WO2008018631A1 - Liposome complex, liposome array, and method for detection of analyte - Google Patents

Liposome complex, liposome array, and method for detection of 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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French (fr)
Japanese (ja)
Inventor
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.

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Abstract

A liposome complex having, on its membrane surface, a substance capable of binding specifically to an analyte and including a pH-dependent fluorescent dye in its inside; a liposome array comprising the liposome complex immobilized on an avidin-modified glass substrate in the form of an array; a method for detecting an analyte at high sensitivity, which can rapidly detect the analyte in multiple samples or multiple components by using the liposome array; and a kit for use in the method.

Description

JP2007/065928  JP2007 / 065928
明 細 書 Specification
リボソーム複合体、リボソームアレイ、及び被検物質を検出する方法 技術分野  Ribosome complex, ribosome array, and method for detecting a test substance
[0001] 本発明は、リボソームを用いる被検物質の高感度検出法に関する。更に詳しくは、 本発明は、リボソームに、被検物質と特異的に結合する物質と、ホスファチジルェタノ ールァミンのピオチン誘導体 (B- cap- PE)とを膜表面に有し、 pH依存性蛍光色素を内 包させ、且つ、 pH依存性蛍光色素を内包するリボソーム内水相の水素イオン濃度が リボソーム外の水素イオン濃度より高くなるように調整されてなるリボソーム複合体、該 リボソーム複合体をアビジン修飾ガラス基板上にアレイ状に固定化したリボソームァ レイ、該リボソームアレイを用いた被検物質の高感度検出法、該方法に用いるキット に関する。  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. 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.
更に本発明は、膜表面にマレイミド基を有するリボソーム複合体 (マレイミド基含有リ ポソーム複合体)、該マレイミド基含有リボソーム複合体が、アビジン修飾ガラス基板 上にアレイ状に固定化されたリボソームアレイ (マレイミド基含有リボソームアレイ）、該 マレイミド基含有リボソームアレイを用いて被検物質を検出する工程において、マレイ ミド基含有リボソーム複合体に被検物質と特異的に結合する抗体又はその Fab'断 片を結合させることで形成されるィムノリボソームアレイ、該ィムノリポソームアレイを用 レ、た被検物質の高感度検出法、該方法に用いるキットに関する。  Furthermore, 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.
背景技術  Background art
[0002] 抗原と抗体間の特異的で厳密な免疫学的相互作用、即ち、免疫反応を利用した検 出及び測定法 (以下、免疫検出法、免疫測定法）は、例えば、タンパク質、ホルモン、 活性ペプチド、ォータコイド、腫瘍マーカー、免疫グロブリン等の生体成分、ジゴキシ ン、フエニトイン、フ； cノバルビタール等の薬剤等の微量成分を高選択的に分析する 方法として広く利用されている。近年、これら生体成分や薬剤等の検出、性質決定、 及び選別のために測定を必要とする試料数は顕著に増加しており、多数の試料ある レヽは多成分を迅速かつ自動的に測定できる方法が要求されてレ、る。  [0002] Specific and exact immunological interaction between an antigen and an antibody, that is, a detection and measurement method using an immune reaction (hereinafter referred to as 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.
[0003] 免疫測定法としては、ラジオィムノアッセィ (RIA法)、酵素標識抗体測定法 (ELISA) 、膜を用いる免疫測定法 (membrane - based immunoassay)等が挙げられる。これらの 方法は、更に、微小流体システム (microfluidic system)、電気化学水晶振動子マイク ロバランス法 (EQCM)や表面プラズモ共嗚 (SPR)現象等と組み合わせることで、その 利用範囲は、益々広がりつつある。しかし、これら免疫測定法は、夫々問題点がある [0003] Radioimmunoassay (RIA method), enzyme-labeled antibody assay (ELISA) And an immunoassay using a membrane (membrane-based immunoassay). These methods can be combined with the microfluidic system, electrochemical quartz crystal microbalance (EQCM), surface plasmo resonance (SPR) phenomenon, etc. is there. However, each of these immunoassays has its own problems
[0004] RIA法は、放射性同位元素を使用しなければならなレヽことから、特別な施設や機器 • が必要であり、廃棄物を処理が問題となる。 ELISA法は、操作上、抗原抗体反応した 標識物質と未反応の標識物質の分離 (B/F分離)が必要な不均一法 (ヘテロジニアス 法、固相系）と B/F分離を必要としない均一法 (ホモジーニアス法）とに分類される。 不均一法は、感度が優れている力 分離 (B/F分離）のために、インキュベーション及 び洗浄工程を繰り返す必要があり、測定に多くの時間を要する。従って、多数の試料 を迅速に処理するのに適していない。均一法は、操作が簡便であり迅速化が可能で 、競合的酵素免疫分析法 (EMIT)等が実用化されているが、抗原抗体反応による信 号の変化に大きく依存し、高分子量物質の測定が困難である。免疫プロット法のよう な膜を用いる免疫測定法は、自動化されたマイクロアレイ法等に適用されるが、測定 感度は、ラベル試薬の量とシグナル強度により制限される。免疫プロット法において 検出に用いる抗体 1分子には、 2~3の酵素分子しか結合することができなレ、 (特許 文献 3)。 [0004] Since the RIA method requires the use of radioisotopes, special facilities and equipment are required, and disposal of waste becomes a problem. 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. Homogeneous methods are easy to operate and can be accelerated, and competitive enzyme immunoassay (EMIT) has been put to practical use. Measurement is difficult. An immunoassay method using a membrane such as an immunoplot method is applied to an automated microarray method or the like, but the measurement sensitivity is limited by the amount of the label reagent and the signal intensity. Only one or two enzyme molecules can bind to one antibody molecule used for detection in the immunoplot method (Patent Document 3).
[0005] これら問題点を解決すべくリボソームを用いた免疫測定法が提案されている。リポソ ームは、同一分子内に親水基と疎水基の両方を有する両親媒性の複合脂質 (レシチ ン、コレステロール、フォスファチジル酸等）を一定の温度以上で緩衝液に懸濁するこ とで調製される脂質二重層からなる小胞体である。リボソームを用レ、る免疫測定法は 、内部に蛍光色素、無機及び有機イオン、酵素分子等を内包し、二官能性架橋剤を 用レ、て膜に抗原または抗体を共有結合させたリボソーム複合体ほたは機能性リポソ ームと呼ばれている）を使用し、リボソーム内水相から放出される蛍光色素等を、蛍光 光度法、電位差測定法などの分析手法により測定するものである。例えば、フルォロ フォア (蛍光色素分子)が内包されたリボソームを検出用ラベルとして使用するリポソ ーム免疫測定法や、リボソーム力 の電気活性種の放出を利用するリボソームを用い たィムノセンサ一についても報告されている。更に、内部に蛍光色素を封入させたリ ポソーム膜に抗原抗体反応を形成させ、これに捕体を作用させてリボソームを破壊し 、放出される蛍光色素を測定し検出することで抗原または抗体量を測定する免疫検 出法が開発されている (特許文献 1、 2)。 In order to solve these problems, an immunoassay method using ribosome has been proposed. 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) and the fluorescent dyes released from the aqueous phase in the ribosome are measured by analytical methods such as fluorometry and potentiometry. For example, 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. There is also a report on the immunosensor. Furthermore, 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).
[0006] 上記のリボソーム複合体を用いる免疫測定法では、抗体あるいは抗原に結合したリ ポソームと、結合していないリボソームとをフロー系で分離後、リボソームを破壌し、放 出された内封物質を測定するか、溶液に懸濁したリボソーム力 免疫反応によってマ 一力一イオン力 Sもれ出るのをイオン選択性電極やポルタンメトリー等により測定する。 これら免疫測定法では、抗体あるいは抗原と結合したリボソームと結合していなレ、リポ ソームを分離する工程 (B/F分離)が必要であり、多試料を同時に検定することが困 難であった。また、リボソームの破壌工程により、液層へ蛍光色素や発色生成物の拡 散が起こるため、感度が低下し、クリア一なシグナルを得ることができなかった。 [0006] In the immunoassay using the above ribosome complex, 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. . In addition, 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.
[0007] Nikoleisら (非特許文献 1、 2)は、リン脂質二重層の物性変化を抗原抗体反応でモ ユタ一している。近年、リボソームを破壌することなく被検物質を検出する方法として、 被検物質を認識する一次抗体と、一次抗体を認識する二次抗体を表面に有し、かつ 、標識物質を内包したリボソームを、リボソームの脂質膜を透過することができる標識 物質と反応させることで被検物質を検出する方法が開発されている (特許文献 3)。  [0007] Nikoleis et al. (Non-patent Documents 1 and 2) monitor changes in physical properties of phospholipid bilayers by antigen-antibody reaction. In recent years, as a method for detecting a test substance without disrupting the ribosome, 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).
[0008] 非特許文献 l :Nikolelis, D. P.; Hianik, T. Krull, U. J. Electroanaylsis 1999, 11, 7-1 5  [0008] Non-patent literature l: Nikolelis, D. P .; Hianik, T. Krull, U. J. Electroanaylsis 1999, 11, 7-1 5
非特許文献 2 : Hianik, Τ·; et al. Gen. Physiol. Biophys. 1998, 17, 239-252 非特許文献 3 :Yanagisawa, H; Hirano, A; Sugawara, M. Anal. Biochem. 2004， 332, Non-Patent Document 2: Hianik, Τ; et al. Gen. Physiol. Biophys. 1998, 17, 239-252 Non-Patent Document 3: Yanagisawa, H; Hirano, A; Sugawara, M. Anal. Biochem. 2004, 332 ,
358-340 358-340
非特許文献 4: Killian, J.A. Biochem. Biophys. Acta 1992, 1113, 391-425  Non-Patent Document 4: Killian, J.A.Biochem.Biophys. Acta 1992, 1113, 391-425
非特許文献 5 : Hirano, A.; Wakabayashi, M.; Matsuno, Y.; Sugawara, M. Biosens. Bi oelectron. 2003, 18, 973—983  Non-Patent Document 5: Hirano, A .; Wakabayashi, M .; Matsuno, Y .; Sugawara, M. Biosens. Bioelectron. 2003, 18, 973–983
非特許文献 6 : Rudnev, V.S.; Ermishkin, L. N.; Fonina, L. A. ; Rovin, Yu. G. Bioche m. Biophys. Acta 1981, 642, 196—202  Non-Patent Document 6: Rudnev, V.S .; Ermishkin, L. N .; Fonina, L. A .; Rovin, Yu. G. Biochem. Biophys. Acta 1981, 642, 196-202
非特許文献 7 : Ishikawa, E.; Imagawa, .; Hashida, D.;Yoshitake, S,; Hamaguchi, Y.; P T/JP2007/065928 Non-Patent Document 7: Ishikawa, E .; Imagawa,.; Hashida, D .; Yoshitake, S ,; Hamaguchi, Y .; PT / JP2007 / 065928
Ueno, T.; J. Immunoassay, 413 (1983) 209—327 Ueno, T .; J. Immunoassay, 413 (1983) 209-327
特許文献 1 :特開平 5— 264551  Patent Document 1: JP-A-5-264551
特許文献 2 :特開平 7— 191033  Patent Document 2: JP-A-7-191033
特許文献; 3：特開 2003— 149246  Patent Literature; 3: JP 2003-149246 A
特許文献 4 :特開 2005— 69823  Patent Document 4: JP-A-2005-69823
特許文献 5：特表 2003— 513225  Patent Document 5: Special Table 2003— 513225
発明の開示 '  Invention Disclosure ''
発明が解決しょうとする課題  Problems to be solved by the invention
[0009] これまで開発されてきたリボソームを用いた検出及ぴ測定法は、多数の試料あるい は多成分を迅速かつ同時に測定するには不十分である。本発明の目的は、リポソ一 ムを破壌または溶解することなぐかつ、 B/F分離の工程を必要とせず、多試料また は多成分の被検物質を迅速に検出することができるリボソーム複合体、該リボソーム 複合体をアビジン修飾ガラス基板上にアレイ状に固定化したリボソームアレイ、該リポ ソームアレイを用いた被検物質の高感度検出法、及び該方法に用いるキットを提供 することにある。更に本発明の目的は、膜表面にマレイミド基を有するリボソーム複合 体 (以下、マレイミド基含有リボソーム複合体)、アビジン修飾ガラス基板上にアレイ状 に固定化したリボソームアレイ (以下、マレイミド基含有リボソームアレイ）、該マレイミド 基含有リボソームアレイを用いて被検物質を検出する工程において、マレイミド基含 有リボソーム複合体に被検物質と特異的に結合することができる抗体又はその Fab' 断片を結合させることで形成されるィムノリボソームアレイ、該ィムノリポソームアレイに よる被検物質の高感度検出法、該方法に用レ、るキットを提供することにある。ここで、 ィムノリボソームアレイとは、マレイミド基含有リボソーム複合体の膜表面部位のマレイ ミド基に対して抗体又はその Fab'断片がチオール基を介して結合することによって 形成されるリボソームアレイである。 [0009] The detection and measurement methods using ribosomes that have been developed so far are insufficient for the rapid and simultaneous measurement of a large number of samples or multiple components. 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. An 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. Here, 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. .
課題を解決するための手段  Means for solving the problem
[0010] 本発明は、以下の 1〜13に関する。 [0010] The present invention relates to the following 1 to 13.
1.以下の (i)〜 (iii)を特徴とするリボソーム複合体：  1. A ribosome complex characterized by the following (i) to (iii):
(i)膜表面に被検物質と特異的に結合する物質と、ホスファチジルエタノールァミンの ビォチン誘導体 (B - cap_PE)とを有し、 (i) a substance that specifically binds to the test substance on the membrane surface and phosphatidylethanolamine A biotin derivative (B-cap_PE),
(ii)リボソーム複合体の内水相は水素イオンがリボソーム外の水素イオン濃度より高く なるように調整され、かつ、 pH依存性蛍光色素が内包されており、  (ii) The inner aqueous phase of the ribosome complex is adjusted so that the hydrogen ions are higher than the concentration of hydrogen ions outside the ribosome, and the pH-dependent fluorescent dye is included,
(iii)膜イオンチャンネル形成物質が添加されることによって、被検物質と特異的に結 合する物質が位置する膜表面部位とは異なる膜表面部位にイオンチャンネルが形成 される、リボソーム複合体。  (iii) A ribosome complex in which an ion channel is formed at a membrane surface site different from the membrane surface site where a substance that specifically binds to a test substance is located by adding a membrane ion channel forming substance.
2. pH依存性蛍光色素力 ¾， -7， -ビス- (カルボキシェチル) -6-カルボキシフルォレセ イン（BCECF)、 8-ヒドロキシフィレン- 1，3，6 -トリスルフォニックアシッド（HPTS)、セミナ フトローダフロース（SNARF)力 なる群力 選択される、上記 1に記載のリボソーム複 合体。  2. pH-dependent fluorescent dye power ¾, -7, -Bis- (carboxyethyl) -6-carboxyfluorescein (BCECF), 8-hydroxyphyllene-1,3,6-trisulfonic acid (HPTS 2) The ribosome complex according to 1 above, wherein a group force consisting of a seminaphtho-loader flow (SNARF) force is selected.
3.膜イオンチャンネル形成物質が、ダラミシジンである上記 2に記載のリボソーム複 合体。  3. The ribosome complex according to 2 above, wherein the membrane ion channel-forming substance is daramicidin.
4.上記 2に記載のリボソーム複合体力、アビジン修飾ガラス基板上にアレイ状に固 定化されたリボソームアレイ。  4. A ribosome array immobilized on an avidin-modified glass substrate in the form of a ribosome complex as described in 2 above.
5.以下の（i)〜 (iii)の工程を含んでなる、上記 4に記載のリボソームアレイを用いる 被検物質を検出する方法：  5. A method for detecting a test substance using the ribosome array according to 4 above, comprising the following steps (i) to (iii):
(i)前記リボソームアレイに被検物質を含む試料を添加し、次レヽで  (i) Add a sample containing the test substance to the ribosome array, and
(ii)膜イオンチャンネル形成物質を添カロし、  (ii) adding a membrane ion channel forming substance,
(iii) pH依存性蛍光色素に由来するリボソームの蛍光色素の強度を測定する。  (iii) Measure the intensity of the ribosomal fluorescent dye derived from the pH-dependent fluorescent dye.
6.膜イオンチャンネル形成物質が、ダラミシジンである上記 5に記載の被検物質を検 出する方法。  6. The method for detecting a test substance according to 5 above, wherein the membrane ion channel-forming substance is daramicidine.
7.上記 4に記載のリボソームアレイと、グラミシジン 含む、被検物質を検出するため のキット。  7. A kit for detecting a test substance comprising the ribosome array described in 4 above and gramicidin.
8.膜表面にマレイミド基を有する、上記 2に記載のリボソーム複合体。  8. The ribosome complex according to 2 above, having a maleimide group on the membrane surface.
9.上記 8に記載のリボソーム複合体力 S、アビジン修飾ガラス基板上にアレイ状に固 定ィ匕されたリボソームアレイ。  9. A ribosome array fixed on the ribosome complex strength S, avidin-modified glass substrate as described in 8 above, in an array.
10.リボソーム複合体の表面のマレイミド基に対して抗体又はその Fab'断片がチォ 一ル基を介して結合した、上記9に記載のリボソームアレイ。 P T/JP2007/065928 10. The ribosome array according to 9 above, wherein an antibody or a Fab ′ fragment thereof is bound to a maleimide group on the surface of the ribosome complex via a phenol group. PT / JP2007 / 065928
11. 以下の（i) ~ (iv)の工程を含んでなる、上記 9に記載のリボソームアレイを用い た抗原抗体反応によって被検物質を検出する方法： 11. A method for detecting a test substance by an antigen-antibody reaction using the ribosome array according to 9 above, comprising the following steps (i) to (iv):
(i)該リボソームアレイに、被検物質と特異的に結合する抗体又はその Fab'断片を 含む試料を添加して上記 10に記載のリポソ一ムアレイを形成させ、  (i) A sample containing an antibody that specifically binds to a test substance or a Fab ′ fragment thereof is added to the ribosome array to form the liposome array as described in 10 above,
(ii)被検物質を含む試料を添加して被検物質と抗体又はその Fab'断片を反応さ せ、次いで、  (ii) A sample containing a test substance is added to react the test substance with an antibody or Fab ′ fragment thereof, and then
(iii)膜イオンチャンネル形成物質を添加して、被検物質と特異的に結合する抗体 又はその Fab'断片が結合するマレイミド基が位置する膜表面部位とは異なる膜表面 部位にイオンチャンネルを形成させ、  (iii) 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 Let
(iv) pH依存性蛍光色素に由来するリボソームの蛍光色素の強度を測定する。 (iv) Measure the intensity of the ribosomal fluorescent dye derived from the pH-dependent fluorescent dye.
12.膜イオンチャンネル形成物質がダラミシジンである上記 11に記載の被検物質を 検出する方法。 12. The method for detecting a test substance according to the above 11, wherein the membrane ion channel forming substance is daramicidin.
13.上記 9に記載のリボソームアレイと、抗体又はその Fab，断片、及びダラミシジンと を含む、抗原抗体反応によって被検物質を検出するためのキット。  13. 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.
発明の効果 The invention's effect
本発明は、リボソーム複合体、リボソームアレイ、該リボソームアレイを用レ、る被検物 質の検出法が、リボソームを溶解または破壊することなぐ被検物質または被検物質 と特異的に結合する物質を蛍光標識することを必要とせず、 B/F分離を必要としなレ、 ことから、従来のリボソームを用いる免疫検出法と比較し、検出測定工程が簡素化さ れ、多試料及び多成分を迅速に検出測定できるという格別な効果を有する。また、被 検物質 (アナライト）の濃度の上昇に伴い、リボソーム複合体に内包されてレ、る蛍光強 度が増幅することから、種々の生物及び化学系試料の高感度検出に適してレ、るとい う有利な効果を併せ持つ。  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.
更に、本発明のマレイミド基含有リボソーム複合体、及び、マレイミド基含有リポソ一 ムアレイを用レ、る被検物質の検出方法は、その検出工程において、被検物質の種類 に応じて、被検物質と特異的に結合する抗体又はその Fab'断片を膜表面に有する ィムノリボソームアレイを適宜形成させることができるので、多様な被検物質を迅速、 かつ簡便に検出することができるという格別な効果を有する。 JP2007/065928 Furthermore, 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. Have JP2007 / 065928
図面の簡単な説明 Brief Description of Drawings
[0012] [図 1]アビジン修飾ガラス基板の作製方法を示す。  [0012] FIG. 1 shows a method for manufacturing an avidin-modified glass substrate.
[図 2]ガラス基板に固定化した pH依存性蛍光色素を内包するリボソームによる免疫検 出法で示す。  [Fig. 2] Shown by an immunodetection method using a ribosome containing a pH-dependent fluorescent dye immobilized on a glass substrate.
[図 3]リボソームアレイによる蛍光検出法を示す。  [Fig. 3] Shows a fluorescence detection method using a ribosome array.
[図 4]アレイ修飾ガラス基板に固定化した力ルセイン内包リボソーム複合体の蛍光発 色を示す。  [Fig. 4] Shows the fluorescence of the force lucein-encapsulated ribosome complex immobilized on an array-modified glass substrate.
[図 5]B_cap - PE量のリボソーム複合体固定化への影響を示す。  FIG. 5 shows the effect of B_cap-PE amount on ribosome complex immobilization.
[図 6]ダラミシジン膜イオンチャンネルの形成による蛍光発色 (A)と、グラミシジン濃度 の蛍光強度への影響を示す。  [Fig. 6] Fluorescence development (A) due to the formation of dalamicidin membrane ion channels and the effect of gramicidin concentration on fluorescence intensity.
[図 7]被検物質の濃度とリボソーム複合体の蛍光強度の時間変化の関係を示す。  FIG. 7 shows the relationship between the test substance concentration and the fluorescence intensity of the ribosome complex over time.
[図 8]リボソーム複合体に内包させる BCECF濃度の蛍光強度への影響を示す。  FIG. 8 shows the effect of the BCECF concentration encapsulated in the ribosome complex on the fluorescence intensity.
[図 9]ダラミシジン膜イオンチャンネルのカチオン選択性を示す。  FIG. 9 shows cation selectivity of dalamicidin membrane ion channel.
[図 10]リボソーム複合体の蛍光強度が被検物質の濃度に依存することを示す。被検 物質、及ぴ被検物質と特異的に結合する物質が、それぞれ、抗- DNP及び DNP-PE ( A)、アビジン及び B- cap- PE(B)の場合を示す。  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.
[図 11]リボソーム複合体の蛍光強度が被検物質の濃度に依存することを示す。被検 物質が抗- BSA、被検物質と特異的に結合する物質が DNP- PEの場合を示す。  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.
, [図 12]被検物質と被検物質と特異的に結合する物質との結合と、 BCEFC由来の蛍 光強度との関係を、過剰試薬免疫アツセィにより検証したことを示す。  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.
[図 13]マレイミド基含有リボソーム複合体アレイを用いた被検物質の検出が、被検物 質濃度依存的であったことを示す。  FIG. 13 shows that detection of a test substance using a maleimide group-containing ribosome complex array was dependent on the test substance concentration.
[図 14]マレイミド基含有リボソームアレイを用レ、る抗原抗体反応による被検物質の検 出における、ダラミシジン濃度の影響を示す。  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.
[図 15]マレイミド基含有リボソームアレイを用いる抗原抗体反応による被検物質の検 出における、被検物質濃度依存性を示す。  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.
[図 16]マレイミド基含有リボソーム複合体をアビジン修飾ガラス基板にアレイ化（固定 ィヒ)してィムノリボソームを調製し、被検物質を検出した場合の蛍光強度を示す。 [図 17]マレイミド基含有リボソーム複合体をアビジン修飾ガラス基板にアレイ化（固定 ィ匕)せずにィムノリポソームを調製し、被検物質を検出した場合の蛍光強度を示す。 発明を実施するための最良の形態 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. BEST MODE FOR CARRYING OUT THE INVENTION
[0013] 本発明における被検物質は、該被検物質と特異的に結合する物質であれば、特に 限定されることはない。 [0013] The test substance in the present invention is not particularly limited as long as it is a substance that specifically binds to the test substance.
[0014] 被検物質と、該被検物質と特異的に結合する物質の関係としては、免疫学的な関 係が挙げられ、具体的には、抗原と抗体の関係が挙げられる。抗原と抗体の関係に おいて、被検物質が抗原である場合、該抗原と特異的に結合する物質は抗体であり ' 、被検物質が抗体である場合は、該抗体と特異的に結合する物質は抗原である。  [0014] 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. In the relationship between an antigen and an antibody, when the test substance is an antigen, the substance that specifically binds to the antigen is an antibody. When the test substance is an antibody, it specifically binds to the antibody. The substance to do is an antigen.
[0015] また、被検物質と特異的に結合する物質は、被検物質と間接的に結合する物質で あってもよい。即ち、該物質は、被検物質と特異的に結合する物質と結合することが できる物質であってもよい。具体的には、被検物質と結合する抗体 (一次抗体)と結 合することのできる抗体 (二次抗体）が挙げられる。  [0015] 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. Specifically, an antibody (secondary antibody) that can bind to an antibody that binds to a test substance (primary antibody) can be mentioned.
[0016] 上記抗体を作製するために用レ、る動物としては、ゥサギ、マウス、ャギ、ゥマ、ゥシ 等が挙げられる。抗体としては、動物に免疫することにより得られるポリクローナル抗 体でも、ハイプリドーマ法で得られるモノクローナル抗体でもよい。  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.
[0017] 本発明における抗原としての被検物質としては、例えば、タンパク質、ホルモン、活 性ペプチド、ォータコイド、腫瘍マーカー、免疫グロプリン等の生体成分、ジゴキシン 、フエニトイン、フエノバルビタール等の薬剤の微量成分が挙げられる力 これら 限 定されることなレ、。被検物質に対する抗体を作製できるものであれば特に制限はなレヽ  [0017] Examples of the test substance as an antigen in the present invention include 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 These are not limited. There is no particular limitation as long as antibodies against the test substance can be produced.
[0018] また、本発明における、被検物質と、該被検物質を特異的に結合する物質としては 、酵素と基質、酵素と阻害剤、ホルモンと受容体、レクチンと糖鎖、 DNAと RNA、 D NAと DNA、血清アルブミンと色素ブルー、酵素と補酵素、タンパク質とコンビナトリ アルリガンドペプチド等の様々な組み合わせを挙げることができる。酵素と補酵素の 組み合わせとして、酸化還元酵素と補酵素 NADHの組み合わせ等が挙げられる。 [0018] In the present invention, 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.
[0019] 本発明におけるリボソームは、その膜表面に被検物質と特異的に結合する物質、ホ スファチジルエタノールァミンのピオチン誘導体 (B-cap-PE)とを有すること、及び、 pH 依存性蛍光色素を内包することができるリボソームであれば特に制限はなレ、。ここで 、ホスファチジルエタノールァミンのビォチン誘導体は、 1,2- dioleoyl- sn- glycero- 3- p hosphoethanolmine-N-(cap biotinyl)のナトリウム塩（以下、 B- cap- PE)が望ましレヽ。 [0019] 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 As long as it is a ribosome that can contain a dependent fluorescent dye, there is no particular limitation. Here, 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).
[0020] 本努明におけるリボソーム複合体は、上記リボソームの膜表面に、被検物質と特異 的に結合する物質、ホスファチジルエタノールァミンのピオチン誘導体 (B-caP_PE)と を有し、その内水相に pH依存性蛍光色素が内包され、及び、リボソーム外よりもリポ ソーム内水相の水素イオン濃度が高くなるよう調製されたものである。例えば、 pH依 存性蛍光色素が 2， - 7， _ビス-（カルボキシェチル) -6 -カルボキシフルォレセイン（BC ECF= 2 ' -7 ' -bis-(carboxyethyl)-(and-6)-carboxyfluorescein)の場合、リボソーム 内水相は pH5.5、被検物質と被検物質と特異的に結合する物質の反応が起こるリポ ソーム外の pHは、 pH5.5以上、好ましくは、 pH7.0以上、更に好ましくは、 pH7.8が望ま しい。 Ribosome complex in [0020] 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. For example, a pH-dependent fluorescent dye is 2, -7, _bis- (carboxyethyl) -6-carboxyfluorescein (BC ECF = 2'-7'-bis- (carboxyethyl)-(and-6 ) -carboxyfluorescein), the aqueous phase inside the ribosome is pH 5.5, and 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.
[0021] 本発明における pH依存性蛍光色素は、細胞蛍光染色に用いられる pH感受性色 素であってリボソームの二重膜を透過できなレ、ものが望ましい。具体的には、カルボ キシフルォレセイン、 2，- 7，-ビス-（カルボキシェチル） -6-カルボキシフルォレセイン (BCECF)、 8 -ヒドロキシフィレン- 1,3,6 -トリスルフォニックアシッド（HPTS)、セミナフト ローダフロース（SNARF) (モレキュラープローブ社)などが挙げられる。細胞蛍光染色 に用レヽられる pH感受性色素には、 BCECFのァセトキシメチル (AM)エステル (BCEC F - AM)などの細胞膜透過性を有する誘導体があるが、本発明における pH依存性蛍 光色素には、これら細胞膜透過性を有する誘導体は含まれない。  [0021] 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. Specifically, 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). Examples of pH-sensitive dyes used for cell fluorescence staining include derivatives having cell membrane permeability such as BCECF acetoxymethyl (AM) ester (BCEC F-AM). In the present invention, pH-dependent fluorescent dyes include: These derivatives having cell membrane permeability are not included.
[0022] 本発明における膜イオンチャンネル形成物質は、脂質二重層膜を貫通して膜孔を 形成するィオノフォア (イオン透過剤）であり、好ましくは、グラミシジン A、 B、 C、または D等のダラミシジン（Gramicidin)である。これらグラミシジンは、 15個のアミノ酸力 な るペプチドであり、そのすベてのアミノ酸は疎水性の側鎖を有している。グラミシジン は、 "head- to- head"の状態で二量体化 (ダイマー）し、脂質二重膜を貫通する膜ィォ ンチャンネルを形成する。この膜イオンチャンネルは、プロトンを含む一価のカチオン に選択性を有する膜貫通チャンネルである。ダラミシジンは、脂質二重膜において、 ダイマー形成とモノマ一^ ·の解離を線り返している（非特許文献 4)。即ち、ダラミシジ ンのダイマーは、脂質二重膜において、モノマーとの平衡状態にある。 [0022] 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.
[0023] 本発明における膜イオンチャンネルは、リボソームの脂質二重膜を貫通する膜孔で 、上記の膜イオンチャンネル形成物質により誘導形成される。従って、膜イオンチャン ネル形成物質としてグラミシジン (Gramicidin)を使用した場合、膜イオンチャンネルは 、リボソームの脂質二重膜を貫通するグラミシジンのダイマーが形成する膜孔である。  [0023] 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.
[0024] 本発明におけるリボソーム複合体は、担体に固定化して用いてもよい。ここで、担体 とは、吸着や触媒活性を示す物質を固定 (担持)する土台となる物質、即ち基板であ り、アルミナやシリカ等が挙げられる。本努明におけるリボソーム複合体が固定化され る担体は、アビジンで修飾された担体であれば特に限定されなレ、が、アビジン修飾ガ ラス基板が好ましい。該ァビジン修飾ガラス基板は、ガラス表面にアビジンが結合し ていることを特徴とするもので、特開 2005- 69δ23 (特許文献 4)、または Yanagisawaら， Anal. Biochem. 2004, 332, 358- 340 (非特許文献 3)に記載の方法により作製すること ができる。アビジン修飾基板を含む担体の形状としては、膜状、チップ状、アレイ状、 ビース状のもの等が挙げられる力、アレイ状のものが好ましい。  [0024] The ribosome complex in the present invention may be used after being immobilized on a carrier. Here, 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 2005-69δ23 (Patent Document 4), or Yanagisawa et al., Anal. Biochem. 2004, 332, 358-340 It can be produced by the method described in (Non-Patent Document 3). 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.
[0025] ここで、アビジン (Avidin)とは、生卵白中に存在する分子量 68、 000、等電点 10〜10 .5で、 4個のサブユニットで構成される低分子の塩基性糖タンパク質である。アビジン の各サブユニットは 1分子のビォチンと特異的に結合することが知られている。本発 明におけるリボソーム複合体は、その膜表面にあるホスファチジルエタノールァミンの ビォチン誘導体 (B- cap - PE)のピオチン残基とアビジンとの特異的結合により、ァビジ ン修飾基板に固定化される。  [0025] Here, 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. .
[0026] 本発明のリボソーム複合体、及び、該リボソーム複合体を固定化したリボソームァレ ィの作製、及ぴ、本発明の検出法とその原理は以下の通りである。 [0026] 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.
(1)リボソーム複合体の作製  (1) Preparation of ribosome complex
L- a-ホスファチジノレコリン（以下、 PC) (12mg)、コレステロール（以下、 Choi) (3.0mg )及び B - cap- PE (O.OOlOmg)を乾燥させて脂質フィルムを形成させ、これを高真空に さらす。得られた脂質フィルムに 10mM MES (2- 2 -モルホリノエタンスルホン酸一水和 物）（ρΗ5·5)及び l,0mM BCECFを含む 10mLの 0.10M NaCl溶液をカ卩えて、 5分間ボ ルテックスすることにより水和し、次いで、 15分間、超音波処理し、 BCECFを内包する リボソームを形成させる。リボソーム外部液力 BCECFを除くために、リボソーム懸濁 液を 3回の遠心分離 (12,000g、 4°C)を行い、沈殿物を目的とするリボソーム複合体と する。この沈殿物を、最終的に、 0.10 NaClを含む 0.72mLの lOraM MES (pH5.5)溶 液 (以下、 MES溶液とする）に拡散させ、使用するまで窒素の存在下 (4°C)で保存す る (保存リボソーム懸濁液)。リボソームアレイを調製する際は、 5.0mlの保存リボソーム 懸濁液を 1.OmLの MES溶液で希釈して用レ、る。 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) (ρΗ5 · 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. To remove ribosome external fluid BCECF, centrifuge the ribosome suspension 3 times (12,000 g , 4 ° C) to make the precipitate a ribosome complex. This precipitate is finally diffused into 0.72 mL of lOraM MES (pH 5.5) solution (hereinafter referred to as MES solution) containing 0.10 NaCl, and in the presence of nitrogen (4 ° C) until use. Store (preserved ribosome suspension). When preparing a ribosome array, dilute 5.0 ml of the stock ribosome suspension with 1.OmL of MES solution.
このようにして作製したリボソーム複合体は、内水相溶液に BCECF(lmM、 pH5.5)を 内包するが、条件や使用形態に応じて BCECF以外の pH依存性蛍光色素、例えば H PTSまたは SNRFなどを内包させることができる。また、リボソーム複合体のアビジン修 飾基板への固定化を検証するために、内水相溶液に力ルセイン (5·0πιΜ ΚΗ ΡΟ /Ν 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. In addition, in order to verify the immobilization of the ribosome complex on the avidin-modified substrate, force lucein (5 · 0πιΜ ΚΗ ΡΟ / Ν
' 2 4 aOH (pH7.4) )を内包するリボソーム複合体を作製することができる。 '2 4 aOH (pH 7.4)) can be produced.
[0027] (2)マレイミド基含有リボソーム複合体の作製 [0027] (2) Preparation of maleimide group-containing ribosome complex
マレイミド基含有リボソーム複合体は、リボソーム複合体の膜表面にマレイミド基を 導入する成分として、 1,2 -ジパルミトイル- sn-グリセ口- 3-ホスホエタノールァミン- N-[4 -(マレイミドフエニル) -ブチレート（以下、 N- MPB-PE)を使用して作製することができ る。具体的には、リボソーム複合体の構成成分である PC、 Choi, B - cap- PE、及び N - MPB- PEの組成比を 80:20:6.7xl0-3:6.7xl0— 3(w/w%)とし、上記「（1)リボソーム複合体 の作製」と同じ方法により作製される。従って、マレイミド基含有リボソーム複合体は、 リボソーム複合体と同じぐ内水相溶液に BCECF(lmVI、 pH5.5)を内包し、条件や使 用形態に応じて BCECF以外の pH依存性蛍光色素、例えば HPTSまたは SNRFなどを 内包させることができる。また、リボソーム複合体のアビジン修飾基板への固定ィ匕を検 証するために、内水相溶液に力ルセイン (5.0mM KH PO /NaOH(pH7.4) )を内包す 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). Specifically, 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 %)) And the same method as in “(1) Preparation of ribosome complex” above. Therefore, 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. In addition, in order to verify the fixation of the ribosome complex to the avidin-modified substrate, force lucein (5.0 mM KH PO / NaOH (pH 7.4)) is included in the inner aqueous phase solution.
2 4  twenty four
るリボソーム複合体を作製することができる。  Ribosome complexes can be made.
[0028] (3)アビジン修飾ガラス基板の作製  [0028] (3) Fabrication of avidin-modified glass substrate
アビジン修飾ガラス基板は、特開 2005- 69823 (特許文献 4)、または Yanagisawaら， Anal. Biochem. 2004, 332, 358- 340 (非特許文献 3)に記載の方法により作製すること ができる（図 1)。カバーガラス（18x24)を 1M NaOH中に 1昼夜、浸漬する。 Milli - Q水 で十分に洗浄後、 70°Cで約 2時間乾燥する。ただちに、カバーガラスの片面に 50(v/v )% 3 -メルカプトプロピルリメトキシシラン（MTS)の無水トルエン溶液を 600ml载せて、 . 60分間、室温で放置する。これにより、カバーガラスをシラン化する。無水トルエンで 洗浄後、 2.0mM N-スクシンィミジル 4-マレイミドブチレート（GMBS)の無水ジメチルス ルホキシド（DMSO)溶液 800mlを载せ、室温で 1時間放置する。これによりカバーガラ スを活性化する。このようにして作製した GMBS基板に 13.5mlの 0.1M NaH PO /NaO 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. After washing with anhydrous toluene, load 800 ml of 2.0 mM N-succinimidyl 4-maleimidobutyrate (GMBS) in anhydrous dimethylsulfoxide (DMSO) and leave at room temperature for 1 hour. This activates the cover glass. 13.5 ml of 0.1M NaH PO / NaO was prepared on the GMBS substrate thus prepared.
2 4  twenty four
Η (ρΗ7.0)溶液 (以下、リン酸溶液と略す）に溶解したアビジン (O.lOmg/ml)をスポット し、室温で 1時間反応させる。 GMBS基板上のスポットを Mili- Q水で洗浄し、次いで、 0 .5Mエタノールァミン溶液（〜pH8.5) 15.0mlを载せて 30分間放置する。  Spot avidin (O.lOmg / ml) dissolved in Η (ρΗ7.0) solution (hereinafter abbreviated as phosphoric acid solution) and allow to react at room temperature for 1 hour. The spot on the GMBS substrate is washed with Mili-Q water, and then placed on 15.0 ml of 0.5 M ethanolamine solution (˜pH 8.5) and left for 30 minutes.
[0029] (4)リボソームアレイの作製  [0029] (4) Preparation of ribosome array
上記（3)で作製した GMBS基板上の各スポットを MES溶液 (pH5.5)で洗浄する。上 記（1)で作製した保存リボソーム懸濁液の希釈液 40ml (保存リボソーム懸濁液を MES 溶液 (pH5.5)で希釈したもの）を GMBS基板上の各スポットに載せ、 20分間反応させ る。次いで、スポットを MES溶液で 3回洗浄する。洗浄は、マイクロピペットで水または MES溶液を添加することで行う。これら一連の工程により、リボソーム複合体がアビジ ン修飾ガラス基板上に固定化される（図 3)。  Wash each spot on the GMBS substrate prepared in (3) above with MES solution (pH 5.5). 40 ml of the diluted stock ribosome suspension prepared in (1) above (diluted stock ribosome suspension diluted with MES solution (pH 5.5)) is placed on each spot on the GMBS substrate and allowed to react for 20 minutes. The The spots are then washed 3 times with MES solution. Wash by adding water or MES solution with a micropipette. Through this series of steps, the ribosome complex is immobilized on an avidin-modified glass substrate (Figure 3).
[0030] (5)マレイミド基含有リボソームアレイの作製  [0030] (5) Preparation of maleimide group-containing ribosome array
上記 (2)で作製したマレイミド基含有リボソーム複合体の保存リボソーム懸濁液を、 上記（3)で作製した GMBS基板上の各スポットに添加することでマレイミド基含有リポ ソームアレイを作製することができる。具体的には、上記「（4)リボソームアレイの作製 Jに記載の方法により、上記（2)で作製したマレイミド基含有リボソーム複合体の保存 リボソーム懸濁液をアビジン修飾ガラス基板上に固定化することで作製する（図 3)。  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).
[0031] (6)ィムノリボソームアレイの形成  [0031] (6) Formation of immunoribosome array
本発明のィムノリボソームアレイは、マレイミド基含有リボソーム複合体の表面のマレ イミド基に対して、抗体又はその Fab'断片がそのチオール基を介して結合したリポソ ーム複合体力 アビジン修飾ガラス基板上にアレイ状に固定化されたリボソームァレ ィであり、マレイミド基含有リボソームアレイを用いた抗原抗体反応によって被検物質 を検出する方法の工程において形成される。具体的には、以下の方法によって形成 される。被検物質と特異的に結合する抗体又はその F¾b'断片を含む溶液をあらかじ め調製する。マレイミド基含有リボソームアレイに被検物質 (アナライト)を含む試料を 添加する前に、マレイミド基含有リボソームアレイの各スポットに、あらかじめ調製した 被検物質と特異的に結合する抗体又はその Fab，断片を含む溶液を添加し、該マレ イミド基含有リボソームアレイをー晚 4°Cにてインキュベートする。これにより、マレイミド 基含有リボソームアレイのスポット中に固定化されているマレイミド基含有リボソーム 複合体の膜表面のマレイミド基に対して、被検物質と特異的に結合する抗体又はそ の Fab' 断片が、そのチオール基を介して結合する。その結果、マレイミド基含有リポ ソームアレイ上に固定化されてレ、るマレイミド基含有リボソーム複合体は、その膜表面 に被検物質と特異的に結合する抗体又はその Fab'断片を有することになる。このよ うにして形成されるマレイミド基含有リボソームアレイをィムノリボソームアレイと呼ぶ。 ィムノリボソームアレイに、被検物質 (アナライト）、次いで、ダラミシジン等の オン チャンネル形成物質を添加することで蛍光を発色させ、この蛍光強度を測定すること で被検物質を検出することができる。 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 F¾b 'fragment Prepare. Before adding a sample containing the test substance (analyte) to the maleimide group-containing ribosome array, 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. As a result, 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) is added to the immunoribosome array, and then 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. .
(7)蛍光検出（図 3) (7) Fluorescence detection (Figure 3)
アビジン修飾ガラス基板上に固定化されているリボソーム複合体は、 l.OmM BCEC Fを内包し、その内水相溶液は 10mM MES (pH5.5)である。該リボソーム複合体がス ポットされているリボソームアレイの各スポットを、 10mM MES (pH7.8) (リボソーム外部 の溶液）に溶解した 0.10M NaCl溶液で洗浄する。次いで、各スポットに、所定の濃度 の被検物質 (アナライト）（アビジンまたは抗- DNP等)を溶解させたリボソーム外水相 溶液 20ml (10mM MES (pH7.8) )を配し、 30分間インキュベートする。次いで、グラミシ ジン Dの l.Omg/mlメタノール溶液をリボソーム外水相溶液で希釈して調製したグラミシ ジン D溶液 10ng/mlの 20mlを各スポットに添加し、室温で 60分間インキュベートする。 リポソ一ムアレイの蛍光画像 (励起波長 488nm、蛍光波長 530nm)を、フルォロイメー ジャー 595 (Fluolmager 595) (モレキュラー ダイナミックス社、サンパレー，カリフオル ニァ州、アメリカ合衆国）により得る。各リボソームスポットの端を除く全てのエリアの平 均蛍光強度をイメージヤー クアント パージヨン 5,0 (Imager Quant version 5.0)で得 て、被検物質 (アナライト)の濃度に対してプロットする。初期に獲得したイメージのデ イスプレイ値を変化させることは、スキャンしたイメージを変えることになる力 初期ィメ ージの平均蛍光強度は、そのような手段では変わらない。 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. Next, 20 ml of 10 ng / ml of gramicidin D solution prepared by diluting a l.Omg / ml methanol solution of gramicidin D with an aqueous ribosome aqueous phase solution is added to each spot and incubated at room temperature for 60 minutes. Fluorescence images of the liposomal array (excitation wavelength 488 nm, fluorescence wavelength 530 nm) are obtained by Fluoromager 595 (Molecular Dynamics, Sunpalay, Calif., USA). Obtain the average fluorescence intensity of all areas excluding the edge of each ribosome spot with Image Yarquat Purgeon 5,0 (Imager Quant version 5.0) and plot it against the concentration of the analyte (analyte). Changing the display value of the initially acquired image is the power that will change the scanned image. The average fluorescence intensity of the page does not change by such means.
アビジン修飾ガラス基板上に固定化されているマレイミド基含有リボソーム複合体 についても、上述したリボソーム複合体と同じく l.OmM BCECFを内包し、その内水相 溶液は 10mM MES (PH5.5)である。従って、マレイミド基含有リボソームアレイ、及びィ ムノリボソームアレイの蛍光検出についても、上述のリボソームアレイの場合と同じ方 法でおこなうことができる。 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.
(8)蛍光検出の原理（図 2、 3) (8) Principle of fluorescence detection (Figs. 2 and 3)
リボソーム複合体（内水相は lmM BCECF(PH5.5))は、アビジン-ピオチン結合を介 してアビジン修飾ガラス基板に固定化され、被検物質を含む試料液 (ρΗ7·8)が添カロ されてインキュベートされる。リボソーム複合体に内包されてレ、る BCECFは、 ρΗが上 昇するとその蛍光強度が増大する特性を有する ρΗ感受性色素 (pKa=6.98)であり、 PH5.5では、その蛍光強度は弱い。アビジン修飾ガラス基板へ添加されたダラミシジ ンが、二量体化してリボソームの脂質二重膜を貫通する膜イオンチャンネルが形成さ れると（開口）、該イオンチャンネルを介して H+イオン力 Sリボソーム複合体内部力ゝら外 部へと放出される。これにより、アビジン修飾ガラス基板に固定化されてレ、るリポソ一 ム複合体の内部の pHが上昇し、内包されている BCECFの蛍光が増大する。 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 (ρΗ7 · 8) containing the test substance is added. Calored and incubated. BCECF, which is encapsulated in the ribosome complex, is a ρΗ sensitive dye (pKa = 6.98) with the property that its fluorescence intensity increases when ρΗ rises, and its fluorescence intensity is weak at PH5.5. When dalamicidin added to an avidin-modified glass substrate dimerizes to form a membrane ion channel that penetrates the lipid bilayer of the ribosome (opening), H + ion force S ribosome complex The internal force is released to the outside. As a result, the pH inside the liposome complex immobilized on the avidin-modified glass substrate increases, and the fluorescence of the encapsulated BCECF increases.
上述したように、ダラミシジンのダイマー（二量体）は、脂質二重膜においてダイマー 形成とモノマーへの解離を繰り返す平衡状態にある。この平衡状態は、リボソーム膜 表面で起こる抗原抗体反応など、被検物質 (アナライト）と該被検物質と特異的に結 合する物質 (レセプター）との結合に起因して生じるリボソーム脂質二重膜の局所的 ひずみによって変化する (非特許文献 5、 6)。被検物質 (アナライト)の濃度が上昇す ると、被検物質 (アナライト）と被検物質と特異的に結合する物質 (レセプター）との結 合頻度が上昇し、それに伴レ、リボソーム脂質二重膜の局所的ひずみが生じる頻度が 高まる。そして、リボソーム二重膜の局所的ひずみにより、ダラミシジンのモノマーダイ マー平衡状態が崩れてグラミシジンダイマー形成が促進される。ダラミシジンダイマ 一が形成されて膜イオンチャンネルが開口すると、リボソーム内水相の水素イオンの リボソーム外への輸送がさらに促進されてリボソーム内水相の pHが上昇し、リポソ一 ムに内包されてレ、る BCECFの蛍光強度が増す。従って、被検物質 (アナライト）の濃 度の上昇に依存してリボソームに内包されてレ、る BCEFCの蛍光強度は増大すること になる。 As described above, 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). When the 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. The frequency of local strains in the lipid bilayer increases. Then, local distortion of the ribosome bilayer disrupts the monomer-dimer equilibrium state of dalamicidin and promotes the formation of gramicidin dimer. When the dalamicidin dimer is formed and the membrane ion channel is opened, the transport of hydrogen ions in the ribosome aqueous phase to the outside of the ribosome is further promoted to increase the pH of the aqueous phase in the ribosome and encapsulate in the liposome. The fluorescence intensity of BCECF increases. Therefore, the concentration of the test substance (analyte) Depending on the degree of increase, the fluorescence intensity of BCEFC encapsulated in ribosomes will increase.
[0034] マレイミド基含有リボソームアレイを用いて抗原抗体反応により被検物質を検出する 方法においても、蛍光検出の原理は、上述のリボソーム複合体の場合に同じである。 該方法は、その工程において、ィムノリボソームアレイが形成された（上記「（6)ィムノ リボソームアレイの形成」）後、ィムノリボソームアレイに、被検物質（アナライト）、次い で、膜イオンチャンネル形成物質を添加することで蛍光を発色させ、この蛍光強度を 測定することで被検物質を検出する方法である。ィムノリボソームアレイが形成された 後、被検物質を含む試料を添加すると、被検物質と抗体又はその Fab'断片との間 で抗原抗体反応が生じ、リボソーム脂質二重膜の局所的ひずみが生じる頻度が高ま る。そこへグラミシジン溶液を添加すると、脂質二重膜においてモノマーダイマー平 衡状態が崩れてグラミシジンダイマー形成が促進される。ダラミシジンダイマーが形 成されて膜イオンチャンネルが開口すると、リボソーム内水相の水素イオンのリポソ一 ム外への輸送がさらに促進されてリボソーム内水相の pHが上昇し、リボソームに内包 されてレ、る BCECFの蛍光強度が増す。従って、被検物質と抗体又はその Fa 断片 との間で抗原抗体反応の頻度に依存してリボソームに内包されている BCEFCの蛍光 強度は増大することになる。  [0034] In the method of detecting a test substance by antigen-antibody reaction using a maleimide group-containing ribosome array, the principle of fluorescence detection is the same as in the case of the above-mentioned ribosome complex. In this method, after an immunoribosome array is formed in the process (“(6) Formation of an immunoribosome array” above), a test substance (analyte) is added to the immunoribosome array, and then a membrane. In this method, fluorescence is developed by adding an ion channel forming substance, and the test substance is detected by measuring the intensity of the fluorescence. When a sample containing a test substance is added after the immunoribosome array is formed, an antigen-antibody reaction occurs between the test substance and the antibody or its Fab 'fragment, and local strain of the ribosomal lipid bilayer membrane is reduced. It occurs more frequently. When a gramicidin solution is added thereto, the monomer dimer equilibrium state is lost in the lipid bilayer membrane, and the formation of gramicidin dimer is promoted. When the dalamicidin dimer is formed and the membrane ion channel is opened, the transport of hydrogen ions in the ribosomal aqueous phase to the outside of the liposome is further promoted to increase the pH of the ribosomal aqueous phase and be encapsulated in the ribosome. The fluorescence intensity of BCECF increases. Therefore, the fluorescence intensity of BCEFC encapsulated in the ribosome increases depending on the frequency of the antigen-antibody reaction between the test substance and the antibody or its Fa fragment.
[0035] なお、上述のリボソーム複合体、及びマレイミド基含有リボソームアレイを用いて抗 原抗体反応により被検物質を検出する方法のいずれの方法においても、被検物質を 含む試料を添加した後に、ダラミシジン溶液を添加することから、リボソーム複合体の 脂質二重膜を貫通する膜イオンチャンネノレは、リボソーム複合体の膜上において、被 検物質と特異的に結合する物質が位置する部位とは異なる部位において形成される (図 3)。  [0035] In any of the methods for detecting a test substance by an antigen-antibody reaction using the above-described ribosome complex and a maleimide group-containing ribosome array, after adding a sample containing the test substance, Because of the addition of daramicidin solution, the membrane ion channel that penetrates the lipid bilayer of the ribosome complex is different from the site where the substance that specifically binds to the test substance is located on the membrane of the ribosome complex. It is formed at the site (Figure 3).
[0036] (9)蛍光強度のキャリブレーション  [0036] (9) Fluorescence intensity calibration
PC、 Choi, B- cap- PE、及ぴ DNP-PE (80:20:6.7xl(f3: 1.6x10— w/w%)で構成される リボソーム複合体について蛍光強度を検出する。該リボソーム複合体は、脂質フィル ムを ρΗ5·5— 7.8の異なる pHに調整した LOmM BCECF及ぴ lOmM MESを含む 0.10M NaClで水酸化することで調製した。リボソームアレイ（5スポット）は、 40mlの各リポソ一 ム懸濁液により調製した。 BCEFCを含まなレ、リボソーム内水相液で各スポットを洗浄 した後、蛍光画像を取得し、その平均蛍光強度を pH値に対してプロットする。 PC, Choi, B- cap- PE,及Pi DNP-PE (80: 20: 6.7xl (f 3:. 1.6x10- the w / w%) in constructed ribosome complex for detecting fluorescence intensity the ribosome The complex was prepared by hydroxylating the lipid film with 0.10M NaCl containing LOmM BCECF and lOmM MES adjusted to different pH of ρΗ5 · 5-7.8. Liposo Prepared by suspension. After washing each spot with an aqueous phase solution containing BCEFC and ribosome, obtain a fluorescence image and plot the average fluorescence intensity against the pH value.
PC、 Choi, B- cap- PE、及び N-MPB-PE (80:20:6.7x10— 3:6,7xl0—3、 w/w%)で構成され るマレイミド基含有リボソーム複合体についても、上述のリボソーム複合体と同じ方法 により蛍光強度を検出する。 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.
[0037] (10)被検物質を検出するためのキット  [0037] (10) Kit for detecting a test substance
本発明の検出法は、上記のように、リボソーム複合体、及ぴ該リボソーム複合体を 固定化したリボソームアレイを用いて、多試料及び多成分を迅速に検出測定すること ができる方法であるため、該方法に基づいたキットを作製することにより、簡便に自動 化を図ることが可能である。本発明のキットの特徵は、上記「(4)リボソームアレイの作 製」により作製したリボソームアレイと、膜イオンチャンネル形成物質を含む、被検物 質 (アナライト）を検出するためのキットである。該膜イオンチャンネル形成物質は、グ ラミシジン A、 B、 C、または D等のグラミシジン (Gramicidin)が望ましい。  Since 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. By preparing a kit based on this method, automation can be easily achieved. 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.
[0038] 更に、本発明の一つであるマレイミド基含有リボソームアレイを用いて抗原抗体反 応により被検物質を検出する方法は、キットを作製することにより、多試料及び多成 分より迅速に被検物質を検出することについて自動化を図ることができる。該キットは 、マレイミド基含有リボソームアレイ、被検物質の種類に応じてあらかじめ調製された 被検物質に特異的に結合する抗体又はその Fab'断片を含む溶液、及び膜イオンチ ヤンネル形成物質を含む、多様な被検物質を迅速、かつ簡便に検出することができ るキットである。該キットの膜イオンチャンネル形成物質としては、ダラミシジン A、 B、 C 、または D等のダラミシジン (Gramicidin)が望ましい。  [0038] Furthermore, the method for detecting a test substance by antigen-antibody reaction using a maleimide group-containing ribosome array, which is one of the present invention, 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.
[0039] 以下に実施例を参照しながら本発明を更に詳細に説明する。なお、これら実施例 は、何ら本 明を限定することを意図するものではない。  [0039] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are not intended to limit the present invention in any way.
実施例 1  Example 1
[0040] [リボソーム複合体のアビジン修飾基板への固定化の検証]  [0040] [Verification of immobilization of ribosome complex to avidin-modified substrate]
上記「（1)リボソーム複合体の作製」におレ、て、 BECEFに代わって蛍光色素力ルセ インを内包するリポソ一ム複合体を作製し、これを用いてリボソーム複合体のアビジン 修飾ガラス基板への固定ィヒの検証をした。具体的には、該リボソーム複合体内水相 力もの蛍光色素力ルセイン漏出の有無を以下の方法により評価した。 In the above “(1) Preparation of 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.
蛍光色素力ルセインを内包するリボソーム複合体を作製し、これを保存リボソーム懸 濁液 (窒素の存在下 (4°C)で保存)とした。上記「（3)アビジン修飾ガラス基板の作製 の方法」に従って作製したアビジン修飾ガラス基板を、 5.0mIVi KH PO /NaOH(pH7.4  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).
2 4  twenty four
)溶液で 3回洗浄した。その後、保存リボソーム懸濁液 22.5mlを、 3,0mlの 5·0πιΜ KH P  ) Washed 3 times with the solution. Then store 22.5ml of stored ribosome suspension, 3,0ml 5 · 0πιΜ KH P
2 2
O /NaOH (pH7.4)に希釈して調製した希釈保存リボソーム懸濁液 0.20mlをアビジンAvidin 0.20 ml of diluted ribosome suspension prepared by diluting in O 2 / NaOH (pH 7.4)
4 Four
修飾ガラス基板に配し、 20分間室温でインキュベートすることで、力ルセインを内包す るリボソーム複合体をアビジン修飾ガラス基板に固定ィ匕した。このアビジン修飾ガラス 基板について、 5.3mM CoClを含む 5.0mM KH PO /NaOH (pH7.4)溶液を添加する By placing on a modified glass substrate and incubating at room temperature for 20 minutes, the ribosome complex encapsulating force lucein was immobilized on the avidin-modified glass substrate. Add 5.0 mM KH PO / NaOH (pH 7.4) solution containing 5.3 mM CoCl to this avidin-modified glass substrate.
2 2 4  2 2 4
前と後の双方において蛍光画像の測定をおこなった (励起波長 488nm、蛍光波長 53 0nm) oその結果、 CoCl溶液を添加する前後において、力ルセイン由来の蛍光が認 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.
2  2
められた（図 4)。力ルセインの蛍光は、 Co2+によって消光する。従って、 CoCl溶液の (Figure 4). The fluorescence of strong lucein is quenched by Co 2+ . Therefore, the CoCl solution
2 添加によっても蛍光が認められたことは、リボソーム複合体からの力ルセイン漏出が 無力つたことを示している。この結果より、上記「（1)リボソーム複合体の作製」に記載 の方法で作製したリボソーム複合体は、内包されてレ、る蛍光色素が漏出することなく 、アビジン修飾ガラス基板に固定化されることが確認された。  2 Fluorescence was also observed after addition, indicating that force lucein leakage from the ribosome complex was ineffective. As a result, the ribosome complex produced by the method described in “(1) Production of ribosome complex” is encapsulated and immobilized on the avidin-modified glass substrate without leakage of the fluorescent dye. It was confirmed.
実施例 2 Example 2
[B- cap- PE量のリボソーム複合体固定化への影響] [Effect of B-cap-PE amount on ribosome complex immobilization]
上記「（1)リボソーム複合体の作製」に記載の方法により、 ImM力ルセインを含む 5.0 mM KH PO /NaOH (pH7.4)溶液を内部に含み、リボソーム二重膜を構成する B- cap  By the method described in “(1) Preparation of ribosome complex” above, a B-cap that contains a 5.0 mM KH PO / NaOH (pH 7.4) solution containing ImM force lusein and forms a ribosome bilayer membrane
2 4  twenty four
-PEの量が重量分率にしてそれぞれ 0、 0.056、 0.55、 5.5、 227、 0.555x10— 5(w/w%)とな るリボソーム複合体を作製し、 B - cap- PE量のリボソーム複合体固定化の影響を検証 した。 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.
上記の各 B - cap - PE量からなるリボソーム複合体を、上記「（1)リボソーム複合体の 作製」の方法に従って作製し、保存リボソーム懸濁液 (窒素の存在下 (4°C)で保存）と した。次いで、各保存リボソーム懸濁液を、実施例 1に記載の方法によりリボソーム複 合体をアビジン修飾基板へ固定化した。固定化した各リボソーム複合体の発する蛍 光を検出したところ（励起波長 514nm、蛍光波長 530nm)、 B - cap-PEの重量分率が 5. 5xl(T5(w/w%)の場合に、力ルセインの蛍光強度が最大であった（図 5)。 Prepare the ribosome complex consisting of each B-cap-PE amount according to the method described in “(1) Preparation of ribosome complex” above, and store it in a storage ribosome suspension (in the presence of nitrogen (4 ° C)). ) Next, the ribosome complex was immobilized on the avidin-modified substrate of each preserved ribosome suspension by the method described in Example 1. When the fluorescence emitted from each immobilized ribosome complex was detected (excitation wavelength: 514 nm, fluorescence wavelength: 530 nm), the weight fraction of B-cap-PE was 5. In the case of 5xl (T 5 (w / w%)), the intensity of fluorescence of force lucein was the highest (FIG. 5).
実施例 3  Example 3
[0042] [グラミシジン濃度の蛍光強度への影響]  [0042] [Effect of gramicidin concentration on fluorescence intensity]
BCECFを内包するリボソーム複合体からの H+の放出力 S、グラミシジン濃度によって 、どのような影響を受けるのか検証した。上記「(4)リボソームアレイの作製」に記載の 方法により作製したリボソームアレイを用いて、上記「（7)蛍光検出」に記載の方法に おいて、添カ卩するグラミシジンの濃度を 0— 1.0ml/ml (0〜60nM)として、リボソームの 蛍光強度を測定した。ダラミシジン濃度が lOng/πύまでは、蛍光強度の増大が認めら れたが、それ以上のダラミシジン濃度では蛍光強度が飽和した（図 6)。この結果は、 ダラミシジンの添加によりリボソームの脂質二重膜に膜イオンチャンネルが形成され て H+イオンが放出され、リボソームの内水相の pHが上昇したことを示している。  We examined how it is affected by the release S of H + from the ribosome complex containing BCECF and the concentration of gramicidin. Using the ribosome array prepared by the method described in “(4) Preparation of ribosome array” above, the concentration of gramicidin added in the method described in “(7) Fluorescence detection” is set to 0 to 1.0. The fluorescence intensity of ribosome was measured as ml / ml (0-60 nM). Fluorescence intensity increased up to dalamicin concentration up to lOng / πύ, but fluorescence intensity was saturated at higher concentrations of daramicidin (Fig. 6). This result indicates that the addition of dalamicidin formed a membrane ion channel in the lipid bilayer of the ribosome and released H + ions, increasing the pH of the ribosome's inner aqueous phase.
実施例 4  Example 4
[0043] [蛍光強度の時間変化] [0043] [Change in fluorescence intensity over time]
ダラミシジンを添加した後のリボソーム複合体の蛍光強度の時間変化を検証した。 具体的には、上記「（1)リボソーム複合体の作製」において、被検物質 (アナライト）と 特異的に結合する物質 (レセプター)を B-cap- PEとするリボソーム複合体を作製し、 上記 Γ (6)蛍光検出」におレ、て、リボソームアレイの各スポットに添加する被検物質 (ァ ナライト）をアビジン、そのアビジンの濃度を 0、 1(Γ6、 10— 7、 lo mUグラミシジン濃度 を 5.31nMとした (図 7)。この検証の結果は、上記「（8)蛍光検出の原理」に記载のよう に、被検物質 (アナライト)の濃度の上昇と、それに伴う該被検物質 (アナライト）と被 検物質と特異的に結合する物質 (レセプター）との結合頻度の上昇により、リボソーム 複合体に内包されてレ、る BCEFCの蛍光強度が増大することを示してレ、る。 The time change of the fluorescence intensity of the ribosome complex after adding daramicidin was verified. Specifically, in the above “(1) Preparation of ribosome complex”, a ribosome complex having B-cap-PE as a substance (receptor) that specifically binds to the test substance (analyte) is prepared. the gamma (6) Slight fluorescence detection "Te, avidin test substances (§ Naraito) to be added to each spot of the ribosome array, the concentration of the avidin 0, 1 (Γ 6, 10- 7, lo mU The gramicidin concentration was 5.31 nM (Fig. 7) The results of this verification were as follows, as described in “(8) Principle of fluorescence detection”, and the increase in the concentration of the analyte (analyte). It shows that the fluorescence intensity of BCEFC encapsulated in the ribosome complex increases due to an increase in the binding frequency of the test substance (analyte) and the substance (receptor) that specifically binds to the test substance. I'm going.
アビジン濃度が Og/mlの場合、グラミシジンを添加して 60分後にリボソームの蛍光強 度が僅かに上昇した（図 7 (D) )。この僅かな蛍光強度の上昇は、ダイマー形成とモノ マ一^■の解離を繰り返す平衡状態にあるグラミシジンがダイマー化した瞬間に開口し た膜イオンチャンネルから放出された H オンに起因する。アビジン濃度が高くなる に伴レ、リポソ一ム複合体の蛍光強度は増大した（図 7 (A)〜(C) )。この結果は、リポ ソーム複合体の脂質二重膜表面におけるアビジンと B - cap- PEのビォチン残基との結 合により、リボソーム脂質二重膜に局所的ひずみが生じ、これに起因してダラミシジン による膜イオンチャンネルが活性化したことを示している。即ち、アビジンと B - cap - PE のビォチン残基との結合頻度が増大したことにより、膜イオンチャンネルを介する H+ のリボソーム外への流出が促進され、 BCEFCの蛍光強度が増大したことを示している 。蛍光強度が最大値に達するまでに 60分を要してレ、る力 これは、リボソーム複合体 内水相の pHが徐々に上昇したことに起因する。 When the avidin concentration was Og / ml, 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. As the avidin concentration increased, 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. This shows that local distortion occurred in the ribosomal lipid bilayer membrane, and this caused the activation of the membrane ion channel by dalamicidin. In other words, the increased binding frequency between avidin and the biotin residue of B-cap-PE promoted the flow of H + out of the ribosome via the membrane ion channel, indicating that the fluorescence intensity of BCEFC increased. Yes. It takes 60 minutes for the fluorescence intensity to reach its maximum value. This is due to the gradual increase in the pH of the aqueous phase in the ribosome complex.
実施例 5  Example 5
[0044] [リボソーム複合体に内包される BCECF濃度の蛍光強度への影響]  [0044] [Effect of BCECF concentration encapsulated in ribosome complex on fluorescence intensity]
リボソーム複合体に内包される BCECFの濃度の蛍光強度の影響を検討した。具体 的には、上記 Γ (1)リボソーム複合体の作製」において、内包させる BCECFの濃度力 SO 〜1.6mMであるリボソーム複合体、該リボソーム複合体を固定化したリボソームアレイ を上記「 (4)リボソームアレイの作製」の方法により作製し、上記「 (4)蛍光検出」の方 法により蛍光強度を測定した。 BCECFの濃度が l.OmMまでは、蛍光強度は上昇した 1 それ以上の濃度では蛍光強度は低下した（図 8)。  The effect of fluorescence intensity on the concentration of BCECF encapsulated in the ribosome complex was examined. Specifically, in the above `` (1) Preparation of ribosome complex '', the ribosome complex having a concentration power SO-1.6 mM of BCECF to be encapsulated and the ribosome array on which the ribosome complex is immobilized are described in the above (4). A ribosome array was prepared, and the fluorescence intensity was measured by the method “(4) Fluorescence detection”. The fluorescence intensity increased until the concentration of BCECF was l.OmM 1 and the fluorescence intensity decreased at higher concentrations (Fig. 8).
実施例 6  Example 6
[0045] [各カチオンの選択性] [0045] [Selectivity of each cation]
グラミシジンによって形成される膜イオンチャンネルは、 H+及び Na+などの一価の力 チオンに対して透過性を示す力 二価のカチオンに対しては透過性を示さなレ、ことが 知られている。そこで、各カチオンの透過性について検討した。具体的には、上記「（ 4)リボソームアレイの作製」の方法で作製したリボソームアレイに、ダラミシジン 10ng/ . ml及び 0.10Mの各カチオン (Na+、 K+、 Ca2+、 NH Mg2+、または Cs+)を含む lOmM ME S溶液（pH5.5)を添加してインキュベートした。これらとの比較として、ダラミシジン 10ng /mlを含まない 0.10Mの各カチオン溶液（lOmM MES溶液 (PH5.5) )についても添加し インキュベートした。リボソーム複合体は、上記「（1)リボソーム複合体の作製」の方法 に従って作製しているので、その内水相の pHは 5.5である。よって、リボソーム複合体 内外においてカチオン濃度の勾配が形成され、リボソームアレイに添加された各カチ オンはリボソーム複合体の内部へ取り込まれる。また、対照区として、上記「（7)蛍光 検出」の方法と同じぐリボソーム複合体の外部溶液の pHが 7.8となるよう 10mM MES ( pH7.8、グラミシジン lOng/ml)をリボソームアレイに添加した実験をおこなった。即ち、 対照区では、リボソーム複合体の内外で H+の勾配が形成される。 It is known that the membrane ion channel formed by gramicidin is permeable to monovalent forces such as H + and Na + . It is not permeable to divalent cations. . Therefore, the permeability of each cation was examined. Specifically, to the ribosome array produced by the method of “(4) Production of ribosome array”, daramicidin 10 ng / .ml and 0.10 M of each cation (Na + , K +, Ca 2+ , NH Mg 2+ , Alternatively, lOmM MES solution (pH 5.5) containing Cs + ) was added and incubated. For comparison with these, 0.10M cation solutions (10 mM MES solution ( PH 5.5)) not containing 10 ng / ml of daramicidin were also added and incubated. Since the ribosome complex is prepared according to the method of “(1) Preparation of ribosome complex” above, the pH of the inner aqueous phase is 5.5. Therefore, a cation concentration gradient is formed inside and outside the ribosome complex, and each cation added to the ribosome array is taken into the ribosome complex. Also, as a control group, 10 mM MES ( An experiment was conducted in which pH7.8, gramicidin lOng / ml) was added to the ribosome array. That is, in the control group, an H + gradient is formed inside and outside the ribosome complex.
対照区以外では、 NH +を添加した場合において、 BCEFCに由来する蛍光強度に  Except for the control group, when NH + is added, the fluorescence intensity derived from BCEFC
4  Four
若干の増大が認められた。これらの結果は、 BCEFCの蛍光強度にとって H+の勾配の 形成が必須であることを示している（図 9)。 A slight increase was observed. These results indicate that the formation of a H + gradient is essential for the fluorescence intensity of BCEFC (Fig. 9).
実施例 7 Example 7
[BCEFC由来の蛍光強度と被検物質 (アナライト)濃度の関係 1]  [Relationship between BCEFC-derived fluorescence intensity and analyte (analyte) concentration 1]
リボソーム複合体膜と外液境界面での被検物質 (アナライト)と被検物質と特異的に 結合する物質 (アナライトレセプター）との結合による膜イオンチャンネル活性化、即 ち、アナライト一アナライトレセプターのグラミシジンのモノマー/ダイマーカイネテツ タス (monomer/dimmer kinetics)に及ぼす影響を以下の実験により検証した。  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.
(i)アナライト (被検物質)が抗- DNP、レセプター力 ¾NPの場合 (i) When the analyte (test substance) is anti-DNP, receptor power ¾NP
アナライトを 2，4-ジニトロフエノール (DNP)に対するマウスモノクローナル抗体 (抗- D NP)、アナライトレセプターを DNPとして実験をおこなった。  Experiments were conducted using an analyte as a mouse monoclonal antibody (anti-DNP) against 2,4-dinitrophenol (DNP) and an analyte receptor as DNP.
上記（1)リボソーム複合体の作製において、リボソーム複合体のアナライトレセプタ 一を DNPとすべく、リボソーム膜力 SPC (L - a -ホスファチジルコリン）、 Choi (コレステロ一 ノレ)、 B—cap—PE、及び DNP— PE(1,2— dipalmitoyト sn— glycero— 3— phosphoethanokmine - N - (2,4 - dinitrophenyl)で構成されるリボソーム複合体を作製した。このようにして作 製したリボソーム複合体を、上記「（3)アビジン修飾ガラス基板の作製」、「 (4)リポソ ームアレイの作製」に記載の方法に従って、アビジン修飾ガラス基板上に固定ィ匕して リボソームアレイとした。次いで、上記 Γ (7)蛍光検出」において、アナライトを抗 -DNP (濃度は 1.2x10一8〜 1.2x10— 6 g/ml)として BCEFC由来の蛍光強度を検出した。対照区 として、 Γ (7)蛍光検出」においてグラミシジンを添カ卩しない実験を採用した。それによ ると、ダラミシジンを添加して 60分後、抗- DNP濃度が高まるにつれ、 BCEFC由来の 蛍光強度も增大した。これに対して、対照区（ダラミシジン無添加）では、 BCEFC由来 の蛍光強度の増加が認められな力 た（図 10 (A) )。なお、この実験において、アナ ライトをゥシ血清アルブミン Ab - 1に対するマウスモノクローナル抗体 (抗 -BSA)とした 場合（抗- BSAの濃度は 1.2x10— 9〜1.2xl0— 6g/ml)、 BCEFC由来の蛍光は検出されな かった (図 11)。これらの結果は、蛍光強度は、リボソーム複合体の脂質二重膜と外 液の境界面での抗- DNPと DNPの特異的結合に起因することを示してレ、る。本発明に おける抗- DNPのダイナミックレンジ (dynamic range)の下限は、水晶振動子を用いる ィムノセンサー及びファイバーオプティカルセンサーに比較して（7xl(f8〜l(T6g/inl) 約 10倍低い。 In the preparation of the above (1) ribosome complex, ribosome membrane strength SPC (L-a-phosphatidylcholine), Choi (cholesterol monore), B-cap-PE, And a ribosome complex composed of DNP-PE (1,2-dipalmitoyto-sn-glycero- 3—phosphoethanokmine-N- (2,4-dinitrophenyl). Then, according to the methods described in “(3) Preparation of avidin-modified glass substrate” and “(4) Preparation of liposome array”, they were immobilized on the avidin-modified glass substrate to obtain a ribosome array. in 7) fluorescence detection ", the analyte anti-DNP (concentration detecting fluorescence intensity derived from the BCEFC as 1.2x10 one 8 ~ 1.2x10- 6 g / ml) as a. control group, gamma (7) fluorescence detection" Do not add gramicidin According to the experiment, 60 minutes after adding dalamicidin, the fluorescence intensity derived from BCEFC increased as the anti-DNP concentration increased, whereas in the control group (without dalamicidin added). In this experiment, when the analyte was a mouse monoclonal antibody (anti-BSA) against ushi serum albumin Ab-1 in this experiment, the increase in fluorescence intensity derived from BCEFC was not observed (Fig. 10 (A)). (anti - BSA concentrations 1.2x10- 9 ~1.2xl0- 6 g / ml) , Do is detected fluorescence from BCEFC (Figure 11). These results indicate that the fluorescence intensity is due to specific binding of anti-DNP and DNP at the interface between the lipid bilayer of the ribosome complex and the outer fluid. The lower limit of the anti-DNP dynamic range in the present invention is about 10 times lower than that of the immunosensor and the fiber optical sensor using a crystal resonator (7xl (f 8 to l (T 6 g / inl)). .
[0047] (ii)アナライト (被検物質)がアビジン、レセプター力 ¾ - cap- PEの場合  [0047] (ii) When the analyte (test substance) is avidin, receptor strength ¾-cap-PE
アナライトをアビジン、アナライトレセプターを B - cap- PEのピオチン残基として実験 をおこなった。上記「（1)リボソーム複合体の作製」に記載の方法によってリボソーム '複合体を作製した。該リボソーム複合体の膜に存在する B- cap- PEのビォチン残基は 、アナライト、及びアビジン修飾ガラス基板上のアビジンのレセプターとなる。上記「（3 )アビジン修飾ガラス基板の作製」、「 (4)リボソームアレイの作製」に記載の方法に従 つて、該リボソーム複合体をアビジン修飾ガラス基板上に固定化してリボソームアレイ とした。次いで、上記「（7)蛍光検出」において、アナライトをアビジン (濃度は 1.0x10— 8〜1.0xl(f6 g/ml)として BCEFC由来の蛍光強度を検出した。対照区として、「（7)蛍 光検出」においてダラミシジンを添加しない実験を採用した。それによると、グラミシジ ンを添加して 60分後、アビジン濃度が高まるにつれ、 BCEFC由来の蛍光強度も増大 した。これに対して、対照区 (グラミシジン無添力 0)では、 BCEFC由来の蛍光強度の 増加が認められな力 た（図 10 (B) )。 The experiment was conducted with the analyte as avidin and the analyte receptor as the piotin residue of B-cap-PE. 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. Subsequently, in the above “(7) Fluorescence detection”, the fluorescence intensity derived from BCEFC was detected with the analyte as avidin (concentration: 1.0 × 10−8 to 1.0xl (f 6 g / ml). As a control, “(7 ) Fluorescence detection ”was adopted in which no dalamicidine was added, according to which the intensity of BCEFC-derived fluorescence increased as the avidin concentration increased 60 minutes after adding gramicidin. In the plot (gramicidin non-addition force 0), the BCEFC-derived fluorescence intensity did not increase (Fig. 10 (B)).
実施例 8  Example 8
[0048] [BCEFC由来の蛍光強度とアナライト一アナライトレセプター間の結合の関係]  [0048] [Relationship between fluorescence intensity derived from BCEFC and binding between analyte and analyte receptor]
更に、 BCECFの蛍光強度が、リボソーム複合体の脂質二重膜と外液の境界面での アナライト一アナライトレセプター間の結合に起因してレ、ることを検証すベぐアビジン (アナライト）、 B- cap- PE (アナライトレセプター）、ピオチン標識 -抗- BSA (ビォチン- 抗- BSA)を用レ、た過剰試薬免疫アツセィを行った。  Furthermore, it is necessary to verify that the fluorescence intensity of BCECF is 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) and piotin-labeled anti-BSA (biotin-anti-BSA) were used to perform an excess reagent immunoassay.
上記「（7)蛍光検出」に記載の蛍光検出を行う前に、ビォチン-抗 -BSA溶液 (10ml) を過剰のアビジン (2.0xl0_6g/ml)を含む MES 10mlと供に 30分間インキュベートした（ 図 12 (B) )。このインキュベートした溶液 (20ml)を、 PC、 Chol、及ぴ B- cap-PEで構成 させるリボソーム複合体を固定化したリボソームアレイ（上記「 (4)リボソームアレイの 作製」に記載の方法で作製)に添加し、蛍光検出を行った。対照区として、ダラミシジ ンを添加しなレヽ蛍光検出（上記「（6)蛍光検出」 )を行った。ピオチン-抗- BSA濃度 (8 .0x10 -9〜 8.0x10— 6g/ml)が高くなるにした力い、蛍光強度は低下した。対照区では、 蛍光強度の上昇が認められな力 た (図 12 (A) )。この結果は、アビジンがビォチン - 抗- BSAと特異的に結合することで、アナライトレセプターである B- cap- PEと結合する アナライトであるアビジンが減少し、それに伴ってリボソーム複合体の蛍光強度も低 下したことを示している。 BCECFの蛍光強度が、リボソーム複合体の脂質二重膜と外 液の境界面でのアナライト一アナライトレセプター間の結合に起因してレ、ることが確 かめられた。 Before performing the fluorescence detection according to the "(7) fluorescence detection", Biochin - and incubated anti -BSA solution (10ml) and excess avidin (2.0xl0 _6 g / ml) MES 10ml and subjected to 30 minutes including (Figure 12 (B)). This incubated solution (20 ml) is a ribosome array with immobilized ribosome complex composed of PC, Chol, and B-cap-PE (see (4) above). And was detected by fluorescence detection. As a control, ray fluorescence detection (above-mentioned “(6) fluorescence detection”) without adding daramicidin was performed. Piochin - anti - BSA Concentration (8 .0x10 - 9 ~ 8.0x10- 6 g / ml) had the force was increases, the fluorescence intensity was reduced. In the control group, no increase in fluorescence intensity was observed (Fig. 12 (A)). This result indicates that avidin specifically binds to biotin-anti-BSA, resulting in a decrease in the analyte avidin that binds to the analyte receptor B-cap-PE, and the fluorescence of the ribosome complex. It also indicates that the strength has decreased. It was confirmed that the fluorescence intensity of BCECF was due to the binding between the analyte-analyte receptor at the interface between the lipid bilayer of the ribosome complex and the outer fluid.
実施例 9  Example 9
[0049] [マレイミド基含有リボソームアレイを使用した抗原抗体反応による被検物質の検出] マレイミド基含有リボソームアレイを使用した抗原抗体反応による被検物質 (アナラ イト）の検出を検討した。具体的には、サブスタンス P (Substance P)及ぴニューロキニ ン Aを被検物質として、以下の (i)〜 (iv)の方法により検討した。  [Detection of test substance by antigen-antibody reaction using maleimide group-containing ribosome array] Detection of a test substance (analyte) by antigen-antibody reaction using a maleimide group-containing ribosome array was examined. Specifically, substance P (substance P) and neurokinin A were used as test substances, and the following methods (i) to (iv) were used.
(i)マレイミド基含有リボソームアレイの作製  (i) Preparation of maleimide group-containing ribosome array
上記「（5)マレイミド基含有リボソームアレイ」の方法により作製した。これにより、マ レイミドリポソーム複合体 (上記「（2)マレイミド基含有リボソーム複合体の作製」により 作製）は、アビジン修飾ガラス基板 (上記「（3)アビジン修飾ガラス基板の作製」により 作製）上に固定化された。マレイミドリポソーム複合体は l.OmM BCECFを内包し、そ の内水相溶液は 10mM MES (pH5.5)である。  It was prepared by the method of “(5) Maleimide group-containing ribosome array”. As a result, the maleimide liposome complex (prepared by “(2) Manufacture of maleimide group-containing ribosome complex” above) is produced on the avidin modified glass substrate (prepared by “(3) Preparation of avidin modified glass substrate” above). To be fixed. The maleimide liposome complex contains l.OmM BCECF, and its inner aqueous phase solution is 10 mM MES (pH 5.5).
[0050] (ii)抗体断片の調製 [0050] (ii) Preparation of antibody fragment
抗-サブスタンス P抗体及び抗-ュユーロキニン A抗体の Fab，断片を非特許文献 7に 記載の方法により調製した。 100 μ gの抗-サブスタンス Ρ抗体 IgG抗体又は 400 μ の 抗-ニューロキュン A IgG抗体を、 0.01M酢酸緩衝液 (ρΗ4·5)を含む 0.10M NaCl溶液 で透析した。透析液に O.lmgのペプシンを加えた後、透析液を 37°Cで 24時間インキュ ペートした後、これを 1M KH PO /KOHにより pH7.0に調整した。次いで、透析液に含  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 μg of anti-substance Ρ antibody IgG antibody or 400 μ of anti-neurocune A IgG antibody was dialyzed against a 0.10 M NaCl solution containing 0.01 M acetate buffer (ρΗ4 · 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.
2 4  twenty four
まれるペプシン消化による F(ab')断片を、ゲルろ過クロマトグラフィー (Sephacyl S- 20  F (ab ') fragments obtained by digestion with pepsin are subjected to gel filtration chromatography (Sephacyl S-20
2  2
0 HR)により精製した。透析液を Seohacyl S- 200 HRに添加し、 F(ab' )断片を 0.01M NaH PO /KOH(pH4.7)を用いて溶出させた。溶出液にフイコール (ficoll)を添カ卩して F0 HR). Add dialysate to Seohacyl S-200 HR and add F (ab ') fragment to 0.01M Elution was carried out using NaH PO / KOH (pH 4.7). Add ficoll to the eluate and add F
2 4 twenty four
(ab，）断片を濃縮した。濃縮された F(ab，）断片 0.80mlに 100 /x lの 0.01Mのシステアミ  The (ab,) fragment was concentrated. Concentrated F (ab,) fragment 0.80ml to 100 / xl 0.01M cysteami
2 2  twenty two
ンを加え 37°Cで 2時間インキュベートした。インキュベート後の溶液を、 0.10M NaH P  And incubated at 37 ° C for 2 hours. After incubation, add 0.10M NaH P
2 2
0 /5m EDTA(pH6.0)で平衡化したゲルろ過クロマトグラフィー（Sephacyl S- 200 HRGel filtration chromatography equilibrated with 0 / 5m EDTA (pH6.0) (Sephacyl S-200 HR
4 Four
)に添加し、 Fab'断片を分離した。得られた Fab'断片を含む溶液 (分画)にフイコー ルを添加し Fab'断片を濃縮した。このようにして得られた Fab'断片溶液を SDS- PAG Eに供したところ、抗 -サブスタンス P抗体の Fab'断片の分子量は〜 55kDa、抗 -二ュ 一口キニン A抗体の Fab'断片の分子量も同じく〜 55kDaであった。 Fab，断片の濃度 は、 OD の吸光度を測定することで求めた (非特許文献 7)。  ) To separate the Fab ′ fragment. To the resulting solution (fraction) containing Fab ′ fragments, phenol was added to concentrate the Fab ′ fragments. When the Fab 'fragment solution thus obtained was subjected to SDS-PAGE, the molecular weight of the Fab' fragment of the anti-substance P antibody was ~ 55 kDa, and the molecular weight of the Fab 'fragment of the anti-two-mouth kinin A antibody. Was also ~ 55 kDa. Fab and fragment concentrations were determined by measuring the absorbance of OD (Non-patent Document 7).
280  280
[0051] (iii)サブスタンス P及びニューロキュン Aの検出（免疫検出法、図 3)  [0051] (iii) Detection of substance P and neurocun A (immunodetection method, Fig. 3)
上記（ii)で調製した Fab'断片溶液（63 μ 1/ml)の 5.0 n 1を 1.0mlの 10mM MES溶液 ( PH5.5)で希釈した。同希釈液の 20 μ ΐを、上記 (i)で作製したマレイミド基含有リポソ ームアレイの各スポットに添加し、マレイミド基含有リボソームアレイを 4°Cでー晚イン キュペートした。一晩のインキュベーションの後、マレイミドマイクロアレイの各スポット を 10mM MES (pH7.8)で洗浄し、各スポットにサブスタンス Pを含む 20 // 1の 10mM ME S (pH7.8)を添加し、マレイミド基含有リボソームアレイを室温で 30分間インキュベート した。 30分間インキュベートの後、マレイミド基含有リボソームアレイの各スポットに 20 lの lOng/mlグラミシジン溶液を添カ卩し、室温で 60分間インキュベートした。そして、リ ポソームアレイの蛍光画像 (励起波長 488nm、蛍光波長 530nm)を、フルォロイメージ ヤー 595 (Fluolmager 595) (モレキュラー ダイナミックス社、サンバレー，カリフォルニ ァ州、アメリカ合衆国）により検出した。  5.0 n 1 of the Fab ′ fragment solution (63 μ 1 / ml) prepared in (ii) above was diluted with 1.0 ml of 10 mM MES solution (PH5.5). 20 μΐ of the same dilution was added to each spot of the maleimide group-containing liposome array prepared in (i) above, and the maleimide group-containing ribosome array was incubated at 4 ° C. After overnight incubation, wash each spot on the maleimide microarray with 10 mM MES (pH 7.8), add 20 // 1 10 mM ME S (pH 7.8) containing substance P to each spot, and add maleimide groups. The containing ribosome array was incubated at room temperature for 30 minutes. After incubation for 30 minutes, 20 l of lOng / ml gramicidin solution was added to each spot of the maleimide group-containing ribosome array and incubated at room temperature for 60 minutes. The fluorescent image of the liposome array (excitation wavelength 488 nm, fluorescence wavelength 530 nm) was detected by Fluoromager 595 (Molecular Dynamics, Sun Valley, Calif., USA).
[0052] ニューロキニン Aの検出については、抗-ニューロキニン A抗体の Fab'断片溶液（0.  [0052] For detection of neurokinin A, Fab 'fragment solution of anti-neurokinin A antibody (0.
21mg/ml)の 1.5 μ Ιを 1.0mlの 10mM MES溶液 (pH5.5)で希釈し、サブスタンス Pと同じ 方法によりおこなった。  21 mg / ml) was diluted with 1.0 ml of 10 mM MES solution (pH 5.5), and the same procedure as in Substance P was performed.
実施例 10  Example 10
[0053] [マレイミド基含有リボソームアレイを用いる抗原抗体反応による被検物質の検出一 被検物質の濃度依存性にっレ、て (図 13) ]  [0053] [Detection of test substance by antigen-antibody reaction using maleimide group-containing ribosome array] (Fig. 13)
マレイミド基含有リボソームアレイを用レ、る抗原抗体反応による被検物質の検出法 の被検物質濃度依存性、即ち、実施例 9による方法で得られる蛍光強度に対する被 検物質の濃度の影響について検討した。具体的には、図 13に示すように、被検物質 サブスタンス P及びニューロキニン Aの濃度を変化させ、実施例 9の方法によりマイクロ アレイリボソームが放出する蛍光強度を測定した。図 13 (a)は、被検物質をサブスタ ンス Pのみとした場合、図 13 (b)は、被検物質を-ユーロキュン Aのみとした場合、（c) は、サブスタンス P及びニューロキ-ン Aの双方を被検物質とした場合を示す。この結 果は、被検物質の濃度は増大するにつれて蛍光強度が増したことを示している。また 、（d)は、被検物質と特異的に結合する物質として、 1)抗 -ニューロキュン A抗体の Fa b'断片、 2)抗-サブスタンス P抗体の Fab'断片、及び 3)抗体断片を使用しない場合 における、マレイミド基含有リボソーム複合体の発する蛍光強度を示す。この結果は、 抗体断片を使用しない場合よりも、抗体断片を使用した場合において蛍光強度が強 く、しかも、 -ユーロキニン A、及びサブスタンス Pともに濃度依存的に、それぞれの Fa b'断片による検出強度が増したことを示している。 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. Specifically, as shown in FIG. 13, 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, and (c) shows the substance P and neurokin. The case where both A are used as test substances is shown. This result shows that the fluorescence intensity increased as the concentration of the test substance increased. In addition, (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 The fluorescence intensity emitted by the maleimide group-containing ribosome complex when is not used. 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.
実施例 11 Example 11
[マレイミド基含有リボソームアレイを用いる抗原抗体反応による被検物質の検出ーグ ラミシジン濃度の影響にっレヽて]  [Detection of test substance by antigen-antibody reaction using maleimide group-containing ribosome array-on the effect of gramicidin concentration]
実施例 10による被検物質の検出方法では、被検物質を添加して抗原抗体反応（ 免疫反応）をおこなった後、グラミシジン溶液（5.31ηΜ、 ρΗ7·8)を添加して蛍光強度 を検出するまで 60分 (室温でインキュベート)を要する（図 3)。そこで、蛍光強度を検 出するまでの時間を短縮すベぐダラミシジン濃度を検討した。具体的には、以下の 検討をおこなった。  In the method for detecting a test substance according to Example 10, an antigen-antibody reaction (immune reaction) is performed by adding a test substance, and then a gramicidin solution (5.31 ηΜ, ρΗ7 · 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.
(i)グラミシジン濃度の影響について（図 14)  (i) Effect of gramicidin concentration (Fig. 14)
マレイミド基含有リボソームアレイ上で抗原抗体反応 (免疫反応)をおこした後に添 加するグラミシジン溶液 (20 1)の濃度をとして、 10—8 g/ml、及び 10—6 g/mlの 2通りを 採用した。即ち、上記 Γ (5)マレイミド基含有リボソームアレイ」の方法により作製した マレイミド基含有リボソームアレイに抗-ニューロキュン A抗体の Fal)'断片をカ卩えてィ ムノリボソームアレイを調製し、被検物質溶液 (ニューロキュン A)を添加した後に加え るグラミシジン溶液 (20 /z l)の濃度を 10— 8 g/ral、及ぴ 10— 6g/mlとした。そして、リポソ一 ムの蛍光強度を 15分毎に 70分まで測定した。その結果、ダラミシジン濃度を 10—6 g/ml に高めると、一定の蛍光強度になる時間が 20分に短縮され、さらに蛍光強度が増し たことが確認された。 As the concentration of the antigen-antibody reaction on the maleimide group-containing ribosome array gramicidin solution added pressure after cause (immune response) (20 1), 10- 8 g / ml, and the two types of 10- 6 g / ml Adopted. In other words, an immunoribosome array was prepared by adding the Fal) ′ fragment of anti-neurocun A antibody to the maleimide group-containing ribosome array prepared by the method of Γ (5) maleimide group-containing ribosome array described above. solution (neuro Kyung a) gramicidin Ru added after the addition of the solution (20 / zl) concentrations 10- 8 g / ral, and as及Pi 10- 6 g / ml. And liposoichi 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.
(ii)被検物質の濃度依存性にっレ、て（図 15)  (ii) Concentration dependence of the test substance (Figure 15)
上記「(i)」の結果から、添加するグラミシジン溶液の濃度を 10— 8 g/m グラミシジン 溶液を添加した後の室温でのインキュベーション時間を 15分とし、 10— 9g/m卜 10—5も I mlのニューロキニン Aについて検出を検討した。具体的には、マレイミド基含有リポソ ームアレイにニューロキニン A溶液（ΙθΛ 10— 8、 10—7、 10— 6 g/πύの各濃度）を添加後、 室温で 30分間インキュベートしてィムノリボソームアレイを調製し、これへ 20 μ 1のダラ ミシジン溶液（10— 8 g/ml)を添加して室温で 15分間インキュベートした。その結果、 - ユーロキニン Aを 10—9 g/mlの濃度力 測定できることを確認した。 From the results of the "(i)", the concentration of the added gramicidin solution 10- 8 g / m gramicidin solution to minutes 15 incubation time at room temperature after addition of, 10 9 g / m Bok 10- 5 We also investigated the detection of neurokinin A in I ml. Specifically, neurokinin A solution to the maleimide group-containing Liposomes Muarei (ΙθΛ 10- 8, 10- 7, 10- 6 g / each concentration of Py) after adding, I Takeno ribosome array was incubated for 30 minutes at room temperature was prepared and incubated for 15 minutes at room temperature by adding it to 20 mu 1 Dara Mishijin solution (10- 8 g / ml). As a result, - it was confirmed that the Yurokinin A measurable concentration force of 10- 9 g / ml.
実施例 12 Example 12
[マレイミド基含有リボソーム複合体をアレイ化（固定化)した場合と、  [When maleimide group-containing ribosome complex is arrayed (immobilized),
アレイィヒ (バルタ）しなレ、場合との比較] Aleich (Balta) Shinare, comparison with case]
実施例 9〜11では、上記「（2)マレイミド基含有リボソーム複合体の作製」により得ら れるマレイミド基含有リボソーム複合体をアビジン修飾ガラス基板にアレイ化（固定化 )したマレイミドアレイを使用している。ここで、マレイミド基含有リボソームをアレイ化し た場合とアレイ化せずにバルタで使用した場合との被検物質の検出について比較し た。  In 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.
(i)アレイ化（固定化）した場合にっレ、て (図 16)  (i) When arrayed (immobilized) (Fig. 16)
実施例 9の「 (i)マレイミド基含有リボソームアレイの作製」に基づレ、て、マレイミド基 含有リボソーム複合体 (40 1)をアビジン修飾ガラス基板に固定ィ匕し、マレイミド基含 有リボソームアレイを作製した。同アレイの各スポットへ抗 -ニューロキュン Αの Fab' 断片 (20 β 1)を添加し、室温で 60分間インキュベートしてィムノリボソームアレイを調製 した。これへ 20 μ 1(0、 10—8、 1(T7、 1(T6 g/mlの各濃度）のニューロキュン Α (被検物質） を添カ卩して室温で 30分間インキュベートした後、 20 μ 1のグラミシジン溶液（10—6 g/ml) をカロえ、 15分後ごとにグラミシジン溶液の添加後 70分まで蛍光強度を測定した。その 結果、ニューロキニン Aの濃度が増大するほど蛍光強度が増すことを確認した。 (ii)アレイィ匕 (パルク)しな！/、場合（図 17) 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 断 片 Fab 'fragment (20β1) was added to each spot of the array and incubated at room temperature for 60 minutes to prepare an immunoribosome array. This to 20 μ 1 (0, 10- 8 , 1 (T 7, 1 (T 6 g / Neuro Kyung at each concentration) of ml Alpha (the analyte) to添Ka卩After incubation for 30 minutes at room temperature , 20 mu 1 of gramicidin solution (10- 6 g / ml) Karoe, fluorescence intensity was measured up to 70 minutes after addition of gramicidine solution after each 15 minutes. As a result, as the concentration of neurokinin a is increased It was confirmed that the fluorescence intensity increased. (ii) Shin Arai (Parc)! /, Case (Figure 17)
上記「（2)マレイミド基含有リボソーム複合体の作製」により得られるマレイミド基含 有リボソーム複合体懸濁液（20 μ 1)を MES緩衝液 (ρΗ5.5)に加え lmlのマレイミド基含 有リボソーム複合体溶液とした。同溶液 (40 /x l)に抗-ニューロキュン Aの Fab，断片（ 20 μ 1)を添加した後に室温で 60分間インキュベートし、ィムノリポソーム溶液を調製し た。このィムノリボソーム溶液 (40 μ ΐ)へ 20 1 (0、 ΙθΛ 10' 10— 6 g/mlの各濃度）の二 ユーロキニン A (被検物質)を添加して室温で 30分間インキュベートした後、 20 1のグ ラミシジン（10—6 g/ml)をカ卩え、 15分後ごとにダラミシジン溶液を添加後 70分まで蛍光 強度を測定した。その結果を上記「 (i)アレイ化（固定化)した場合について」の結果と 比較すると、アレイ化した場合の方がニューロキニン Aの濃度依存性が明確であった 。アレイィヒしない場合 (バルタ法)では、より高レ、濃度でのみ検出強度に変化が認めら れた。 Add the maleimide group-containing ribosome complex suspension (20 μ 1) obtained in “(2) Preparation of maleimide group-containing ribosome complex” above to MES buffer (ρΗ5.5), 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 μ1) was added and incubated at room temperature for 60 minutes to prepare an immunoliposome solution. After incubation for 30 minutes at room temperature the I Takeno ribosome solution (40 μ ΐ) to 20 1 with the addition of two Yurokinin A (analyte) of (0, each concentration of ΙθΛ 10 '10- 6 g / ml ), 20 1 of grayed Ramishijin (10- 6 g / ml) to Ka卩E, the fluorescence intensity was measured Daramishijin solution until 70 minutes after the addition after each 15 minutes. When the result was compared with the result of “(i) When arrayed (immobilized)”, the concentration dependency of neurokinin A was clearer when arrayed. When the array was not used (Balta method), the detected intensity changed only at higher levels and concentrations.
産業上の利用可能性 Industrial applicability
本発明のリボソーム複合体、リボソームアレイ、該リボソームアレイを用レ、る被検物 質を検出する方法、及ぴ該方法のためのキットは、抗原や抗体等の生体成分、薬剤 等の検出測定を含め、様々な生物及び化学系の分析に適用が可能である。  The ribosome complex of the present invention, a ribosome array, a method for detecting a test substance using the ribosome array, and a kit for the method include detection and measurement of biological components such as antigens and antibodies, drugs, etc. It can be applied to various biological and chemical analysis.
また、本発明のマレイミド基含有リボソーム複合体、及び、マレイミド基含有リポソ一 ムアレイを用いる被検物質の検出方法は、その検出工程において、被検物質の種類 に応じて、被検物質と特異的に結合する抗体又はその Fa 断片を適宜選択するこ とによって、被検物質と特異的に結合する物質を膜表面に有するィムノリボソームァ レイを形成させることができるので、多様な被検物質を迅速、かつ簡便に検出するこ とが可能となる。  Further, 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. By appropriately selecting an antibody or its Fa fragment that binds to the protein, 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.

Claims

27 - 請求の範囲 27-Claims
[1] 以下の (i)〜 (iii)を特徴とするリボソーム複合体：  [1] A ribosome complex characterized by the following (i) to (iii):
(i)膜表面に被検物質と特異的に結合する物質と、ホスファチジルエタノールァミンの ビォチン誘導体（B - cap- PE)とを有し、  (i) having a substance that specifically binds to the test substance on the membrane surface, and a phosphatidylethanolamine biotin derivative (B-cap-PE),
(ii)リボソーム複合体の内水相は水素イオン力 Sリボソーム外の水素イオン濃度より高く なるように調整され、かつ、 pH依存性蛍光色素が内包されており、  (ii) The internal aqueous phase of the ribosome complex is adjusted to be higher than the hydrogen ion concentration outside the S ribosome, and contains a pH-dependent fluorescent dye,
(iii)膜イオンチャンネル形成物質が添加されることによって、被検物質と特異的に結 合する物質が位置する膜表面部位とは異なる膜表面部位にイオンチャンネルが形成 される、リボソーム複合体。  (iii) A ribosome complex in which an ion channel is formed at a membrane surface site different from the membrane surface site where a substance that specifically binds to a test substance is located by adding a membrane ion channel forming substance.
[2] pH依存性蛍光色素が 2 ' -7 ' -ビス- (カルボキシェチル) -6-カルボキシフルォレセィ ン（BCECF)、 8-ヒドロキシフィレン- 1,3,6-トリスルフォニックアシッド（HPTS)、セミナフ トローダフロース（SNARF)からなる群から選択される、請求項 1に記載のリボソーム複 合体。  [2] The pH-dependent fluorescent dye is 2'-7'-bis- (carboxyethyl) -6-carboxyfluorescein (BCECF), 8-hydroxyphyllene-1,3,6-trisulfonic acid 2. The ribosome complex according to claim 1, wherein the ribosome complex is selected from the group consisting of (HPTS) and seminaphtholoader flow (SNARF).
[3] 膜イオンチャンネル形成物質が、ダラミシジンである請求項 2に記載のリボソーム複 合体。  [3] The ribosome complex according to [2], wherein the membrane ion channel forming substance is daramicidine.
[4] 請求項 2に記載のリボソーム複合体が、アビジン修飾ガラス基板上にアレイ状に固 定化されたリボソームアレイ。  [4] A ribosome array in which the ribosome complex according to claim 2 is immobilized in an array on an avidin-modified glass substrate.
[5] 以下の（i)〜（iii)の工程を含んでなる、請求項 4に記載のリボソームアレイを用いる 被検物質を検出する方法： [5] The method for detecting a test substance using the ribosome array according to claim 4, comprising the following steps (i) to (iii):
(i)前記リボソームアレイに被検物質を含む試料を添加し、次レヽで  (i) Add a sample containing the test substance to the ribosome array, and
(ii)膜イオンチャンネル形成物質を添加し、  (ii) adding a membrane ion channel forming substance,
(iii) pH依存性蛍光色素に由来するリボソームの蛍光色素の強度を測定する。  (iii) Measure the intensity of the ribosomal fluorescent dye derived from the pH-dependent fluorescent dye.
[6] 膜イオンチャンネル形成物質が、ダラミシジンである請求項 5に記載の被検物質を 検出する方法。  6. The method for detecting a test substance according to claim 5, wherein the membrane ion channel forming substance is daramicidin.
[7] 請求項 4に記載のリボソームアレイと、ダラミシジンとを含む、被検物質を検出するた めのキット。  [7] A kit for detecting a test substance, comprising the ribosome array according to claim 4 and daramicidin.
[8] 膜表面にマレイミド基を有する、請求項 2に記載のリボソーム複合体。  8. The ribosome complex according to claim 2, which has a maleimide group on the membrane surface.
[9] 請求項 8に記載のリボソーム複合体が、アビジン修飾ガラス基板上にアレイ状に固 28 定化されたリボソームアレイ。 [9] The ribosome complex according to claim 8 is immobilized in an array on an avidin-modified glass substrate. 28 Stylized ribosome array.
[10] マレイミド基に対して抗体又はその Fab，断片がチオール基を介して結合した、請 求項 9に記載のリボソームアレイ。 [10] The ribosome array according to claim 9, wherein an antibody or Fab or fragment thereof is bound to a maleimide group via a thiol group.
[11] 以下の (i)〜 (iv)の工程を含んでなる、請求項 9に記載のリボソームアレイを用いた 抗原抗体反応によって被検物質を検出する方法： [11] The method for detecting a test substance by an antigen-antibody reaction using the ribosome array according to claim 9, comprising the following steps (i) to (iv):
(i)該リボソームアレイに、被検物質と特異的に結合する抗体又はその Fab'断片を 含む試料を添加して請求項 10に記載のリボソームアレイを形成させ、  (i) A sample containing an antibody that specifically binds to a test substance or a Fab ′ fragment thereof is added to the ribosome array to form the ribosome array according to claim 10;
(ii)被検物質を含む試料を添加して被検物質と抗体又はその FalD'断片とを反応さ せ、次いで、  (ii) A sample containing the test substance is added to react the test substance with the antibody or FalD 'fragment thereof, and then
(iii)膜イオンチャンネル形成物質を添加して、被検物質と特異的に結合する抗体 又はその Fab' 断片が結合するマレイミド基が位置する膜表面部ィ έとは異なる膜表面 部位にイオンチャンネルを形成させ、  (iii) A membrane ion channel-forming substance is added, and the ion channel is located on a membrane surface portion different from the membrane surface portion where the maleimide group to which the antibody or its Fab 'fragment specifically binds to the test substance is located. Form
(iv) pH依存性蛍光色素に由来するリボソームの蛍光色素の強度を測定する。  (iv) Measure the intensity of the ribosomal fluorescent dye derived from the pH-dependent fluorescent dye.
[12] 膜イオンチャンネル形成物質がダラミシジンである請求項 11に記載の被検物質を 検出する方法。 12. The method for detecting a test substance according to claim 11, wherein the membrane ion channel forming substance is daramicidin.
[13] 請求項 9に記載のリボソームアレイと、抗体又はその Fab'断片、及びダラミシジンと を含んでなる、抗原抗体反応によって被検物質を検出するためのキット。  [13] A kit for detecting a test substance by an antigen-antibody reaction, comprising the ribosome array according to claim 9, an antibody or a Fab ′ fragment thereof, and daramicidin.
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