WO2009084596A1 - 検出対象の検出方法及び定量方法 - Google Patents
検出対象の検出方法及び定量方法 Download PDFInfo
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- WO2009084596A1 WO2009084596A1 PCT/JP2008/073626 JP2008073626W WO2009084596A1 WO 2009084596 A1 WO2009084596 A1 WO 2009084596A1 JP 2008073626 W JP2008073626 W JP 2008073626W WO 2009084596 A1 WO2009084596 A1 WO 2009084596A1
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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
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
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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
- G01N33/54326—Magnetic particles
- G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
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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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Definitions
- the present invention relates to a detection method and a quantification method of a detection target.
- a latex agglutination method has been performed as a method for detecting a detection target in a specimen.
- the latex agglutination method is a method for detecting the antigen in a fluid such as a biological sample by mixing a latex carrying an antibody or a fragment thereof that specifically binds to the antigen with the fluid to determine the degree of latex aggregation.
- This is a method for detecting or quantifying an antigen by measuring (for example, see Patent Document 1).
- an antigen added as a specimen crosslinks a plurality of latex-bound antibodies to promote latex agglutination. Since the procedure is simple as described above, the antigen can be detected easily and rapidly. However, when the antigen is in a trace amount, the crosslinking is difficult to occur, so that the latex does not sufficiently aggregate. For this reason, it was difficult to detect a trace amount of antigen.
- ELISA and CLEIA enzyme substrate reactions
- a primary antibody that specifically binds to an antigen is bound to the antigen
- a secondary antibody having an enzyme is bound to the primary antibody.
- the antigen is detected or quantified by adding the enzyme substrate and measuring the degree of reaction catalyzed by the enzyme.
- this method is composed of multiple steps such as a step of incubating the sample and each reagent (ST110, ST130), a step of washing the system (ST120), and a step of measuring luminescence (ST140).
- the operation is complicated. Moreover, the time required for each stage is extremely long, and is not suitable for large-scale processing.
- the present invention has been made in view of the above circumstances, and an object of the present invention is to provide a detection method and a quantification method of a detection target capable of detecting and quantifying the detection target quickly, inexpensively, simply and with high accuracy.
- the present inventors show that aggregation of the stimulus-responsive polymer is inhibited when approaching a charged or hydrophilic substance, and that the degree of aggregation of the stimulus-responsive polymer can be detected with high accuracy by utilizing magnetic force.
- the headline and the present invention were completed. Specifically, the present invention has the following configuration.
- a method for detecting a detection target in a specimen A first binding substance in which a first substance containing a stimulus-responsive polymer and a particulate magnetic substance and a first affinity substance for the detection target are combined with the specimen, and the mixture is stimulated. After applying the condition that the responsive polymer aggregates, add magnetic force, A method comprising the step of measuring the generated magnetic field and detecting the detection object based on a degree of increase of the magnetic field after the magnetic force is applied. [2] The method according to [1], further comprising a step of further adding a first substance to the mixture before applying the magnetic force.
- the first bound substance and the specimen are mixed with a second bound substance in which a charged or hydrophilic second substance and a second affinity substance for the detection target are bound, The method according to [1] or [2], wherein the first affinity substance and the second affinity substance can simultaneously bind to the detection target at different sites of the detection target.
- a method for quantifying a detection target in a sample A first binding substance in which a first substance containing a stimulus-responsive polymer and a particulate magnetic substance and a first affinity substance for the detection target are combined with the sample, and the mixture is stimulated.
- a method comprising: measuring a generated magnetic field, and calculating an amount of the detection target in the specimen based on a correlation formula between the amount of the detection target and the magnetic field under the predetermined condition. [5] The method according to [4], further comprising the step of further adding a first substance to the mixture before applying the magnetic force. [6] The first bound substance and the analyte are mixed with a second bound substance in which a charged or hydrophilic second substance and a second affinity substance for the detection target are bound, The method according to [4] or [5], wherein the first affinity substance and the second affinity substance can simultaneously bind to the detection target at different sites of the detection target.
- the first affinity substance binds to the binding target. Then, the charge portion or hydrophilic portion to be detected approaches the stimulus-responsive polymer bound to the first affinity substance. Thereby, since the charge portion or the hydrophilic portion is arranged in the vicinity of the stimulus-responsive polymer, the aggregation of the first substance by the stimulus-responsive polymer in response to the stimulus is inhibited depending on the amount of the detection target.
- the first substance when a magnetic force is applied, the first substance exhibits ferromagnetism when it is in an aggregated state and has a large residual magnetism, but exhibits superparamagnetism when it is in a non-aggregated state. It has the characteristic of having no residual magnetism.
- the degree of increase in the magnetic field after the magnetic force is applied depends on the degree of aggregation of the first substance. Therefore, since the degree of increase in the magnetic field after the magnetic force is applied depends on the amount of the detection target, the detection target can be detected based on the degree of increase in the magnetic field. Further, the detection target can be quantified based on the correlation equation between the detection target amount and the magnetic field.
- any of the above procedures can be performed without using any special reagent, and is inexpensive and simple.
- the detection target can be detected or quantified quickly and with high accuracy.
- the magnetic field to be measured is not greatly influenced by the contaminants in the detection target, it is not always necessary to perform a preliminary procedure such as removing the contaminants before measurement, and the detection target can be detected more accurately and more quickly. Detection or quantification can be performed.
- First Embodiment Detection Method In the detection method of the present invention, first, the first binding substance and the specimen are mixed, and this mixture is subjected to conditions under which the stimulus-responsive polymer aggregates. First, the 1st combination used here is demonstrated in detail.
- the first binding substance is a combination of the first substance containing the stimulus-responsive polymer and the first affinity substance for the detection target.
- the first substance used in the present invention is a substance containing a stimulus-responsive polymer, and this stimulus-responsive polymer undergoes a structural change in response to an external stimulus and is a polymer capable of adjusting aggregation and dispersion.
- the stimulus include, but are not limited to, temperature change, light irradiation, acid or base addition (pH change), electric field change, and the like.
- a temperature-responsive polymer that can be aggregated and dispersed by temperature change can be used.
- the temperature-responsive polymer include a polymer having a lower critical solution temperature (hereinafter also referred to as LCST) and a polymer having an upper critical solution temperature (hereinafter also referred to as UCST).
- LCST lower critical solution temperature
- UCST upper critical solution temperature
- a polymer having a lower critical solution temperature with an LCST of 37 ° C. is completely dispersed in an aqueous solution having a temperature lower than the LCST, and can be agglomerated immediately when the water temperature is raised above the LCST.
- a polymer having an upper critical solution temperature with a UCST of 5 ° C. is completely dispersed in an aqueous solution having a temperature exceeding the UCST, and can be agglomerated immediately when the water temperature is lowered below the UCST.
- Examples of the polymer having a lower critical solution temperature used in the present invention include Nn-propylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-acryloylpyrrolidine, N-acryloylpiperidine, N -N substitution such as acryloylmorpholine, Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-ethylmethacrylamide, N, N-dimethylmethacrylamide, N-methacryloylpyrrolidine, N-methacryloylpiperidine, N-methacryloylmorpholine Polymer composed of (meth) acrylamide derivative; hydroxypropyl cellulose, polyvinyl alcohol partially acetylated product, polyvinyl methyl ether, (polyoxyethylene-polyoxypro Len) block copolymers, polyoxyethylene alkylamine derivatives such as polyoxyethylene laurylamine
- copolymers comprising these polymers and at least two of these monomers can also be used.
- a copolymer of N-isopropylacrylamide and Nt-butylacrylamide can also be used.
- another copolymerizable monomer may be copolymerized with the polymer within a range having a lower critical solution temperature.
- a polymer having the upper critical solution temperature used in the present invention a polymer comprising at least one monomer selected from the group consisting of acryloylglycinamide, acryloylnipecotamide, acryloylasparagineamide, acryloylglutamineamide, and the like can be used. Moreover, the copolymer which consists of these at least 2 types of monomers may be sufficient.
- These polymers include other copolymerizable monomers such as acrylamide, acetylacrylamide, biotinol acrylate, N-biotinyl-N′-methacryloyl trimethylene amide, acryloyl sarcosine amide, methacryl sarcosine amide, acryloylmethyl uracil, etc. May be copolymerized within the range having the upper critical solution temperature.
- a pH-responsive polymer that can be aggregated and dispersed by pH change can be used as the stimulus-responsive polymer.
- the pH at which the pH-responsive polymer undergoes a structural change is not particularly limited, but it can suppress a decrease in detection and quantification accuracy due to denaturation of the first bound substance, the second bound substance, and the specimen when stimulating.
- the pH is preferably 4 to 10, and more preferably 5 to 9.
- Examples of such a pH-responsive polymer include polymers containing groups such as carboxyl, phosphoric acid, sulfonyl and amino as functional groups. More specifically, (meth) acrylic acid, maleic acid, styrene sulfonic acid, 2-acrylamido-2-methylpropane sulfonic acid, phosphorylethyl (meth) acrylate, aminoethyl methacrylate, aminopropyl (meth) acrylamide, dimethylamino
- a monomer having a dissociating group such as propyl (meth) acrylamide may be polymerized, and the monomer having such a dissociating group and other vinyl monomers such as methyl (meta ) (Meth) acrylates such as acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, vinyl esters such as vinyl acetate and vinyl propionate, vinyl compounds such as styrene, vinyl chloride and N-viny
- the particulate magnetic material used here may be composed of polyhydric alcohol and magnetite.
- the polyhydric alcohol is not particularly limited as long as it is an alcohol structure having at least two hydroxyl groups in the structural unit and capable of binding to iron ions, and examples thereof include dextran, polyvinyl alcohol, mannitol, sorbitol, and cyclodextrin. It is done.
- Japanese Patent Application Laid-Open No. 2005-82538 discloses a method for producing a particulate magnetic material using dextran.
- the compound which has an epoxy like a glycidyl methacrylate polymer and forms a polyhydric alcohol structure after ring-opening can also be used.
- the fine particle magnetic material (magnetic fine particles) prepared using such a polyhydric alcohol preferably has an average particle size of 0.9 nm or more and less than 1000 nm so as to have good dispersibility.
- the average particle size is preferably 2.9 nm or more and less than 200 nm, in particular, in order to increase the detection sensitivity of the target detection target.
- the first affinity substance may be, for example, a monoclonal antibody that recognizes different antigenic determinants to be detected.
- the antibody used herein may be any type of immunoglobulin molecule, and may be an immunoglobulin molecule fragment having an antigen binding site such as Fab.
- the antibody may be a monoclonal antibody or a polyclonal antibody.
- the first binding substance is prepared by binding the first substance and the first affinity substance.
- the binding method is not particularly limited.
- substances having affinity for each other on both the first substance side for example, stimulus-responsive polymer portion
- the first affinity substance for example, first antibody
- avidin and biotin, glutathione and glutathione S-transferase are bound, and the first substance and the first affinity substance are bound via these substances.
- the biotin binding to the stimulus-responsive polymer is performed by adding biotin or the like to a polymerizable functional group such as methacryl or acryl as described in International Publication WO 01/009141. It can be carried out by forming a monomer and copolymerizing it with other monomers.
- the binding of avidin or the like to the first affinity substance can be performed according to a conventional method.
- the biotin-binding stimulus-responsive polymer and the avidin-bonded first affinity substance are mixed, the first affinity substance and the stimulus-responsive polymer are bonded through the bond between avidin and biotin.
- a monomer having a functional group such as carboxyl, amino or epoxy is copolymerized with another monomer during polymerization of the polymer, and through this functional group, an antibody affinity substance (e.g., according to methods well known in the art)
- an antibody affinity substance e.g., according to methods well known in the art
- a method of binding melon gel, protein A, protein G to a polymer can be used.
- a first bound product of the stimulus-responsive polymer and the first antibody against the antigen to be detected is produced.
- a monomer having a functional group such as carboxyl, amino, or epoxy may be copolymerized with another monomer at the time of polymerization of the polymer, and the first antibody against the antigen to be detected may be directly bonded to these functional groups according to a conventional method. Good.
- the first affinity substance and the stimulus-responsive polymer may be bound to the fine-particle magnetic substance.
- the first bound substance may be purified by subjecting the first substance to conditions where the stimulus-responsive polymer aggregates and then separating by centrifugation.
- the first bound substance is purified by a method in which a particulate magnetic substance is bound to the stimulus-responsive polymer, and further the first affinity substance is bound, and then the magnetic substance is recovered by applying a magnetic force. Also good.
- the fine magnetic substance can be bonded to the stimulus-responsive polymer via a reactive functional group, or a polymerizable unsaturated bond is introduced into the active hydrogen or polyhydric alcohol on the polyhydric alcohol in the magnetic substance.
- the graft polymerization may be carried out by a method known in the art such as graft polymerization (for example, ADV.Polym.Sci., Vol.4, p111, 1965, J. Polymer Sci., Part-A, 3, p1031). 1965).
- the stimulus-responsive polymer is the charge part or hydrophilic part of the detection target.
- the dispersion is inhibited by aggregation.
- the stimulus-responsive polymer aggregates without being inhibited from aggregation.
- the first conjugate 10 contains a stimulus-responsive polymer 11, and this stimulus-responsive polymer 11 binds to the first antibody 13 against the detection target 50 via avidin 15 and biotin 17.
- the first bonded material 10 includes a particulate magnetic material 19, and the stimulus-responsive polymer 11 is bonded to the surface of the magnetic material 19.
- the detection target 50 can approach the magnetic substance 19 via the first antibody 13, and at this time, the positively charged portion of the detection target 50 is positioned in the vicinity of the magnetic substance 19.
- the positively charged portion of the detection target 50 is positioned near the magnetic material 19.
- the present invention is not limited to this, and the negatively charged portion or the hydrophilic portion is positioned near the magnetic material 19. There may be.
- the container containing the mixed solution may be transferred to a constant temperature bath at a temperature at which the temperature-responsive polymer aggregates.
- temperature-responsive polymers There are two types of temperature-responsive polymers: a polymer having an upper critical solution temperature (hereinafter sometimes abbreviated as “UCST”) and a polymer having a lower critical solution temperature (hereinafter sometimes abbreviated as “LCST”).
- UCST upper critical solution temperature
- LCST lower critical solution temperature
- the temperature-responsive polymer can be agglomerated by transferring the container containing the mixed solution to a constant temperature bath of 37 ° C. or higher. .
- the temperature-responsive polymer can be agglomerated by moving the container containing the mixed solution to a thermostatic bath of less than 5 ° C. .
- an acid solution or an alkali solution may be added to a container containing a mixed solution.
- an acid solution or an alkali solution is added to a container containing a dispersion mixture outside the pH range where the pH-responsive polymer causes a structural change, and the pH-responsive polymer causes a structural change inside the container. Change to the range.
- an acid solution may be added to a container containing a mixed solution that is dispersed at pH 5 or higher so that the pH is 5 or lower. .
- an alkaline solution may be added to a container containing a mixed solution that is dispersed at a pH lower than 10 so that the pH becomes 10 or higher.
- the pH at which the pH-responsive polymer undergoes a structural change is not particularly limited, but is preferably pH 4 to 10, and more preferably pH 5 to 9.
- a photoresponsive polymer when a photoresponsive polymer is used, light having a wavelength capable of aggregating the polymer may be irradiated to a container containing the mixed solution.
- the preferred light for aggregation varies depending on the type and structure of the photoresponsive functional group contained in the photoresponsive polymer, but in general, ultraviolet light or visible light having a wavelength of 190 to 800 nm can be suitably used.
- the strength is preferably 0.1 to 1000 mW / cm 2 .
- it is preferable that a photoresponsive polymer is a thing which is hard to produce dispersion
- the measurement accuracy can be improved by shortening the irradiation time.
- the stimulus-responsive polymer 11 When the mixture of the first binding substance 10 and the specimen is placed under such conditions, when the detection target 50 exists, the stimulus-responsive polymer 11 is agglutinated and dispersed by the positively charged portion of the detection target 50 (see FIG. 2 (A)). On the other hand, when the detection target 50 does not exist, the stimulus-responsive polymer 11 aggregates without being inhibited from aggregation (FIG. 2B).
- the aggregation of the temperature-responsive polymer may be performed after the first bound substance and the detection target are bound, or may be performed in parallel, but the latter is preferable because the processing time can be shortened.
- the lower critical solution temperature is determined as follows. First, a sample is put into a cell of an absorptiometer and the sample is heated at a rate of 1 ° C./min. During this time, the change in transmittance at 550 nm is recorded. Here, when the transmittance when the polymer is transparently dissolved is 100% and when the transmittance when the polymer is completely aggregated is 0%, the temperature at which the transmittance is 50% is obtained as LCST.
- the upper critical solution temperature it is determined as follows. The sample is cooled at a rate of 1 ° C./min and the change in transmittance at 550 nm is recorded as in the case of the lower critical solution temperature.
- the transmittance when the polymer is transparently dissolved 100% and the transmittance when the polymer is completely aggregated is 0%
- the temperature at which the transmittance is 50% is obtained as UCST.
- FIG. 3 is a schematic configuration diagram of the inspection device 60.
- the inspection device 60 includes a magnetic force addition system 70 and a magnetic field measurement system 80.
- the magnetic force addition system 70 includes a support cylinder 71, and a sample tube 75 is inserted into the support cylinder 71 along the axial direction of the support cylinder 71. Inside the sample tube 75, the sample M pushed out from the syringe pump 77 moves.
- the sample M is a mixture of the first binding substance and the specimen.
- Helmholtz coils 73a and 73b are supported at both ends of the support cylinder 71 in the axial direction.
- the Helmholtz coils 73a and 73b are electrically connected to an AC power source 74.
- an AC current is supplied from the AC power source 74 to the Helmholtz coils 73a and 73b, an AC magnetic field is generated inside the support cylinder 71.
- the sample M pushed into the support cylinder 71 is moved out of the support cylinder 71 after the magnetic force is applied.
- the Helmholtz coils 73a and 73b are a pair of cylindrical coils having the same radius and the same number of turns arranged at intervals in the axial direction and connected in series to each other, and have a more uniform magnetic field than that produced by a single coil. Suitable when desired.
- the winding directions of the coils 73a and 73b are the same, and the generated magnetic fields have the same polarity.
- the magnetic field measurement system 80 includes a SQUID magnetic sensor 81, and this magnetic sensor 81 is placed on a heat-resistant container 83.
- a SQUID superconducting quantum interference device
- the magnetic sensor 81 is positioned below the support cylinder 71 and receives a magnetic field generated from the sample M passing through the magnetic sensor 81. At this time, when the detection target exists in the sample M, the first binding substance is dispersed, so the magnetic field does not increase significantly. However, when the detection target does not exist in the sample M, Since the first conjugate is agglomerated, it significantly increases the magnetic field.
- the magnetic sensor 81 transmits the magnetic field signal to the SQUID driving circuit 84, and the SQUID driving circuit 84 converts the magnetic field signal into a voltage signal and transmits it to the lock-in amplifier 85.
- the voltage signal received by the lock-in amplifier 85 is amplified and then output to the recorder 86.
- the detection target does not exist in the sample, and in the case where no significant increase is observed. It can be determined that the detection target exists in the sample.
- the range of “significant increase” is set in advance according to the conditions of the system used for detection.
- Detection target examples of targets that can be detected by the above detection methods include substances used for clinical diagnosis. Specifically, human immunoglobulin G, human immunoglobulin M, human immunogen contained in body fluid, urine, sputum, feces, etc.
- Globulin A human immunoglobulin E, human albumin, human fibrinogen (fibrin and their degradation products), ⁇ -fetoprotein (AFP), C-reactive protein (CRP), myoglobin, carcinoembryonic antigen, hepatitis virus antigen, human villi Gonadotropins (hCG), human placental lactogen (HPL), HIV virus antigens, allergens, bacterial toxins, bacterial antigens, enzymes, hormones (eg, human thyroid stimulating hormone (TSH), insulin, etc.), drugs and the like.
- the first affinity substance binds to the binding target. Then, the charge portion or hydrophilic portion to be detected approaches the stimulus-responsive polymer bound to the first affinity substance. Thereby, since the charge portion or the hydrophilic portion is arranged in the vicinity of the stimulus-responsive polymer, the aggregation of the first substance by the stimulus-responsive polymer in response to the stimulus is inhibited depending on the amount of the detection target.
- the first substance when a magnetic force is applied, the first substance exhibits ferromagnetism when it is in an aggregated state and has a large residual magnetism, but exhibits superparamagnetism when it is in a non-aggregated state. It has the characteristic of having no residual magnetism.
- the degree of increase in the magnetic field after the magnetic force is applied depends on the degree of aggregation of the first substance. Therefore, since the degree of increase in the magnetic field after the magnetic force is applied depends on the amount of the detection target, the detection target can be detected based on the degree of increase in the magnetic field.
- any of the above procedures can be performed without using any special reagent, and is inexpensive and simple.
- the system since the system only measures the magnetic field and does not use a reaction catalyzed by an enzyme, the detection target can be detected quickly and with high accuracy.
- the magnetic field to be measured is not greatly affected by the contaminants in the detection target, it is not always necessary to perform a preliminary procedure such as removing the contaminants before the measurement. It is possible to detect a specimen in a sample or the like.
- Second Embodiment Quantification Method In the quantification method of the present invention, first, the first binding substance and the specimen are mixed, and after the mixture is placed under a predetermined condition in which the stimulus-responsive polymer aggregates, a magnetic force is applied. Subsequently, the generated magnetic field is measured, and the amount of the detection target in the sample is calculated based on a correlation equation under a predetermined condition between the amount of the detection target and the magnetic field. Since the procedure of the first half is similar to the detection method described above, the description is omitted.
- a correlation equation between the amount of the detection target and the magnetic field under the same condition as the predetermined condition is created.
- the measurement of the amount of the detection target and the magnetic field constituting the correlation equation may be performed as long as it relates to the amount of the detection target of two or more points. However, the detection of three or more points is possible because a highly reliable correlation equation is obtained. It is preferably related to the amount of the object.
- the correlation equation between the amount of the detection target and the magnetic field is not only an expression indicating a direct correlation between the amount of the detection target and the magnetic field, but also a parameter (for example, voltage) reflecting the amount of the detection target and the magnetic field.
- the correlation equation may be used.
- the degree of increase in the magnetic field after the magnetic force is applied depends on the amount of the detection target, so that the detection can be performed by substituting the magnetic field measurement value into the correlation equation between the detection target amount and the magnetic field.
- the target can be quantified.
- this procedure is inexpensive and simple, and the detection target can be quantified quickly and with high accuracy. Further, it is not always necessary to perform a preliminary procedure of removing contaminants before measurement, and the detection target in the whole blood sample or the like can be quantified with high accuracy and more quickly.
- This embodiment is different from the first and second embodiments in that it includes a step of further adding a first substance to a mixture before applying a magnetic force.
- the mixture is subjected to a condition where the stimulus-responsive polymer aggregates in a state where the first substance is further added to the first binding substance and the specimen. Then, the first substance is further aggregated into the aggregate of the first combined substance, and the aggregate is enlarged. Thereafter, when a magnetic force is applied, the enlarged aggregates are strongly magnetized and have a stronger remanence.
- the first substance is added to the mixture of the first binding substance and the specimen.
- the present invention is not limited to this, and the order of adding the first binding substance, the specimen, and the first substance is arbitrary. It's okay.
- the first substance may be added alone, or may be added in a state of being complexed with another substance, for example, in a state of a first bound substance in which the first affinity substance is bound. .
- the following operational effects can be obtained. Since the first substance is further added to the first bound substance and the specimen, the formed aggregates are enlarged. As a result, a stronger magnetic field is generated after the magnetic force is applied, so that the difference in the amount of detection target is amplified and detected. Therefore, the detection target can be detected or quantified with higher accuracy.
- the second binding substance is a combination of a charged or hydrophilic second substance and a second affinity substance for the detection target.
- the charged second substance is, for example, a polymer compound having a charge, and is preferably a polyanion or a polycation.
- the polyanion means a substance having a plurality of anion groups
- the polycation means a substance having a plurality of cation groups.
- Examples of polyanions include nucleic acids such as DNA and RNA. These nucleic acids have the properties of polyanions due to the presence of a plurality of phosphodiester groups along the nucleic acid urn.
- polyanions include polypeptides containing many carboxyls (polypeptides composed of amino acids such as glutamic acid and aspartic acid), polyacrylic acid, polymethacrylic acid, polymers containing acrylic acid or methacrylic acid as polymerization components, carboxy Polysaccharides such as methylcellulose, hyaluronic acid, and heparin are also included.
- examples of the polycation include polylysine, polyarginine, polyornithine, polyalkylamine, polyethyleneimine, and polypropylethyleneimine.
- the number of functional groups of the polyanion (carboxyl) or polycation (amino) is preferably 25 or more.
- the hydrophilic second substance is, for example, a water-soluble polymer compound, and contains a polymer containing an ether bond such as polyethylene glycol, polypropylene glycol, polyethylene oxide, or polypropylene oxide, and an alcoholic hydroxyl group such as polyvinyl alcohol.
- a polymer containing an ether bond such as polyethylene glycol, polypropylene glycol, polyethylene oxide, or polypropylene oxide
- an alcoholic hydroxyl group such as polyvinyl alcohol.
- examples thereof include polymers, water-soluble polysaccharides such as dextran, cyclodextrin, agarose and hydroxypropylcellulose.
- These charged or hydrophilic substances may have a functional group for binding the second affinity substance in the polymer chain or at the terminal.
- the second affinity substance can bind to the detection target at the same time as the first affinity substance at a site different from the first affinity substance.
- the first affinity substance and the second affinity substance may be, for example, monoclonal antibodies that recognize different antigenic determinants to be detected.
- the second binding substance is created by directly or indirectly binding the second substance and the second affinity substance.
- substances that are compatible with each other for example, avidin and biotin, glutathione, and glutathione S-transferase
- the second substance side and the second affinity substance eg, second antibody
- the second substance and the second affinity substance are indirectly bound via these substances.
- the second substance and the second affinity substance may be bound via a functional group.
- a functional group the method of Gosh et al .: Bioconjugate Chem., 1, 71-76, 1990). Specifically, there are the following two methods.
- a mercapto group also known as a sulfhydryl group
- EMCS 6-maleimidohexanoic acid succinimide ester
- a mercapto group is introduced at the 5 ′ end of the nucleic acid in the same manner as in the first method, and N, N-1,2-phenylenedioxide, which is a homobifunctional reagent, is further introduced into this mercapto group.
- N, N-1,2-phenylenedioxide which is a homobifunctional reagent
- maleimide By reacting with maleimide, a maleimide group is introduced at the 5 ′ end of the nucleic acid, while a mercapto group is introduced into the antibody.
- these two substances are bonded through a mercapto group and a maleimide group.
- nucleic Acids Research Vol. 15 5275 (1987) and Nucleic Acids Research Vol. 16 3671 (1988) are known. Yes. These techniques can be applied to the binding of nucleic acids and antibodies.
- a mercapto group is first introduced into the 5'-terminal hydroxyl group of an oligonucleotide by reacting the oligonucleotide with cystamine, carbodiimide and 1-methylimidazole. After purifying the oligonucleotide with a mercapto group introduced, it is reduced with dithiothreitol, followed by the addition of 2,2'-dipyridyl disulfide to introduce a pyridyl group via a disulfide bond at the 5 'end of the oligonucleotide. To do.
- a mercapto group is introduced by reacting iminothalylene.
- the oligonucleotide into which the pyridyl disulfide is introduced and the protein into which the mercapto group is introduced are mixed, and the protein and the oligonucleotide are bound by specifically reacting the pyridyl group and the mercapto group.
- dithio-bis-propionic acid-N- Hydroxysuccinimide ester (abbreviation: dithio-bis-propionyl-NHS) is reacted.
- dithiothreitol is added to reduce the disulfide bond in the dithio-bis-propionyl-NHS molecule and introduce a mercapto group at the 3 'end of the oligonucleotide.
- a heterobifunctional cross-linking agent as shown in JP-A-5-48100 is used.
- a heterobicycle having a first reactive group (succinimide) that can react with a functional group (for example, an amino group) in a protein and a second reactive group (for example, maleimide) that can react with a mercapto group By reacting the protein with a functional cross-linking agent, a second reactive group is introduced into the protein to obtain a protein reagent activated in advance. The protein reagent thus obtained is covalently bound to the mercapto group of the thiolated polynucleotide.
- a second conjugate can be produced by the same operation as described above by introducing a mercapto group into these ends or the like.
- the above-mentioned second binding substance is mixed with the first binding substance and the specimen, and this mixture is subjected to conditions under which the stimulus-responsive polymer aggregates. Then, when the detection target is present, the stimulus-responsive polymer is aggregated and inhibited by the charge in the second binding substance, whereas when the detection target is not present, the stimulus-responsive polymer is not aggregated and inhibited. Aggregate. This phenomenon will be described with reference to FIGS.
- the second conjugate 20 includes a second substance 21 having a negative charge or hydrophilicity, and this second substance 21 is bound to the second antibody 23 against the detection target 50. Yes.
- the first antibody 13 and the second antibody 23 can bind to the detection target 50 simultaneously at different parts of the detection target 50.
- the stimulus-responsive polymer 11 is Aggregation is inhibited by the charge or hydrophilic portion in the second bonded product 20 and dispersed (FIG. 5A).
- the degree of inhibition of aggregation of the first conjugate 10 is greater than that of the above-described embodiment (see FIG. 2A).
- the stimulus-responsive polymer 11 aggregates without being inhibited by aggregation (FIG. 5B).
- the aggregation of the temperature-responsive polymer may be performed after the first bound substance and the second bound substance are bound to the detection target, or may be performed in parallel, but the processing time can be shortened.
- the latter is preferable in this respect.
- the former is preferable when the conditions under which the temperature-responsive polymer is aggregated are significantly different from the conditions under which the first bound substance and the second bound substance bind to the detection target.
- the following operational effects can be obtained.
- the detection target exists, the first affinity substance and the second affinity substance are bound to the binding target. Therefore, the stimulus-responsive polymer bound to the first affinity substance and the second affinity substance The bound second substance approaches.
- a charged part or a hydrophilic part is arrange
- the detection target can be quantified by measuring the degree of aggregation inhibition.
- aggregation inhibition depends on the charged part or hydrophilic part of the second substance, and the degree of dependence on the detection target species is greatly reduced. For this reason, detection or quantification of any detection target can be performed, and certainty and versatility can be improved.
- TM-LPDP temperature-responsive polymer surface-modified magnetic particle
- TM-LPDP a temperature-responsive polymer surface-modified magnetic particle
- GSH glutathione
- NHS-PEG weight average molecular weight 40000
- PBS buffer Commercially available PBS (8.1 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 2.7 mM KCl, 137 mM NaCl, pH 7.4, manufactured by Nippon Gene Co., Ltd.) with purified water 1 / 10 (V / V) diluted.
- Boric acid buffer Borate buffer manufactured by Polysciences, 100 mM boric acid, pH 8.5.
- Purified water Water purified with “Direct-Q” (trade name) manufactured by MILLIPORE.
- TM-LAm Thermo-Max LAm Amine manufactured by Magnabeat Co., Ltd.
- TM-LAm Thermo-Max LAm Amine manufactured by Magnabeat Co., Ltd.
- TM-LAm 2 mL
- borate buffer solution was added to replace the solvent, and TM-LAm was sufficiently dispersed to obtain a boric acid buffer solution containing magnetic fine particles.
- the dispersion was reheated to 42 ° C., the aggregate was collected with a magnet, the supernatant was removed, and then the temperature-responsive polymer surface-modified magnetic particles containing a protected thiol group were dispersed in a PBS buffer.
- a combined product was prepared (particle content: 0.3% by mass).
- a neodymium permanent magnet 73 (manufactured by Neomag Co., Ltd.) having dimensions of 5 mm ⁇ 9 mm ⁇ 2 mm was attached outside the optical path of a conventionally used semi-micro cell for a spectrophotometer. This cell was placed in an ultraviolet-visible spectrophotometer V-660DS (manufactured by JASCO Corporation) equipped with a cell temperature controller, and kept at 37 ° C. for 10 minutes or more.
- the above-mentioned mixture was dispensed into a cell, zero-corrected according to the instruction manual attached to the spectrophotometer, and immediately measured continuously for 1000 seconds at a bandwidth of 2.0 nm using light having a wavelength of 420 nm. The result is shown in FIG.
- FIG. 7 is a schematic diagram of a measurement system 160 used in this embodiment. Note that an AC magnetic field applying device applied to the sample is omitted.
- 151 is a slide substrate on which a sample is mounted
- 152 is a wire
- 153 is a drive motor
- 154 is a control device
- 155 is a cryostat (low temperature holding container)
- 156 is a magnetic shield box
- 157 is a low temperature holding container 155
- a SQUID (ultra high sensitivity) magnetic sensor disposed at the top and in the vicinity of the slide substrate 151
- 158 is a drive circuit
- 159 is an amplifier
- 161 is a personal computer
- 163 is an XY pen recorder.
- the measurement system 160 includes (1) a SQUID (ultra high sensitivity) magnetic sensor 157 and a drive circuit 158 for measuring magnetic signals of temperature-responsive magnetic nanoparticles, and (2) a SQUID (ultra high sensitivity) magnetic sensor 157 at a low temperature. From a cryostat (low temperature holding container) 155 for holding, (3) a substrate moving mechanism comprising a drive motor 153, a wire 152 and a control device 154, (4) a magnetic shield box 156 for blocking magnetic noise such as geomagnetism Composed. The drive circuit 158 is used for reducing noise.
- each well plate is placed on the slide substrate 151, and moved to a position immediately above the SQUID (ultra high sensitivity) magnetic sensor 157 by a moving mechanism installed outside the magnetic shield box 156, and a magnetic signal when passing through the magnetic sensor 157 is measured. And recorded.
- FIG. 8 the vertical axis in FIG. 8 is a magnetic flux as a magnetic signal.
- the excitation magnetic field was 88 ⁇ T
- the excitation frequency was 100 Hz
- the sample moving speed was 10 mm / sec
- the lift-off distance between the SQUID magnetic sensor 157 and the sample
- the measured value of the magnetic signal was greatly different depending on the content of glutathione to be detected.
- the detection target can be detected with high accuracy even when using a sample whose detection target is difficult to detect by turbidity measurement (FIG. 6), such as a chyle sample having high turbidity among serum samples. . Therefore, according to the method of this example, it has been found that the detection target can be detected with high accuracy for a wide range of whole blood samples.
- FIG. 9 shows a graph showing a correlation equation between the glutathione content and the average value of the magnetic signal (pT).
- a stimulus-responsive polymer is essential, but the polymer is not limited to a polymer, and a stimulus-responsive low molecule may be used.
- Examples of such small molecules include Japanese Patent No. 3693979, Japanese Patent No. 3916330, Japanese Patent Application Laid-Open No. 2002-85957, Japanese Patent No. 4071738, Japanese Patent No. 2869684, Japanese Patent No. 2927601, and Japanese Patent No. 3845249. Examples thereof include small molecules disclosed in Japanese Patent Laid-Open No. 2006-242597.
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Abstract
Description
具体的には、本発明は以下の構成を有する。
刺激応答性ポリマー及び微粒子状の磁性物質を含有する第1の物質と前記検出対象に対する第1の親和性物質とが結合した第1の結合物と、前記検体とを混合し、この混合物を刺激応答性ポリマーが凝集する条件下においた後、磁力を付加し、
発生する磁界を測定して、磁力の付加後における磁界の増加の程度に基づいて、前記検出対象を検出する工程を含む方法。
[2]磁力の付加の前に、第1の物質を前記混合物に更に添加する工程を更に含む[1]記載の方法。
[3]第1の結合物及び前記検体を、有電荷又は親水性の第2の物質と前記検出対象に対する第2の親和性物質とが結合した第2の結合物と混合し、
第1の親和性物質と第2の親和性物質が、前記検出対象の異なる部位において、同時に前記検出対象に結合できる[1]又は[2]記載の方法。
[4]検体中の検出対象を定量する方法であって、
刺激応答性ポリマー及び微粒子状の磁性物質を含有する第1の物質と前記検出対象に対する第1の親和性物質とが結合した第1の結合物と、前記検体とを混合し、この混合物を刺激応答性ポリマーが凝集する所定条件下においた後、磁力を付加し、
発生する磁界を測定し、前記検出対象の量と磁界との前記所定条件下における相関式に基づいて、前記検体中の検出対象の量を算出することを含む方法。
[5]磁力の付加の前に、第1の物質を前記混合物に更に添加する工程を更に含む[4]記載の方法。
[6]第1の結合物及び前記検体を、有電荷又は親水性の第2の物質と前記検出対象に対する第2の親和性物質とが結合した第2の結合物と混合し、
第1の親和性物質と第2の親和性物質が、前記検出対象の異なる部位において、同時に前記検出対象に結合できる[4]又は[5]記載の方法。
ここで、第1の物質は、磁力が付加されると、凝集状態にある場合には強磁性を示して大きな残留磁気を有するようになるが、非凝集状態にある場合には超常磁性を示して残留磁気を有しないという特性を備える。つまり、磁力の付加後における磁界の増加の程度は、第1の物質の凝集の程度に依存することになる。
よって、磁力の付加後における磁界の増加の程度が検出対象の量に依存することになるので、磁界の増加の程度に基づいて検出対象を検出できる。また、検出対象量及び磁界の相関式に基づいて検出対象を定量できる。
11 刺激応答性ポリマー
13 第1の抗体(第1の親和性物質)
15 アビジン
17 ビオチン
19 磁性物質
20 第2の結合物
21 第2の物質
23 第2の抗体(第2の親和性物質)
50 検出対象
〔混合・凝集〕
本発明の検出方法では、まず、第1の結合物及び検体を混合し、この混合物を刺激応答性ポリマーが凝集する条件下におく。まず、ここで用いる第1の結合物について詳細に説明する。
第1の結合物は、刺激応答性ポリマーを含有する第1の物質と、検出対象に対する第1の親和性物質とが結合したものである。
本発明で用いられる第1の物質は刺激応答性ポリマーを含有する物質であり、この刺激応答性ポリマーは、外的な刺激に応答して構造変化を起こし、凝集及び分散を調整できるポリマーである。刺激としては、特に限定されないが、温度変化、光の照射、酸又は塩基の添加(pHの変化)、電場変化等が挙げられる。
ここで用いる微粒子状の磁性物質は、多価アルコールとマグネタイトとで構成されてよい。この多価アルコールは、構成単位に水酸基を少なくとも2個有し且つ鉄イオンと結合可能なアルコール構造体である限りにおいて特に限定されず、例えば、デキストラン、ポリビニルアルコール、マンニトール、ソルビトール、シクロデキストリンが挙げられる。例えば特開2005-82538公報には、デキストランを用いた微粒子状の磁性物質の製造方法が開示されている。また、グリシジルメタクリレート重合体のようにエポキシを有し、開環後多価アルコール構造体を形成する化合物も使用できる。このような多価アルコールを用いて調製された微粒子状の磁性物質(磁性微粒子)は、良好な分散性を有するように、その平均粒径が0.9nm以上1000nm未満であることが好ましい。平均粒径は、特に目的とする検出対象の検出感度を高めるためには、2.9nm以上200nm未満であることが好ましい。
第1の親和性物質は、例えば、検出対象の異なる抗原決定基を認識するモノクローナル抗体であってよい。ここで用いる抗体は、いかなるタイプの免疫グロブリン分子であってもよく、Fab等の抗原結合部位を有する免疫グロブリン分子断片であってもよい。また、抗体は、モノクローナル抗体でもポリクローナル抗体でもよい。
第1の結合物は、第1の物質と第1の親和性物質とを結合することによって作製する。この結合方法は、特に限定されないが、例えば、第1の物質側(例えば刺激応答性ポリマー部分)及び第1の親和性物質(例えば、第1の抗体)側の双方に、互いに親和性の物質(例えば、アビジン及びビオチン、グルタチオン及びグルタチオンSトランスフェラーゼ)を結合させ、これら物質を介して第1の物質及び第1の親和性物質を結合させる。
磁力の付加及び磁場の測定は、常法に従って行えばよい。以下、一態様について説明するが、この態様に本発明が限定されるものではない。
以上の検出方法で検出できる対象としては、臨床診断に利用される物質が挙げられ、具体的には、体液、尿、喀痰、糞便中等に含まれるヒトイムノグロブリンG、ヒトイムノグロブリンM、ヒトイムノグロブリンA、ヒトイムノグロブリンE、ヒトアルブミン、ヒトフィブリノーゲン(フィブリン及びそれらの分解産物)、α-フェトプロテイン(AFP)、C反応性タンパク質(CRP)、ミオグロビン、ガン胎児性抗原、肝炎ウイルス抗原、ヒト絨毛性ゴナドトロピン(hCG)、ヒト胎盤性ラクトーゲン(HPL)、HIVウイルス抗原、アレルゲン、細菌毒素、細菌抗原、酵素、ホルモン(例えば、ヒト甲状腺刺激ホルモン(TSH)、インスリン等)、薬剤等が挙げられる。
本発明の第1実施形態によれば、以下のような作用効果が得られる。
ここで、第1の物質は、磁力が付加されると、凝集状態にある場合には強磁性を示して大きな残留磁気を有するようになるが、非凝集状態にある場合には超常磁性を示して残留磁気を有しないという特性を備える。つまり、磁力の付加後における磁界の増加の程度は、第1の物質の凝集の程度に依存することになる。
よって、磁力の付加後における磁界の増加の程度が検出対象の量に依存することになるので、磁界の増加の程度に基づいて検出対象を検出できる。
本発明の定量方法では、まず、第1の結合物及び検体を混合し、この混合物を刺激応答性ポリマーが凝集する所定条件下においた後、磁力を付加する。続いて、発生する磁界を測定し、検出対象の量と磁界との所定条件下における相関式に基づいて、検体中の検出対象の量を算出する。前半部分の手順は前述した検出方法と類似するので、説明を省略する。
上記所定条件と同一の条件における、検出対象の量と磁界との相関式を作成する。この相関式を構成する検出対象の量と磁界との測定は、2点以上の検出対象の量に関するものであればよいが、信頼性の高い相関式が得られる点で、3点以上の検出対象の量に関するものであることが好ましい。
磁場測定値を、作成した相関式に代入することによって、検体中の検出対象の量を算出できる。
本発明の第2実施形態によれば、以下の作用効果が得られる。
第1実施形態と同様に、磁力の付加後における磁界の増加の程度が検出対象の量に依存することになるので、検出対象量及び磁界の相関式に磁場測定値を代入することで、検出対象を定量できる。
しかも、この手順は安価且つ簡便であり、迅速且つ高精度に検出対象の定量を行うことができる。また、測定前に夾雑物を除去するといった予備手順を必ずしも行う必要がなく、高精度且つより迅速に全血試料等の中の検出対象の定量を行うことができる。
本実施形態は、磁力の付加の前に、第1の物質を混合物に更に添加する工程を含む点で第1~2実施形態とは異なる。
第1の結合物及び検体に更に第1の物質を添加したので、形成される凝集体が肥大化する。これにより、磁力付加後により強い磁界が発生するため、検出対象の量の差異が増幅されて検出される。よって、より高精度に検出対象を検出又は定量できる。
本実施形態は、第1の結合物及び検体を第2の結合物と混合する点で、前記実施形態とは異なる。以下、具体的に説明する。
第2の結合物は、有電荷又は親水性の第2の物質と検出対象に対する第2の親和性物質とが結合したものである。
有電荷の第2の物質は、例えば電荷を有する高分子化合物であり、ポリアニオン又はポリカチオンであることが好ましい。ポリアニオンとは複数のアニオン基を有する物質を意味し、ポリカチオンとは複数のカチオン基を有する物質を意味する。ポリアニオンの例として、DNA及びRNA等の核酸が挙げられる。これらの核酸は、核酸骨恪に沿って複数個のホスホジエステル基が存在することにより、ポリアニオンの性質を有する。また、ポリアニオンには、多数のカルボキシルを含むポリペプチド(グルタミン酸、アスパラギン酸等のアミノ酸からなるポリペプチド)、ポリアクリル酸、ポリメタクリル酸、及びアクリル酸やメタクリル酸を重合成分として含有するポリマー、カルボキシメチルセルロース、ヒアルロン酸、及びヘパリン等の多糖等も含まれる。一方、ポリカチオンの例としては、ポリリジン、ポリアルギニン、ポリオルニチン、ポリアルキルアミン、ポリエチレンイミンやポリプロピルエチレンイミン等が挙げられる。なお、ポリアニオン(カルボキシル)やポリカチオン(アミノ)の官能基数は、25個以上が好ましい。
第2の親和性物質は、第1の親和性物質とは異なる部位において、第1の親和性物質と同時に検出対象に結合できるものである。第1の親和性物質及び第2の親和性物質は、例えば、検出対象の異なる抗原決定基を認識するモノクローナル抗体であってよい。
第2の結合物は、第2の物質と第2の親和性物質とを直接又は間接に結合することによって作製する。特に限定されないが、例えば、第2の物質側及び第2の親和性物質(例えば、第2の抗体)側の双方に、互いに親和性の物質(例えば、アビジン及びビオチン、グルタチオン及びグルタチオンSトランスフェラーゼ)を結合させ、これら物質を介して第2の物質及び第2の親和性物質を間接的に結合させる。
検出対象が存在すると、この結合対象に第1の親和性物質及び第2の親和性物質が結合するため、第1の親和性物質に結合した刺激応答性ポリマーと、第2の親和性物質に結合した第2の物質が接近する。これにより、有電荷部分又は親水性部分が刺激応答性ポリマーの近傍に配置されるため、刺激に応答した刺激応答性ポリマーの凝集が阻害される。従って、この凝集阻害の有無を観察することで、検出対象の存否を検出できる。また、凝集阻害の程度を測定することで、検出対象を定量できる。
また、凝集阻害は、第2の物質の有電荷部分又は親水性部分に依存し、検出対象種への依存の程度が大幅に低下する。このため、あらゆる検出対象の検出又は定量を行うことができ、確実性及び汎用性を向上できる。
本発明の実施例で用いた代表的な試薬は次のとおりである。
PBSバッファー:10倍濃度の市販のPBS(8.1mM Na2HPO4、1.5mM KH2PO4、2.7mM KCl、137mM NaCl、pH7.4、ニッポンジーン(株)製)を精製水で1/10(V/V)に希釈して用いた。
ホウ酸緩衝液:ポリサイエンス社製Borate buffer、100mM ホウ酸、pH8.5。
精製水:MILLIPORE社製「Direct-Q」(商品名)で精製した水。
アミノ基結合-温度応答性ポリマー表面修飾磁性粒子として、マグナビート(社)製のTherma-Max LAm Amine(以後、TM-LAmと称する)0.4質量%を用いた。TM-LAm 2mLを2mLマイクロチューブにとり、このマイクロチューブを42℃に加熱することで、TM-LAmを凝集させ、磁石で回収した後、上清を除去した。除去後のマイクロチューブにホウ酸緩衝液2mLを加えて溶媒を置換し、TM-LAmを充分に分散させることで、磁性微粒子含有ホウ酸緩衝液を得た。
[試料の調製]
Pro MedDx LLC.(10 Commerce Way North, MA 02766)製のヒト正常血清のうち、顕著な乳び検体(Scan#:1228761)に、還元型グルタチオン(和光純薬工業(株)製)を12μg/mL及び6μg/mLとなるよう溶解したもの、及びグルタチオンを含まないものをそれぞれ試料とした。
(混合)
1.5mLチューブに、上述した第1の結合物のPBSバッファー分散液500μLをとり、0.5M EDTA溶液(pH8、ニッポンジーン社製)10μLを添加し、混合することで溶液を作製した。この溶液に上記の試料200μLを加え、4℃で12時間に亘り撹拌した。その後、チューブに、NHS-PEG又はPEGを700μL(200μM)加え、4℃で24時間に亘り撹拌した。この撹拌物400μLにPBSバッファー800μLを加え、混合物を得た。
従来汎用されている分光光度計用セミミクロセルの光路外に、寸法5mm×9mm×2mmのネオジム永久磁石73(ネオマグ(株)製)を取り付けた。このセルを、セル温度制御機が設けられた紫外可視分光光度計V-660DS(日本分光(株)製)内に設置し、37℃のもと10分間以上保持した。
図7は、本実施例で用いる計測システム160の模式図である。なお、試料に印加される交流磁界印加装置は省略されている。図7において、151は試料が搭載されるスライド基板、152はワイヤ、153は駆動モータ、154は制御装置、155はクライオスタット(低温保持容器)、156は磁気シールドボックス、157は低温保持容器155の上部であり且つスライド基板151の近傍に配置されるSQUID(超高感度)磁気センサ、158は駆動回路、159はアンプ、161はパーソナルコンピュータ、163はX-Yペンレコーダである。
Claims (6)
- 検体中の検出対象を検出する方法であって、
刺激応答性ポリマー及び微粒子状の磁性物質を含有する第1の物質と前記検出対象に対する第1の親和性物質とが結合した第1の結合物と、前記検体とを混合し、この混合物を刺激応答性ポリマーが凝集する条件下においた後、磁力を付加し、
発生する磁界を測定して、磁力の付加後における磁界の増加の程度に基づいて、前記検出対象を検出する工程を含む方法。 - 磁力の付加の前に、第1の物質を前記混合物に更に添加する工程を更に含む請求項1記載の方法。
- 第1の結合物及び前記検体を、有電荷又は親水性の第2の物質と前記検出対象に対する第2の親和性物質とが結合した第2の結合物と混合し、
第1の親和性物質と第2の親和性物質が、前記検出対象の異なる部位において、同時に前記検出対象に結合できる請求項1又は2記載の方法。 - 検体中の検出対象を定量する方法であって、
刺激応答性ポリマー及び微粒子状の磁性物質を含有する第1の物質と前記検出対象に対する第1の親和性物質とが結合した第1の結合物と、前記検体とを混合し、この混合物を刺激応答性ポリマーが凝集する所定条件下においた後、磁力を付加し、
発生する磁界を測定し、前記検出対象の量と磁界との前記所定条件下における相関式に基づいて、前記検体中の検出対象の量を算出することを含む方法。 - 磁力の付加の前に、第1の物質を前記混合物に更に添加する工程を更に含む請求項4記載の方法。
- 第1の結合物及び前記検体を、有電荷又は親水性の第2の物質と前記検出対象に対する第2の親和性物質とが結合した第2の結合物と混合し、
第1の親和性物質と第2の親和性物質が、前記検出対象の異なる部位において、同時に前記検出対象に結合できる請求項4又は5記載の方法。
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102483409A (zh) * | 2009-05-29 | 2012-05-30 | Jnc株式会社 | 检出对象的检出方法和定量方法 |
JP2012189395A (ja) * | 2011-03-09 | 2012-10-04 | Jnc Corp | 検出対象の検出方法および定量方法 |
JP2013228280A (ja) * | 2012-04-26 | 2013-11-07 | Hitachi Ltd | 交流磁場を用いた磁気的免疫検査方法及び検査装置 |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7981688B2 (en) | 2007-03-08 | 2011-07-19 | University Of Washington | Stimuli-responsive magnetic nanoparticles and related methods |
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US9080933B2 (en) | 2009-11-09 | 2015-07-14 | University Of Washington Through Its Center For Commercialization | Stimuli-responsive polymer diagnostic assay comprising magnetic nanoparticles and capture conjugates |
US20110117668A1 (en) * | 2009-11-09 | 2011-05-19 | University Of Washington Through Its Center For Commercialization | Self-powered smart diagnostic devices |
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US20170000375A1 (en) * | 2015-07-01 | 2017-01-05 | Verily Life Sciences Llc | Magnetic Nanoparticle Detection and Separation by Magnetic Relaxation Time |
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Citations (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5811575B2 (ja) | 1976-08-16 | 1983-03-03 | 帝国臓器製薬株式会社 | 抗原−抗体反応の測定法 |
JPH0548100A (ja) | 1991-08-20 | 1993-02-26 | Fujitsu Ltd | 半導体装置の製造方法 |
JP2869684B2 (ja) | 1992-03-01 | 1999-03-10 | 科学技術振興事業団 | 刺激応答性コレステロール系化合物 |
JP2927601B2 (ja) | 1992-03-01 | 1999-07-28 | 科学技術振興事業団 | クラウン環を有する刺激応答性コレステロール系化合物 |
WO2001009141A1 (fr) | 1999-07-29 | 2001-02-08 | National Institute Of Advanced Industrial Science And Technology | Derives de biotine polymerisables, polymere de la biotine et polymere reagissant a la stimulation de l'avidine |
JP2002085957A (ja) | 2000-09-18 | 2002-03-26 | Honda Motor Co Ltd | ハイドロゲル |
JP2005082538A (ja) | 2003-09-09 | 2005-03-31 | Chisso Corp | 刺激応答性ポリマー固定化磁性微粒子及びこれを用いた吸着材 |
JP3693979B2 (ja) | 2002-05-13 | 2005-09-14 | 独立行政法人科学技術振興機構 | グリコシドアミノ酸誘導体から成るヒドロゲル化剤およびヒドロゲル |
JP2006242597A (ja) | 2005-02-28 | 2006-09-14 | Fuji Photo Film Co Ltd | 磁性体ナノ粒子の凝集・分散制御方法、磁性体ナノ粒子の捕集方法及び磁性体ナノ粒子含有液の処理方法 |
JP3845249B2 (ja) | 2000-06-01 | 2006-11-15 | 独立行政法人科学技術振興機構 | 金属ポルフィリン−コレステロール誘導体から成るゲル化剤 |
JP2007071832A (ja) * | 2005-09-09 | 2007-03-22 | Shiseido Co Ltd | 修飾金粒子 |
JP3916330B2 (ja) | 1998-03-13 | 2007-05-16 | 独立行政法人科学技術振興機構 | 糖ベンジリデン誘導体から成るゲル化剤 |
JP2007244374A (ja) * | 2006-01-13 | 2007-09-27 | Japan Science & Technology Agency | 核酸応答性ゲルおよびその製造方法ならびにその利用 |
JP4071738B2 (ja) | 2004-01-07 | 2008-04-02 | 學校法人浦項工科大學校 | ヒドロゲル化剤として用いられる2’−デオキシウリジン誘導体 |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE69620898T2 (de) | 1995-09-01 | 2002-11-07 | University Of Washington, Seattle | Interaktive molekulare konjugate |
JPH11311625A (ja) | 1998-04-28 | 1999-11-09 | Nikken Seibutsu Igaku Kenkyusho:Kk | 簡便な抗体価測定方法及び抗パルボウイルス抗体検出用参照抗体 |
JP2000346844A (ja) | 1999-03-31 | 2000-12-15 | Sekisui Chem Co Ltd | 免疫測定法及び測定キット |
US6676904B1 (en) | 2000-07-12 | 2004-01-13 | Us Gov Sec Navy | Nanoporous membrane immunosensor |
DE60138195D1 (de) * | 2000-08-21 | 2009-05-14 | Nat Inst Of Advanced Ind Scien | Magnetische partikel mit kritischer lösungstemperatur am unteren bereich |
DE10254430A1 (de) * | 2002-11-21 | 2004-06-03 | Süd-Chemie AG | LCST-Polymere |
US8198020B2 (en) | 2003-08-22 | 2012-06-12 | Potentia Pharmaceuticals, Inc. | Compositions and methods for enhancing phagocytosis or phagocyte activity |
CA2656203C (en) | 2006-06-30 | 2014-07-22 | Chisso Corporation | Kit for detection/quantification of analyte, and method for detection/quantification of analyte |
JP5205807B2 (ja) * | 2007-05-17 | 2013-06-05 | 株式会社日立製作所 | 磁気信号計測装置 |
-
2008
- 2008-12-25 WO PCT/JP2008/073626 patent/WO2009084596A1/ja active Application Filing
- 2008-12-25 CA CA2710681A patent/CA2710681C/en not_active Expired - Fee Related
- 2008-12-25 US US12/810,454 patent/US9714942B2/en not_active Expired - Fee Related
- 2008-12-25 EP EP08868061.6A patent/EP2237036B1/en active Active
- 2008-12-25 CN CN200880123562.8A patent/CN101952724B/zh not_active Expired - Fee Related
- 2008-12-25 JP JP2009548069A patent/JP5193228B2/ja active Active
Patent Citations (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5811575B2 (ja) | 1976-08-16 | 1983-03-03 | 帝国臓器製薬株式会社 | 抗原−抗体反応の測定法 |
JPH0548100A (ja) | 1991-08-20 | 1993-02-26 | Fujitsu Ltd | 半導体装置の製造方法 |
JP2869684B2 (ja) | 1992-03-01 | 1999-03-10 | 科学技術振興事業団 | 刺激応答性コレステロール系化合物 |
JP2927601B2 (ja) | 1992-03-01 | 1999-07-28 | 科学技術振興事業団 | クラウン環を有する刺激応答性コレステロール系化合物 |
JP3916330B2 (ja) | 1998-03-13 | 2007-05-16 | 独立行政法人科学技術振興機構 | 糖ベンジリデン誘導体から成るゲル化剤 |
WO2001009141A1 (fr) | 1999-07-29 | 2001-02-08 | National Institute Of Advanced Industrial Science And Technology | Derives de biotine polymerisables, polymere de la biotine et polymere reagissant a la stimulation de l'avidine |
JP3845249B2 (ja) | 2000-06-01 | 2006-11-15 | 独立行政法人科学技術振興機構 | 金属ポルフィリン−コレステロール誘導体から成るゲル化剤 |
JP2002085957A (ja) | 2000-09-18 | 2002-03-26 | Honda Motor Co Ltd | ハイドロゲル |
JP3693979B2 (ja) | 2002-05-13 | 2005-09-14 | 独立行政法人科学技術振興機構 | グリコシドアミノ酸誘導体から成るヒドロゲル化剤およびヒドロゲル |
JP2005082538A (ja) | 2003-09-09 | 2005-03-31 | Chisso Corp | 刺激応答性ポリマー固定化磁性微粒子及びこれを用いた吸着材 |
JP4071738B2 (ja) | 2004-01-07 | 2008-04-02 | 學校法人浦項工科大學校 | ヒドロゲル化剤として用いられる2’−デオキシウリジン誘導体 |
JP2006242597A (ja) | 2005-02-28 | 2006-09-14 | Fuji Photo Film Co Ltd | 磁性体ナノ粒子の凝集・分散制御方法、磁性体ナノ粒子の捕集方法及び磁性体ナノ粒子含有液の処理方法 |
JP2007071832A (ja) * | 2005-09-09 | 2007-03-22 | Shiseido Co Ltd | 修飾金粒子 |
JP2007244374A (ja) * | 2006-01-13 | 2007-09-27 | Japan Science & Technology Agency | 核酸応答性ゲルおよびその製造方法ならびにその利用 |
Non-Patent Citations (7)
Title |
---|
ADV. POLYM. SCI., vol. 4, 1965, pages 111 |
GHOSH ET AL., BIOCONJUGATE CHEM., vol. 1, 1990, pages 71 - 76 |
J. POLYMER SCI., vol. 3, 1965, pages 1031 |
NUCLEIC ACID RESEARCH, vol. 16, 1988, pages 3671 |
NUCLEIC ACIDS RESEARCH, vol. 15, 1987, pages 5275 |
NUCLEIC ACIDS RESEARCH, vol. 16, 1988, pages 3671 |
See also references of EP2237036A4 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102483409A (zh) * | 2009-05-29 | 2012-05-30 | Jnc株式会社 | 检出对象的检出方法和定量方法 |
CN102483409B (zh) * | 2009-05-29 | 2016-05-18 | Jnc株式会社 | 检出对象的检出方法和定量方法 |
JP2012189395A (ja) * | 2011-03-09 | 2012-10-04 | Jnc Corp | 検出対象の検出方法および定量方法 |
JP2013228280A (ja) * | 2012-04-26 | 2013-11-07 | Hitachi Ltd | 交流磁場を用いた磁気的免疫検査方法及び検査装置 |
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CN101952724A (zh) | 2011-01-19 |
EP2237036A1 (en) | 2010-10-06 |
JP5193228B2 (ja) | 2013-05-08 |
JPWO2009084596A1 (ja) | 2011-05-19 |
US9714942B2 (en) | 2017-07-25 |
EP2237036A4 (en) | 2010-12-29 |
US20100330688A1 (en) | 2010-12-30 |
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