WO2022131208A1 - 脂質二重層を有する膜構造体の検出方法 - Google Patents

脂質二重層を有する膜構造体の検出方法 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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membrane structure
sample
lipid bilayer
virus
test strip
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PCT/JP2021/045817
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English (en)
French (fr)
Japanese (ja)
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憲祐 齊藤
逸明 楊
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株式会社堀場製作所
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Publication of WO2022131208A1 publication Critical patent/WO2022131208A1/ja

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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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PCT/JP2021/045817 2020-12-14 2021-12-13 脂質二重層を有する膜構造体の検出方法 WO2022131208A1 (ja)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06341991A (ja) * 1992-09-16 1994-12-13 Navix Inc 標識トラップアッセイ−非分離結合アッセイ方法
JPH11337553A (ja) * 1989-12-18 1999-12-10 Princeton Biomeditech Corp 免疫化学的標識およびこれを含有する水性懸濁液、ならびに該免疫化学的標識の製造方法
JP2005214670A (ja) * 2004-01-27 2005-08-11 Denka Seiken Co Ltd 簡便な検出法、検出装置及び検出キットとその製法
WO2010001598A1 (ja) * 2008-06-30 2010-01-07 積水メディカル株式会社 結合アッセイ用多孔性固相及びこれを用いた結合アッセイ法
JP2015230280A (ja) * 2014-06-06 2015-12-21 凸版印刷株式会社 分析対象物の検出方法及びラテラルフロー用テストストリップ
JP2017211396A (ja) * 2015-08-28 2017-11-30 栄研化学株式会社 免疫学的測定用試薬組成物およびその用途
JP2019536066A (ja) * 2016-11-18 2019-12-12 カルス インコーポレイテッド ラテラルフロー型アッセイストリップ用の濃縮キット
WO2020111223A1 (ja) * 2018-11-30 2020-06-04 旭化成株式会社 乳房炎の原因菌の検出方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11337553A (ja) * 1989-12-18 1999-12-10 Princeton Biomeditech Corp 免疫化学的標識およびこれを含有する水性懸濁液、ならびに該免疫化学的標識の製造方法
JPH06341991A (ja) * 1992-09-16 1994-12-13 Navix Inc 標識トラップアッセイ−非分離結合アッセイ方法
JP2005214670A (ja) * 2004-01-27 2005-08-11 Denka Seiken Co Ltd 簡便な検出法、検出装置及び検出キットとその製法
WO2010001598A1 (ja) * 2008-06-30 2010-01-07 積水メディカル株式会社 結合アッセイ用多孔性固相及びこれを用いた結合アッセイ法
JP2015230280A (ja) * 2014-06-06 2015-12-21 凸版印刷株式会社 分析対象物の検出方法及びラテラルフロー用テストストリップ
JP2017211396A (ja) * 2015-08-28 2017-11-30 栄研化学株式会社 免疫学的測定用試薬組成物およびその用途
JP2019536066A (ja) * 2016-11-18 2019-12-12 カルス インコーポレイテッド ラテラルフロー型アッセイストリップ用の濃縮キット
WO2020111223A1 (ja) * 2018-11-30 2020-06-04 旭化成株式会社 乳房炎の原因菌の検出方法

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