WO2008018631A1 - Complexe de liposome, réseau de liposome, et procédé de détection d'un analyte - Google Patents

Complexe de liposome, réseau de liposome, et procédé de détection d'un analyte Download PDF

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Publication number
WO2008018631A1
WO2008018631A1 PCT/JP2007/065928 JP2007065928W WO2008018631A1 WO 2008018631 A1 WO2008018631 A1 WO 2008018631A1 JP 2007065928 W JP2007065928 W JP 2007065928W WO 2008018631 A1 WO2008018631 A1 WO 2008018631A1
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Prior art keywords
ribosome
array
test substance
substance
complex
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PCT/JP2007/065928
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English (en)
Japanese (ja)
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Masao Sugawara
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Nihon University
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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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/5432—Liposomes or microcapsules
    • 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
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552—Glass or silica
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7786—Fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G01N21/80—Indicating pH value

Definitions

  • Ribosome complex Ribosome complex, ribosome array, and method for detecting a test substance
  • the present invention relates to a highly sensitive detection method for a test substance using ribosome. More specifically, the present invention comprises a substance that specifically binds to a test substance on a ribosome and a phosphatidylethanolamine piotin derivative (B-cap-PE) on the membrane surface, and a pH-dependent fluorescent dye.
  • B-cap-PE phosphatidylethanolamine piotin derivative
  • a ribosome complex in which the hydrogen ion concentration in the aqueous phase of the ribosome containing the pH-dependent fluorescent dye is adjusted to be higher than the hydrogen ion concentration outside the ribosome, and the ribosome complex is modified with avidin The present invention relates to a ribosome array immobilized in an array on a glass substrate, a highly sensitive detection method for a test substance using the ribosome array, and a kit used in the method.
  • the present invention relates to a ribosome complex having a maleimide group on the membrane surface (maleimide group-containing liposome complex), a ribosome array in which the maleimide group-containing ribosome complex is immobilized in an array on an avidin-modified glass substrate ( Maleimide group-containing ribosome array), and in the step of detecting the test substance using the maleimide group-containing ribosome array, an antibody or its Fab ′ fragment that specifically binds to the test substance is bound to the maleimide group-containing ribosome complex.
  • the present invention relates to an immunoribosome array formed by binding, a method for highly sensitive detection of a test substance using the immunoliposome array, and a kit used in the method.
  • an immunodetection method or an immunoassay method includes, for example, proteins, hormones. It is widely used as a method for highly selective analysis of biological components such as active peptides, otachoids, tumor markers, immunoglobulins, and trace components such as drugs such as digoxin, phenytoin, and c-nobarbital. In recent years, the number of samples that need to be measured for the detection, characterization, and sorting of these biological components and drugs has increased significantly, and many samples can quickly and automatically measure multiple components. A method is required.
  • Radioimmunoassay (RIA method), enzyme-labeled antibody assay (ELISA) And an immunoassay using a membrane (membrane-based immunoassay).
  • RIA method enzyme-labeled antibody assay
  • ELISA enzyme-labeled antibody assay
  • EQCM electrochemical quartz crystal microbalance
  • SPR surface plasmo resonance
  • the ELISA method requires a heterogeneous method (heterogeneous method, solid phase system) and B / F separation that require separation of the labeled material that reacted with the antigen and antibody (B / F separation). Not classified as homogeneous (homogeneous).
  • the heterogeneous method requires a lot of time for measurement because it requires repeated incubation and washing steps for force separation (B / F separation) with excellent sensitivity. Therefore, it is not suitable for processing a large number of samples quickly.
  • Liposomes suspend amphiphilic complex lipids (lecithin, cholesterol, phosphatidylic acid, etc.) that have both hydrophilic and hydrophobic groups in the same molecule in a buffer solution above a certain temperature. It is an endoplasmic reticulum composed of a lipid bilayer prepared in (1).
  • the immunoassay method using a ribosome is a ribosome complex that contains fluorescent dyes, inorganic and organic ions, enzyme molecules, etc. inside, and uses a bifunctional cross-linking agent to covalently bind an antigen or antibody to the membrane.
  • the body is called a functional liposome
  • the fluorescent dyes released from the aqueous phase in the ribosome are measured by analytical methods such as fluorometry and potentiometry.
  • analytical methods such as fluorometry and potentiometry.
  • a liposomal immunoassay that uses a ribosome encapsulated in a fluorophore (fluorescent dye molecule) as a detection label, or a ribosome that utilizes the release of electroactive species by ribosome force.
  • fluorophore fluorescent dye molecule
  • an antigen-antibody reaction is formed in a liposomal membrane encapsulating a fluorescent dye inside, a trap is applied to this to destroy the ribosome, and the amount of antigen or antibody is detected by measuring and detecting the released fluorescent dye.
  • Immunodetection methods have been developed to measure urine (Patent Documents 1 and 2).
  • a liposome bound to an antibody or an antigen and an unbound ribosome are separated by a flow system, and then the ribosome is disrupted and released. The substance is measured, or the ribosome force suspended in the solution is measured by an ion selective electrode or portammetry, etc.
  • These immunoassays require a process (B / F separation) that separates liposomes that are not bound to antibodies or antigen-bound ribosomes, making it difficult to simultaneously assay multiple samples.
  • the ribosome disruption process diffuses fluorescent dyes and colored products into the liquid layer, resulting in a decrease in sensitivity and a clear signal could not be obtained.
  • Non-patent Documents 1 and 2 monitor changes in physical properties of phospholipid bilayers by antigen-antibody reaction.
  • a ribosome having a primary antibody that recognizes the test substance and a secondary antibody that recognizes the primary antibody on the surface and encapsulating the labeling substance Has been developed to detect a test substance by reacting with a labeling substance capable of permeating the lipid membrane of ribosome (Patent Document 3).
  • Non-patent literature l Nikolelis, D. P .; Hianik, T. Krull, U. J. Electroanaylsis 1999, 11, 7-1 5
  • Non-Patent Document 2 Hianik, ⁇ ; et al. Gen. Physiol. Biophys. 1998, 17, 239-252
  • Non-Patent Document 3 Yanagisawa, H; Hirano, A; Sugawara, M. Anal. Biochem. 2004, 332 ,
  • Non-Patent Document 4 Killian, J.A.Biochem.Biophys. Acta 1992, 1113, 391-425
  • Non-Patent Document 5 Hirano, A .; Wakabayashi, M .; Matsuno, Y .; Sugawara, M. Biosens. Bioelectron. 2003, 18, 973–983
  • Non-Patent Document 6 Rudnev, V.S .; Ermishkin, L. N .; Fonina, L. A .; Rovin, Yu. G. Biochem. Biophys. Acta 1981, 642, 196-202
  • Non-Patent Document 7 Ishikawa, E .; Imagawa,.; Hashida, D .; Yoshitake, S ,; Hamaguchi, Y .; PT / JP2007 / 065928
  • Patent Document 1 JP-A-5-264551
  • Patent Document 2 JP-A-7-191033
  • Patent Literature; 3 JP 2003-149246 A
  • Patent Document 4 JP-A-2005-69823
  • Patent Document 5 Special Table 2003— 513225
  • An object of the present invention is to provide a ribosome complex that can rapidly detect multi-samples or multi-component test substances without disrupting or dissolving liposomes and without requiring a B / F separation step. And a ribosome array in which the ribosome complex is immobilized in an array on an avidin-modified glass substrate, a high-sensitivity detection method for a test substance using the liposome array, and a kit used in the method.
  • a further object of the present invention is to provide a ribosome complex having a maleimide group on the membrane surface (hereinafter referred to as a maleimide group-containing ribosome complex), a ribosome array immobilized on an avidin-modified glass substrate (hereinafter referred to as a maleimide group-containing ribosome array). ), In the step of detecting a test substance using the maleimide group-containing ribosome array, an antibody capable of specifically binding to the test substance or a Fab ′ fragment thereof is bound to the maleimide group-containing ribosome complex.
  • the immunoribosome array formed by the method described above, a highly sensitive detection method for a test substance using the immunoliposome array, and a kit for use in the method.
  • the immunoribosome array is a ribosome array formed by binding of an antibody or its Fab 'fragment via a thiol group to the maleimide group of the membrane surface portion of the maleimide group-containing ribosome complex. .
  • the present invention relates to the following 1 to 13.
  • a ribosome complex characterized by the following (i) to (iii):
  • pH-dependent fluorescent dye power 3 ⁇ 4, -7, -Bis- (carboxyethyl) -6-carboxyfluorescein (BCECF), 8-hydroxyphyllene-1,3,6-trisulfonic acid (HPTS 2)
  • BECF carboxyethyl -6-carboxyfluorescein
  • HPTS 2 8-hydroxyphyllene-1,3,6-trisulfonic acid
  • a method for detecting a test substance using the ribosome array according to 4 above comprising the following steps (i) to (iii):
  • a kit for detecting a test substance comprising the ribosome array described in 4 above and gramicidin.
  • a membrane ion channel forming substance is added to form an ion channel on a membrane surface site different from the membrane surface site where the maleimide group to which the antibody or its Fab 'fragment specifically binds to the test substance is located
  • a kit for detecting a test substance by an antigen-antibody reaction comprising the ribosome array according to 9 above, and an antibody or Fab, fragment thereof, and daramicidin.
  • the present invention relates to a ribosome complex, a ribosome array, and a substance that specifically binds to a test substance or a test substance in which the detection method of the test substance using the ribosome array does not dissolve or destroy the ribosome. Therefore, the detection measurement process is simplified compared to the conventional immunodetection method using ribosome, and multiple samples and multiple components can be obtained. It has the special effect of being able to detect and measure quickly. In addition, as the concentration of the analyte (analyte) increases, the fluorescence intensity contained in the ribosome complex increases and is suitable for highly sensitive detection of various biological and chemical samples. It also has the advantageous effect of being.
  • the detection method of the test substance using the maleimide group-containing ribosome complex and the maleimide group-containing liposome array of the present invention is based on the type of the test substance in the detection step.
  • An immunoribosome array that has an antibody that specifically binds to the membrane or its Fab ′ fragment on the membrane surface can be formed as appropriate, making it possible to detect various test substances quickly and easily.
  • FIG. 1 shows a method for manufacturing an avidin-modified glass substrate.
  • FIG. 2 Shown by an immunodetection method using a ribosome containing a pH-dependent fluorescent dye immobilized on a glass substrate.
  • FIG. 3 Shows a fluorescence detection method using a ribosome array.
  • FIG. 4 Shows the fluorescence of the force lucein-encapsulated ribosome complex immobilized on an array-modified glass substrate.
  • FIG. 5 shows the effect of B_cap-PE amount on ribosome complex immobilization.
  • FIG. 6 Fluorescence development (A) due to the formation of dalamicidin membrane ion channels and the effect of gramicidin concentration on fluorescence intensity.
  • FIG. 7 shows the relationship between the test substance concentration and the fluorescence intensity of the ribosome complex over time.
  • FIG. 8 shows the effect of the BCECF concentration encapsulated in the ribosome complex on the fluorescence intensity.
  • FIG. 9 shows cation selectivity of dalamicidin membrane ion channel.
  • FIG. 10 shows that the fluorescence intensity of the ribosome complex depends on the concentration of the test substance.
  • the cases where the test substance and the substance that specifically binds to the test substance are anti-DNP and DNP-PE (A), avidin and B-cap-PE (B), respectively.
  • FIG. 11 shows that the fluorescence intensity of the ribosome complex depends on the concentration of the test substance. This shows the case where the test substance is anti-BSA and the substance that specifically binds to the test substance is DNP-PE.
  • FIG. 12 shows that the relationship between the binding between a test substance and a substance that specifically binds to the test substance and the fluorescence intensity derived from BCEFC was verified by excess reagent immunoassay.
  • FIG. 13 shows that detection of a test substance using a maleimide group-containing ribosome complex array was dependent on the test substance concentration.
  • FIG. 14 shows the effect of dalamicidin concentration on detection of a test substance by antigen-antibody reaction using a maleimide group-containing ribosome array.
  • FIG. 15 shows the concentration dependence of a test substance in detection of a test substance by an antigen-antibody reaction using a maleimide group-containing ribosome array.
  • FIG. 16 shows the fluorescence intensity when iminoribosomes were prepared by arraying (immobilizing) maleimide group-containing ribosome complexes on an avidin-modified glass substrate and detecting the test substance.
  • FIG. 17 shows the fluorescence intensity when iminoliposomes were prepared without arraying (immobilizing) maleimide group-containing ribosome complexes on an avidin-modified glass substrate and the test substance was detected.
  • test substance in the present invention is not particularly limited as long as it is a substance that specifically binds to the test substance.
  • the relationship between a test substance and a substance that specifically binds to the test substance includes an immunological relationship, and specifically includes a relationship between an antigen and an antibody.
  • the substance that specifically binds to the antigen is an antibody.
  • the test substance is an antibody, it specifically binds to the antibody.
  • the substance to do is an antigen.
  • the substance that specifically binds to the test substance may be a substance that binds indirectly to the test substance. That is, the substance may be a substance that can bind to a substance that specifically binds to the test substance.
  • an antibody (secondary antibody) that can bind to an antibody that binds to a test substance (primary antibody) can be mentioned.
  • Examples of animals used for producing the above-mentioned antibodies include rabbits, mice, goats, horses, and rabbits.
  • the antibody may be a polyclonal antibody obtained by immunizing an animal or a monoclonal antibody obtained by the hyperidoma method.
  • test substance as an antigen in the present invention examples include biological components such as proteins, hormones, active peptides, otacoids, tumor markers, immunoglobulins, and trace components of drugs such as digoxin, phenytoin, and phenobarbital.
  • biological components such as proteins, hormones, active peptides, otacoids, tumor markers, immunoglobulins, and trace components of drugs such as digoxin, phenytoin, and phenobarbital.
  • the power that can be mentioned are not limited. There is no particular limitation as long as antibodies against the test substance can be produced.
  • the test substance and the substance that specifically binds the test substance include an enzyme and a substrate, an enzyme and an inhibitor, a hormone and a receptor, a lectin and a sugar chain, DNA and RNA.
  • examples include various combinations of DNA and DNA, serum albumin and dye blue, enzyme and coenzyme, protein and combinatorial ligand peptide, and the like.
  • Examples of the combination of an enzyme and a coenzyme include a combination of an oxidoreductase and a coenzyme NADH.
  • the ribosome in the present invention has a substance that specifically binds to a test substance on its membrane surface, a phosphatidylethanolamine piotin derivative (B-cap-PE), and a pH
  • a phosphatidylethanolamine piotin derivative B-cap-PE
  • a pH As long as it is a ribosome that can contain a dependent fluorescent dye, there is no particular limitation.
  • the biotin derivative of phosphatidylethanolamine is preferably a sodium salt of 1,2-dioleoyl-sn-glycero-3-phosphoethanolmine-N- (cap biotinyl) (hereinafter B-cap-PE).
  • the present TsutomuAkira is the membrane surface of the ribosome, and a test substance that specifically binds to substances, phosphatidylethanolamine ⁇ Minh Piochin derivative (B-ca P _PE), its The pH-dependent fluorescent dye is encapsulated in the inner aqueous phase, and the hydrogen ion concentration in the liposomal aqueous phase is higher than that outside the ribosome.
  • the aqueous phase inside the ribosome is pH 5.5
  • the pH outside the liposome where the reaction between the test substance and the substance that specifically binds to the test substance occurs is pH 5.5 or higher, preferably pH 7 0.0 or more, more preferably pH 7.8 is desirable.
  • the pH-dependent fluorescent dye in the present invention is preferably a pH-sensitive dye used for cell fluorescent staining and cannot pass through the ribosome double membrane.
  • carboxyfluorescein, 2, -7, -bis- (carboxyethyl) -6-carboxyfluorescein (BCECF), 8-hydroxyphyllene-1,3,6-trisul Examples include phonic acid (HPTS) and semi-naphtho rhoda flow (SNARF) (Molecular Probes).
  • pH-sensitive dyes used for cell fluorescence staining include derivatives having cell membrane permeability such as BCECF acetoxymethyl (AM) ester (BCEC F-AM).
  • pH-dependent fluorescent dyes include: These derivatives having cell membrane permeability are not included.
  • the membrane ion channel-forming substance in the present invention is a ionophore (ion permeable agent) that penetrates the lipid bilayer membrane to form a membrane pore, and preferably daramicidine such as gramicidin A, B, C, or D (Gramicidin). These gramicidins are peptides with 15 amino acid strengths, and all the amino acids have hydrophobic side chains. Gramicidin dimerizes in a “head-to-head” state, forming a membrane channel that penetrates the lipid bilayer. This membrane ion channel is a transmembrane channel having selectivity for monovalent cations including protons.
  • Daramicidin turns back on dimer formation and monomer dissociation in lipid bilayers (Non-patent Document 4). That is, Dalamishiji The dimer is in equilibrium with the monomer in the lipid bilayer.
  • the membrane ion channel in the present invention is a membrane pore penetrating the lipid bilayer of ribosome, and is induced and formed by the above-mentioned membrane ion channel forming substance. Therefore, when gramicidin is used as a membrane ion channel-forming substance, the membrane ion channel is a membrane pore formed by a gramicidin dimer that penetrates the lipid bilayer of the ribosome.
  • the ribosome complex in the present invention may be used after being immobilized on a carrier.
  • the carrier is a base material for fixing (supporting) a substance exhibiting adsorption or catalytic activity, that is, a substrate, and examples thereof include alumina and silica.
  • the carrier on which the ribosome complex is immobilized in this effort is not particularly limited as long as it is a carrier modified with avidin, but an avidin-modified glass substrate is preferred.
  • the avidin-modified glass substrate is characterized in that avidin is bonded to the glass surface. JP 2005-69 ⁇ 23 (Patent Document 4), or Yanagisawa et al., Anal. Biochem.
  • the shape of the carrier including the avidin-modified substrate is preferably a force, such as a film shape, a chip shape, an array shape, or a bead shape, or an array shape.
  • avidin is a low molecular weight basic glycoprotein composed of four subunits having a molecular weight of 68,000 and an isoelectric point of 10 to 10.5 present in raw egg white. It is. Each subunit of avidin is known to bind specifically to one molecule of biotin.
  • the ribosome complex in the present invention is immobilized on the avidin-modified substrate by specific binding between the avidin and the biotin derivative of phosphatidylethanolamine biotin derivative (B-cap-PE) on the membrane surface. .
  • the ribosome complex of the present invention and the preparation of the ribosome array on which the ribosome complex is immobilized, and the detection method of the present invention and its principle are as follows.
  • L-a-phosphatidinorecholine (hereinafter PC) (12 mg), cholesterol (hereinafter Choi) (3.0 mg) and B-cap-PE (O.OOlOmg) were dried to form a lipid film. Expose to high vacuum. Place 10 mL of 0.10 M NaCl solution containing 10 mM MES (2-2-morpholinoethanesulfonic acid monohydrate) ( ⁇ 5 ⁇ 5) and l, 0 mM BCECF on the obtained lipid film, and vortex for 5 minutes. Hydrated, then sonicated for 15 minutes and encapsulate BCECF Form ribosomes.
  • PC L-a-phosphatidinorecholine
  • Choi cholesterol
  • B-cap-PE O.OOlOmg
  • the ribosome complex thus prepared contains BCECF (lmM, pH 5.5) in the inner aqueous phase solution, but depending on conditions and usage, pH-dependent fluorescent dyes other than BCECF, such as HPTS or SNRF Etc. can be included.
  • BCECF pH-dependent fluorescent dyes other than BCECF, such as HPTS or SNRF Etc.
  • force lucein 5 ⁇ 0 ⁇ ⁇ ⁇ / ⁇
  • the maleimide group-containing ribosome complex is a component that introduces a maleimide group into the membrane surface of the ribosome complex.
  • 1,2-Dipalmitoyl-sn-glycease-3-phosphoethanolamine-N- [4- (maleimide group) Enil) -butyrate hereinafter referred to as N-MPB-PE.
  • N-MPB-PE 1,2-Dipalmitoyl-sn-glycease-3-phosphoethanolamine-N- [4- (maleimide group) Enil
  • N-MPB-PE 1,2-Dipalmitoyl-sn-glycease-3-phosphoethanolamine-N- [4- (maleimide group) Enil) -butyrate
  • the composition ratio of PC, Choi, B-cap-PE, and N-MPB-PE, which are constituents of the ribosome complex is 80: 20: 6.7xl0 -3 : 6.7xl0— 3 (w / w %)
  • the maleimide group-containing ribosome complex contains BCECF (lmVI, pH 5.5) in the same inner aqueous phase solution as the ribosome complex, and a pH-dependent fluorescent dye other than BCECF, depending on the conditions and usage, For example, HPTS or SNRF can be included.
  • force lucein 5.0 mM KH PO / NaOH (pH 7.4) is included in the inner aqueous phase solution.
  • Ribosome complexes can be made.
  • the avidin-modified glass substrate can be produced by the method described in JP-A-2005-69823 (Patent Document 4) or Yanagisawa et al., Anal. Biochem. 2004, 332, 358-340 (Non-Patent Document 3) (FIG. 1). Immerse the cover glass (18x24) in 1M NaOH overnight. After thoroughly washing with Milli-Q water, dry at 70 ° C for about 2 hours. Immediately, on one side of the cover glass 50 (v / v ) Place 600 ml of anhydrous toluene solution of% 3-mercaptopropyltrimethoxysilane (MTS) and leave at room temperature for 60 minutes. Thereby, the cover glass is silanized.
  • Patent Document 4 Immerse the cover glass (18x24) in 1M NaOH overnight. After thoroughly washing with Milli-Q water, dry at 70 ° C for about 2 hours. Immediately, on one side of the cover glass 50 (v / v ) Place 600 ml of
  • GMBS N-succinimidyl 4-maleimidobutyrate
  • DMSO dimethylsulfoxide
  • a maleimide group-containing liposome array can be prepared by adding the preserved ribosome suspension of the maleimide group-containing ribosome complex prepared in (2) above to each spot on the GMBS substrate prepared in (3) above. . Specifically, preservation of the maleimide group-containing ribosome complex prepared in (2) above is immobilized on an avidin-modified glass substrate by the method described in “(4) Preparation of ribosome array J” above. (Fig. 3).
  • the immunoribosome array of the present invention has a liposomal complex force on an avidin-modified glass substrate in which an antibody or a Fab ′ fragment thereof is bound via a thiol group to a maleimide group on the surface of a maleimide group-containing ribosome complex.
  • the ribosome array is immobilized in the form of an array in the method step of detecting a test substance by an antigen-antibody reaction using a maleimide group-containing ribosome array. Specifically, it is formed by the following method. In advance, a solution containing an antibody that specifically binds to the test substance or its F3 ⁇ 4b 'fragment Prepare.
  • each spot of the maleimide group-containing ribosome array has an antibody or Fab or fragment thereof that specifically binds to the test substance prepared in advance. Is added, and the maleimide group-containing ribosome array is incubated at ⁇ 4 ° C. As a result, an antibody that specifically binds to the test substance or its Fab ′ fragment binds to the maleimide group on the membrane surface of the maleimide group-containing ribosome complex immobilized in the spot of the maleimide group-containing ribosome array. Bind through its thiol group.
  • the maleimide group-containing ribosome complex immobilized on the maleimide group-containing liposome array has an antibody or Fab ′ fragment thereof that specifically binds to the test substance on the membrane surface.
  • the maleimide group-containing ribosome array formed in this way is called an immunoribosome array.
  • a test substance analyte
  • fluorescence is developed by adding an on-channel forming substance such as dalamicidin, and the test substance can be detected by measuring the fluorescence intensity. .
  • the ribosome complex immobilized on the avidin-modified glass substrate contains l.OmM BCEC F, and its inner aqueous phase solution is 10 mM MES (pH 5.5). Each spot of the ribosome array in which the ribosome complex is spotted is washed with a 0.10 M NaCl solution dissolved in 10 mM MES (pH 7.8) (solution outside the ribosome). Next, 20 ml (10 mM MES (pH 7.8)) of an aqueous ribosome solution in which a test substance (analyte) of a predetermined concentration (such as avidin or anti-DNP) is dissolved is placed in each spot for 30 minutes. Incubate.
  • a test substance analyte
  • a predetermined concentration such as avidin or anti-DNP
  • the maleimide group-containing ribosome complex immobilized on the avidin-modified glass substrate also contains l.OmM BCECF in the same manner as the ribosome complex described above, and its inner aqueous phase solution is 10 mM MES ( PH 5.5). is there. Therefore, the fluorescence detection of the maleimide group-containing ribosome array and the immunoribosome array can be performed in the same manner as in the case of the ribosome array described above.
  • the ribosome complex (lmM BCECF ( PH 5.5) in the inner aqueous phase) is immobilized on an avidin-modified glass substrate via an avidin-piotine bond, and a sample solution ( ⁇ 7 ⁇ 8) containing the test substance is added. Calored and incubated.
  • the daramicidin dimer (dimer) is in an equilibrium state in which dimer formation and dissociation into monomers are repeated in the lipid bilayer membrane.
  • This equilibrium state is a ribosomal lipid duplex caused by the binding between a test substance (analyte) and a substance (receptor) that specifically binds to the test substance, such as an antigen-antibody reaction occurring on the surface of the ribosome membrane. It varies depending on the local strain of the film (Non-Patent Documents 5 and 6).
  • concentration of the test substance (analyte) increases, the binding frequency between the test substance (analyte) and the substance that specifically binds to the test substance (receptor) increases.
  • PC, Choi, B- cap-PE, and N-MPB-PE regard to (80: 20:: 6.7x10- 3 6,7xl0- 3, w / w%) maleimide group-containing ribosome complex that consists in, Fluorescence intensity is detected by the same method as the ribosome complex described above.
  • the detection method of the present invention is a method capable of rapidly detecting and measuring multiple samples and multiple components using a ribosome complex and a ribosome array on which the ribosome complex is immobilized, as described above.
  • the feature of the kit of the present invention is a kit for detecting a test substance (analyte) comprising the ribosome array produced by the above “(4) Production of ribosome array” and a membrane ion channel forming substance.
  • the membrane ion channel-forming substance is preferably gramicidin such as gramicidin A, B, C, or D.
  • the method for detecting a test substance by antigen-antibody reaction using a maleimide group-containing ribosome array is more rapid than a multi-sample and a multi-component by preparing a kit. It is possible to automate the detection of the test substance.
  • the kit includes a maleimide group-containing ribosome array, a solution containing an antibody or Fab ′ fragment thereof that specifically binds to a test substance prepared in advance according to the type of the test substance, and a membrane ion channel forming substance. This kit can detect various test substances quickly and easily.
  • the membrane ion channel forming substance of the kit is preferably dalamicidin such as daramicidin A, B, C or D.
  • ribosome complex instead of BECEF, a liposome complex containing a fluorescent dye-powered lusein was prepared, and this was used to create an avidin-modified glass substrate for the ribosome complex. I verified the fixed rigs. Specifically, the aqueous phase in the ribosome complex The presence or absence of leaking fluorescent dye strength lucein was evaluated by the following method.
  • a ribosome complex encapsulating the fluorescent dye power lusein was prepared and used as a storage ribosome suspension (stored in the presence of nitrogen (4 ° C)).
  • the avidin-modified glass substrate produced according to the above “(3) Method for producing an avidin-modified glass substrate” was added to 5.0 mIVi KH PO / NaOH (pH 7.4).
  • Fluorescence images were measured both before and after (excitation wavelength 488 nm, fluorescence wavelength 530 nm) o As a result, fluorescence derived from force lucein was recognized before and after adding the CoCl solution.
  • Each amount of -PE is the weight fraction 0, 0.056, 0.55, 5.5, 227, to prepare a 0.555x10- 5 (w / w%) and Do that ribosome complexes, B - cap-PE amount of ribosome complexes The effect of immobilization was verified.
  • the time change of the fluorescence intensity of the ribosome complex after adding daramicidin was verified.
  • a ribosome complex having B-cap-PE as a substance (receptor) that specifically binds to the test substance (analyte) is prepared.
  • the gramicidin concentration was 5.31 nM (Fig.
  • the fluorescence intensity of the ribosome slightly increased 60 minutes after adding gramicidin (Fig. 7 (D)). This slight increase in fluorescence intensity is attributed to H ions released from the membrane ion channel opened at the moment when gramicidin in an equilibrium state in which dimer formation and monomer dissociation repeatedly undergo dimerization.
  • the fluorescence intensity of the liposomal complex increased (FIGS. 7 (A) to (C)). This result shows the binding of avidin to the biotin residue of B-cap-PE on the lipid bilayer surface of the liposome complex.
  • the membrane ion channel is activated by the binding of the test substance (analyte) and the substance that specifically binds to the test substance (analyte receptor) at the interface between the ribosome complex and the outer fluid, ie, the analyte
  • the effect of the analyte receptor gramicidin on monomer / dimmer kinetics was verified by the following experiment.
  • ribosome membrane strength SPC L-a-phosphatidylcholine
  • Choi cholesterol monore
  • B-cap-PE B-cap-PE
  • DNP-PE 1,2-dipalmitoyto-sn-glycero- 3—phosphoethanokmine-N- (2,4-dinitrophenyl
  • a ribosome ′ complex was prepared by the method described in “(1) Preparation of ribosome complex” above.
  • the B-cap-PE biotin residue present in the membrane of the ribosome complex serves as a receptor for analyte and avidin on an avidin-modified glass substrate.
  • the ribosome complex was immobilized on an avidin-modified glass substrate according to the methods described in “(3) Preparation of avidin-modified glass substrate” and “(4) Preparation of ribosome array” to obtain a ribosome array.
  • BCECF fluorescence intensity due to the binding between the analyte-analyte receptor at the interface between the lipid bilayer membrane of the ribosome complex and the outer fluid.
  • B-cap-PE analyte receptor
  • piotin-labeled anti-BSA biotin-anti-BSA
  • test substance by antigen-antibody reaction using maleimide group-containing ribosome array
  • substance P substance P
  • neurokinin A neurokinin A
  • maleimide liposome complex prepared by “(2) Manufacture of maleimide group-containing ribosome complex” above
  • avidin modified glass substrate prepared by “(3) Preparation of avidin modified glass substrate” above.
  • the maleimide liposome complex contains l.OmM BCECF, and its inner aqueous phase solution is 10 mM MES (pH 5.5).
  • Fabs and fragments of anti-substance P antibody and anti-eurokinin A antibody were prepared by the method described in Non-Patent Document 7. 100 ⁇ g of anti-substance ⁇ antibody IgG antibody or 400 ⁇ of anti-neurocune A IgG antibody was dialyzed against a 0.10 M NaCl solution containing 0.01 M acetate buffer ( ⁇ 4 ⁇ 5). After adding O.lmg of pepsin to the dialysate, the dialysate was incubated at 37 ° C for 24 hours, and then adjusted to pH 7.0 with 1M KH PO / KOH. Next, it is contained in the dialysate.
  • Detection method of test substance by antigen-antibody reaction using maleimide group-containing ribosome array Of the test substance concentration that is, the influence of the test substance concentration on the fluorescence intensity obtained by the method of Example 9.
  • the fluorescence intensity emitted by the microarray ribosome was measured by the method of Example 9 by changing the concentrations of the test substance substance P and neurokinin A.
  • Fig. 13 (a) shows the case where the substance to be tested is substance P only
  • Fig. 13 (b) shows the case where the substance to be tested is -eurokun A only
  • (c) shows the substance P and neurokin. The case where both A are used as test substances is shown.
  • (d) is a substance that specifically binds to the test substance: 1) Fa b 'fragment of anti-neurocun A antibody, 2) Fab' fragment of anti-substance P antibody, and 3) antibody fragment
  • This result shows that the fluorescence intensity is stronger when the antibody fragment is used than when the antibody fragment is not used, and both -eurokinin A and substance P are concentration-dependently detected by each Fa b 'fragment. Indicates an increase.
  • an antigen-antibody reaction is performed by adding a test substance, and then a gramicidin solution (5.31 ⁇ , ⁇ 7 ⁇ 8) is added to detect fluorescence intensity. 60 minutes (incubation at room temperature) is required (Fig. 3). Therefore, we examined the concentration of daramicidin, which shortens the time to detect the fluorescence intensity. Specifically, the following examination was conducted.
  • the fluorescence intensity was measured every 15 minutes up to 70 minutes. As a result, increasing the Daramishijin concentration 10- 6 g / ml, the time constant of the fluorescence intensity is reduced to 20 minutes, it was confirmed that the further fluorescence intensity increased.
  • Examples 9 to 11 a maleimide array in which the maleimide group-containing ribosome complex obtained by “(2) Preparation of maleimide group-containing ribosome complex” was arrayed (immobilized) on an avidin-modified glass substrate was used. Yes. Here, the detection of the test substance was compared between when the maleimide group-containing ribosome was arrayed and when it was used in Balta without being arrayed.
  • Example 9 Based on “(i) Preparation of maleimide group-containing ribosome array” in Example 9, the maleimide group-containing ribosome complex (40 1) was immobilized on an avidin-modified glass substrate, and the maleimide group-containing ribosome array was obtained. Was made. An anti-neurocun ⁇ ⁇ Fab 'fragment (20 ⁇ 1) was added to each spot of the array and incubated at room temperature for 60 minutes to prepare an immunoribosome array.
  • maleimide group-containing ribosome complex suspension (20 ⁇ 1) obtained in “(2) Preparation of maleimide group-containing ribosome complex” above to MES buffer ( ⁇ 5.5), and add 1 ml of maleimide group-containing ribosome. A complex solution was obtained. To the same solution (40 / xl), an anti-neurocun A Fab fragment (20 ⁇ 1) was added and incubated at room temperature for 60 minutes to prepare an immunoliposome solution.
  • the ribosome complex of the present invention includes detection and measurement of biological components such as antigens and antibodies, drugs, etc. It can be applied to various biological and chemical analysis.
  • the detection method of the test substance using the maleimide group-containing ribosome complex and the maleimide group-containing liposome array of the present invention is specific to the test substance in the detection step depending on the type of the test substance.
  • an immunoribosome array having a substance that specifically binds to the test substance on the membrane surface can be formed. Detection can be performed quickly and easily.

Abstract

L'invention concerne un complexe de liposome ayant, sur sa surface membranaire, une substance capable de se lier spécifiquement à un analyte et comprenant un colorant fluorescent pH dépendant à l'intérieur ; un réseau de liposome comprenant le complexe de liposome immobilisé sur un substrat en verre modifié à l'avidine sous la forme d'un réseau ; un procédé de détection d'un analyte à haute sensibilité, qui peut rapidement détecter l'analyte dans des échantillons multiples ou des composants multiples en utilisant le réseau de liposome ; et un kit d'utilisation du procédé.
PCT/JP2007/065928 2006-08-11 2007-08-09 Complexe de liposome, réseau de liposome, et procédé de détection d'un analyte WO2008018631A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2682859B2 (ja) * 1987-07-27 1997-11-26 コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガニゼーション レセプター膜

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2682859B2 (ja) * 1987-07-27 1997-11-26 コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガニゼーション レセプター膜

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HORIE M.: "pH Izonsei Keiko Shikiso o Naiho shita Liposome to Gramicidin o Mochiiru Keiko Immuno Assay-ho no Kaihatsu", THE JAPAN SOCIETY FOR ANALYTICAL CHEMISTRY, DAI 55 NENKAI KOEN YOSHISHU, 6 September 2006 (2006-09-06), pages 197 + ABSTR. NO. H3022, XP003020747 *
SUGAWARA M.: "Nanoteku no Iryo Oyo -Nanomedicine Channel Tanpakushitsu Sokutei Gijutsu", BIONICS, vol. 3, no. 7, 1 July 2006 (2006-07-01), pages 36 - 41, XP003020746 *
VAMVAKAKI V.: "Fluorescence detection of enzymatic activity within a liposome based nano-biosensor", BIOSENSORS AND BIOELECTRONICS, vol. 21, no. 2, 2005, pages 384 - 388, XP004981164 *

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