WO2010071045A1 - イムノアッセイ用キャピラリー及びそれを用いたキャピラリーイムノアッセイ法 - Google Patents
イムノアッセイ用キャピラリー及びそれを用いたキャピラリーイムノアッセイ法 Download PDFInfo
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- WO2010071045A1 WO2010071045A1 PCT/JP2009/070485 JP2009070485W WO2010071045A1 WO 2010071045 A1 WO2010071045 A1 WO 2010071045A1 JP 2009070485 W JP2009070485 W JP 2009070485W WO 2010071045 A1 WO2010071045 A1 WO 2010071045A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
Definitions
- the present invention relates to an immunoassay capillary for detecting a target protein with high sensitivity in only one step of sucking up a sample by capillary force by an antigen-antibody reaction and a capillary immunoassay method using the same.
- An immunoassay in which a target protein contained in a sample is measured using an antigen-antibody reaction using an antibody capable of specifically binding to the target protein. For example, after adding a sample containing a target protein to a carrier such as a microtiter plate or a magnetic particle to which a primary antibody capable of binding to the target protein is bound, and binding the target protein and the primary antibody, A sandwich immunoassay or the like in which a secondary antibody capable of binding is further bound to a target protein and a labeling substance bound to the secondary antibody is detected is widely used.
- a microchannel device in which a capillary made of glass or plastic is embedded in at least a part of the channel Japanese Patent No. 4073023 (Patent Document 1)
- Patent Document 1 Japanese Patent No. 4073023
- capillaries can be used for immune reactions.
- a capillary immunoassay in which an antigen-antibody reaction is performed in a square capillary having an inner wall of about 100 ⁇ m on the side has been developed using such a technique (for example, Henares TG et al., Analytica Chimica Acta 589 (2007)).
- p.173-179 Non-Patent Document 1).
- the presence of an antigen is detected by sandwich immunoassay between a primary antibody and a secondary antibody by sequentially circulating an antigen solution, an enzyme-bound secondary antibody, and a substrate solution through a capillary having an inner wall bound to the primary antibody. Is detected.
- the amount of antibody required can be reduced.
- An object of the present invention is to provide a capillary and a method for performing an immunoassay simply and in a short time in one step without performing the washing step necessary for the conventional capillary immunoassay.
- an insoluble layer made of oxidase bound to the first antibody is formed on the inner wall surface of the capillary, Furthermore, a hydrophilic polymer layer containing a second antibody conjugated with peroxidase is laminated thereon, Provided is an immunoassay capillary in which the first antibody and the second antibody can bind to the same antigen.
- the present invention also activates the inner wall surface of the capillary, binds the oxidase to the activated inner wall surface, binds the first antibody to the oxidase to form an insoluble layer, and further, There is also provided a method for producing a capillary for immunoassay as described above, comprising a step of laminating a layer of a hydrophilic polymer containing a second antibody bound to peroxidase.
- the present invention further includes a step of introducing into the above-described immunoassay capillary a sample to which the oxidase substrate, a dye catalyzed by the peroxidase, and a hydrogen peroxide scavenger are added.
- a capillary immunoassay method for detecting a protein of interest suspected of being present in a sample is also provided.
- the present invention also provides a micro-channel device in which a channel is formed in a branched or lattice shape inside, and the above-described capillary for immunoassay is embedded in at least a part of the channel.
- the washing step necessary for the immunoassay using the conventional capillary is not required, and the immunoassay can be performed easily and rapidly in one step.
- the reaction time can be shortened and better detection sensitivity of the target protein can be realized.
- Example 1 the fluorescence image at the time of detecting the target protein using the capillary for immunoassay of this invention and the measurement result of the fluorescence intensity obtained from it are shown.
- concentration of the target protein is shown.
- Example 2 the graph which plotted the measurement result of the fluorescence intensity at the time of detecting HIV-1 (p24) antigen using the capillary for immunoassay of this invention with respect to the density
- the capillary for immunoassay of the present invention is a capillary for detecting and measuring a target protein based on an antigen-antibody reaction.
- the “antigen-antibody reaction” refers to a binding reaction between a certain protein and an antibody that specifically recognizes and binds to the protein. Therefore, it is recognized by those skilled in the art that the “antigen”, that is, the target protein may be a protein that can specifically bind to the antibody, and does not necessarily intend a substance that shows immunogenicity by binding to the antibody. .
- the antigen-antibody reaction performed in the capillary for immunoassay of the present invention is a “sandwich method” in which the presence of a target protein is detected using two different antibodies that can specifically bind to the target protein. Therefore, the “first antibody” and the “second antibody” in the present specification can bind to the same antigen (target protein) via different recognition sites.
- the “first antibody” and the “second antibody” are conveniently referred to as “first antibody” and “second antibody” depending on whether they bind to oxidase or peroxidase, respectively. Those skilled in the art will recognize that either may be used for the “first antibody” or “second antibody”.
- the target protein that can be detected and measured using the capillary for immunoassay of the present invention is not particularly limited as long as it can produce an antibody against the protein.
- the target protein include a disease marker protein that can serve as an index for diagnosis of a disease that can be present in a sample such as blood, urine, cerebrospinal fluid, and cell lysate, and candidate compounds in the search for useful pharmaceuticals.
- cancer markers eg carcinoembryonic antigen (CEA), ⁇ -fetoprotein (AFP), human chorionic gonadotropin (hCG), basic fetoprotein (BFP), squamous cell carcinoma associated antigen (SCC antigen), BCA225 , CA15-3, CA19-9, CA50, CA54 / 61, CA72-4, CA125, CA130, CA602, pancreatic cancer-associated glycoprotein antigen (DUPAN-2), KMO-1, NCC-ST-439, sialyl Le x- i antigen (SLX), cytokeratin 19 fragment (CYFRA), tissue polypeptide antigen (TPA), immunosuppressive acidic protein (IAP), prostate specific antigen (PSA), neuron specific enolase (NSE), ferritin, elastase 1, p53 Antibody, gastrin releasing peptide precursor (ProGRP), prostatic acid phosphatase (PAP), ⁇ -seminoprotein ( ⁇ -
- Inflammatory markers such as C-reactive protein (CRP)
- arteriosclerotic disease markers such as homocysteine
- renal function markers such as cystatin C
- HIV markers p24 antigen
- hepatitis markers HBs antigen
- HBe antigen HBe antibody
- immunoglobulins such as IgG, IgA, IgE, IgD, and IgM, which are known to be examined as markers for various diseases, can also be detected and measured as target proteins.
- the capillary of the present invention is preferably composed of a material that can transmit light (fluorescence, visible light) or color generated by an immunoassay.
- materials include glass and plastics such as polystyrene, polyethylene, polypropylene, polyethylene terephthalate, polymethyl methacrylate (PMMA), polymethyl acrylate, polydimethylsiloxane (PDMS).
- the capillary of the present invention may have any shape such as a square or a circular cross section (surface perpendicular to the longitudinal direction), but it is easier to detect light or color generated by immunoassay.
- a square including a square and a rectangle is preferable, and a square is more preferable.
- one side of its outer cross section is preferably 200 ⁇ m to 2 mm, more preferably 200 ⁇ m to 1 mm.
- one side of the cross section of the lumen of the capillary is preferably 50 ⁇ m to 1 mm, more preferably 50 ⁇ m to 300 ⁇ m.
- Commercially available capillaries can be used.
- Square Flexible Fused Silica Capillary Tubing (Polymicro Technologies) is commercially available as a glass capillary whose outer cross section is a square of 300 ⁇ m ⁇ 300 ⁇ m and whose inner cross section is a square of 100 ⁇ m ⁇ 100 ⁇ m.
- the length of the capillary can be appropriately selected according to the type of target protein to be measured.
- Capillaries are usually provided with a length of about 10 cm to 1 m. After forming the insoluble layer and the hydrophilic polymer layer described below on the capillary having such a length, the capillaries are formed to a desired length. It may be cut.
- the capillaries preferably have a length of about 0.5 to 5 mm, more preferably about 0.5 to 2 mm, in view of appropriate reaction volume when used.
- an insoluble layer made of oxidase bound to the first antibody is formed on the inner wall surface of the capillary for immunoassay of the present invention.
- the term “insoluble layer” refers to a layer formed from an oxidase bound to the first antibody, in which the oxidase bound to the first antibody does not dissociate from the inner wall surface of the capillary even when contacted with water. That means.
- the insoluble layer may be formed on at least a part of the inner wall surface of the immunoassay capillary, and is preferably formed on the entire inner wall surface.
- the bond between the inner wall surface of the capillary and the oxidase is a covalent bond.
- a method for forming a covalent bond is known per se. For example, it can be performed by activating the surface of glass or plastic to form an appropriate covalent bond with the functional group of the amino acid of the protein (oxidase).
- the activation treatment of glass or plastic include functionalization with a compound having at least one functional group such as amino group, methacryl group, carboxyl group, isocyanate group, epoxy group, aldehyde group, and SH group.
- the oxidase is not particularly limited as long as it is an enzyme that oxidizes a substrate using oxygen as an electron acceptor to generate hydrogen peroxide.
- the oxidase include glucose oxidase, galactose oxidase, amino acid oxidase, glutamate oxidase, lactate oxidase, pyruvate oxidase, oxalate oxidase, alcohol oxidase, glycerol oxidase, histamine oxidase, urate oxidase, xanthine oxidase, choline oxidase, cholesterol oxidase and the like.
- An oxidase whose substrate is easily available is preferably used, and glucose oxidase, amino acid oxidase and the like are preferable.
- the first antibody is not particularly limited as long as it can specifically bind to the target protein.
- the antibody may be either a monoclonal antibody or a polyclonal antibody.
- the antibody class may be any of IgG, IgM, and the like.
- the first antibody may be an antibody fragment as long as it has the ability to bind to the target protein. Examples of antibody fragments include Fab fragments, F (ab ′) fragments, F (ab) 2 fragments, sFv fragments, and the like.
- the antibody may be derived from any mammal and bird such as human, mouse, rabbit, rat, goat, sheep, chicken and the like.
- the antibody may be one produced by recombinant genetic engineering.
- the first antibody can be obtained by a method known in the art depending on the target protein.
- the first antibody can be obtained from a hybridoma prepared by a method known per se.
- the hybridoma immunizes an appropriate mammal (eg, mouse, rat, etc.) with an antigen (target protein) mixed with an appropriate adjuvant as desired.
- antibody-producing cells such as spleen cells, lymph node cells, and B lymphocytes of the animal can be obtained by fusing with myeloma cells derived from an appropriate mammal (eg, mouse, rat, etc.).
- antibody-producing cells and myeloma cells are derived from the same animal species.
- Cell fusion can be performed by, for example, the PEG method in which antibody-producing cells and myeloma cells are fused in the presence of polyethylene glycol or the like in an appropriate medium.
- a hybridoma is selected in a selective medium such as HAT medium, and screening is performed according to a conventional method (for example, enzyme immunoassay (EIA)) for the ability to produce an antibody that recognizes the target protein of the hybridoma.
- EIA enzyme immunoassay
- a hybridoma producing an antibody capable of binding to the target protein is cloned according to a conventional method (for example, limiting dilution method), and a hybridoma producing an antibody is selected. And an antibody can be obtained from the obtained hybridoma.
- the first antibody a commercially available antibody can be used according to the target protein.
- the first antibody and the oxidase are bound directly or indirectly.
- the first antibody and the oxidase can be bound by two kinds of binding substances that specifically bind to each other and are interposed between them.
- two binding substances that specifically bind to each other include biotin and streptavidin or avidin, hapten and anti-hapten antibody, nickel and histidine tag ((His) 6 tag), glutathione and glutathione-S-transferase, etc. It is done.
- the hapten and anti-hapten antibody include dinitrophenol (DNP) and anti-DNP antibody, biotin and anti-biotin antibody, and the like.
- One of the two binding substances can be bound to oxidase and the other can be bound to the first antibody.
- the formation of the bond between the binding substance and the oxidase or the first antibody is carried out by a method known per se (for example, a reactive functional group such as N-hydroxysuccinimidyl (NHS) group, maleimide group, and the like) (Covalent bond between amino (NH 2 ) group, mercapto (SH) group, etc., or expression of recombinant protein by genetic engineering technique).
- the oxidase and the first antibody can be bound to each other by contacting the oxidase bound to the binding substance thus obtained with the first antibody bound to the paired binding substance.
- the first antibody is preferably bound to oxidase by orienting the first antibody so that the antigen recognition site of the first antibody is not blocked.
- one of the above binding substances is preferably bound to a portion other than the antigen recognition site of the first antibody, preferably an Fc fragment.
- a first antibody alignment substance can be interposed between the first antibody and the oxidase instead of the binding substance.
- the first antibody orientation substance include substances that can specifically bind to a portion other than the antigen recognition site of the antibody, preferably an Fc fragment, such as protein A and protein G. Protein A is preferred because it has less nonspecific adsorption.
- the binding between the first antibody alignment substance and the oxidase can be performed by a method known per se.
- a reactive functional group such as NHS group or maleimide group is introduced into one of the first antibody alignment substance or oxidase, and the amino group of the other protein, mercapto
- the first antibody alignment substance and the oxidase can be bound.
- the first antibody alignment substance and the oxidase can be bound by introducing an aldehyde group into the oxidase and forming a covalent bond between the aldehyde group and the amino group or SH group of the first antibody alignment substance.
- a hydrophilic polymer layer containing a second antibody bound to peroxidase is laminated on the insoluble layer.
- the hydrophilic polymer is not particularly limited as long as it is a polymer that can be solubilized by contact with water.
- examples of such hydrophilic polymers include polyethylene glycol, dextran, and hydroxyethyl cellulose.
- Polyethylene glycol having any molecular weight can be used, and examples thereof include those having a molecular weight of 2 to 4 million. Of these, those having a molecular weight of 2,000 to 100,000 are preferable, and those having a molecular weight of 6,000 to 50,000 are more preferable in terms of easy handling.
- the hydrophilic polymer layer is preferably composed of a buffer solution containing a hydrophilic polymer.
- the buffer is not particularly limited as long as it can maintain a pH that does not affect the peroxidase and the second antibody contained in the hydrophilic polymer layer, and includes a buffer that can maintain pH 6-9.
- a buffer phosphate buffered saline (PBS), Tris-HCl buffer, HEPES buffer, borate buffer, and the like can be used.
- the concentration of polyethylene glycol in the above buffer solution may be in a range where the buffer solution containing polyethylene glycol has a viscosity to be retained on the insoluble layer.
- Such kinematic viscosity is preferably 1000 to 2000 cSt.
- the concentration of polyethylene glycol in the buffer that can give such kinematic viscosity is preferably 100 to 300 g / L, although it depends on the molecular weight of polyethylene glycol.
- the peroxidase bound to the second antibody contained in the hydrophilic polymer layer is not particularly limited, and any of enzymes derived from plants, animals and microorganisms and recombinant enzymes obtained by genetic engineering techniques can be used. Also good. Horseradish peroxidase (HRP) is preferably used because it is widely used in the biochemical field.
- HRP horseradish peroxidase
- the second antibody bound to the peroxidase contained in the hydrophilic polymer layer is not particularly limited as long as it specifically binds to the same target protein to which the first antibody binds and binds via a different site of the antigen. Not. Other properties and production methods of the second antibody are the same as those described for the first antibody.
- the peroxidase and the second antibody can be bound by a method known per se.
- peroxidase is modified with a reactive functional group such as NHS group or maleimide group, and this is reacted with amino (NH 2 ) group, mercapto (SH) group or the like of the amino acid of the second antibody, so that the peroxidase is converted into the second form.
- a reactive functional group such as NHS group or maleimide group
- amino (NH 2 ) group, mercapto (SH) group or the like of the amino acid of the second antibody so that the peroxidase is converted into the second form.
- an antibody to which HRP is bound is often commercially available, and such a commercially available HRP-labeled antibody can be used as the second antibody in the present invention.
- the capillary for immunoassay of the present invention can be prepared by a method known per se. That is, the capillary for immunoassay comprises (1) activation treatment of the inner wall surface of the capillary, (2) binding of oxidase to the activated inner wall surface, and (3) binding of the first antibody to the oxidase to form an insoluble layer. (4) Furthermore, it can be obtained by a method comprising a step of laminating a layer of a hydrophilic polymer containing a second antibody bound to peroxidase on the formed insoluble layer. Between each of these steps, a washing step using an appropriate buffer may be included.
- the inner wall surface of the capillary can be activated by the compound having a functional group as described above according to a method known per se.
- the inner wall surface of the capillary is treated with a silane coupling agent, dried to introduce an amino group, and this amino group is reacted with an aldehyde group-containing compound such as glutaraldehyde.
- an aldehyde group-containing compound such as glutaraldehyde.
- Silane coupling agents include ⁇ -aminopropyltrimethoxysilane, aminoalkoxysilanes such as 3-aminopropyltriethoxysilane, epoxy silanes such as 3-glycidoxypropyltrimethoxysilane, 3-trimethoxysilylpropyl methacrylate, etc. And chloroalkylsilanes such as (chloromethyl) triethoxysilane.
- the oxidase binding efficiency in the next step can be improved by further treating the inner wall surface of the capillary activated with an aldehyde group with a polyethyleneimine and an aldehyde group-containing compound.
- the inner wall surface of the capillary can be activated by treating the inner wall surface of the capillary with a silane coupling agent and introducing the epoxy group after drying.
- the oxidase can be bound via a covalent bond.
- the aqueous solution containing oxidase preferably has a pH of 8-9. In order to obtain such a pH, an appropriate buffer may be used.
- the concentration of the oxidase in the aqueous solution is preferably 3 to 5 mg / mL.
- the aqueous solution containing oxidase is preferably reacted at a temperature of 25 ° C. to 40 ° C. for about 30 minutes to 2 hours.
- Binding of first antibody to oxidase By introducing an aqueous solution containing the first antibody into the capillary obtained by (2) and reacting for an appropriate time, the first antibody is bound to oxidase, An insoluble layer can be formed.
- a modifier for binding the first antibody alignment material to the oxidase is introduced into the capillary, then the first antibody alignment material is introduced, and then the first antibody is further introduced.
- the first antibody can be bound to the oxidase.
- the aqueous solution containing the first antibody preferably has a pH of 7 to 7.4. In order to obtain such a pH, an appropriate buffer may be used.
- the concentration of the first antibody in the aqueous solution is preferably 10 to 100 ⁇ g / mL.
- the aqueous solution containing the first antibody is preferably reacted at a temperature of 25 ° C. to 40 ° C. for about 30 minutes to 2 hours after binding of protein A to oxidase. .
- bovine serum albumin BSA
- skim milk or the like can be introduced and coated to suppress nonspecific protein adsorption.
- hydrophilic polymer layer Subsequent to the step (3), a hydrophilic polymer in which a second antibody bound to peroxidase is dissolved is continuously introduced into the capillary to thereby obtain the hydrophilic polymer.
- This layer can be laminated on the insoluble layer formed on the inner wall surface of the capillary.
- the hydrophilic polymer may be dissolved in the appropriate buffer so as to have the appropriate viscosity.
- concentration of the second antibody bound to peroxidase in the hydrophilic polymer is preferably 10 to 100 ⁇ g / mL.
- the rate at which the hydrophilic polymer is continuously introduced into the capillary is preferably several tens of ⁇ l / min. Within this range, the hydrophilic polymer layer can be satisfactorily formed.
- the thus prepared capillary for immunoassay can be cooled ( ⁇ 20 to 10 ° C.) and stored until use.
- the present invention also provides a capillary immunoassay method comprising detecting a target protein suspected of being present in a sample using the above-described capillary for immunoassay.
- the sample is not particularly limited as long as it is a sample suspected of having the target protein.
- Examples of the sample include liquids derived from biological samples such as blood, urine, and cerebrospinal fluid, cell lysates, and aqueous solutions.
- the immunoassay method of the present invention can be performed by introducing a sample into the above-described capillary for immunoassay.
- the sample can be introduced by capillary action by bringing the sample into contact with one end of the capillary (sample inlet).
- the sample introduced into the capillary is usually about 0.05 to 0.1 ⁇ l depending on the size of the capillary.
- the introduced sample is preferably kept in the capillary at a temperature of 5 to 1 hour, more preferably 10 to 30 minutes, 20 to 40 ° C., more preferably 25 to 38 ° C. for reaction.
- the sample is added with the oxidase substrate, a dye that is catalyzed by peroxidase and becomes detectable, and a hydrogen peroxide scavenger.
- the substrate for the oxidase is appropriately selected depending on the type of oxidase used.
- the oxidase substrate is usually added to the sample so as to have a concentration of 10 to 50 mM, although it depends on the type.
- the dye that can be detected by being catalyzed by peroxidase may be a fluorescent dye, a coloring dye, or a chemiluminescent dye.
- coloring dyes include 3,3′-diaminobenzidine (DAB), 3,3 ′, 5,5′-tetramethylbenzidine (TMB), 3-amino-9-ethylcarbazole (AEC), methyl green, N- Examples thereof include a combination of an ethyl-m-toluidine derivative (for example, N-hydroxysulfopropyl derivative (TOOS)) and 4-aminoantipyrine (4-AA).
- the fluorescent dye include 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red), p-hydroxyphenylacetic acid, thiamine and the like.
- chemiluminescent dyes include luminol.
- the dye that can be detected by being catalyzed by peroxidase is preferably added to the sample so as to be usually 0.1 to 1 mM, although it depends on the type.
- the hydrogen peroxide scavenger is not particularly limited as long as it is a substance capable of decomposing hydrogen peroxide into water by reacting with hydrogen peroxide. Ascorbic acid, glutathione, histidine, uric acid, transition metal ions (Fe 2+ , Ti 3+ , Co 2+, etc.) and sodium hypochlorite (NaOCl).
- the hydrogen peroxide scavenger is preferably added to the sample so as to be 0.1 to 100 mM, more preferably 1 to 10 mM, depending on the type.
- the capillary immunoassay method of the present invention will be described.
- the first antibody and the secondary antibody form a complex near the inner wall surface of the capillary via the target protein.
- the oxidase substrate added to the sample is oxidized by the oxidase to which the first antibody is bound to generate hydrogen peroxide.
- This hydrogen peroxide, together with peroxidase bound to the second antibody bound to the first antibody via the target protein, converts the dye added to the sample into a detectable form. Therefore, the presence of the antigen can be detected in one step.
- the first antibody and the second antibody cannot be bound to each other, so that they are separated from each other in the capillary lumen.
- the oxidase present on the inner wall surface generates hydrogen peroxide from the substrate added to the sample, but this hydrogen peroxide is used for the peroxidase that is bound to the second antibody present at a distance. Without being trapped by the hydrogen peroxide scavenger added in the sample, it is decomposed into water. Thus, the dye added to the sample is not converted to a detectable form.
- the detection device can be a fluorescence microscope, a fluorometer, a CCD camera, a photodiode, a photomultiplier tube with a suitable excitation light source when the dye is a fluorescent dye.
- the detection device may be a spectrophotometer, a CCD camera, a photodiode, or a photomultiplier tube when the above dye is a coloring dye.
- the detection device can be a CCD camera, a photodiode, a photomultiplier tube when the dye is a chemiluminescent dye. These detection devices are preferably connected to an analysis device having software capable of digitizing the detected signal from the dye.
- the capillary immunoassay method of the present invention is based on two types of reactions, an antigen-antibody reaction and an enzyme reaction. Since the antigen-antibody reaction is a reaction between proteins having a very large molecular weight, it is usually a very slow reaction (usually several hours). However, since the capillary immunoassay method of the present invention is a reaction carried out in a capillary having a very small capacity (usually several tens to several hundreds of nl), the distance over which the molecules diffuse is short, and the antigen-antibody reaction is comparative. Proceeds quickly (about 10 to 20 minutes). On the other hand, an enzyme reaction is usually a reaction that proceeds very rapidly (usually several tens of seconds to several minutes).
- the enzyme reaction proceeds relatively slowly (about 10 to 20 minutes). Therefore, it becomes possible to keep the reaction rate balance of these two kinds of reactions within a preferable range, and the immunoassay method of the present invention is a single step of sucking up the sample solution by capillary force and is shorter than the conventional immunoassay. In the reaction time, it is possible to detect a target protein at a low concentration of about 1 ng / ml using a small amount of sample.
- the present invention also provides a micro-channel device in which a penetrating channel is formed in a branched or lattice shape inside, and the above-described immunoassay capillary is embedded in at least a part of the channel.
- the shape, material, manufacturing method, and the like of the channel of the microchannel device are the same as those described in JP-A-2005-140681.
- FIG. 3 shows an example of a preferable embodiment of the microchannel device of the present invention.
- FIG. 3 shows a microchannel device 1 in which a channel having a width of 300 ⁇ m is formed.
- a part of the microchannel device includes an immunoassay capillary 2 having a first antibody and a second antibody that can bind to AFP, an immunoassay capillary 3 having a first antibody and a second antibody that can bind to CEA, and hCG.
- An immunoassay capillary 4 having a first antibody and a second antibody that can be bound is embedded.
- a sample can be introduced into each immunoassay capillary by capillary force by dropping the sample into the sample dropping port 5 of the microchannel device and sucking the sample from the sample suction port 6. Then, AFP, CEA and hCG can be detected in one step by measuring the fluorescence intensity at a certain point (for example, the dotted line) of each capillary for immunoassay.
- Protein A (0.5 mg / mL; Sigma P6031) aqueous solution (phosphate buffer pH 9) is introduced into the capillary and reacted at room temperature for 40 minutes to bind protein A to glucose oxidase modified with aldehyde groups. It was. 6). The inside of the capillary was washed with pure water. 7. A 0.1 M NaBH 4 aqueous solution was introduced into the capillary and allowed to react at room temperature for 40 minutes. 8). The capillary was washed with pure water and then washed with Tris-HCl buffer pH 7.4. 9.
- anti-human IgG (Funakoshi, E80-104 Human ELISA quantitation kit) diluted 100 times with Tris-HCl buffer pH 7.4 was introduced into a capillary and reacted at room temperature for 40 minutes to obtain a protein.
- the first antibody was bound to A. 10.
- the capillary was washed with Tris buffer pH 7.4.
- a tris buffer pH 7.4 solution of 1% bovine serum albumin (BSA) was introduced into the capillary and allowed to stand at room temperature for 2 hours or longer to perform nonspecific adsorption suppression coating on the inner wall surface of the capillary. . 12
- BSA bovine serum albumin
- Tris-HCl in which horseradish peroxidase (HRP) -labeled anti-human IgG (Funakoshi, E80-104 Human ELISA quantitation kit) was dissolved in polyethylene glycol (PEG; molecular weight 20000) at 300 mg / mL. Dilute 100-fold with buffer pH 7.4.
- HRP horseradish peroxidase
- PEG polyethylene glycol
- the solution is aspirated at a rate of 30 ⁇ L / min, and the second antibody bound to peroxidase is deposited on the insoluble layer consisting of glucose oxidase bound to the first antibody.
- a layer of polyethylene glycol containing was laminated. 2.
- the immunoassay capillary can be stored at -10 ° C if not used immediately.
- Example 1 The following components were dissolved in Tris-HCl buffer pH 9 to prepare a sample solution. Dyes catalyzed by peroxidase and made detectable: 0.8 mM Amplex Red (Invitrogen A36006); Oxidase substrate: 28 mM glucose; Hydrogen peroxide scavenger: 10 mM ascorbic acid; Target protein: human IgG (0, 0.1, 1, 10, 1000 and 5000 ng / mL; Funakoshi, E80-104 Human ELISA quantitation kit).
- Dyes catalyzed by peroxidase and made detectable 0.8 mM Amplex Red (Invitrogen A36006); Oxidase substrate: 28 mM glucose; Hydrogen peroxide scavenger: 10 mM ascorbic acid; Target protein: human IgG (0, 0.1, 1, 10, 1000 and 5000 ng / mL; Funakoshi, E80-104 Human ELISA quantitation kit).
- the sample solution (0.05 ⁇ l) was introduced into the capillary by capillary force. With the sample solution introduced into the capillary, the sample solution was allowed to stand at room temperature for 30 minutes.
- FIG. 1B shows a fluorescence signal measured at a point about 2.5 mm from the sample solution inlet of the capillary (dotted line portion in FIG. 1A).
- FIG. 2 is a graph in which the fluorescence intensity ( ⁇ F) at each antigen concentration is plotted against the logarithm of the concentration of the target protein, using a signal with zero antigen concentration as a background signal. From these results, it can be seen that by measuring using the capillary for immunoassay of the present invention, a result of increasing the fluorescence intensity almost in proportion to the concentration of the target protein was obtained.
- the target protein can be detected and quantified by immunoassay using a small amount of sample solution easily in a single step, in a relatively short time, without requiring a washing step. Was confirmed.
- Production Example 2 Preparation of capillary for immunoassay for HIV antigen measurement Surface modification of square capillary The inner surface of the square capillary was modified by the same procedure as in Production Example 1A.
- Anti-HIV-1 (p24) antibody (0.5 mg / mL; Anogen MO-140002D2) aqueous solution (phosphate buffer pH 9) was introduced into the capillary, reacted at room temperature for 40 minutes, and modified with an aldehyde group Anti-HIV-1 (p24) antibody was conjugated to glucose oxidase. 6). The inside of the capillary was washed with pure water. 7. A 0.1 M NaBH 4 aqueous solution was introduced into the capillary and allowed to react at room temperature for 40 minutes. 8). The capillary was washed with Tris buffer pH 7.4. 9.
- a tris buffer pH7.4 solution of 1% bovine serum albumin (BSA) was introduced into the capillary and allowed to stand at room temperature for 2 hours or longer to perform nonspecific adsorption suppression coating on the inner wall surface of the capillary. . 12
- the capillary was washed with Tris buffer pH 7.4.
- HRP horseradish peroxidase
- p24 horseradish peroxidase
- Tris-HCl in which polyethylene glycol (PEG; molecular weight 20000) was dissolved to 300 mg / mL Dilute 100-fold with buffer pH 7.4.
- PEG polyethylene glycol
- the solution is aspirated at a rate of 30 ⁇ L / min, and the second antibody bound to peroxidase is deposited on the insoluble layer composed of glucose oxidase bound to the first antibody.
- a layer of polyethylene glycol containing was laminated. 2.
- the immunoassay capillary can be stored at -10 ° C if not used immediately.
- Example 2 The following components were dissolved in Tris-HCl buffer pH 9 to prepare a sample solution. Dyes catalyzed by peroxidase and made detectable: 0.8 mM Amplex Red (Invitrogen A36006); Oxidase substrate: 28 mM glucose; Hydrogen peroxide scavenger: 10 mM ascorbic acid; Target protein: HIV-1 p24 Antigen (0, 0.1, 1, 10, 100 and 1000 ng / mL; Biodesign International, R18301).
- Dyes catalyzed by peroxidase and made detectable 0.8 mM Amplex Red (Invitrogen A36006); Oxidase substrate: 28 mM glucose; Hydrogen peroxide scavenger: 10 mM ascorbic acid; Target protein: HIV-1 p24 Antigen (0, 0.1, 1, 10, 100 and 1000 ng / mL; Biodesign International, R18301).
- the sample solution (0.05 ⁇ l) was introduced into the capillary by capillary force. With the sample solution introduced into the capillary, the sample solution was allowed to stand at room temperature for 30 minutes.
- FIG. 4 shows a graph in which the fluorescence intensity ( ⁇ F) at each antigen concentration is plotted against the logarithm of the concentration of the target protein, using a signal with zero antigen concentration as a background signal.
- ⁇ F fluorescence intensity
- HIV antigens can be detected and quantified by immunoassay using a small amount of sample solution in a simple manner, in a relatively short time, without requiring a washing step. Was confirmed.
- the present invention relates to Japanese Patent Application No. 2008-324059 filed on Dec. 19, 2008, which is incorporated herein by reference in its entirety. It is.
Abstract
Description
このような技術を利用して、近年、内壁が一辺100μm程度の角型キャピラリーの中で、抗原-抗体反応を行うキャピラリーイムノアッセイが開発されている(例えばHenares T.G.ら、Analytica Chimica Acta 589 (2007) p.173-179(非特許文献1))。この技術は、内壁面に1次抗体を結合させたキャピラリーに、抗原溶液、酵素結合2次抗体及び基質溶液を順次流通させることにより、1次抗体と2次抗体とのサンドイッチイムノアッセイにより抗原の存在を検出するものである。
このような少量の測定系を用いることにより、必要とされる抗体の量を低減することが可能となった。
さらにその上に、ペルオキシダーゼと結合した第2抗体を含む親水性高分子の層が積層されてなり、
前記第1抗体と前記第2抗体とが、同じ抗原に対して結合可能である
イムノアッセイ用キャピラリーを提供する。
本発明は、また、キャピラリーの内壁面を活性化処理し、活性化処理された内壁面へオキシダーゼを結合させ、前記オキシダーゼに第1抗体を結合させて不溶性層を形成し、さらにその上に、ペルオキシダーゼと結合した第2抗体を含む親水性高分子の層を積層する工程を含む、上記のイムノアッセイ用キャピラリーの製造方法も提供する。
本発明は、また、内部に分枝状又は格子状に流路が形成され、上記のイムノアッセイ用キャピラリーが前記流路の少なくとも一部に埋設されてなる微小流路デバイスも提供する。
本発明のイムノアッセイ用キャピラリーは、抗原-抗体反応に基づいて目的タンパク質を検出及び測定するためのキャピラリーである。本明細書において、「抗原-抗体反応」とは、あるタンパク質と、該タンパク質を認識して特異的に結合する抗体との結合反応のことをいう。よって、「抗原」、すなわち目的タンパク質は、抗体に特異的に結合し得るタンパク質であればよく、抗体に結合することにより免疫原性を示す物質を必ずしも意図しないことが、当業者により認識される。
疾患マーカータンパク質としては、癌マーカー(例えば癌胎児抗原(CEA)、α-フェトプロテイン(AFP)、ヒト絨毛性ゴナドトロピン(hCG)、塩基性フェトプロテイン(BFP)、扁平上皮癌関連抗原(SCC抗原)、BCA225、CA15-3、CA19-9、CA50、CA54/61、CA72-4、CA125、CA130、CA602、膵癌関連糖タンパク抗原(DUPAN-2)、KMO-1、NCC-ST-439、シアリルLex-i抗原(SLX)、サイトケラチン19フラグメント(CYFRA)、組織ポリペプチド抗原(TPA)、免疫抑制酸性タンパク質(IAP)、前立腺特異抗原(PSA)、神経特異エノラーゼ(NSE)、フェリチン、エラスターゼ1、p53抗体、ガストリン放出ペプチド前駆体(ProGRP)、前立腺酸性ホスファターゼ(PAP)、γ-セミノプロテイン(γ-Sm)、Dpyr、ポリアミン、BJPなど)、糖尿病マーカー(例えばインスリンなど)、肥満マーカー(例えばレプチン、アディポネクチンなど)、炎症マーカー(例えばC反応性タンパク質(CRP)など)、動脈硬化性疾患マーカー(例えばホモシステインなど)、腎機能マーカー(例えばシスタチンCなど)、HIVマーカー(p24抗原)、肝炎マーカー(HBs抗原、HBs抗体、HBe抗原、HBe抗体)などが挙げられる。また、種々の疾患のマーカーとして検査対象であることが知られているIgG、IgA、IgE、IgD、IgMなどの免疫グロブリンを目的タンパク質として検出及び測定することもできる。
本発明のキャピラリーは、イムノアッセイにより発生する光(蛍光、可視光)又は色を透過し得る材料で構成されるものが好ましい。そのような材料としては、ガラス、及びポリスチレン、ポリエチレン、ポリプロピレン、ポリエチレンテレフタレート、ポリメタクリル酸メチル(PMMA)、ポリアクリル酸メチル、ポリジメチルシロキサン(PDMS)などのプラスチックが挙げられる。
キャピラリーが方形である場合、その外形の横断面の一辺は、200μm~2mmが好ましく、より好ましくは200μm~1mmである。また、キャピラリーの内腔の横断面の一辺は、50μm~1mmが好ましく、より好ましくは50μm~300μmである。
上記のキャピラリーは、市販されたものを用いることができる。例えば、外形の横断面が300μm×300μmの正方形であり、内腔の横断面が100μm×100μmの正方形であるガラス製キャピラリーとして、Square Flexible Fused Silica Capillary Tubing(Polymicro Technologies)が市販されている。
本発明のイムノアッセイ用キャピラリーの内壁面には、第1抗体と結合したオキシダーゼからなる不溶性層が形成されている。
本明細書において「不溶性層」とは、第1抗体と結合したオキシダーゼが、水と接触してもキャピラリーの内壁面との結合が解離しない、第1抗体と結合したオキシダーゼから形成される層のことをいう。
上記の不溶性層は、イムノアッセイ用キャピラリーの内壁面の少なくとも一部分に形成されていればよく、好ましくは該内壁面の全体に形成される。
抗体は、ヒト、マウス、ウサギ、ラット、ヤギ、ヒツジ、ニワトリなどのいずれの哺乳類及び鳥類に由来するものであってもよい。また、抗体は、組換え遺伝子工学により産生されたものであってもよい。
細胞融合は、例えば適切な培地中で抗体産生細胞と骨髄腫細胞とをポリエチレングリコールなどの存在下で融合させるPEG法などにより行うことができる。細胞融合後、HAT培地などの選択培地でハイブリドーマを選択し、ハイブリドーマの目的タンパク質を認識する抗体を産生する能力について、常法(例えば酵素免疫測定法(EIA))に従ってスクリーニングを行う。次いで、目的タンパク質に結合可能な抗体を産生するハイブリドーマを常法(例えば限界希釈法)に従ってクローニングし、抗体を産生するハイブリドーマが選択される。そして、得られたハイブリドーマから、抗体を得ることができる。
本発明のキャピラリーは、上記の不溶性層の上に、ペルオキシダーゼと結合した第2抗体を含む親水性高分子の層が積層されている。
上記の親水性高分子は、水と接触して可溶化し得る高分子であれば特に限定されない。このような親水性高分子としては、ポリエチレングリコール、デキストラン、ヒドロキシエチルセルロースなどが挙げられる。
ポリエチレングリコールは、いずれの分子量のものであっても用いることができ、200~400万の分子量のものが挙げられる。中でも、取り扱いが容易である点で、2000~10万の分子量のものが好ましく、6000~5万の分子量のものがより好ましい。
特に、HRPが結合した抗体は、市販で入手可能である場合が多く、そのような市販のHRP標識抗体を本発明において第2抗体として用いることができる。
本発明のイムノアッセイ用キャピラリーは、それ自体公知の方法を用いて作製できる。すなわち、イムノアッセイ用キャピラリーは、(1)キャピラリーの内壁面を活性化処理し、(2)活性化処理された内壁面へオキシダーゼを結合させ、(3)オキシダーゼに第1抗体を結合させて不溶性層を形成し、(4)さらに、形成された不溶性層の上に、ペルオキシダーゼと結合した第2抗体を含む親水性高分子の層を積層する工程を含む方法により得ることができる。これらの各工程の間には、適切な緩衝液を用いる洗浄工程を含み得る。
キャピラリーの内壁面は、それ自体公知の方法に従って、上記のような官能基を有する化合物により活性化処理できる。
キャピラリー内壁面をアルデヒド基で活性化処理する場合、シランカップリング剤でキャピラリーの内壁面を処理し、乾燥させてアミノ基を導入し、このアミノ基をグルタルアルデヒドなどのアルデヒド基含有化合物と反応させてアルデヒド基を導入することにより、キャピラリーの内壁面を活性化処理できる。
シランカップリング剤としては、γ-アミノプロピルトリメトキシシラン、3-アミノプロピルトリエトキシシランなどのアミノアルコキシシラン、3-グリシドキシプロピルトリメトキシシランなどのエポキシシラン、3-トリメトキシシリルプロピルメタクリレートなどのメタクリルシラン、(クロロメチル)トリエトキシシランなどのクロロアルキルシランなどが挙げられる。
なお、アルデヒド基で活性化処理されたキャピラリーの内壁面を、ポリエチレンイミン及びアルデヒド基含有化合物でさらに処理することにより、次の工程におけるオキシダーゼの結合効率を向上させることができる。
また、キャピラリー内壁面をエポキシ基で活性化処理する場合、シランカップリング剤でキャピラリーの内壁面を処理し、乾燥させてエポキシ基を導入することにより、キャピラリーの内壁面を活性化処理できる。
(1)の工程により活性化処理されたキャピラリーに、オキシダーゼを含む水溶液を導入し、適切な時間反応させることにより、キャピラリーの内壁面に共有結合を介してオキシダーゼを結合させることができる。
オキシダーゼを含む水溶液は、pHが8~9であることが好ましい。このようなpHとするために、適切な緩衝剤を用い得る。
水溶液中のオキシダーゼの濃度は、好ましくは3~5mg/mLである。
オキシダーゼを含む水溶液は、30分~2時間程度、25℃~40℃の温度で反応させることが好ましい。
(2)により得られたキャピラリーに、第1抗体を含む水溶液を導入して、適切な時間反応させることにより、オキシダーゼに第1抗体を結合させて、不溶性層を形成することができる。上記の第1抗体配向物質を用いる場合、第1抗体配向物質をオキシダーゼと結合させるための修飾剤をキャピラリーに導入し、次いで第1抗体配向物質を導入し、さらに第1抗体を導入することにより、オキシダーゼに第1抗体を結合させ得る。
第1抗体を含む水溶液は、pHが7~7.4であることが好ましい。このようなpHとするために、適切な緩衝剤を用い得る。
水溶液中の第1抗体の濃度は、好ましくは10~100μg/mLである。
第1抗体を含む水溶液は、第1抗体配向物質としてプロテインAを用いる場合、プロテインAをオキシダーゼと結合させた後に、30分~2時間程度、25℃~40℃の温度で反応させることが好ましい。
(3)の工程に続いて、キャピラリーに、ペルオキシダーゼと結合した第2抗体を溶解した親水性高分子を連続的に導入することにより、親水性高分子の層を、キャピラリーの内壁面に形成された不溶性層の上に積層できる。
親水性高分子は、上記の適切な粘度を有するように上記の適切な緩衝液に溶解したものであってもよい。
親水性高分子中のペルオキシダーゼと結合した第2抗体の濃度は、好ましくは10~100μg/mLである。
親水性高分子をキャピラリーに連続的に導入する速度としては、数十μl/分であることが好ましく、この範囲であれば、親水性高分子の層が良好に形成できる。
本発明は、上記のイムノアッセイ用キャピラリーを用いて、試料中での存在が疑われる目的タンパク質を検出することを含むキャピラリーイムノアッセイ法も提供する。
上記の試料は、目的タンパク質の存在が疑われる試料であれば特に限定されない。試料としては、血液、尿、髄液などの生体試料に由来する液体、細胞破砕液、水溶液などが挙げられる。
キャピラリーに導入される試料は、キャピラリーの大きさにもよるが、通常、0.05~0.1μl程度である。
オキシダーゼの基質は、用いられるオキシダーゼの種類により適宜選択される。
オキシダーゼの基質は、その種類にもよるが、通常、10~50mMの濃度になるように試料に添加されるのが好ましい。
過酸化水素捕捉剤は、その種類にもよるが0.1~100mM、より好ましくは1~10mMになるように試料中に添加されるのが好ましい。
これらの検出装置は、色素からの検出されたシグナルを数値化できるソフトウェアを有する解析装置に接続されていることが好ましい。
一方、酵素反応は、通常、非常に迅速に進行する反応である(通常、数十秒~数分)。しかし、本発明のキャピラリーイムノアッセイ法においては、試料に過酸化水素捕捉剤が適切な量で添加されているので、酵素反応が比較的ゆっくりと進行する(約10~20分程度)。
よって、これらの2種類の反応の反応速度の均衡を好ましい範囲に保つことが可能になり、本発明のイムノアッセイ法は、毛細管力で試料溶液を吸い上げるだけの1工程で、従来のイムノアッセイよりも短い反応時間で、少量の試料を用いて、1ng/ml程度までの低い濃度の目的タンパク質の検出を可能にできる。
本発明は、内部に分枝状又は格子状に貫通状流路が形成され、上記のイムノアッセイ用キャピラリーが該流路の少なくとも一部に埋設されてなる微小流路デバイスも提供する。
微小流路デバイスの流路の形状、材料、製造方法などについては、特開2005-140681号公報に記載されることと同様である。
A.角型キャピラリーの表面修飾
1.内腔の一辺が100μmで、外形の一辺が300μmの角型キャピラリー(Polymicro technologies社製、WWP100375)に1 N NaOHを導入し、30分間静置した。
2.十分な量の純水を通して洗浄後、アセトンを導入して水分を除去した。
3.キャピラリーを70℃のオーブンに入れて、30分乾燥させた。
4.30%(v/v)の3-アミノプロピルトリエトキシシラン(APTES)のアセトン溶液をキャピラリーに導入して、室温にて40分静置した。
5.アセトンで洗浄後、70℃のオーブンに入れて、60分乾燥させた。
6.2.5% (v/v)グルタルアルデヒド水溶液を導入して、室温にて40分間反応させた。
7.純水でキャピラリー内を洗浄した。
8.5%ポリエチレンイミン(v/v)水溶液(リン酸緩衝液 pH9)を導入し、室温にて40分間反応させ、アミノ基を多数有するポリエチレンイミンを、キャピラリーの内壁面に固定した。
9.リン酸緩衝液pH9でキャピラリー内を洗浄した。
10.2.5% (v/v)グルタルアルデヒド水溶液をキャピラリーに導入し、40分間反応させた。
11.リン酸緩衝液pH9でキャピラリー内を洗浄した。
1.グルコースオキシダーゼ(5 mg/mL;Sigma G7141)水溶液(リン酸緩衝液pH9)を、上記のA.のようにして作製した、内壁面がグルタルアルデヒドで修飾されたキャピラリーに導入し、室温にて40分間反応させた。
2.リン酸緩衝液pH9でキャピラリー内を洗浄した。
3.2.5% (v/v)グルタルアルデヒド水溶液を導入し、室温にて40分間反応させた。
4.リン酸緩衝液pH9でキャピラリー内を洗浄した。
5.プロテインA(0.5 mg/mL;Sigma P6031)水溶液(リン酸緩衝液 pH9)を、キャピラリー内に導入し、室温にて40分間反応させて、アルデヒド基で修飾されたグルコースオキシダーゼにプロテインAを結合させた。
6.キャピラリー内を純水で洗浄した。
7.0.1 M NaBH4水溶液をキャピラリー内に導入し、室温にて40分間反応させた。
8.キャピラリー内を純水で洗浄後、Tris-HCl緩衝液pH7.4で洗浄した。
9.第1抗体として、Tris-HCl緩衝液pH7.4で100倍に希釈した抗ヒトIgG(フナコシ, E80-104 Human ELISA quantitation kit)をキャピラリー内に導入し、室温にて40分間反応させて、プロテインAに第1抗体を結合させた。
10.トリス緩衝液pH7.4でキャピラリー内を洗浄した。
11.1%ウシ血清アルブミン(BSA)のトリス緩衝液pH7.4溶液をキャピラリー内に導入し、室温にて2時間以上静置することにより、キャピラリーの内壁面の非特異吸着抑制コーティングを行った。
12.トリス緩衝液pH7.4でキャピラリー内を洗浄した。
1.第2抗体として、セイヨウワサビペルオキシダーゼ(HRP)標識抗ヒトIgG(フナコシ, E80-104 Human ELISA quantitation kit)を、300mg/mL になるようにポリエチレングリコール(PEG;分子量20000)を溶解させたTris-HCl緩衝液pH7.4で100倍に希釈した。この溶液を、キャピラリーに20μL/分の速度で導入した直後に、30μL/分の速度で吸引し、第1抗体と結合したグルコースオキシダーゼからなる不溶性層の上に、ペルオキシダーゼと結合した第2抗体を含むポリエチレングリコールの層を積層した。
2.このイムノアッセイ用キャピラリーは、すぐに使用しない場合は-10℃で保存できる。
Tris-HCl緩衝液pH9に、以下の成分を溶解して、試料溶液を調製した。
ペルオキシダーゼにより触媒されて検出可能になる色素:
0.8 mM Amplex Red (Invitrogen社 A36006);
オキシダーゼの基質:28 mMグルコース;
過酸化水素捕捉剤:10 mMアスコルビン酸;
目的タンパク質:ヒトIgG (0、0.1、1、10、1000及び5000 ng/mL;フナコシ, E80-104 Human ELISA quantitation kit)。
その後、そのデジタル画像を、Image-J(米国国立衛生研究所(NIH)が頒布するフリーソフトウェア:参照URL http://rsbweb.nih.gov/ij/)などのソフトウェアで解析し、蛍光信号を定量化した。
さらに、抗原濃度ゼロの信号をバックグラウンド信号とした、各抗原濃度における蛍光強度(ΔF)を、目的タンパク質の濃度の対数に対してプロットしたグラフを、図2に示す。
これらの結果から、本発明のイムノアッセイ用キャピラリーを用いて測定することにより、目的タンパク質の濃度にほぼ比例して、蛍光強度が増加する結果が得られたことがわかる。
A.角型キャピラリーの表面修飾
製造例1のAと同様の手順で角型キャピラリーの内表面を修飾した。
1.グルコースオキシダーゼ(5 mg/mL;Sigma G7141)水溶液(リン酸緩衝液pH9)を、上記のA.のようにして作製した、内壁面がグルタルアルデヒドで修飾されたキャピラリーに導入し、室温にて40分間反応させた。
2.リン酸緩衝液pH9でキャピラリー内を洗浄した。
3.2.5% (v/v)グルタルアルデヒド水溶液を導入し、室温にて40分間反応させた。
4.リン酸緩衝液pH9でキャピラリー内を洗浄した。
5.抗HIV-1(p24)抗体(0.5 mg/mL;Anogen MO-140002D2)水溶液(リン酸緩衝液 pH9)を、キャピラリー内に導入し、室温にて40分間反応させて、アルデヒド基で修飾されたグルコースオキシダーゼに抗HIV-1(p24)抗体を結合させた。
6.キャピラリー内を純水で洗浄した。
7.0.1 M NaBH4水溶液をキャピラリー内に導入し、室温にて40分間反応させた。
8.トリス緩衝液pH7.4でキャピラリー内を洗浄した。
9.1%ウシ血清アルブミン(BSA)のトリス緩衝液pH7.4溶液をキャピラリー内に導入し、室温にて2時間以上静置することにより、キャピラリーの内壁面の非特異吸着抑制コーティングを行った。
12.トリス緩衝液pH7.4でキャピラリー内を洗浄した。
1.第2抗体として、セイヨウワサビペルオキシダーゼ(HRP)標識抗HIV-1(p24)抗体(Anogen MO-140002T2HRP)を、300mg/mL になるようにポリエチレングリコール(PEG;分子量20000)を溶解させたTris-HCl緩衝液pH7.4で100倍に希釈した。この溶液を、キャピラリーに20μL/分の速度で導入した直後に、30μL/分の速度で吸引し、第1抗体と結合したグルコースオキシダーゼからなる不溶性層の上に、ペルオキシダーゼと結合した第2抗体を含むポリエチレングリコールの層を積層した。
2.このイムノアッセイ用キャピラリーは、すぐに使用しない場合は-10℃で保存できる。
Tris-HCl緩衝液pH9に、以下の成分を溶解して、試料溶液を調製した。
ペルオキシダーゼにより触媒されて検出可能になる色素:
0.8 mM Amplex Red (Invitrogen社 A36006);
オキシダーゼの基質:28 mMグルコース;
過酸化水素捕捉剤:10 mMアスコルビン酸;
目的タンパク質:HIV-1 p24 Antigen (0、0.1、1、10、100及び1000 ng/mL;Biodesign International, R18301)。
その後、そのデジタル画像を、Image-J(米国国立衛生研究所(NIH)が頒布するフリーソフトウェア:参照URL http://rsbweb.nih.gov/ij/)などのソフトウェアで解析し、蛍光信号を定量化した。
これらの結果から、本発明のイムノアッセイ用キャピラリーを用いて測定することにより、HIV抗原の濃度に応じて、蛍光強度が増加する結果が得られたことがわかる。また、本発明のキャピラリーは、キャピラリー内に導入されている抗体に対する抗原を特異的に測定でき、該抗体と特異的には反応しない抗原に対しては、用量応答反応性がないこともわかる。
2、3、4 本発明のイムノアッセイ用キャピラリー
5 試料滴下口
6 試料吸引口
Claims (11)
- キャピラリーの内壁面に、第1抗体と結合したオキシダーゼからなる不溶性層が形成され、
さらにその上に、ペルオキシダーゼと結合した第2抗体を含む親水性高分子の層が積層されてなり、
前記第1抗体と前記第2抗体とが、同じ抗原に対して結合可能である
イムノアッセイ用キャピラリー。 - 前記親水性高分子が、分子量2000~10万のポリエチレングリコールである請求項1に記載のイムノアッセイ用キャピラリー。
- 前記オキシダーゼがグルコースオキシダーゼであり、前記ペルオキシダーゼがセイヨウワサビペルオキシダーゼである請求項1に記載のイムノアッセイ用キャピラリー。
- キャピラリーの長手方向に垂直な横断面が、正方形及び矩形を含む方形であり、その内腔の一辺が50μm~1mmの長さである請求項1に記載のイムノアッセイ用キャピラリー。
- キャピラリーの内壁面を活性化処理し、
活性化処理された内壁面へオキシダーゼを結合させ、
前記オキシダーゼに第1抗体を結合させて不溶性層を形成し、
さらにその上に、ペルオキシダーゼと結合した第2抗体を含む親水性高分子の層を積層する
工程を含む、請求項1に記載のイムノアッセイ用キャピラリーの製造方法。 - キャピラリーの内壁面の活性化処理が、アミノ基、メタクリル基、カルボキシル基、イソシアネート基、エポキシ基、アルデヒド基、SH基から選択される少なくとも1つの官能基を有する化合物による活性化である請求項5に記載の方法。
- 請求項1に記載のイムノアッセイ用キャピラリーに、前記オキシダーゼの基質と、前記ペルオキシダーゼに触媒されて検出可能になる色素と、過酸化水素捕捉剤とが添加された試料を導入する工程を含む、試料中での存在が疑われる目的タンパク質を検出するためのキャピラリーイムノアッセイ法。
- 前記過酸化水素捕捉剤が、アスコルビン酸、グルタチオン、ヒスチジン、尿酸、遷移金属イオン、次亜塩素酸ソーダ(NaOCl)から選択される請求項7に記載のキャピラリーイムノアッセイ法。
- 前記目的タンパク質が、癌マーカー、糖尿病マーカー、肥満マーカー、炎症マーカー、動脈硬化性疾患マーカー、腎機能マーカー、HIVマーカー及び肝炎マーカーから選択される疾患マーカーである請求項7に記載のキャピラリーイムノアッセイ法。
- 前記基質、色素及び過酸化水素捕捉剤が、それぞれ、10~50mM、0.1~1mM及び0.1~100mMの濃度になるように試料に添加される請求項7に記載のキャピラリーイムノアッセイ法。
- 内部に分枝状又は格子状に流路が形成され、請求項1に記載のイムノアッセイ用キャピラリーが前記流路の少なくとも一部に埋設されてなる微小流路デバイス。
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US8563260B2 (en) | 2013-10-22 |
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