer in the sample. For example, the reagent is dissolved in an organic solvent such as DMSO. After that, it may be contained in a state where it is infiltrated into the conjugate portion and dried. Examples of the material of the conjugate portion include those made of cellulose, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, cotton cloth, etc., and more specifically, for example, Glass Fiber Pad (manufactured by Millipore). And so on. The conjugate portion may be integrated with the sample pad (sample receiving portion) or may be independent of each other.
　サンプルパッドとコンジュゲート部は、後述する展開膜（メンブレン）上に設けられていてもよい。また、展開膜は、展開部を有し、該展開部は、展開膜の全部であってもよく、一部であってもよい。 The sample pad and the conjugate portion may be provided on a developing membrane (membrane) described later. Further, the developing film has a developing portion, and the developing portion may be the whole or a part of the developing film.
　展開膜上の検出部が設けられたテストラインと、任意により設けられてもよいコントロールラインの形状は、捕捉物質が固定化されている限り、特に限定されず、例えば、ライン状、円状、帯状等が挙げられる。該形状のうち、幅が0.5 mm～2.0 mmのライン状であることが好ましい。固定化される捕捉物質の量は、所望する脂質二重層を有する膜構造体を検出し得る限り特に限定されないが、例えば、捕捉物質が抗体である場合、ライン1 cmあたりの抗体量は、0.01～20μgである。また、テストラインとコントロールラインを設ける位置については、サンプル中の検出を所望する物質（例：脂質二重層を有する膜構造体）を検出し得る限り、特に限定されず、例えば、サンプルの展開方向に対して垂直、平行等に設けられてもよい。より具体的には、平行に設ける場合、サンプルを滴下するポジションからの距離がテストラインとコントロールラインが同じになるように、該テストラインとコントロールラインとを左右並列に配置してもよい（図2（a）の240aと240b（テストライン/コントロールライン）、及び図2（b）の340aと340ｂ（テストライン/コントロールライン）を参照）。 The shape of the test line provided with the detection portion on the developing membrane and the control line optionally provided is not particularly limited as long as the trapping substance is immobilized, and is, for example, line-shaped, circular, or circular. Examples include strips. Of the shapes, it is preferable that the width is a line shape of 0.5 mm to 2.0 mm. The amount of the capture substance to be immobilized is not particularly limited as long as a membrane structure having a desired lipid bilayer can be detected. For example, when the capture substance is an antibody, the amount of antibody per 1 cm of line is 0.01. It is ~ 20 μg. Further, the positions where the test line and the control line are provided are not particularly limited as long as a substance (eg, a membrane structure having a lipid bilayer) desired to be detected in the sample can be detected, and for example, the development direction of the sample. It may be provided perpendicular to, parallel to, or the like. More specifically, when they are provided in parallel, the test line and the control line may be arranged in parallel on the left and right so that the distance from the position where the sample is dropped is the same as the test line and the control line (Fig.). See 240a and 240b (test line / control line) in 2 (a) and 340a and 340b (test line / control line) in Figure 2 (b)).
　吸水パッドは、毛細管現象で展開膜（メンブレン）を移動してきたサンプルを吸収し、常に一定の流れを生じさせるための部材である。吸水パッドの材質としては、セルロース、不織布、綿布、セルロースアセテート等が挙げられ、セルロース素材の繊維状のものが好ましく、より具体的には、例えば、Cellulose Fiber Sample Pad（Millipore社製）等が挙げられる。 The water absorption pad is a member that absorbs the sample that has moved through the developing membrane due to the capillary phenomenon and constantly generates a constant flow. Examples of the material of the water absorption pad include cellulose, non-woven fabric, cotton cloth, cellulose acetate and the like, and a fibrous cellulose material is preferable, and more specifically, for example, Cellulose Fiber Sample Pad (manufactured by Millipore) and the like. Be done.
　バッキングシートは、（i）サンプルパッド（サンプル受入れ部）、（ii）フィルター、（iii）展開膜（メンブレン）、任意により（iv）吸収パッドの4部材を固定化するための粘着性を備えたシートであり、該シートとしては、例えば、バッキングシートAR9020（Adhesives Research社製）が挙げられる。 The backing sheet has adhesiveness for immobilizing four members: (i) sample pad (sample receiving part), (ii) filter, (iii) developing membrane (membrane), and optionally (iv) absorption pad. It is a sheet, and examples of the sheet include a backing sheet AR9020 (manufactured by Adhesives Research).
　本発明の検出方法の工程（1）は、サンプル中に存在する脂質二重層を有する膜構造体と、蛍光色素等の標識試薬とを接触させて、該膜構造体を標識する工程である。該工程は、サンプル中に存在する脂質二重層を有する膜構造体と、蛍光色素等の標識試薬とを接触させて、該膜構造体を標識することができれば特に限定されないが、テストストリップ上等において行われてもよく、別の場所（例：チューブ内等）で行われてもよい。テストストリップ上であれば、例えば、テストストリップ上の後述するような標識試薬を含むコンジュゲート部で行われてもよい。 The step (1) of the detection method of the present invention is a step of contacting a membrane structure having a lipid bilayer existing in a sample with a labeling reagent such as a fluorescent dye to label the membrane structure. The step is not particularly limited as long as the membrane structure having the lipid bilayer present in the sample can be brought into contact with a labeling reagent such as a fluorescent dye to label the membrane structure, but on a test strip or the like. It may be done in, or in another place (eg, in a tube, etc.). If it is on a test strip, it may be performed, for example, in a conjugate section containing a labeling reagent as described later on the test strip.
　本発明の検出方法において使用されるサンプルとしては、生体サンプルであって液状のものが好ましく、該生体サンプルとしては、例えば、動物又は細胞から採取したサンプルが挙げられる。必要に応じて、細胞を破砕、溶解等することで、細胞内の成分（例：エクソソーム等）を抽出又は単離してもよい。また、前記サンプルは、本発明の方法により検出の対象となる脂質二重層を有する膜構造体（被験膜構造体）を含むことが既知のサンプルであってもよいし、被験膜構造体が含まれるか不明なサンプルであってもよい。また、サンプルは、自体公知の方法により、適宜、溶媒（水、生理食塩水、緩衝液、有機溶媒等）で希釈してもよいし、各種カラム（例：シリカモノリス等）等を用いて、精製あるいは濃縮してもよい。 The sample used in the detection method of the present invention is preferably a biological sample that is liquid, and examples of the biological sample include samples collected from animals or cells. If necessary, intracellular components (eg, exosomes, etc.) may be extracted or isolated by disrupting, lysing, or the like. Further, the sample may be a sample known to contain a membrane structure having a lipid bilayer (test membrane structure) to be detected by the method of the present invention, or may include a test membrane structure. It may be an unknown sample. Further, the sample may be appropriately diluted with a solvent (water, physiological saline, buffer solution, organic solvent, etc.) by a method known per se, or using various columns (eg, silica monolith, etc.). It may be purified or concentrated.
　かかる動物又は細胞としては、例えば、マウス、ラット、ハムスター、モルモット、イヌ、サル、オランウータン、チンパンジー、ヒト等の哺乳動物又は該動物に由来する細胞が挙げられる。動物由来の試料としては、例えば、血液、血清、血漿、唾液、尿、涙、汗、乳汁、鼻汁、精液、胸水、消化管分泌液、脳脊髄液、組織間液、及びリンパ液等が挙げられ、好ましくは血液、血清又は血漿である。これらの試料は、自体公知の方法により得ることができ、例えば、血清や血漿は、常法に従って被験動物から採血し、液性成分を分離することにより調製することができ、脳脊髄液は、脊椎穿刺等の公知の手段により採取することができる。 Examples of such animals or cells include mammals such as mice, rats, hamsters, guinea pigs, dogs, monkeys, oran wootans, chimpanzees, and humans, or cells derived from the animals. Examples of animal-derived samples include blood, serum, plasma, saliva, urine, tears, sweat, milk, nasal juice, semen, pleural effusion, gastrointestinal secretions, cerebrospinal fluid, intertissue fluid, lymph fluid and the like. , Preferably blood, serum or plasma. These samples can be obtained by a method known per se, for example, serum or plasma can be prepared by collecting blood from a test animal according to a conventional method and separating the humoral component, and cerebrospinal fluid can be prepared. It can be collected by a known means such as spinal puncture.
　上記被験膜構造体としては、例えば、エンベロープウイルス、細菌、細胞、エクソソーム（直径40～100 nm）、マイクロベシクル（直径100～1000 nm）、アポトーシス小体（直径1000～5000 nm）等が挙げられる。
　上記エンベロープウイルスとしては、DNAウイルスであっても、RNAウイルスであってもよい。DNAウイルスとしては、例えば、ヘルペスウイルス科（Family Herpesviridae）ウイルス、ポックスウイルス科（Family Poxviridae）ウイルス、ヘパドナウイルス科（Family Hepadnaviridae）ウイルス等が挙げられる。RNAウイルスとしては、例えば、フラビウイルス科（Family Flaviviridae）ウイルス、トガウイルス科（Family Togaviridae）ウイルス、コロナウイルス科（Family Coronaviridae）ウイルス、D型肝炎ウイルス（Hepatitis D virus）、オルトミクソウイルス科（Family Orthomyxoviridae）ウイルス、パラミクソウイルス科（Family Paramyxoviridae）ウイルス、ラブドウイルス科（Family Rhabdoviridae）ウイルス、ブニヤウイルス科（Family Bunyaviridae）ウイルス、フィロウイルス科（Family Filoviridae）ウイルス、レトロウイルス科（Family Retroviridae）ウイルス等が挙げられる。 Examples of the test membrane structure include enveloped viruses, bacteria, cells, exosomes (diameter 40 to 100 nm), microvesicles (diameter 100 to 1000 nm), apoptotic bodies (diameter 1000 to 5000 nm), and the like. ..
The enveloped virus may be a DNA virus or an RNA virus. Examples of the DNA virus include a herpesvirus family (Family Herpesviridae) virus, a Poxviridae virus, a hepadnaviridae virus, and the like. Examples of RNA viruses include Flavivirus family (Family Flaviviridae) virus, Togavirus family (Family Togaviridae) virus, Coronavirus family (Family Coronaviridae) virus, Hepatitis D virus (Hepatitis D virus), and Orthomixovirus family (Family). Orthomyxoviridae virus, Family Paramyxoviridae virus, Family Rhabdoviridae virus, Family Bunyaviridae virus, Family Filoviridae virus, Family Retroviridae virus, etc. Can be mentioned.
　ヘルペスウイルス科ウイルスとしては、例えば、バリセロウイルス属（Genus Varicellovirus）ウイルス（例：水痘帯状疱疹ウイルス（Varicella Zoster virus）等）、シンプレックスウイルス属（Genus Simplexvirus）ウイルス（例：単純ヘルペスウイルス2型（Herpes simplex virus 2）等）、ロゼオロウイルス属（Genus Roseolovirus）ウイルス（例：ヒトヘルペスウイルス6,7型（human Herpesvirus 6, 7）等）等が挙げられる。ポックスウイルス科ウイルスとしては、例えば、オルトポックスウイルス属（Genus Orthopoxvirus）ウイルス（例：痘瘡ウイルス（Variola virus）、牛痘ウイルス（Cowpox virus）、ワクシニアウイルス（Vaccinia virus）等）、パラポックスウイルス属（Genus Parapoxvirus）ウイルス（例：オーフウイルス（Orf virus）等）等が挙げられる。ヘパドナウイルス科ウイルスとしては、例えば、オルトヘパドナウイルス属（Genus Orthohepadnavirus）ウイルス（例：B型肝炎ウイルス（Hepatitis B virus）等）、アビヘパドナウイルス属（Genus Avihepadnavirus）ウイルス（例：アヒルB型肝炎ウイルス（Duck hepatitis B virus）等）、ロスガチョウB型肝炎ウイルス（Ross's goose hepatitis B virus）等が挙げられる。 Examples of the herpesvirus family virus include Varisellovirus virus (eg, Varicella Zoster virus, etc.) and Simplexvirus virus (eg, simple herpesvirus type 2 (eg, simple herpesvirus type 2). Herpes simplex virus 2), etc.), Roseolovirus virus (eg, human Herpesvirus 6, 7), etc.). Examples of poxvirus family viruses include Genus Orthopoxvirus virus (eg, Variola virus, Cowpox virus, Vaccinia virus, etc.) and Parapox virus (Genus). Parapoxvirus) virus (eg, Orf virus, etc.) and the like. Examples of the hepadonavirus family virus include orthohepadnavirus (genus Orthohepadnavirus) virus (eg, Hepatitis B virus), Abihepadnavirus (Genus Avihepadnavirus) virus (eg, duck B). Hepatitis virus (Duck hepatitis B virus), etc.), Ross's goose hepatitis B virus, etc. can be mentioned.
　フラビウイルス科ウイルスとしては、例えば、ヘパシウイルス属（Genus Hepacivirus）ウイルス（例：C型肝炎ウイルス（Hepatitis C virus）等）、フラビウイルス属（Genus Flaviviridae）ウイルス（例：日本脳炎ウイルス（Japanese encephalitis virus）、ジカウイルス（Zika virus）など)等が挙げられる。トガウイルス科ウイルスとしては、例えば、ルビウイルス属（Genus Rubivirus）ウイルス（例：風疹ウイルス（Rubella virus）など）等が挙げられる。コロナウイルス科ウイルスとしては、例えば、ベータコロナウイルス属（Genus Betacoronavirus）ウイルス（例：SARSコロナウイルス（Severe acute respiratory syndrome coronavirus：SARS-CoV）、SARSコロナウイルス2（SARS-CoV-2））、MERSコロナウイルス（Middle East respiratory syndrome coronavirus）など）等が挙げられる。オルトミクソウイルス科ウイルスとしては、例えば、A型インフルエンザウイルス（Influenzavirus A）、B型インフルエンザウイルス（Influenzavirus B）、C型インフルエンザウイルス（Influenzavirus C）等が挙げられる。パラミクソウイルス科ウイルスとしては、例えば、モルビリウイルス属（Genus Morbillivirus）ウイルス（例：麻疹ウイルス（Measles morbillivirus）等）、オルトニューモウイルス属（Genus Orthopneumovirus）ウイルス（例：RSウイルス（Respiratory syncytial virus）など）等が挙げられる。ラブドウイルス科ウイルスとしては、例えば、リッサウイルス属（Genus Lyssavirus）ウイルス（例：狂犬病ウイルス（Rabies virus）等）等が挙げられる。ブニヤウイルス科ウイルスとしては、例えば、ナイロウイルス属（Genus Nairovirus）ウイルス（例：クリミア・コンゴ出血熱ウイルス（Crimean-Congo hemorrhagic fever virus）など）等が挙げられる。フィロウイルス科ウイルスとしては、例えば、エボラウイルス属（Genus Ebolavirus）ウイルス（例：ブンディブギョエボラウイルス（Bundibugyo ebolavirus）、レストンエボラウイルス（Reston ebolavirus）、スーダンエボラウイルス（Sudan ebolavirus）、タイフォレストエボラウイルス（Tai Forest ebolavirus）、ザイールエボラウイルス（Zaire ebolavirus）等）、マールブルグウイルス属（Genus Marburgvirus）ウイルス（例：マールブルグウイルス（Marburg marburgvirus）など）等が挙げられる。レトロウイルス科ウイルスとしては、例えば、レンチウイルス属（Genus Lentivirus）ウイルス（例：ヒト免疫不全ウイルス（Human Immunodeficiency Virus）など）等が挙げられる。 Flaviviridae viruses include, for example, Hepacivirus virus (eg, Hepatitis C virus, etc.), Flaviviridae virus (eg, Japanese encephalitis virus). , Zikavirus, etc.). Examples of the Togavirus family virus include a Genus Rubivirus virus (eg, Rubella virus, etc.) and the like. Examples of the coronavirus family virus include beta coronavirus (Genus Betacoronavirus) virus (eg, SARS coronavirus (Severe acute respiratory syndrome coronavirus: SARS-CoV), SARS coronavirus 2 (SARS-CoV-2)), and MERS. Corona virus (Middle East respiratory syndrome corona virus, etc.) and the like can be mentioned. Examples of the orthomixovirus family virus include influenza A virus (Influenzavirus A), influenza B virus (Influenzavirus B), influenza C virus (Influenzavirus C), and the like. Examples of the paramyxovirus family virus include a genus Morbillivirus virus (eg, Measles morbillivirus), a Genus Orthopneumovirus virus (eg, RS virus (Respiratory syncytial virus)). Etc.) etc. Examples of the rhabdoviridae virus include Lyssavirus virus (eg, Rabies virus, etc.) and the like. Examples of the Bunyaviridae virus include Nairovirus (Genus Nairovirus) virus (eg, Crimean-Congo hemorrhagic fever virus) and the like. Examples of the phyllovirus family virus include Ebolavirus (Genus Ebolavirus) virus (eg, Bundibugyoebolavirus), Reston ebolavirus, Sudanebolavirus, and Thai Forest Ebolavirus. (Tai Forest ebolavirus), Zaire ebolavirus, etc.), Genus Marburgvirus virus (eg, Marburgmarburgvirus, etc.) and the like. Examples of the retrovirus family virus include Lentivirus (Genus Lentivirus) virus (eg, Human Immunodeficiency Virus) and the like.
　細菌としては、例えば、シュードモナス属（緑膿菌等）、大腸菌属（大腸菌等）、クレブシエラ属（肺炎桿菌、クレブシエラ・オキシトカ等）、ヘモフィルス属（インフルエンザ菌、パラインフルエンザ菌等）、ボルデテラ属（百日咳菌、気管支敗血症菌等）、セラチア属（セラチア・マルセッセンス等）、プロテウス属（プロテウス・ミラビリス等）、エンテロバクター属（エンテロバクター・クロアカ等）、カンピロバクター属（カンピロバクター・ジェジュニなど）、シトロバクター属、ビブリオ属（腸炎ビブリオ、コレラ菌等）、モルガネラ属（モルガネラ・モルガニ等）、サルモネラ属（チフス菌、パラチフス菌等）、シゲラ属（赤痢菌等）、アシネトバクター属（アシネトバクター・バウマニー、アシネトバクター・カルコアセチカス等）、レジオネラ属（レジオネラ・ニューモフィラ等）、バクテロイデス属（バクテロイデス・フラジリス等）、ナイセリア属（淋菌、髄膜炎菌等）、モラキセラ属（モラキセラ・カタラーリス等）、クラミジア属（クラミジア・トラコマティス、クラミジア・シッタシー等）及びヘリコバクター属（ヘリコバクター・ピロリ等）が挙げられる。マイコプラズマとしては、M. gallisepticum、M. genitalium、M. hominis、M. hyopneumoniae、M. laboratorium、M. mycoides、M. ovipneumoniae、M. pneumonia、黄色ブドウ球菌等が挙げられる。 Bacteria include, for example, Pseudomonas (Pyogenic bacteria, etc.), Escherichia coli (E. coli, etc.), Klebsiella (Pneumococcus, Klebsiella, Oxytoca, etc.), Hemophilus (Influenza, Parainfluenza, etc.), Bordetella (Pertussis). Bacteria, Bronchial septicemia, etc.), Serratia (Serratia, Marsessens, etc.), Proteus (Proteus, Mirabilis, etc.), Enterobacter (Enterobacter, Croaka, etc.), Campylobacter (Campylobacter, Jejuni, etc.), Citrobacter, Vibrio (Enterobacter vibrio, Cholera, etc.), Morganella (Morganella, Morgani, etc.), Serratia (Tihus, Palatifus, etc.), Shigera (Erythrophylla, etc.) ), Regionella (Regionella, Pneumophylla, etc.), Bacteroides (Bacteroides, Fraziris, etc.), Nyceria (Gyco, enterobacteriella, etc.), Moraxella (Moraxella, Catalaris, etc.), Chlamydia (Chlamydia trachomatis, etc.) Chlamydia sittercy, etc.) and the genus Helicobacter (Helicobacter, Pyrroli, etc.) can be mentioned. Examples of mycoplasma include M. gallisepticum, M. genitalium, M. hominis, M. hyopneumoniae, M. laboratorium, M. mycoides, M. ovipneumoniae, M. pneumonia, Staphylococcus aureus and the like.
　また、被験膜構造体としての細胞は、原核細胞であっても真核細胞であってもよく、目的に応じて適宜選択することができ、例えば、酵母、昆虫細胞、動物細胞等が用いられるが、中でも動物細胞が好ましい。動物細胞としては、例えば、哺乳動物（例：マウス、ラット、ハムスター、モルモット、イヌ、サル、オランウータン、チンパンジー、ヒト等）の細胞が挙げられる。細胞種としては、例えば、神経細胞、オリゴデンドロサイト、赤血球、単核細胞（例：リンパ球（NK細胞、B細胞、T細胞、単球、樹状細胞等））、顆粒球（例：好酸球、好中球、好塩基球）、巨核球、上皮細胞（例：網膜色素上皮細胞等）、内皮細胞（例：血管内皮細胞、肝類洞内皮細胞等）、筋肉細胞、線維芽細胞（例：皮膚細胞等）、毛細胞、肝細胞、胃粘膜細胞、腸細胞、脾細胞、膵細胞(例：膵外分泌細胞等)、脳細胞、肺細胞、腎細胞及び脂肪細胞等の分化した細胞、あるいはこれらの未成熟細胞等が挙げられる。 Further, the cell as the test membrane structure may be a prokaryotic cell or a eukaryotic cell, and may be appropriately selected depending on the intended purpose. For example, yeast, insect cells, animal cells and the like are used. However, animal cells are particularly preferable. Examples of animal cells include cells of mammals (eg, mice, rats, hamsters, guinea pigs, dogs, monkeys, orangutans, chimpanzees, humans, etc.). Examples of cell types include nerve cells, oligodendrocytes, erythrocytes, mononuclear cells (eg, lymphocytes (NK cells, B cells, T cells, monospheres, dendritic cells, etc.)), granulocytes (eg, favorable). Acid globules, neutrophils, basal spheres), macronuclear cells, epithelial cells (eg, retinal pigment epithelial cells, etc.), endothelial cells (eg, vascular endothelial cells, hepatic sinus endothelial cells, etc.), muscle cells, fibroblasts Differentiation of (eg, skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (eg, pancreatic exocrine cells, etc.), brain cells, lung cells, kidney cells, fat cells, etc. Examples thereof include cells and these immature cells.
　脂質二重層を有する膜構造体を標識し得る試薬としては、蛍光色素又は可視色素が挙げられる。
　蛍光色素は、具体的には、例えば、二価のマンガン、鉛、アンチモン、セリウム、三価のセリウム、三価のクロム、二価又は三価の鉄、三価又は四価のチタン、銅、銀、二価のサマリウム、二価又は三価のユーロピウム、三価のテルビウム、三価のジスプロシウム、三価のホルミウム、三価のエルビウム、ウラニル化合物、ルテニウム化合物、錫化合物、タリウム化合物、ビスマス化合物、タングステン酸化合物、モリブデン酸化合物、硫黄、バナジウム化合物、ランタン化合物、プラセオジム化合物、ネオジム化合物、プロメチウム化合物、ガドリニウム化合物、ツリウム化合物、イッテルビウム化合物、ルテチウム化合物、有機蛍光色素化合物、有機可視色素化合物等が挙げられる。 Reagents that can label membrane structures with lipid bilayers include fluorescent dyes or visible dyes.
Specific examples of the fluorescent dye include divalent manganese, lead, antimony, cerium, trivalent cerium, trivalent chromium, divalent or trivalent iron, trivalent or tetravalent titanium, and copper. Silver, divalent sumalium, divalent or trivalent europium, trivalent terbium, trivalent dysprosium, trivalent holmium, trivalent erbium, uranyl compound, lutetium compound, tin compound, thulium compound, bismus compound, Examples thereof include tungonic acid compound, molybdic acid compound, sulfur, vanadium compound, lanthanum compound, praseodymium compound, neodymium compound, promethium compound, gadrinium compound, thulium compound, ytterbium compound, lutetium compound, organic fluorescent dye compound, organic visible dye compound and the like. ..
　有機蛍光色素化合物、有機可視色素化合物としては、例えば、DY630、DY631、DY633、DY635、DY636、DY650、DY651（以上、Dyomics GmbH社製）、Oyster643、Oyster656（以上、Denovo Biolabels社製）5-カルボキシ-フルオレセイン、6-カルボキシ-フルオレセイン、5,6-ジカルボキシ-フルオレセイン、6-カルボキシ-2',4,4',5',7,7'-ヘキサクロロフルオレセイン、6-カルボキシ-2',4,7,7'-テトラクロロフルオレセイン、6-カルボキシ-4',5'-ジクロロ-2',7'-ジメトキシフルオレセイン、ナフトフルオレセイン、5-カルボキシ-ローダミン、6-カルボキシ-ローダミン、5,6-ジカルボキシ-ローダミン、ローダミン 6G、テトラメチルローダミン、X-ローダミン、及びAlexa Fluor 350、Alexa Fluor 405、Alexa Fluor 430、Alexa Fluor 488、Alexa Fluor 500、Alexa Fluor 514、Alexa Fluor 532、Alexa Fluor 546、Alexa Fluor 555、Alexa Fluor 568、Alexa Fluor 594、Alexa Fluor 610、Alexa Fluor 633、Alexa Fluor 635、Alexa Fluor 647、Alexa Fluor 660、Alexa Fluor 680、Alexa Fluor 700、Alexa Fluor 750、BODIPY FL、BODIPY TMR、BODIPY 493/503、BODIPY 530／550、BODIPY 558／568、BODIPY 564／570、BODIPY 576／589、BODIPY 581／591、BODIPY 630/650、BODIPY 650/665（以上、インビトロジェン社製）、FM3-25、FM2-10、FM1-43、FM1-84、FM4-64、FM5-95（以上、バイオティウム社製）メトキシクマリン、エオジン、NBD、ピレン、Cy5、Cy5.5、Cy7等が挙げられる。 Examples of the organic fluorescent dye compound and the organic visible dye compound include DY630, DY631, DY633, DY635, DY636, DY650, DY651 (above, manufactured by Dyomics GmbH), Oyster643, Oyster656 (above, manufactured by Denovo Biolabels) 5-carboxy. -Fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein, 6-carboxy-2', 4,4', 5', 7,7'-hexachlorofluorescein, 6-carboxy-2', 4, 7,7'-Tetrachlorofluorescein, 6-carboxy-4', 5'-dichloro-2', 7'-dimethoxyfluorescein, naphthofluorescein, 5-carboxy-rhodamine, 6-carboxy-rhodamine, 5,6-di Carboxy-Rhodamine, Rhodamine 6G, Tetramethyl Rhodamine, X-Rhodamine, and Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor. 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, BODIPY FL, BODIPYTM / 503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665 (above, made by Invitrogen), FM3-25, FM2 -10, FM1-43, FM1-84, FM4-64, FM5-95 (above, manufactured by Biotium) methoxycoumarin, eodin, NBD, pyrene, Cy5, Cy5.5, Cy7 and the like.
　本発明の検出方法の工程（2）は、所定の脂質二重層を有する膜構造体を含む画分を分離する工程であり、該画分を分離することができれば特に限定されない。該分離は、毛細管現象によって為され、工程（1）により標識された膜構造体を含むサンプルを、任意によりフィルター（例：血球分離フィルター等）を通過させ、不要な夾雑物を除いた後、展開膜（メンブレン）において、該サンプル中に含まれる物質の質量、該メンブレンに対する吸着力、あるいは電荷により、それぞれの物質を分離する。該分離の程度は、後述する工程（3）のテストライン上に存在する捕捉分子（例：抗体等）と、検出を所望する脂質二重層を有する膜構造体との結合が阻害されなければ、分離の程度は問題とはならない。 The step (2) of the detection method of the present invention is a step of separating a fraction containing a membrane structure having a predetermined lipid bilayer, and is not particularly limited as long as the fraction can be separated. The separation is performed by a capillary phenomenon, and a sample containing the membrane structure labeled in step (1) is optionally passed through a filter (eg, blood cell separation filter, etc.) to remove unnecessary contaminants, and then the separation is performed. In the developing membrane, each substance is separated by the mass of the substance contained in the sample, the adsorbing force on the membrane, or the electric charge. The degree of separation is such that the binding between the capture molecule (eg, antibody, etc.) present on the test line in step (3) described later and the membrane structure having the lipid bilayer desired to be detected is not inhibited. The degree of separation does not matter.
　本発明の工程（2）で使用される展開膜（メンブレン）は、テストライン上に存在する捕捉分子（例：抗体等）と、検出を所望する脂質二重層を有する膜構造体との結合が阻害されない程度に展開できれば特に限定されない。具体的には、例えば、ポリメチルメタクリル酸メチル（PMMA）、ポリカーボネート、ポリプロピレン、ポリエチレン、ポリメチルペンテン、ポリスチレン、ポリテトラフルオロエチレン、ABS樹脂、ポリジメチルシロキサン、ニトロセルロース、シリコンの樹脂、それらの高分子化合物を含む共重合体あるいは複合体、シリカ（シリカモノリス）、石英ガラス、パイレックス（登録商標）ガラス、ソーダガラス、ホウ酸ガラス、ケイ酸ガラス、ホウケイ酸ガラス、セラミックス及びその複合体等が挙げられる。また、展開膜には、検出を所望する膜構造体と特異的に結合する捕捉分子が固定されている。 The developing membrane (membrane) used in the step (2) of the present invention has a bond between a capture molecule (eg, an antibody, etc.) existing on the test line and a membrane structure having a lipid bilayer that is desired to be detected. It is not particularly limited as long as it can be deployed to the extent that it is not hindered. Specifically, for example, polymethylmethyl methacrylate (PMMA), polycarbonate, polypropylene, polyethylene, polymethylpentene, polystyrene, polytetrafluoroethylene, ABS resin, polydimethylsiloxane, nitrocellulose, silicon resin, and high of them. Examples include copolymers or composites containing molecular compounds, silica (silica monolith), quartz glass, Pyrex (registered trademark) glass, soda glass, borate glass, silicate glass, borosilicate glass, ceramics and their composites. Be done. Further, a capture molecule that specifically binds to a membrane structure desired to be detected is immobilized on the developed membrane.
　本発明の検出方法の工程（3）は、工程（2）により分画された検出を所望する所定の脂質二重層を有する膜構造体を含む画分中の該膜構造体を、展開膜に固定化された捕捉分子が捕捉し、検出することができれば特に限定されない。
　上記捕捉は、検出を所望する所定の脂質二重層を有する膜構造体の表面抗原（例：膜タンパク質等）と特異的に結合することができる抗体又はその断片（以下、「抗体等」と称することがある。）、アプタマー等を用いて行われる。 In step (3) of the detection method of the present invention, the membrane structure in the fraction containing the membrane structure having a predetermined lipid bilayer layer for which detection desired to be fractionated by step (2) is desired is used as a developing membrane. It is not particularly limited as long as it can be captured and detected by the immobilized capture molecule.
The capture is referred to as an antibody or a fragment thereof (hereinafter referred to as "antibody or the like" that can specifically bind to a surface antigen (eg, a membrane protein or the like) of a membrane structure having a predetermined lipid bilayer that is desired to be detected. It may be done using an aptamer or the like.
　上記表面抗原について、エンベロープウイルスの表面抗原としては、例えば、HBV表面（HBsAg）、コア（HBcAg）抗原等のB型肝炎ウイルス抗原、ヘマグルチニン抗原（HA）、ノイラミニダーゼ抗原（NA）等のインフルエンザウイルス抗原、糖タンパク質（GP）等のエボラウイルス抗原、HIVエンベロープタンパク質gp41、gp120等のHIV抗原、スパイク（S）、エンベロープ（E）、膜（M）及びヌクレオカプシド（N）タンパク質等のSARS-Cov-2ウイルス抗原等が挙げられる。 Regarding the above surface antigens, the surface antigens of the envelope virus include, for example, hepatitis B virus antigens such as HBV surface (HBsAg) and core (HBcAg) antigens, and influenza virus antigens such as hemagglutinin antigen (HA) and neurominidase antigen (NA). , Ebola virus antigen such as glycoprotein (GP), HIV antigen such as HIV envelope protein gp41, gp120, SARS-Cov-2 such as spike (S), envelope (E), membrane (M) and nucleocapsid (N) protein Examples include viral antigens.
　細胞の表面抗原としては、例えば、CD3（T細胞）、CD19（B細胞）、NK1.1（NK細胞）、CD11c（樹状細胞）、CD11b（単球）、FcγR1（好塩基球）、Gr1（顆粒球）等が挙げられる。 Examples of cell surface antigens include CD3 (T cells), CD19 (B cells), NK1.1 (NK cells), CD11c (dendritic cells), CD11b (monocytes), FcγR1 (basophils), and Gr1. (Granulocytes) and the like.
　細胞外小胞のうち、アポトーシス小体の表面抗原としては、例えば、アネキシンV等が挙げられる。 Among extracellular vesicles, examples of surface antigens of apoptotic bodies include Annexin V and the like.
　細胞外小胞のうち、マイクロベシクルの表面抗原としては、例えば、セレクチン、インテグリン、フロチリン、CD40等が挙げられる。 Among extracellular vesicles, examples of surface antigens of microvesicles include selectin, integrin, floritrin, CD40 and the like.
　細胞外小胞のうち、エクソソームの表面抗原としては、例えば、テトラスパニン、細胞接着分子、抗原提示関連タンパク質、サイトカイン受容体等が挙げられる。また、癌患者の体液中のエクソソームの膜に多く存在することが知られているタンパク質等が挙げられる。テトラスパニンとしては、例えば、CD9、CD37、CD63、CD81等が挙げられる。細胞接着分子としては、例えば、インテグリン、CEAファミリーメンバー、ICAM-1（intercellular adhesion molecule-1）、CD31等が挙げられる。抗原提示関連タンパク質としては、例えば、MHC I、MHC II等が挙げられる。サイトカイン受容体としては、例えば、EGFRvIII等が挙げられる。癌患者の体液中のエクソソームの膜に多く存在することが知られているタンパク質としては、例えば、CD9、CD24、CD91、CD147、caveolin-1、glypican-1、EpCAM（Epithelial cell adhesion molecule）等が挙げられる。 Among extracellular vesicles, examples of surface antigens of exosomes include tetraspanin, cell adhesion molecule, antigen presentation-related protein, cytokine receptor and the like. In addition, proteins known to be abundant in the membrane of exosomes in the body fluids of cancer patients can be mentioned. Examples of tetraspanin include CD9, CD37, CD63, CD81 and the like. Examples of cell adhesion molecules include integrin, CEA family member, ICAM-1 (intercellular adhesion molecule-1), CD31 and the like. Examples of the antigen-presenting-related protein include MHC I, MHC II and the like. Examples of the cytokine receptor include EGFRvIII and the like. Examples of proteins known to be abundant in the membrane of exosomes in the body fluids of cancer patients include CD9, CD24, CD91, CD147, caveolin-1, glypican-1, and EpCAM (Epithelial cell adhesion molecule). Can be mentioned.
　エクソソーム膜タンパク質のうち、糖タンパク質であるCD9、CD37、CD63、インテグリン、CEAファミリーメンバー、ICAM-1、CD31、MHC I、MHC II、EGFRvIII、CD24、CD147、glypican-1、EpCAMが好ましく、CEAファミリーメンバーがより好ましい。CEAファミリーメンバーとしては、例えば、CEA、CEACAM 1、CEACAM 3、CEACAM 4、CEACAM 6、CEACAM 7、CEACAM 8等が挙げられ、好ましくはCEA、CEACAM 1、CEACAM 6である。 Among the exosome membrane proteins, glycoproteins CD9, CD37, CD63, integrin, CEA family members, ICAM-1, CD31, MHCI, MHCII, EGFRvIII, CD24, CD147, glypican-1, EpCAM are preferable, and the CEA family Members are more preferred. Examples of CEA family members include CEA, CEACAM1, CEACAM3, CEACAM4, CEACAM6, CEACAM7, CEACAM8, and the like, preferably CEA, CEACAM1, and CEACAM6.
　本発明の検出方法において用いる、検出対象である被験膜構造体に特異的親和性のある捕捉物質は、被験膜構造体に特異的親和性を有する限り、特に限定されない。該捕捉物質は、化学結合又は物理結合によって、テストラインに固定化されている。また、脂質二重層を認識する捕捉物質は、脂質二重層に特異的親和性を有する限り、特に限定されない。該捕捉物質は、化学結合又は物理結合によって、コントロールラインに固定化されている。
　テストラインに固定化されている被験膜構造体に特異的親和性のある捕捉物質と、コントロールラインに固定化されている脂質二重層を認識する捕捉物質は、同一のものであってもよく、異なるものであってもよいが、異なるものであるほうが好ましい。
　上記捕捉物質としては、例えば、アミノ酸、核酸、ポリペプチド、ポリヌクレオチド、脂質、リン脂質、糖質、多糖、低分子化合物、無機物質、及びこれらの融合体等が挙げられる。 The capture substance used in the detection method of the present invention and having a specific affinity for the test membrane structure to be detected is not particularly limited as long as it has a specific affinity for the test membrane structure. The trapping material is immobilized on the test line by a chemical bond or a physical bond. Further, the capture substance that recognizes the lipid bilayer is not particularly limited as long as it has a specific affinity for the lipid bilayer. The trapping material is immobilized on the control line by a chemical bond or a physical bond.
The capture substance having a specific affinity for the test membrane structure immobilized on the test line and the capture substance recognizing the lipid bilayer immobilized on the control line may be the same. It may be different, but it is preferable that it is different.
Examples of the capture substance include amino acids, nucleic acids, polypeptides, polynucleotides, lipids, phospholipids, carbohydrates, polysaccharides, low molecular weight compounds, inorganic substances, and fusions thereof.
　より具体的には、例えば、タンパク質、タンパク質断片、結合ドメイン、標的結合ドメイン、結合タンパク質、結合タンパク質断片、抗体、抗原、エピトープ、ハプテン、ビオチン、ビオチン誘導体、アビジン、ストレプトアビジン、基質、酵素、補因子、受容体、受容体断片、ホルモン等が挙げられる。 More specifically, for example, protein, protein fragment, binding domain, target binding domain, binding protein, binding protein fragment, antibody, antigen, epitope, hapten, biotin, biotin derivative, avidin, streptavidin, substrate, enzyme, supplement. Factors, receptors, receptor fragments, hormones and the like can be mentioned.
　例えば、上記抗体であれば、公知の手段を用いてポリクローナル又はモノクローナル抗体として得ることができる。あるいは、市販品を用いてもよい。本発明に用いる抗体の由来は、特に限定されるものではないが、好ましくは哺乳動物由来である。哺乳動物由来のモノクローナル抗体としては、ハイブリドーマに産生されるもの、及び遺伝子工学的手法により抗体遺伝子を含む発現ベクターで形質転換した宿主に産生されるもののいずれでもよい。抗体産生ハイブリドーマは、自体公知の方法により作製することができ、例えば、被験膜構造体の膜に含まれるタンパク質又はその一部を抗原として使用して、該抗原を通常の免疫方法にしたがって免疫し、得られる免疫細胞を通常の細胞融合法によって公知の親細胞と融合させ、通常のスクリーニング法により、モノクローナルな抗体産生細胞をスクリーニングすることによって作製できる。 For example, the above antibody can be obtained as a polyclonal or monoclonal antibody by using known means. Alternatively, a commercially available product may be used. The origin of the antibody used in the present invention is not particularly limited, but is preferably derived from mammals. The monoclonal antibody derived from a mammal may be either one produced by a hybridoma or one produced by a host transformed with an expression vector containing an antibody gene by a genetic engineering technique. An antibody-producing hybridoma can be produced by a method known per se, for example, using a protein contained in the membrane of a test membrane structure or a part thereof as an antigen, and immunizing the antigen according to a conventional immunization method. It can be produced by fusing the obtained immune cells with known parent cells by a conventional cell fusion method and screening monoclonal antibody-producing cells by a conventional screening method.
　抗体の断片としては、同一の相補性決定領域を含むものであれば特に制限されず、軽鎖の断片と軽鎖の断片とをリンカー等を介して融合したもの（例：scFv（一本鎖抗体）等）も包含される。具体的な抗体又はその断片には、例えば、Fab、Fab'、F（ab'）2等の断片、scFv、scFv-Fc、ミニボディー、ダイアボディー等の遺伝子工学的に作製されたコンジュゲート分子、あるいはポリエチレングリコール（PEG）等のタンパク質安定化作用を有する分子等で修飾されたそれらの誘導体などが挙げられる。 The antibody fragment is not particularly limited as long as it contains the same complementarity determining regions, and is a fusion of a light chain fragment and a light chain fragment via a linker or the like (eg, scFv (single chain). Antibodies) etc.) are also included. Specific antibodies or fragments thereof include, for example, fragments such as Fab, Fab', F (ab') 2, and genetically engineered conjugated molecules such as scFv, scFv-Fc, minibody, and diabody. , Or derivatives thereof modified with a molecule having a protein stabilizing action such as polyethylene glycol (PEG).
　テストライン、及びテストラインにおける蛍光（発光）の検出又は測定方法に特に限定はないが、一般的な光ファイバ型蛍光検出器、マイクロプレートリーダー、蛍光顕微鏡、カメラ（例：CCDカメラ等）を備えた検出器（例：Celvinなど）等を用いれば検出又は測定できる。また、ELISA法での方法と同様に、標的の被験膜構造体の濃度が既知の試料（標準品）を用いて検量線を作成し、該検量線を用いて、試料中の被験膜構造体の濃度を定量することもできる。 The test line and the method for detecting or measuring fluorescence (emission) in the test line are not particularly limited, but are equipped with a general optical fiber type fluorescence detector, a microplate reader, a fluorescence microscope, and a camera (eg, CCD camera, etc.). It can be detected or measured by using a detector (eg, Celvin, etc.). Further, as in the method of the ELISA method, a calibration curve is prepared using a sample (standard product) having a known concentration of the target test membrane structure, and the calibration curve is used to prepare the test membrane structure in the sample. It is also possible to quantify the concentration of.
2．本発明の判定方法
　本発明は、以下：
　対象における疾患を判定（検出）するための方法であって、
（1）前記対象由来のサンプル中に存在する脂質二重層を有する膜構造体を標識する工程、
（2）所定の脂質二重層を有する膜構造体を含む画分を分離する工程、及び
（3）前記画分中の前記所定の脂質二重層を有する膜構造体を検出する工程
を含む、方法
を提供する。 2. The determination method of the present invention The present invention is as follows:
It is a method for determining (detecting) a disease in a subject.
(1) A step of labeling a membrane structure having a lipid bilayer present in a sample derived from the subject.
A method comprising (2) a step of separating a fraction containing a membrane structure having a predetermined lipid bilayer, and (3) a step of detecting a membrane structure having the predetermined lipid bilayer in the fraction. I will provide a.
　本発明の判定方法は、対象がウイルスや細菌感染、あるいはがん等の疾患に罹患しているか否かの判定（対象におけるウイルスや細菌感染、あるいはがん等の疾患の検出）のための方法である。判定は、上記「1．本発明の検出方法」において記載したようなウイルス、細菌、エクソソーム等の脂質二重層を有する膜構造体の表面抗原を検出することにより為される。
　例えば、ウイルスや細菌であれば、それらの存在を検出することで、対象（被験者）が該ウイルスや細菌に感染している（あるいは感染している可能性がある）と判定し得る。
　また、例えば、エクソソームであれば、特定のがんに罹患した対象由来のエクソソームの脂質二重層の表面には、特定の表面抗原（例：EGFRIII（膠芽腫）、Del-1（乳がん）、Fibronectin（乳がん）、CD147（大腸がん）、GPC-1（膵臓がん）、EphA2（膵臓がん）、EGFR、EpCAM、MUC1、WNT2、GPC1（膵臓がん）、CD91（肺がん）、CD24、EpCAM（卵巣がん）、CD24、EpCAM、CA-125（卵巣がん）、Survivin（前立腺がん）、PD-L1（咽頭がん）、TACSTD2(膀胱がん)、PD-L1（メラノーマ）、CD63、Caveolin-1（メラノーマ）等）が（有意に）発現し得る。そのため、エクソソーム自体の表面抗原（例：CD9等）と、特定の疾患に起因する上記エクソソームの表面抗原との両方を検出することで、該特定の疾患を判定（特定の疾患に罹患している、あるいは罹患している可能性があると判定）し得る。例えば、本発明の判定方法をラテラルフロー用のテストストリップ上で行う場合、エクソソーム自体の表面抗原（例：CD9等）をコントロールラインにて検出し、特定の疾患に起因する上記エクソソームの表面抗原をテストラインにて検出することで判定を行ってもよい。また、本発明の方法の工程（3）で検出した対象（被験者）由来の所定の脂質二重層を有する膜構造体の量と、健常者の所定の脂質二重層を有する膜構造体の量とを比較すること（工程（4））で、特定の疾患（例：がん等）に罹患している、あるいは罹患している可能性があると判定してもよい。より具体的には、例えば、対象（被験者）由来のCD9とCD147の共陽性のエクソソームの量が、健常者と比較して（有意に）多い場合、該対象が大腸がんに罹患している（あるいは罹患している可能性がある）と判定し得る。該比較は、例えば、蛍光強度や自体公知の方法によって求めたカットオフ値等を用いて行ってもよい。
　本発明の判定方法は、医師等における確定診断を補助するものであってもよく、従って、該判定方法は、判定（診断）を補助するための方法ともいえる。本発明の判定方法は、上記「1．本発明の検出方法」において記載した内容を必要に応じて適宜、援用するものとする。 The determination method of the present invention is a method for determining whether or not a subject has a virus or bacterial infection or a disease such as cancer (detection of a virus or bacterial infection in the subject or a disease such as cancer). Is. The determination is made by detecting the surface antigen of the membrane structure having a lipid bilayer such as a virus, a bacterium, an exosome, etc. as described in the above "1. Detection method of the present invention".
For example, in the case of a virus or a bacterium, it can be determined that the subject (subject) is infected with (or may be infected with) the virus or the bacterium by detecting the presence of the virus or the bacterium.
In addition, for example, in the case of exosomes, specific surface antigens (eg, EGFRIII (collagenoma), Del-1 (breast cancer), etc. Fibronectin (breast cancer), CD147 (colon cancer), GPC-1 (pancreatic cancer), EphA2 (pancreatic cancer), EGFR, EpCAM, MUC1, WNT2, GPC1 (pancreatic cancer), CD91 (lung cancer), CD24, EpCAM (ovarian cancer), CD24, EpCAM, CA-125 (ovarian cancer), Survivin (prostatic cancer), PD-L1 (pharyngeal cancer), TACSTD2 (bladder cancer), PD-L1 (melanoma), CD63, Caveolin-1 (melanoma), etc.) can be expressed (significantly). Therefore, the specific disease is determined (affected by a specific disease) by detecting both the surface antigen of the exosome itself (eg, CD9, etc.) and the surface antigen of the exosome caused by the specific disease. , Or it may be determined that it may be affected). For example, when the determination method of the present invention is performed on a test strip for lateral flow, the surface antigen of the exosome itself (eg, CD9, etc.) is detected on the control line, and the surface antigen of the exosome caused by a specific disease is detected. Judgment may be made by detecting on the test line. Further, the amount of the membrane structure having a predetermined lipid double layer derived from the subject (subject) detected in the step (3) of the method of the present invention and the amount of the membrane structure having a predetermined lipid double layer of a healthy person. By comparing (step (4)), it may be determined that the patient has or may have a specific disease (eg, cancer, etc.). More specifically, for example, if the amount of co-positive exosomes of CD9 and CD147 derived from a subject (subject) is (significantly) higher than that of a healthy subject, the subject has colorectal cancer. It can be determined (or may be affected). The comparison may be performed using, for example, the fluorescence intensity, the cutoff value obtained by a method known per se, or the like.
The determination method of the present invention may assist a definitive diagnosis by a doctor or the like, and therefore, the determination method can be said to be a method for assisting a determination (diagnosis). As the determination method of the present invention, the contents described in the above "1. Detection method of the present invention" shall be appropriately incorporated as necessary.
3．本発明のキット
　本発明のキットの構成の一例を、図1（a）に模式的に示した図を参照して説明する。図1（a）に示すように、当該キットは、本発明の検出方法を実施するためのものであって、試薬容器100と、テストストリップ200とを有して構成される。試薬容器100には、標識試薬110が収容されている。該標識試薬110は、サンプルS1中に存在する上記した膜構造体を標識するための試薬である。テストストリップ200は、サンプル受入れ部210と、フィルター部220と、検出部240とを少なくとも有し、図1（a）の例では、これらが支持体層200b上に配置されている。支持体層200bの材料、厚さ、テストストリップ200の全体的な積層構造、各部の長さ、全体の長さなどは、従来公知のテストストリップを参照することができる。 3. 3. Kit of the present invention An example of the configuration of the kit of the present invention will be described with reference to the figure schematically shown in FIG. 1 (a). As shown in FIG. 1 (a), the kit is for carrying out the detection method of the present invention, and includes a reagent container 100 and a test strip 200. The reagent container 100 contains the labeling reagent 110. The labeling reagent 110 is a reagent for labeling the above-mentioned membrane structure present in the sample S1. The test strip 200 has at least a sample receiving unit 210, a filter unit 220, and a detecting unit 240, which are arranged on the support layer 200b in the example of FIG. 1 (a). For the material and thickness of the support layer 200b, the overall laminated structure of the test strip 200, the length of each part, the overall length, and the like, conventionally known test strips can be referred to.
　当該キットの各部の構成例、及び、その使用方法は次のとおりである。
　先ず、図1（a）に太い矢印で示すように、サンプルS1が試薬容器100中に注入されると、該サンプルS1中の膜構造体が標識試薬110によって標識される。以下、標識された膜構造体を含んだサンプルを、「標識されたサンプルS2」、又は、単に「サンプルS2」と呼ぶ。
　図1（a）の例では、簡単に説明するために、サンプルS1が試薬容器100中に注入された例を示しているが、ウエルなどの別の混合用容器を用いて、サンプルS1と標識試薬110とを混合してもよい。 The configuration example of each part of the kit and the usage method thereof are as follows.
First, as shown by the thick arrow in FIG. 1 (a), when the sample S1 is injected into the reagent container 100, the membrane structure in the sample S1 is labeled with the labeling reagent 110. Hereinafter, the sample containing the labeled membrane structure is referred to as "labeled sample S2" or simply "sample S2".
In the example of FIG. 1 (a), for the sake of brevity, an example in which the sample S1 is injected into the reagent container 100 is shown, but another mixing container such as a well is used to label the sample S1 with the sample S1. It may be mixed with the reagent 110.
　次に、標識されたサンプルS2は、ピペット等を用いて、テストストリップ200のサンプル受入れ部210に供給（例えば、滴下）される。
　サンプル受入れ部210は、標識されたサンプルS2を受入れる部分であって、供給されるサンプルS2を十分な量だけ一時的に保持し得、かつ、テストストリップ中に放出し得るように構成された部分である。サンプル受入れ部210の構造自体は、従来公知のテストストリップにおけるサンプル受入れ部の構造を参照することができ、例えば、セルロース、ガラス繊維、ポリウレタン、ポリアセテート、酢酸セルロース、ナイロン、綿布等を素材としたものが挙げられ、より詳細には、例えば、Cellulose Absorbent Pad（Pall社製）等が例示される。 Next, the labeled sample S2 is supplied (for example, dropped) to the sample receiving portion 210 of the test strip 200 using a pipette or the like.
The sample receiving section 210 is a portion that receives the labeled sample S2 and is configured to temporarily hold a sufficient amount of the supplied sample S2 and release it into the test strip. Is. The structure of the sample receiving portion 210 itself can refer to the structure of the sample receiving portion in a conventionally known test strip, and is made of, for example, cellulose, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, cotton cloth or the like. In more detail, for example, Cellulose Absorbent Pad (manufactured by Pall) and the like are exemplified.
　サンプル受入れ部210で受け入れられたサンプルS2は、図1（a）に太い破線の矢印で示すように、テストストリップ200中を進み、フィルター部220を通過し、検出部240に到達する。サンプルS2がテストストリップ200中を進むための駆動力（推進力や吸引力）は、特に限定はされず、例えば、重力、毛管現象が例示され、これらを組み合わせたものであってもよい。 The sample S2 received by the sample receiving unit 210 travels through the test strip 200, passes through the filter unit 220, and reaches the detection unit 240, as shown by the thick broken line arrow in FIG. 1 (a). The driving force (propulsive force or suction force) for the sample S2 to travel through the test strip 200 is not particularly limited, and for example, gravity and capillarity are exemplified, and a combination thereof may be used.
　図1（a）の例では、好ましい態様として、フィルター部220と検出部240との間に展開部230が設けられる。展開部230は、展開膜（メンブレン）の全部であってもよく一部であってもよく、テストライン上に存在する捕捉分子（例：抗体等）と、検出を所望する脂質二重層を有する膜構造体との結合が阻害されない程度の展開のために設けられる部分である。また、図1（a）の例では、好ましい態様として、サンプルS2が検出部240を通過するように構成されている。 In the example of FIG. 1 (a), as a preferred embodiment, the expansion unit 230 is provided between the filter unit 220 and the detection unit 240. The developing portion 230 may be the whole or a part of the developing membrane, and has a capture molecule (eg, an antibody, etc.) present on the test line and a lipid bilayer layer desired to be detected. It is a part provided for development to the extent that the bond with the membrane structure is not hindered. Further, in the example of FIG. 1 (a), as a preferred embodiment, the sample S2 is configured to pass through the detection unit 240.
　フィルター部220は、サンプル受入れ部210で受け入れたサンプルS2中の標識された膜構造体を、選択的に通過させる部分である。該フィルター部220が存在することで、サンプルS2中に含まれる夾雑物を除去でき、展開部230における分離が容易となる。該フィルター部220の構成は、夾雑物を除去し得る限り、特に限定されず、従来公知のフィルターの構成を参照することができる。また、サンプル受入部210とフィルター部220は個々に存在してもよく、一体として存在してもよい。 The filter unit 220 is a portion that selectively passes the labeled membrane structure in the sample S2 received by the sample receiving unit 210. The presence of the filter unit 220 makes it possible to remove impurities contained in the sample S2, and facilitates separation in the developing unit 230. The configuration of the filter unit 220 is not particularly limited as long as impurities can be removed, and conventionally known filter configurations can be referred to. Further, the sample receiving unit 210 and the filter unit 220 may exist individually or as a unit.
　検出部240は、サンプルS2中の標識された膜構造体を、該標識に基づいて検出するように構成された部分である。該検出部240の構成は、「1．本発明の検出方法」にて詳述した通りであり、テストラインと、任意によりコントロールラインを有する。 The detection unit 240 is a portion configured to detect the labeled membrane structure in the sample S2 based on the label. The configuration of the detection unit 240 is as described in detail in "1. Detection method of the present invention", and has a test line and optionally a control line.
4．本発明のテストストリップ
　本発明のテストストリップの構成の一例を、図1（b）に模式的に示した図を参照して説明する。図1（b）に示すように、当該テストストリップ300は、本発明の検出方法を実施するためのものであって、サンプル受入れ部310と、コンジュゲート部350、フィルター部320と、検出部340とを少なくとも有し、図1（b）の例では、これらが支持体層300b上に配置されている。当該テストストリップ300と、図1（a）に示したキットのテストストリップ200との差異は、当該テストストリップ300がコンジュゲート部350を有するという点にある。コンジュゲート部350が設けられているので、ユーザーは、当該テストストリップ300だけを用いて、本発明の検出方法を実施することができる。該テストストリップ300に含まれる、サンプル受入れ部310、フィルター部320、展開部330、検出部340、支持体層300bは、それぞれ、上記したキットにおけるサンプル受入れ部210、フィルター部220、展開部230、検出部240、支持体層200bと同様である。支持体層200bの材料、厚さ、テストストリップ200の全体的な積層構造、各部の長さ、全体の長さなどは、従来公知のテストストリップを参照することができる。 Four. Test Strip of the Present Invention An example of the configuration of the test strip of the present invention will be described with reference to the figure schematically shown in FIG. 1 (b). As shown in FIG. 1 (b), the test strip 300 is for carrying out the detection method of the present invention, and has a sample receiving unit 310, a conjugate unit 350, a filter unit 320, and a detection unit 340. In the example of FIG. 1 (b), these are arranged on the support layer 300b. The difference between the test strip 300 and the test strip 200 of the kit shown in FIG. 1 (a) is that the test strip 300 has a conjugate portion 350. Since the conjugate portion 350 is provided, the user can carry out the detection method of the present invention using only the test strip 300. The sample receiving unit 310, the filter unit 320, the expanding unit 330, the detecting unit 340, and the support layer 300b included in the test strip 300 are the sample receiving unit 210, the filter unit 220, and the expanding unit 230, respectively. This is the same as the detection unit 240 and the support layer 200b. For the material and thickness of the support layer 200b, the overall laminated structure of the test strip 200, the length of each part, the overall length, and the like, conventionally known test strips can be referred to.
　先ず、図1（b）に太い矢印で示すように、サンプルS1が、ピペット等を用いて、テストストリップ300のサンプル受入れ部310に供給（例えば、滴下）される。サンプル受入れ部310で受け入れられたサンプルS1は、図1（b）に太い破線の矢印で示すように、テストストリップ300中を進み、コンジュゲート部350を通過する。コンジュゲート部350は、サンプルS1中の膜構造体を標識するように構成されている。換言すると、サンプルS1がコンジュゲート部350を通過すると、標識された膜構造体を含んだサンプル（即ち、サンプルS2）となる。サンプルS2がフィルター部320を通過して検出部340に到達する構成は、上記したキットのテストストリップと同様である。 First, as shown by the thick arrow in FIG. 1 (b), the sample S1 is supplied (for example, dropped) to the sample receiving portion 310 of the test strip 300 using a pipette or the like. The sample S1 received by the sample receiving section 310 travels through the test strip 300 and passes through the conjugate section 350, as shown by the thick dashed arrow in FIG. 1 (b). The conjugate portion 350 is configured to label the membrane structure in sample S1. In other words, when sample S1 passes through the conjugate section 350, it becomes a sample containing the labeled membrane structure (ie, sample S2). The configuration in which the sample S2 passes through the filter unit 320 and reaches the detection unit 340 is the same as the test strip of the above-mentioned kit.
　以下、本発明を実施例に基づいてさらに詳細に説明する。本発明はこれらの実施例に何ら制限されるものではない。
（1）テストストリップの作製
　メンブレン（HiFlow Plus HF120 (Milipore社)）に、抗体塗布機で、抗CD9マウスモノクローナル抗体をコントロールラインに、抗CD147マウスモノクローナル抗体をテストラインに、1μl/cmで塗布する。コントロールラインとテストラインに関しては、サンプル溶液を滴下するポジションからの距離がテストラインとコントロールラインが同じになるように、該テストラインと該コントロールラインとを左右並列に配置する（図2（a）及び（b）を参照）。
　抗体を塗布したメンブレン、吸収パッド（CFSP203000（Milipore社））、サンプルパッド（Cellulose Absorbent Pad #111（Pall社））を、バッキングシート（AdhesiveResearch社）に貼り付ける。該バッキングシートを、カッティング機器を用いて5mm幅に裁断した後、ハウジングケースに入れて、テストストリップとする。 Hereinafter, the present invention will be described in more detail based on examples. The present invention is not limited to these examples.
(1) Preparation of test strip Apply anti-CD9 mouse monoclonal antibody to the control line and anti-CD147 mouse monoclonal antibody to the test line at 1 μl / cm using an antibody coater on the membrane (HiFlow Plus HF120 (Milipore)). .. Regarding the control line and the test line, the test line and the control line are arranged in parallel on the left and right so that the distance from the position where the sample solution is dropped is the same as the test line and the control line (Fig. 2 (a)). And (b)).
Attach the antibody-coated membrane, absorption pad (CFSP203000 (Milipore)), and sample pad (Cellulose Absorbent Pad # 111 (Pall)) to the backing sheet (Adhesive Research). The backing sheet is cut to a width of 5 mm using a cutting device, and then placed in a housing case to form a test strip.
（2）サンプル溶液の調製
　検出対象である脂質二重層を有する膜構造体（エクソソーム）含むサンプルとして、大腸がん患者由来の血清を使用する。該サンプルである大腸がん患者由来の血清中に、FM4-64を添加して、検出対象である脂質二重層を有する膜構造体を染色する。
（3）テストストリップにおける検出
　FM4-64を添加したサンプルを、テストストリップ1枚あたり100μl滴下し、テストストリップ上に展開する。展開した結果、テストライン及びコントロールラインのいずれもFM4-64に起因する蛍光が確認される。従って、作製したテストストリップにより、CD９とCD147の共陽性のエクソソームが検出され得ることを確認する。
　該テストラインにおけるFM4-64に起因する蛍光の強度と、同様の方法により検出を行った健常者由来の血清におけるテストラインのFM4-64に起因する蛍光強度とを比較する。該比較の結果から、作製したテストストリップが十分に使用可能であるものであることが分かる。 (2) Preparation of sample solution Serum derived from a colorectal cancer patient is used as a sample containing a membrane structure (exosome) having a lipid bilayer to be detected. FM4-64 is added to the serum derived from the colorectal cancer patient, which is the sample, to stain the membrane structure having the lipid bilayer to be detected.
(3) Detection on test strips 100 μl of the sample to which FM4-64 was added was dropped per test strip and developed on the test strips. As a result of unfolding, fluorescence caused by FM4-64 is confirmed on both the test line and the control line. Therefore, it is confirmed that co-positive exosomes of CD9 and CD147 can be detected by the prepared test strip.
The fluorescence intensity due to FM4-64 in the test line is compared with the fluorescence intensity due to FM4-64 in the test line in the serum derived from a healthy subject detected by the same method. From the results of the comparison, it can be seen that the prepared test strips are sufficiently usable.
　本発明の脂質二重層を有する膜構造体を検出する方法は、所望する脂質二重層を有する膜構造体を高精度且つ簡便に検出するために利用可能である。また、本発明の疾患の判定方法は、疾患特異的なタンパク質を発現する脂質二重層を有する膜構造体を効率的に検出することができ、信頼性の高い疾患の判定に利用可能である。 The method of the present invention for detecting a membrane structure having a lipid bilayer can be used to detect a desired membrane structure having a lipid bilayer with high accuracy and convenience. In addition, the disease determination method of the present invention can efficiently detect a membrane structure having a lipid bilayer expressing a disease-specific protein, and can be used for highly reliable disease determination.
　本出願は、２０２０年１２月１４日付で日本国に出願された特願２０２０－２０７１１４を基礎としており、ここで言及することにより、その内容のすべてが本明細書に組み込まれるものである。 This application is based on Japanese Patent Application No. 2020-207114 filed in Japan on December 14, 2020, the entire contents of which are incorporated herein by reference.
　S1、S2　　サンプル
　100　　試薬容器
　110　　標識試薬
　200、300　　テストストリップ S1, S2 Sample 100 Reagent Container 110 Labeled Reagent 200, 300 Test Strip

Claims (3)

  1. 　脂質二重層を有する膜構造体を検出する方法であって、
    （1）サンプル中に存在する脂質二重層を有する膜構造体を標識する工程、
    （2）所定の脂質二重層を有する膜構造体を含む画分を分離する工程、及び
    （3）前記画分中の前記所定の脂質二重層を有する膜構造体を検出する工程
    を含む、方法。     A method for detecting a membrane structure having a lipid bilayer.
    (1) A step of labeling a membrane structure having a lipid bilayer present in a sample,
    A method comprising (2) a step of separating a fraction containing a membrane structure having a predetermined lipid bilayer, and (3) a step of detecting a membrane structure having the predetermined lipid bilayer in the fraction. ..
  2. 　脂質二重層を有する膜構造体を検出するキットであって、当該キットは、
    　サンプル中に存在する前記膜構造体を標識するための標識試薬が収容された試薬容器と、
    　前記標識試薬によって標識された膜構造体を含んだサンプルから、該膜構造体を検出するテストストリップとを有し、
    　前記テストストリップは、
    　　前記標識された膜構造体を含んだサンプルを受入れるサンプル受入れ部と、
    　　前記サンプル受入れ部で受け入れたサンプル中の前記標識された膜構造体を選択的に通過させるフィルター部と、
    　　前記フィルター部を通過した前記標識された膜構造体を、前記標識に基づいて検出する検出部と
    を有するものである、前記キット。     It is a kit for detecting a membrane structure having a lipid bilayer, and the kit is
    A reagent container containing a labeling reagent for labeling the membrane structure present in the sample, and
    It has a test strip for detecting the membrane structure from a sample containing the membrane structure labeled with the labeling reagent.
    The test strip
    A sample receiving unit that receives a sample containing the labeled membrane structure, and a sample receiving unit.
    A filter unit that selectively passes the labeled membrane structure in the sample received by the sample receiving unit, and a filter unit.
    The kit comprising a detection unit for detecting the labeled membrane structure that has passed through the filter unit based on the label.
  3. 　脂質二重層を有する膜構造体を検出するテストストリップであって、当該テストストリップは、
    　前記膜構造体を含んだサンプルを受入れるサンプル受入れ部と、
    　前記サンプル受入れ部で受け入れたサンプル中の前記膜構造体を標識するためのコンジュゲート部と、
    　前記コンジュゲート部によって標識された膜構造体を選択的に通過させるフィルター部と、
    　前記フィルター部を通過した前記標識された膜構造体を、前記標識に基づいて検出する検出部と
    を有する、前記テストストリップ。     A test strip for detecting a membrane structure having a lipid bilayer, and the test strip is
    A sample receiving unit that receives a sample containing the membrane structure,
    A conjugate section for labeling the membrane structure in the sample received by the sample receiving section, and a conjugate section.
    A filter unit that selectively passes the membrane structure labeled by the conjugate unit, and a filter unit.
    The test strip comprising a detection unit for detecting the labeled membrane structure that has passed through the filter unit based on the label.
PCT/JP2021/045817 2020-12-14 2021-12-13 Method for detecting membrane structural body having lipid bilayer WO2022131208A1 (en)

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Publication number Priority date Publication date Assignee Title
JPH11337553A (en) * 1989-12-18 1999-12-10 Princeton Biomeditech Corp Immune chemical label, water suspension containg it, and manufacture of immune chemical label
JPH06341991A (en) * 1992-09-16 1994-12-13 Navix Inc Unseparated bonding assay method for label trap assay
JP2005214670A (en) * 2004-01-27 2005-08-11 Denka Seiken Co Ltd Simple detection method, detection device, detection kit and its manufacturing method
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JP2015230280A (en) * 2014-06-06 2015-12-21 凸版印刷株式会社 Detection method of analysis object and test strip for lateral flow
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