JPWO2006088192A1 - Analytical carrier and measuring method using the same - Google Patents

Abstract

The present invention relates to a carrier for measuring a measurement object in a sample and a capture molecule-binding carrier, a measurement method using the carrier and the capture molecule-binding carrier, a measurement object measurement reagent comprising the carrier or the capture molecule-binding carrier, A method for producing the carrier or the capture molecule-binding carrier is provided. A support having an aggregate region of two or more polymers having different stereoregularities on the surface, a carrier containing a molecular binding substance fixed to the aggregate region of the support, and a capture molecule for binding the molecule to the carrier The present invention relates to a capture molecule-binding carrier bound via a substance, a reagent containing the carrier or the capture molecule-binding carrier, and a method for measuring a measurement object using the same.

Description

  The present invention relates to a carrier for measuring a measurement object and a capture molecule-binding carrier, a measurement reagent containing the carrier or the capture molecule-binding carrier, a method for producing the carrier and the capture molecule-binding carrier, the carrier and the capture molecule-binding carrier. The present invention relates to a measuring method of a measuring object using the above.

  In recent years, methods for analyzing various substances expressed in the living body have been developed and used for diagnosing diseases and drug discovery. In addition, in order to understand the relationship between genetic information and the physiological functions of cells, analysis of in vivo substances is necessary, and a simple and accurate measurement method for trace amounts of substances is required.

  As a method for measuring a substance, a measurement method using a specific reaction such as an antigen-antibody reaction and a measurement method using an enzyme reaction are known. Specifically, a substance such as an antibody that specifically binds to a measurement object such as an antigen is fixed to a support, and the measurement object and a substance that specifically binds to the measurement object are bound and bound. The measurement object can be measured by various methods, for example, a method for measuring the amount of enzyme bound by binding an antibody-enzyme complex (ELISA method), a method for measuring a change in dielectric constant (surface plasmon resonance method), and a change in weight. It is measured by a measurement method (quartz crystal method) and a method of measuring the amount of generated heat (a micro differential calorimetry method) (see, for example, Patent Documents 1 and 2). Further, as another aspect, a substance such as an enzyme that specifically binds the measurement target is fixed to the support, the measurement target is bound, and a reaction product generated by the reaction between the measurement target and the enzyme or This can be done by detecting a change in the amount of substance caused by the conversion, for example, an optical change.

  In these measurement methods, it is necessary to bind a substance that specifically binds to a measurement target such as an antibody or an enzyme to the support. Conventionally, for example, antibody binding methods include a method in which protein A or G is directly immobilized on a support and an antibody is bound to the immobilized protein A or G, and the support and protein A or G are immobilized with a crosslinking agent. Methods for binding antibodies to A or G have been reported (see, for example, Patent Documents 2 and 3). As an enzyme binding method, for example, a method of directly fixing an enzyme to a support and a method of fixing the support and the enzyme with a crosslinking agent are generally known.

  However, a method for improving measurement accuracy and sensitivity, a method for improving convenience of measurement, a method for improving storage stability of a support on which protein A, G or enzyme is immobilized, protein A, G or enzyme is immobilized. There is a need for a simpler method for preparing a support. Prior art documents related to the present invention include the following. Patent Document 1: Japanese Patent Application Laid-Open No. 2004-37209, Patent Document 2: Japanese Patent Application Laid-Open No. 2004-117026, and Patent Document 3: Japanese Patent Application Laid-Open No. 2002-340766.

  The present invention has been made in view of the above circumstances, a carrier and a capture molecule-binding carrier for measuring a measurement object in a sample, a measurement method using the carrier and the capture molecule-binding carrier, the carrier or the capture molecule An object of the present invention is to provide a reagent for measuring a measurement object containing a binding carrier, a method for producing the carrier or the capturing molecule binding carrier.

  As a result of diligent investigations to solve these problems, the present inventors formed an aggregate of two or more polymers having different stereoregularities on the surface of the support, and a molecular binding substance on the surface of the aggregate And it discovered that said problem could be solved by fixing a capture molecule further if necessary, and completed this invention. In this polymer aggregate, the molecular binding substance can be easily fixed at a high density and without a cross-linking agent, and the denaturation of the molecular binding substance is suppressed even when stored for a long period of time. Decrease can be suppressed, and accurate and reproducible measurement can be performed.

The present invention relates to the following 1 to 23.
1. Measures an object to be measured in a sample, including a support having an aggregate region of two or more polymers having different stereoregularities on the surface, and a molecular binding substance fixed to the aggregate region of the support. Carrier to do.
2. 2. The carrier according to 1 above, wherein the aggregate comprises an aggregate that forms a sterically complementary association.
3. 3. The carrier according to 1 or 2 above, wherein the two or more polymers having stereoregularity include a syndiotactic polymer and an isotactic polymer.
4). 4. The carrier according to any one of 1 to 3 above, wherein the aggregate is an aggregate containing a helical structure or a van der Waals structure.
5. 5. The carrier according to any one of 1 to 4 above, wherein at least one of the polymers is polymethacrylate.
6). 6. The carrier according to 5 above, wherein the polymethacrylate is polymethyl methacrylate, polyethyl methacrylate, polyisopropyl methacrylate or poly n-propyl methacrylate.
7). 7. The carrier according to any one of 1 to 6 above, wherein the support is in the form of a plate, chip, bead, fiber or membrane.
8). 8. The carrier according to any one of 1 to 7 above, wherein the carrier is subjected to a blocking treatment with a blocking agent.
9. 9. The carrier according to any one of 1 to 8 above, wherein the molecular binding substance is an enzyme.
10. 9. The carrier according to any one of 1 to 8 above, wherein the molecule binding substance is a capture molecule binding protein.
11. 11. The carrier according to 10 above, wherein the capture molecule binding protein is protein A, protein G, or protein L.
12 12. A capture molecule-binding carrier for measuring a measurement object in a sample, wherein a capture molecule is bound to the carrier according to 10 or 11 via the capture molecule-binding protein.
13. A reagent for measuring a measurement object, comprising the carrier according to any one of 1 to 12 above.
14 A measurement object measurement kit comprising the carrier according to any one of 1 to 12 above.
15. A kit for measuring an object to be measured in a sample, comprising a reagent comprising a support having on its surface an aggregate region of two or more polymers having different stereoregularities, and a reagent containing a substance for molecular binding.
16. 16. The measurement kit according to 15 above, further comprising a reagent containing a capture molecule.
17. The step of bringing the carrier described in 10 or 11 above into contact with a capture molecule to form a capture molecule-bound carrier, contacting the capture molecule-bound carrier with the measurement object, and supporting the measurement object via the capture molecule A method for measuring a measurement object in a sample, comprising a step of binding to a carrier and a step of measuring the measurement object bound to the carrier.
18. The step of bringing the capture molecule into contact with the measurement object to form the measurement object-bound capture molecule, the carrier according to 10 or 11 above and the measurement object-bound capture molecule are brought into contact with each other and the measurement object is bound to the carrier A method for measuring a measurement object in a sample, comprising the steps of: measuring the measurement object bound to the carrier.
19. The step of bringing the carrier according to 9 above into contact with the measurement target, causing the measurement target to react with the enzyme immobilized on the carrier, and the step of measuring the amount of change in the substance that increases or decreases due to the reaction A method for measuring a measurement object in a sample.
20. The method includes the steps of contacting the carrier and the measurement object according to 12 above, and binding the measurement object to the carrier via a capture molecule, and measuring the measurement object bound to the carrier. Measuring method of the measurement object.
21. A carrier for measuring an object to be measured in a sample, characterized in that a substance for molecular binding is immobilized on an aggregate region of a support having two or more polymer aggregate regions having different stereoregularities on the surface. Manufacturing method.
22. Immobilizing a molecular binding substance on an aggregate area of a support having two or more polymer aggregate areas having different stereoregularities on the surface, and binding a capture molecule to the molecular binding substance. A method for producing a capture molecule-binding carrier for measuring an object to be measured in a sample.
23. Different stereoregulations used as a carrier for immobilizing a molecular binding substance on an aggregate region of two or more polymers having different stereoregularities on a support surface and measuring a measurement object in a sample A polymer-attached support having an aggregate region of two or more polymers having properties on the surface.

  According to the present invention, a carrier having improved storage stability, a capture molecule-bound carrier and a measurement reagent, a method for measuring a measurement object in a measurement sample with improved measurement accuracy or sensitivity, and a simple carrier and capture molecule-bound carrier A method is provided.

FIG. 1 is a schematic diagram showing one embodiment of a carrier formation reaction, a capture molecule binding carrier formation reaction, and a measurement object binding reaction when a capture molecule binding protein is used as a molecule binding substance. FIG. 2 is a conceptual diagram showing one mode of a carrier formation reaction when an enzyme is used as a molecule binding substance and a reaction between the enzyme on the carrier and a measurement object (substrate). FIG. 3 shows a calibration curve representing the relationship between the amount of HSA antigen binding and the concentration of HSA antigen. In the figure, ◯ represents the HSA antigen binding amount of Example 1, □ represents Comparative Example 6, and Δ represents Comparative Example 9. FIG. 4 is a graph showing the measurement results of the fixed amount of β-galactosidase using a QCM chip. In the figure, ◯ represents the amount of β-galactosidase bound in Example 4 and ● represents Comparative Example 12. FIG. 5 is a graph showing the activity evaluation of β-galactosidase immobilized on a maxisorp plate. In the figure, the bold line ◯ represents the result for Example 5, the thin line □ represents the result for Comparative Example 13, and the broken line Δ represents the result for Comparative Example 14. FIG. 6 is a graph showing the activity evaluation of β-galactosidase immobilized on a maxi soap plate for 7 days. In the figure, the thick line ◯ represents the results for Example 5, the thin line □ represents the results for Comparative Example 13, and the wavy line Δ represents the results for Comparative Example 14.

  The support used in the present invention is not particularly limited as long as it can be used as a solid phase, and is a material used for measurement, diagnosis, inspection, etc. by a method using specific binding such as antigen-antibody reaction, enzyme reaction, etc. Can be used, and is appropriately selected depending on the method for measuring the solid-phased measurement object. For example, when measurement is performed by a spectroscopic technique, a support material is selected according to the light transmittance used.

  Examples of the support material include, but are not limited to, crystal, polymer, glass, ceramics, and the like. Examples of the quartz include various crystal forms used as a crystal resonator. Depending on the application, various known cuts may be applied. Examples of the polymer of the support material include polymethyl methacrylate, polyethyl methacrylate, poly n-propyl methacrylate, polyisopropyl methacrylate, poly n-butyl methacrylate, polyisobutyl methacrylate, polymethacrylate such as polymethacrylic acid, such as polymethyl acrylate, Examples include polyacrylates such as polyethyl acrylate, poly n-butyl acrylate, polyisobutyl acrylate, and polyacrylic acid, for example, polycarbonate such as bisphenol A polycarbonate, polyacrylonitrile, polystyrene, polyethylene, polypropylene, and ABS. Examples of the glass include soda lime glass, coning glass, and quartz glass.

  The support may be appropriately provided with a metal thin film (for example, a gold thin film), an electrode, a pre-coating or the like according to the purpose of measuring the measurement object.

  The support is, for example, a measurement plate provided for immunoassay such as 96 or 384 well plate, for example, beads such as Luminex beads, for example, fine particles used for immunoturbidimetry, magnetic particles, fine particles such as gold colloid, for example Membranes used for immunochromatography, Western blotting, etc., for example, quartz crystal chips, chips used for surface plasmon resonance, chips having multiple antibody binding regions on one flat plate, flowed on a support by microfabrication technology A chip such as a biochip constructed by integrating a channel, a reaction field and a detection part, for example, a biostrand of Protein System Science, a hollow fiber or an array of a plurality of such bundles (JP 2001-133453), glucose Molded body made of hollow fiber used for sensors 4), fibers such as gel-filled hollow fibers (Japanese Patent Laid-Open No. 2003-079377), cellulosic molded products (Japanese National Publication No. 10-511144), and the like.

  In the present invention, the aggregate of two or more polymers having different stereoregularity is an aggregate of two or more molecules of polymer, and all or part of the polymers of two or more molecules are different in stereoregularity. Refers to an assembly that has The polymer may be a block in a block copolymer. Moreover, the aggregate of two or more polymers having different stereoregularity may be formed by associating two or more blocks having different stereoregularity in one molecule. That is, in the polymer having two or more blocks having stereoregularity and other blocks having different stereoregularity, the association includes an intramolecular association formed by the association of these two or more blocks. Mergers are also included.

  The monomer which is a raw material of the polymer used for the polymer aggregate can be selected from known ones. For example, monomers include alkenes such as propylene, 1-butene, 1-pentene, 3-methyl-1-butene, 4-methyl-1-pentene, 4,4-dimethyl-1-pentene, styrene, 2-methyl Styrenes such as styrene, 3-methylstyrene, 2,4-dimethylstyrene, 2,5-dimethylstyrene, 3,4-dimethylstyrene, 3,5-dimethylstyrene, butadienes such as butadiene and isoprene, methacrylic acid, Methyl methacrylate, butyl acrylate, N-isopropylacrylamide, N, N-diisopropylacrylamide, acrylic acid derivatives such as acrylonitrile or methacrylic acid derivatives, vinyl ethers such as butyl vinyl ether, vinyl halides such as vinyl chloride, propylene oxide, Styrene Oki Oxiranes such as de, but can be appropriately selected from such aldehydes such as acetaldehyde, but are not limited thereto. These monomers may be used alone or in combination.

  The stereoregularity of the polymer can be controlled by appropriately selecting experimental conditions such as catalyst, temperature, solvent, etc., using the above monomer as a raw material. Any known method can be used as the polymerization method. For example, anionic polymerization, coordination anionic polymerization, radical polymerization and the like can be mentioned. It is also possible to separate a polymer having stereoregularity by separating a polymer obtained by polymerizing the above monomer as a raw material by an appropriate known means.

  In this specification, the stereoregularity includes regularity regarding the arrangement of substituents with respect to the polymer main chain, and regularity obtained by using only one of the enantiomers as a monomer. In the former regularity, a polymer having a substituent on either side of the polymer main chain is called an isotactic polymer (hereinafter abbreviated as it-polymer), and the substituent sandwiches the polymer main chain. The polymer that is regularly exchanged in the above is called a syndiotactic polymer (hereinafter abbreviated as st-polymer). A polymer in which both properties are irregularly mixed is called an atactic polymer.

  Examples of monomers that give it-polymers include, for example, propylene, 1-butene, 1-pentene, 3-methyl-1-butene, 4-methyl-1-pentene, 4,4-dimethyl-1-pentene, styrene, Methacrylic acid derivatives such as 2-methylstyrene, 3-methylstyrene, 2,4-dimethylstyrene, 2,5-dimethylstyrene, 3,4-dimethylstyrene, 3,5-dimethylstyrene, butadiene, methacrylic acid, and methyl methacrylate , Butyl acrylate, butyl vinyl ether, N, N-diisopropylacrylamide, propylene oxide, acetaldehyde, and the like.

  Examples of the monomer that gives the st-polymer include, but are not limited to, methacrylic acid derivatives such as butadiene and methyl methacrylate, N-isopropylacrylamide, and the like.

  The polymer having stereoregularity in the present invention is not limited to the polymer having ideal stereoregularity, and may have a somewhat disordered structure in the molecule, and a polymer in which a certain regularity continues for a certain length. It may be.

  Regarding stereoregularity, the it-polymer isotacticity (% mm; triplet display) is generally 70 or more, preferably 80 or more, more preferably 90 or more, and the st-polymer syndiotacticity ( % Rr; triplet display) is generally 70 or more, preferably 80 or more, more preferably 90 or more.

  The number average molecular weight (Mn) of the polymer is generally 1,000 or more, preferably 5,000 or more, and more preferably 10,000 or more. The weight average molecular weight (Mw) of the polymer is generally 1,000 or more, preferably 5,000 or more, and more preferably 10,000 or more. The ratio of the weight average molecular weight to the number average molecular weight (Mw / Mn) is 1 or more, generally 5 or less, preferably 2 or less, more preferably 1.5 or less.

  As described above, the polymer having stereoregularity in the present invention includes a polymer obtained by polymerizing one kind of enantiomer as a polymerization unit. Examples of the combination of the polymer and the polymer having different stereoregularity from the polymer include a combination of poly D-lactic acid and poly L-lactic acid.

  In the present invention, among the aggregates, an aggregate in which the polymers are sterically complemented is preferable. In this specification, an aggregate in which polymers are sterically complementary is called a stereo complex. Stereocomplementary association means that the free energy of the polymer forming the stereocomplex is associated with a minimum value or a local value so that the free energy is stabilized, and that the polymer is associated in a close packed manner. Including. Examples of the polymer forming the stereocomplex include polymethacrylate and polylactic acid.

  The polymethacrylate used in the present invention is not limited as long as it is a polymer obtained by polymerizing a monomer selected from the group consisting of methacrylic acid and polymethacrylic acid ester, and even if it is a homopolymer having one kind of polymerized unit. The polymerization unit may be a plurality of types of copolymers. Specific examples of the polymethacrylate of the present invention include polymethyl methacrylate (hereinafter abbreviated as PMMA), polyethyl methacrylate (hereinafter abbreviated as PMMA), poly n-propyl methacrylate, polyisopropyl methacrylate, and the like. It is not limited to these. Polymethacrylate is available from Polymer Source, Inc., Polyscience, Inc., Aldrich, Nacalai, and others.

  Examples of stereocomplexes include a structure formed between a polymer having stereoregularity and a polymer having different stereoregularity from the polymer and having various length ratios with respect to the length of the polymer. Preferably, the polymer has a structure other than the structure in which a helix is formed by van der Waals force acting between each other (hereinafter referred to as a helix structure) and the structure in which a helix is formed based on van der Waals aggregation ( (Hereinafter referred to as van der Waals structure). As a structure that forms a helix, specifically, a structure that forms a double helix (hereinafter referred to as a double helix structure: Serizawa et al., J. Am. Chem. Soc. 2000, Vol. 122, p.1891) But) is not limited to these.

  The double helix structure is a structure in which an it-polymer is surrounded by a st-polymer that is twice as long, and Van der Waals forces are engaged between them. The it-polymer and st-polymer in the double helix structure may be the same or different. For example, PMMA may be used for both the it-polymer and the st-polymer, PMMA may be used for one of the it-polymer and the st-polymer, and PMMA may be used for the other. As the Van der Waals structure, a structure in which it-polymer and st-polymer of the same length are associated is known.

  There is no restriction | limiting in particular in the formation method of a stereo complex. For example, it can be prepared by dissolving and mixing a polymer to be associated with an appropriate solvent, and may be prepared by separating a desired stereocomplex as necessary. Preferably, the alternate lamination (Layer-by-Layer) method (hereinafter abbreviated as LbL method) described in J. Am. Chem. Soc., 2000 Vol. 122, p.1891 Used. In this method, the stereocomplex layer is prepared by performing at least one cycle of creating a stereocomplex layer including the step of immersing the support in the syndiotactic polymethacrylate solution and the step of immersing the support in the isotactic polymethacrylate solution, and includes the stereocomplex. Forming a polymer film. More specifically, it-polymer and st-polymer were separately dissolved in an appropriate solvent (hereinafter referred to as a film-forming solvent), and the support was immersed in the it-polymer solution to adsorb the it-polymer. After that, if necessary, it is washed and dried, and subsequently immersed in the st-polymer solution to form a single-layer stereocomplex consisting of it-polymer and st-polymer and van der Waals contact structure. The Thereafter, the support is washed and dried to remove excess st-polymer and leave a stereocomplex layer. By repeating this cycle, a stereo complex layer corresponding to the number of cycles is laminated, and a polymer film is formed. Within the cycle, the immersion order of the it-polymer and st-polymer may be reversed. As long as the capture molecule-binding protein can be immobilized, the number of cycles, that is, the number of stereo complex layers is not limited, and may be one time or two or more times. In order to reliably cover the entire support, the number of cycles is 2 times or more, preferably 5 times or more. Although there is no restriction | limiting in particular in the upper limit of the number of cycles, From an economical viewpoint, it is 50 times or less, Preferably it is 30 times or less.

  The film forming solvent can be selected from water, a polar organic solvent, or a mixture thereof. Examples of the polar organic solvent include acetonitrile, dimethylformamide, acetone, alcohols, dimethyl sulfoxide, dioxane and the like. The water may contain a buffer. The polymer concentration is not limited as long as it is a concentration suitable for forming a film, and is generally 0.1 mg / mL or more, preferably 0.5 mg / mL or more, more preferably 1 mg / mL or more. 20 mg / mL or less, preferably 10 mg / mL or less, more preferably 1 to 5 mg / mL or less.

  The film forming temperature is generally 4 ° C. or higher, preferably 10 ° C. or higher, more preferably 20 ° C. or higher, generally 50 ° C. or lower, preferably 40 ° C. or lower, more preferably 30 ° C. or lower. The immersion time is generally 1 minute or longer, preferably 3 minutes or longer, more preferably 5 minutes or longer. The upper limit is not particularly required, but is preferably 60 minutes or less, more preferably 30 minutes or less.

  As described above, as the support having an aggregate region of two or more polymers having different stereoregularities on the surface, as described above, a part or all of the surface of the above support has two or more different stereoregularities. Although it means what was covered with the support body of a polymer, for example, it may be composed of an assembly itself of two or more polymers having different stereoregularities. In this case, the support and the polymer aggregate formed on the support can be prepared as a single body at the same time, which is preferable in that it does not have to be prepared separately.

  In the case of an intramolecular aggregate, since it can be easily formed into a form other than film formation, it is also suitable for use with beads or the like. Examples of the polymer forming the intramolecular aggregate include a block copolymer having an isotactic block and a syndiotactic block, a block polymerized using one type of enantiomer as a polymerization unit, and another type of enantiomer And a block copolymer having a polymer polymerized as a polymer unit.

  The block copolymer may be produced by bonding an isotactic block and a syndiotactic block directly or via a linker, or may be produced by block polymerization. Specific examples include the following methods. When synthesizing a stereoregular polymethacrylate by low-temperature living anionic polymerization, a polymer having a hydroxyl group, a carboxyl group, an amino group or the like at one end can be synthesized by appropriately selecting a reaction terminator. A block copolymer in which an isotactic block and a syndiotactic block coexist in one molecule can be synthesized by condensation by ester or amide formation between these end groups or bonding to an appropriate linker. These form hairpins by interaction and form a stable intramolecular stereocomplex.

  In addition, the block copolymer may be produced by polymerizing another type of enantiomer following a block polymerized using one type of enantiomer as a polymerization unit. It may be produced by bonding a block polymerized with a polymer unit as a type and a block polymerized with another type of enantiomer as a polymer unit directly or via a linker. Specifically, after synthesizing one polylactic acid block of an enantiomer by ring-opening polymerization under an appropriate condition, the enantiomer of the other lactic acid is added and further polymerization reaction is performed, thereby poly-D-lactic acid. A block copolymer comprising a block and a poly L-lactic acid block can be synthesized. In addition, specifically, when synthesizing the poly-D-lactic acid block and the poly-L-lactic acid block, by selecting an appropriate reaction stopper in the same manner as described above, as a result of the same condensation reaction or binding reaction as described above, Block copolymers can be obtained. The block copolymer forms a stable intramolecular stereocomplex by intramolecular interaction.

  The molecular binding substance in the present invention means a substance that can be fixed to an aggregate region of a support having two or more polymer aggregate regions having different stereoregularities on the surface. The molecule-binding substance fixed to the aggregate region of the support has affinity for the substance to be measured and can be directly bound to the object to be measured. Can be combined. In such a case, the measurement object can be bound to the molecule binding substance via the capture molecule. Examples of such molecular binding substances include protein A, protein G or protein L, Fc receptor, or a protein for binding binding molecules such as a combination thereof. As a molecular binding substance that can directly bind to an object to be measured. And enzymes.

  When an enzyme is used as the molecular binding substance of the present invention, for example, an oxidoreductase, a transferase, a hydrolase, a desorption enzyme, an isomerase, a synthetic enzyme, etc. can be preferably used.

As the oxidoreductase, those using CH—OH as a donor, those using an aldehyde or oxo group as a donor, those using CH—CH as a donor, those using CH—NH ₂ as a donor, CH— Those having NH as a donor, those acting on reduced nicotinamide adenine dinucleotide (hereinafter abbreviated as NADH) or reduced nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NADPH), nitrogen compounds , Donors with diphenol or related compounds, those with hydrogen peroxide as acceptors, those that take in molecular oxygen and act on a pair of donors, superoxide as an acceptor Those that use sulfur-containing groups as donors, those that use heme as donors, those that use hydrogen molecules as donors, and take in molecular oxygen Acting one donor (oxygenases), which acts on the CH group or a CH ₂ group, which the iron-sulfur protein and donors, such as those for the reduction flavodoxin and donor and the like.

  Examples of those using CH-OH as a donor include alcohol dehydrogenase, glucose-6-phosphate dehydrogenase, glycerol dehydrogenase, glycerol-3-phosphate dehydrogenase, 3α-hydroxysteroid dehydrogenase, 7α-hydroxysteroid dehydrogenase, 12α-hydroxy. Steroid dehydrogenase, isocitrate dehydrogenase, L-lactate dehydrogenase, 3-hydroxybutyrate dehydrogenase, pyranose oxidase, glycerol oxidase, alcohol oxidase, choline oxidase, galactose oxidase, glucose oxidase, cholesterol oxidase, L-α-glycerophosphate oxidase, lactate oxidase, D-lactate dehydrogenase, male Dehydrogenase, isopropyl maleate dehydrogenase, phosphogluconate dehydrogenase, fructose dehydrogenase, D-3- hydroxybutyrate dehydrogenase, mannitol dehydrogenase, glucuronyl sulfonates reductase and the like.

  Examples of those having an aldehyde or oxo group as a donor include pyruvate oxidase, glyceraldehyde-3-phosphate dehydrogenase, xanthine oxidase, pyruvate dehydrogenase, and formaldehyde dehydrogenase.

  Examples of those using CH-CH as a donor include acyl-CoA dehydrogenase and bilirubin oxidase.

Examples of those using CH-NH ₂ as a donor include glutamate synthetase, alanine dehydrogenase, tyramine oxidase, amine oxidase, putrescine oxidase, glutamate dehydrogenase, leucine dehydrogenase, L-amino acid oxidase, and cytochrome P450.

  Examples of those using CH—NH as a donor include sarcosine oxidase, sarcosine dehydrogenase, Δ1-piperidein-2-carboxylic acid reductase, and the like.

  Examples of substances that act on NADH or NADPH include glutathione reductase, diaphorase, NADH-flavin mononucleotide (hereinafter abbreviated as FMN) oxidoreductase, NADPH-FMN oxidoreductase, and dihydrolipoamide reductase.

  Examples of those using a nitrogen compound as a donor include uricase.

  As what uses diphenol or a related compound as a donor, ascorbate oxidase etc. are mention | raise | lifted, for example.

  Examples of those having hydrogen peroxide as a receptor include peroxidase and glutathione peroxidase.

  Examples of compounds that take up molecular oxygen and act on a pair of donors include p-hydroxybenzoate 4-monooxygenase, salicylic acid 1-monooxygenase, steroid 11β-monooxygenase, phenylalanine 4-monooxygenase, and dopamine β-monooxygenase. Etc.

  Examples of the substance that acts as a receptor for superoxide include superoxide dismutase and the like.

  Examples of donors that contain sulfur-containing groups include sulfite reductase, hypotaurine dehydrogenase, sulfite oxidase, thiol oxidase, glutathione-homocysteine transhydrogenase, protein disulfide reductase, glutathione-cysteine transhydrogenase, glutathione dehydrogenase, etc. Can be given.

  Examples of those using heme as a donor include cytochrome c oxidase, nitrate reductase and the like.

  Examples of those using hydrogen molecules as a donor include hydrogen dehydrogenase and cytochrome c3 hydrogenase.

  Examples of those that take up molecular oxygen and act on a single donor (oxygenase) include catechol 1,2-dioxygenase, catechol 2,3-dioxygenase, tryptophan 2,3-dioxygenase, lipoxygenase, ascorbate 2 , 3-dioxygenase, arginine 2-monooxygenase, lysine 2-monooxygenase, tryptophan 2-monooxygenase, lactate 2-monooxygenase, Renilla luciferin 2-monooxygenase, Cypridina luciferin 2-monooxygenase, firefly luciferin 2-mono Examples include oxygenase.

Examples of those acting on the CH group or CH ₂ group include xanthine dehydrogenase, nicotinate dehydrogenase and the like.

  Examples of those using iron-sulfur protein as a donor include ferrodoxine-oxidized NAD reductase and ferrodoxine-oxidized NADP reductase.

  Examples of those using reduced flavodoxin as a donor include nitrogenase and the like.

  Examples of transferases include those that transfer one carbon group, those that transfer aldehydes or ketones, acyltransferases, glycosyltransferases, those that transfer alkyl groups or allyl groups other than methyl groups, and groups that contain nitrogen. Examples thereof include those that transfer, those that transfer a group containing phosphorus, and those that transfer a group containing sulfur.

  Examples of those that transfer one carbon group include nicotinamide methyltransferase, histamine methyltransferase, and homocysteine methyltransferase.

  Examples of those that transfer aldehyde or ketone include transketolase and transaldolase.

  Examples of the acyltransferase include amino acid acetyltransferase, glucosamine acetyltransferase, choline acetyltransferase, phosphate acetyltransferase, acetyl CoA acyltransferase, galactoside acetyltransferase, diacylglycerol acyltransferase, lysolecithin acyltransferase, sphingosine acyltransferase, glycine acetyltransferase, Examples thereof include glutamate acetyltransferase, histone acetyltransferase, peptidyltransferase, citrate synthase, γ-glutamyltransferase, phosphotransacetylase, and transglutaminase.

  Examples of glycosyltransferases include phosphorylase, purine nucleotide phosphorylase, sucrose phosphorylase, maltose phosphorylase, glycogen synthase, cellulose synthase, chitin synthase, UDP glucuronosyltransferase, UDP galactose-collagen galactosyltransferase, GDP fucose-glycoprotein fucosyltransferase, etc. Can be given.

  Examples of those that transfer an alkyl group or an allyl group other than a methyl group include dimethylallyl transferase, terpenoid-allyl transferase, aminopropyl transferase, farnesyl transferase, phosphoserine sulfide relase, and the like.

  Examples of those capable of transferring a group containing nitrogen include aspartate aminotransferase, alanine aminotransferase, diamino acid aminotransferase, acetylornithine aminotransferase, and the like.

  Examples of those that transfer phosphorus-containing groups include creatine kinase, myokinase, hexokinase, phosphoglucomutase, phosphoglyceromutase, pyruvate kinase, glycerol kinase, acetate kinase, phosphofructokinase, phosphoglycerate kinase, polynucleotide phosphorylase Phospholipase A2, adenosine kinase, thymidine kinase, protein kinase, adenylate kinase, nucleoside monophosphate kinase, nucleoside diphosphate kinase, nicotinamide mononucleotide adenylyltransferase, FMN adenylyltransferase, DNA nucleotidyltransferase, RNA Nucleotidyl transferase, ethanolamine phosphotransferase, phosphatid Serine synthase, and the like.

  Examples of those capable of transferring a group containing sulfur include allyl sulfotransferase, estrone sulfotransferase, chondroitin sulfotransferase, and acetyl CoA transferase.

  Hydrolytic enzymes include those that act on ester bonds, those that act on glycosyl compounds, those that act on ether bonds, those that act on peptide bonds, those that act on CN bonds other than peptides, acid anhydrides Examples thereof include those that act on CC bonds, those that act on halides, those that act on PN bonds, and those that act on S—N bonds.

  Examples of those that act on the ester bond include carboxyesterase, allyl esterase, lysophospholipase, cholesterol esterase, phospholipase A2, phospholipase C, phospholipase D, sphingomyelinase, alkaline phosphatase, acid phosphatase, lipoprotein lipase, 5 ′ nucleotidase, Examples include 3 ′ nucleotidase, glucose 6-phosphate phosphatase, fructose bisphosphatase, phosphodiesterase, allyl sulfatase, sterol sulfatase, exodeoxyribonuclease, exoribonuclease, endodeoxyribonuclease, endoribonuclease and the like.

  Examples of those that act on glycosyl compounds include α-amylase, β-amylase, cellulase, dextranase, chitinase, chitosanase, lysozyme, α-glucosidase, β-glucosidase, β-galactosidase, β-glucuronidase, β-fucosidase, β-N-acetylglucosaminidase, endo 1,3-β-glucanase, endo 1,3-β-galactanase, endo 1,4-β-galactanase, glucan 1,6-α-glucosidase, neuraminidase, invertase, nucleo Sidase, isopullulanase and the like can be mentioned.

  Examples of those acting on the ether bond include adenosylmethionine hydrolase.

  Examples of agents that act on peptide bonds include aminopeptidase, carboxypeptidase, prolyl aminopeptidase, purine iminopeptidase, pyroglutamyl peptidase, dipeptidylpeptidase, dipeptidylcarboxypeptidase, trypsin, chymotrypsin, thrombin, plasmin, blood coagulation factor, Examples include kallikrein, elastase, cathepsin, papain, phytin, bromelain, chymopapain, cysteine proteinase, pepsin, chymosin, aspartic proteinase, collagenase and the like.

  Examples of those that act on CN bonds other than peptides include urease, creatinine amide hydrolase, creatinine deaminase, creatinase, creatininase, asparaginase, glutaminase, aminoacylase, N-acetylmuramoylalanine amidase and the like.

  Examples of substances that act on acid anhydrides include apyrase and ATP pyrophosphatase.

  Examples of the substance that acts on the C—C bond include oxaloacetase and the like.

  Examples of substances that act on halides include alkylhalidase, haloacetate dehalogenase, and fluoroacetate dehalogenase.

  Examples of the substance acting on the PN bond include phosphoamidase and the like.

  Examples of those that act on the S—N bond include sulfoglucosamine sulfohydrolase.

  As the desorption enzyme, those acting on carbon-carbon bonds, those acting on carbon-oxygen bonds, those acting on carbon-nitrogen bonds, those acting on carbon-sulfur bonds, those acting on phosphorus-oxygen bonds Etc.

  Examples of substances that act on carbon-carbon bonds include N-acetylneuraminate aldolase, oxaloacetate decarboxylase, pyruvate decarboxylase, glutamate decarboxylase, ornithine decarboxylase, ribulose bisphosphate carboxylase, phosphoenolpyruvate carboxylase, fructose Examples thereof include bisphosphate aldolase and phenylserine aldolase.

  Examples of those that act on the carbon-oxygen bond include enolase, aconitase, carbonic anhydratase, tryptophan synthetase, threonine synthase, and nitrile hydratase.

  Examples of those that act on the carbon-nitrogen bond include aspartate ammonia lyase, arginosuccinate lyase, and uroporphyrinogen I synthase.

  Examples of those that act on the carbon-sulfur bond include cystathionine γ lyase, cystathionine β lyase, methionine γ lyase, and the like.

  Examples of the substance that acts on the phosphorus-oxygen bond include adenylate cyclase and guanylate cyclase.

  Examples of the isomerase include racemase and epimerase, cis-trans isomerase, intramolecular oxidoreductase, intramolecular transferase, intramolecular desorbase, and the like.

  Examples of racemase and isomerase include mutarotase, alanine racemase, serine racemase, amino acid racemase, and lactate racemase.

  Examples of the cis-trans isomerase include maleate isomerase and linoleate isomerase.

  Examples of the intramolecular oxidoreductase include phosphoglucose isomerase, xylose isomerase, mannose isomerase, and glucuronic acid isomerase.

  Examples of the intramolecular transferase include lysolecithin acyl mutase, phosphoglycerate phosphomutase, lysine 2,3-aminomutase, methyl aspartate mutase and the like.

  Examples of the intramolecular elimination enzyme include flavonone lyase.

  Synthetic enzymes include those that produce carbon-oxygen bonds, those that produce carbon-sulfur bonds, those that produce carbon-nitrogen bonds, those that produce carbon-carbon bonds, and those that produce phosphate esters. can give.

  Examples of those that generate a carbon-oxygen bond include tyrosyl-tRNA synthetase, leucyl-tRNA synthetase, methionyl-tRNA synthetase, lysyl-tRNA synthetase, and glutamyl-tRNA synthetase.

  Examples of those that generate a carbon-sulfur bond include acyl-CoA synthetase, acetyl-CoA synthetase, succinyl-CoA synthetase, and the like.

  Examples of those that generate carbon-nitrogen bonds include glutamine synthetase, asparagine synthetase, oxidized nicotinamide adenine dinucleotide synthetase, glutathione synthetase, pantothenic acid synthetase, guanosine monophosphate synthetase, urea carboxylase, glutamate-cysteine Examples include ligase.

  Examples of those that generate carbon-carbon bonds include acetyl CoA carboxylase, pyruvate carboxylase, and propionyl CoA carboxylase.

  Examples of those that produce phosphate esters include polydeoxyribonucleotide synthetase and polyribonucleotide synthetase.

  The capture molecule in the present invention means a molecule that has binding affinity for both the molecule binding substance and the measurement target and can separate the measurement target and the contaminant contained in the sample. In the present invention, when the molecule binding substance is a protein for binding a capture molecule such as protein A, protein G or protein L, or Fc receptor, it is preferable to use a capture molecule that can bind to the protein for binding a capture molecule. Examples of capture molecules in the present invention include antibodies and fragments thereof. Other examples include a molecule in which a ligand is bound to an antibody or antibody fragment, a complex containing an antibody or antibody fragment, a protein, a nucleic acid, a lipid, a sugar, a sugar chain, an aptamer (Nature, 1992, Vol. 355, p.850). ), And a molecule containing a site for capturing a measurement target such as a polymer template (Nature, 1993, Vol.361, p.645) and a site for binding to a molecule-binding substance. . The binding site to the molecular binding substance is selected according to the molecular binding substance. For example, when the molecular binding substance is protein A or protein G, Fc can be selected as the site, and when it is protein L, Fab can be selected.

  When the capture molecule is an antibody, the measurement target is an antigen specific to the antibody, a hapten, or a complex thereof. Here, the antigen complex refers to a substance containing other components in addition to the antigen, such as an antibody or an enzyme. A hapten complex refers to a substance containing other components in addition to a hapten, such as an antibody or an enzyme. In the embodiment of the antigen-antibody complex, an antibody specific for another epitope that is not occupied in the antigen-antibody complex is bound to a molecule-binding substance.

As long as it binds to the substance for molecular binding and the measurement target, the antibody as a capture molecule may be a whole molecule or a fragment. Fragments include Fab, F (ab) ₂ , Fab ′, F (ab ′) ₂ , Fab / c having one Fab and complete Fc, Fc that binds a molecule that binds the substance to be measured, and those Combinations are listed. In order to bind to protein A and protein G, it preferably has Fc. On the other hand, in order to bind to protein L, it is preferable to have Fab. Fc bound to a molecule that binds the substance to be measured can be produced by a method using a known covalent bond or a known genetic engineering technique.

  There is no restriction | limiting in the antibody as a capture molecule used by this invention, A mouse antibody, a rat antibody, a rabbit antibody, a sheep antibody, a chimeric antibody, a humanized antibody, a human antibody etc. are mentioned. There is no restriction on the immunoglobulin class of the antibody, and examples include IgG such as IgG1, IgG2, IgG3, and IgG4, IgM, IgA, and IgE. The antibody may be a polyclonal antibody or a monoclonal antibody, and a monoclonal antibody is preferable in that a homogeneous antibody can be stably produced. Polyclonal and monoclonal antibodies can be prepared by methods well known to those skilled in the art. When the molecule binding substance is an enzyme, no capture molecule is required.

  The present invention is characterized in that a molecular binding substance and optionally a capture molecule are immobilized on an aggregate region of a support having an aggregate region of two or more polymers having different stereoregularities on the surface. The present invention relates to a method for producing a carrier for measuring an object to be measured. The present invention also includes a step of fixing a molecular binding substance to an aggregate region of a support having two or more polymer aggregate regions having different stereoregularities on the surface, and optionally a capture molecule as the molecular binding material. The manufacturing method of the support | carrier for measuring the measuring object in a sample including the process couple | bonded with.

  There is no particular limitation on the method for fixing the substance for molecular binding on the aggregate of two or more polymers having different stereoregularities, and various known methods can be used. For example, a method in which a solution obtained by dissolving a substance for molecular binding is brought into contact with the aggregate is preferable. Examples of the contact method include a method of coating or spotting a solution on the aggregate, and examples of the coating method include dip coating, spin coating, gravure coating, spray coating and the like. As the solution for dissolving the molecular binding substance, an aqueous medium described later is suitable. The concentration of the molecular binding substance contained in the solution may be any concentration, but it is preferably a concentration that achieves binding of the molecular binding substance on the aggregate in a saturated state. The concentration of the substance for molecular binding contained in the solution is generally 0.01 μmol / L or more, preferably 0.1 μmol / L or more, more preferably 0.2 μmol / L or more, and the substance is contained in the solution. The upper limit is not set as long as it dissolves with an appropriate viscosity, but it is generally 1 mmol / L or less, preferably 100 μmol / L or less, more preferably 50 μmol / L or less. The temperature at the time of contact is preferably a temperature at which the molecular binding substance is not denatured, generally 0 ° C. or higher, preferably 4 ° C. or higher, more preferably 15 ° C. or higher, generally 70 ° C. or lower, preferably 50 ° C. or lower. More preferably, it is 40 ° C. or lower. The contact time is not particularly limited and can be appropriately selected depending on the temperature at the time of contact. Generally, immediately after contact, it is preferably 10 minutes or more, more preferably 30 minutes or more, and generally 48 hours or less, preferably 24 hours or less. More preferably, it is 2 hours or less. Since the carrier of the present invention is a carrier in which the denaturation of the molecular binding substance fixed on the polymer aggregate is suppressed and the storage stability is thereby improved, the carrier remains long in contact with the molecular binding substance. An aspect in which the time is left is also possible.

  In particular, when an enzyme is used as the molecular binding substance, the method for immobilizing the enzyme on an aggregate of two or more polymers having different stereoregularities is not particularly limited, and various known methods can be used. For example, a method in which a solution obtained by dissolving an enzyme is brought into contact with the aggregate. As a contact method, the above-mentioned contact method is mentioned, for example. As the solution for dissolving the enzyme, an aqueous medium described later is suitable. The concentration of the enzyme contained in the lysate may be any concentration, but is preferably a concentration that achieves binding of the enzyme in a saturated state on the aggregate. The concentration of the enzyme contained in the lysate is generally 0.05 μmol / L or more, preferably 0.5 μmol / L or more, more preferably 1 μmol / L or more, and the protein dissolves in the solution with an appropriate viscosity. Although there is no upper limit as much as possible, it is generally 100 μmol / L or less, preferably 10 μmol / L or less, more preferably 5 μmol / L or less. The temperature at the time of contact is preferably a temperature at which the enzyme does not denature, generally 0 ° C or higher, preferably 4 ° C or higher, more preferably 15 ° C or higher, generally 70 ° C or lower, preferably 50 ° C or lower, more preferably Is 40 ° C. or lower. The contact time is not particularly limited and can be appropriately selected depending on the temperature at the time of contact. Generally, immediately after contact, it is preferably 10 minutes or more, more preferably 30 minutes or more, and generally 48 hours or less, preferably 24 hours or less. More preferably, it is 2 hours or less. Since the carrier of the present invention is a carrier in which the denaturation of the enzyme immobilized on the polymer aggregate is suppressed, thereby improving the storage stability, an embodiment in which the carrier is left in contact with the enzyme for a long time is also possible It is.

  For example, when the above-described capture molecule-binding protein is used as the molecule-binding substance, the capture molecule can be further bound to the capture molecule-binding protein. However, such a method is not particularly limited, and any known method can be used. Can be used. For example, a method in which a solution in which a capture molecule is dissolved and a capture molecule-binding protein are brought into contact with each other can be mentioned. As the contact method and conditions, for example, the contact method and conditions for immobilizing the aforementioned molecular binding substance may be used.

  The carrier of the present invention is a carrier for measuring a measurement object in a sample that binds to a capture molecule when the molecular binding substance is the aforementioned protein for binding a capture molecule, and has different stereoregularity on the surface. And a support having an aggregate region of two or more polymers having a molecular binding substance fixed to the aggregate region of the support. Further, the capture molecule can be bound to the carrier through a molecule binding substance as the case may be. In the present specification, such a carrier is particularly referred to as a capture molecule binding carrier. A capture molecule binding carrier is also a kind of a suitable carrier of the present invention.

  In addition, when the molecular binding substance is an enzyme, the carrier of the present invention (enzyme-immobilized carrier) can be a carrier for measuring an object to be measured in a sample by an enzyme reaction or a continuous enzyme reaction. And a support having an aggregate region of two or more polymers having different stereoregularities on the surface, and an enzyme immobilized on the aggregate region of the support. The enzyme-immobilized carrier of the present invention can also be used for sensors, immunochromatography, bioreactors and the like.

  The carrier and capture molecule-binding carrier of the present invention are preferably those subjected to a blocking treatment with a blocking agent. The blocking agent preferably occupies a region not occupied by the molecular binding substance. By this blocking, a substance other than the measurement target contained in the measurement sample is adsorbed to the polymer aggregate and the support, and / or the measurement target is bound to the carrier at a part other than the molecular binding substance or the capture molecule. Measurement accuracy and reproducibility can be improved.

  As a blocking agent, a substance having adsorptivity to a water-absorbing substrate such as bovine serum albumin, casein, gelatin, skim milk, polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene glycol, or a commercially available blocking agent such as Block Ace (Dainippon Pharmaceutical). Agents and the like. The blocking agent can be used by contacting a solution obtained by dissolving it in an appropriate solvent such as an aqueous solvent described later with the carrier of the present invention and the capture molecule-binding carrier. The content of the blocking agent contained in the solution is generally 0.1% by weight or more, preferably 1% by weight or more, and there is no upper limit as long as it can be dissolved in the solvent, but it is preferably 10% by weight or less. . Examples of the method for fixing the blocking agent include physical adsorption or chemical bonding which are generally used. The temperature and time conditions for the blocking treatment may be the contact conditions for immobilizing the aforementioned molecular binding substance.

  From a practical point of view, a carrier not bound to a capture molecule is prepared and stored in advance, and the capture molecule corresponding to the measurement object is bound immediately before the measurement to obtain a capture molecule-bound carrier, which is used for measurement. This aspect is preferred. Also preferred is an embodiment in which a carrier subjected to blocking treatment with a blocking agent or a capture molecule-binding carrier is prepared and stored in advance and can be used immediately for measurement of a measurement object. The carrier of the present invention, in which the molecular binding substance is fixed to the support through the association of two or more polymers having different stereoregularities, is excellent in stability and can be stored even in a dried state. Suitable for use in the above embodiments. The storage temperature in the dry state is preferably 2 ° C to 40 ° C, more preferably 4 ° C to 30 ° C.

  In addition, the present invention provides a surface used for a carrier for measuring a measurement object in a sample by immobilizing a molecular binding substance on an aggregate region of two or more polymers having different stereoregularities of a support. A polymer-attached support having an aggregate region of two or more polymers having different stereoregularity.

  The polymer-attached support is used for measurement by immobilizing a molecule binding substance before measurement and further binding a capture molecule.

  In the present invention, when the substance for molecular binding is, for example, protein A (one of the proteins for binding capture molecules), protein A can be directly immobilized on a polymer aggregate membrane containing a polymethacrylate stereocomplex. it can. As a result, the carrier can be easily prepared. In addition, by providing the polymer aggregate film, it is possible to suppress deterioration of the performance of the carrier after protein A fixation over time, and storage stability is improved. Compared to the conventional immobilization method, it is considered that the denaturation of protein A is suppressed by using the direct interaction between the polymer-associated membrane and protein A. Furthermore, the amount of measurement object binding per unit area can be increased, resulting in improved measurement sensitivity. Although not bound by any theory, protein A does not grow three-dimensionally (SK mode) but layered (FM mode) on the polymer film. It is also considered that the antibody is bound efficiently. In addition, by controlling the orientation of protein A, the binding surface of protein A with the antibody can be presented to the antibody so that the effect of binding of the antibody can be obtained, which also facilitates the binding of the antibody. It is done.

  In the present invention, when the molecular binding substance is an enzyme, the enzyme can be directly immobilized on the polymer aggregate film containing a polymethacrylate stereocomplex. As a result, the carrier can be easily prepared. In another aspect, by providing the polymer aggregate membrane, it is possible to suppress the deterioration of the performance of the carrier after enzyme immobilization with time, and the storage stability is improved. Compared with the conventional fixing method, it is considered that the denaturation of the enzyme is suppressed by using the direct interaction between the polymer-associated membrane and the enzyme. In yet another aspect, the enzyme reaction efficiency per unit area can be increased, resulting in improved measurement sensitivity or product production efficiency. While not being bound by any theory, the area on which the substrate and the enzyme are bound to each other because the enzyme grows in layers (FM mode) instead of three-dimensional growth (SK mode) on the polymer film. This is thought to be due to the fact that the substrate is in efficient contact with the enzyme. In addition, it is considered that the orientation of the enzyme can be controlled so that the binding surface of the enzyme with the substrate can be presented to the substrate so that the effect of binding of the substrate can be obtained, thereby facilitating the binding of the substrate.

  The present invention also relates to a method for measuring an object to be measured in a sample using the carrier of the present invention. The sample used in the measurement method of the present invention is not particularly limited as long as it enables the measurement method of the present invention. For example, biological fluids derived from humans or animals other than humans (blood, serum, plasma, urine) , Feces, sputum, saliva, intraperitoneal fluid, etc.), liquids separated and extracted from plants and microorganisms, liquids separated and extracted from food and food ingredients, liquids generated from the manufacturing process of chemicals, rivers and seawater, air, Examples thereof include a liquid separated and extracted from the environment such as soil, a liquid artificially added to a buffer solution or the like so that a measurement object has a known concentration or a certain constant concentration.

  The measurement method of the present invention comprises a step of bringing the aforementioned carrier into contact with the measurement object in the reaction solution and reacting the measurement object with the enzyme immobilized on the carrier, and measuring a substance that increases or decreases by the reaction. The process of carrying out.

  Furthermore, the measurement method of the present invention comprises a step of bringing the aforementioned carrier and a capture molecule into contact with each other in a reaction solution to form a capture molecule-binding carrier, and contacting the capture molecule-bound carrier with a measurement object via the capture molecule. A step of binding the measurement object to a carrier and a step of measuring the measurement object bonded to the carrier.

  Further, the measurement method of the present invention comprises a step of bringing a capture molecule and a measurement object into contact with each other in a reaction solution to form a measurement object-bound capture molecule, and contacting the above-mentioned carrier with the measurement object-bound capture molecule. And a step of measuring a measurement object bound to the carrier.

  Further, the method of the present invention comprises a step of bringing a capture molecule-binding carrier and a measurement object into contact with each other in a reaction solution, and binding the measurement object to the carrier via the capture molecule, and measuring the measurement object bound to the carrier. The process of carrying out.

  The reaction solution used in the measurement method of the present invention is not particularly limited as long as the carrier and the measurement object, or the carrier and the capture molecule, or the solvent capable of reacting the capture molecule binding carrier and the measurement object are used. Examples thereof include an aqueous medium, an organic solvent, and a supercritical fluid, and an aqueous medium is preferable. Examples of the aqueous medium include deionized water, distilled water, and a buffer solution, and a buffer solution is preferable. The buffer used for the buffer is not particularly limited as long as it has a buffering capacity, but has a pH of 1 to 11, for example, lactate buffer, citrate buffer, acetate buffer, succinate buffer, phthalate buffer, phosphate buffer. Agent, triethanolamine buffer, diethanolamine buffer, lysine buffer, barbitur buffer, tris (hydroxymethyl) aminomethane buffer, imidazole buffer, malic acid buffer, oxalic acid buffer, glycine buffer, boron Acid buffer, carbonate buffer, Good buffer and the like can be mentioned. Examples of the good buffer include 2-morpholinoethanesulfonic acid (MES), bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetamido) iminodiacetic acid (ADA), Piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N- [tris (hydroxymethyl) methyl] -2-aminoethanesulfonic acid (TES), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethane Phosphonic acid (HEPES), 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), N- [tris (hydroxymethyl) methyl] -2-hydroxy-3- Aminopropanesulfonic acid (TAPSO), piperazine-N, N′-bis (2-hydroxypropanesulfonic acid) (POPSO), 3- [4- (2-hydroxyethyl) -1-piperazinyl] -2-hydroxypropanesulfone Acid (HEPPSO), 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid [(H) EPPS], N- [tris (hydroxymethyl) methyl] glycine (Tricine), N, N- Bis (2-hydroxyethyl) glycine (Bicine), N-tris (hydroxymethyl) methyl -3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-3-amino-2-hydroxypropanesulfonic acid (CAPSO), N-cyclohexyl-3-amino Examples thereof include propanesulfonic acid (CAPS). The concentration of the buffer solution is not particularly limited as long as it is suitable for measurement, but is preferably 0.001 to 2.0 mol / L, more preferably 0.005 to 1.0 mol / L, and 0.01 to 0.00. 1 mol / L is particularly preferable.

    Examples of the organic solvent include ethanol, methanol, acetonitrile, dimethylformamide, dioxane, hexane, benzene, isooctane and the like. Examples of the supercritical fluid include supercritical carbon dioxide, supercritical water, supercritical ethanol, and supercritical methanol.

  The reaction temperature is not particularly limited as long as a specific reaction can be performed, but is generally 0 ° C. or higher, preferably 4 ° C. or higher, more preferably 15 ° C. or higher, and generally 70 ° C. or lower. Preferably it is 50 degrees C or less, More preferably, it is 40 degrees C or less. The reaction time is not particularly limited and can be appropriately selected depending on the reaction temperature. Generally, immediately after the reaction, it is preferably 1 minute or longer, more preferably 5 minutes or longer, generally 24 hours or shorter, preferably 4 hours or shorter, more preferably Is 2 hours or less.

  The measurement method of the present invention can be used for both quantitative and qualitative applications. For quantitative use, the physical property change to be measured may be measured quantitatively and converted to the amount of the combined measurement object. In addition, for example, by fixing different capture molecules to a plurality of capture molecule binding regions on the carrier, quantitatively measuring the amount of change in physical properties in each region, and converting it to the amount of bound measurement object, It is also possible to quantify the measurement object at once.

  As a qualitative use, there is a use in which a measurement target is identified by immobilizing different capture molecules in a plurality of regions on a carrier and detecting to which region the measurement target is bound. Alternatively, there may be mentioned a use in which a measurement target is identified by fixing different molecular binding substances in a plurality of regions on the support and detecting whether the measurement target substance is bound to the regions. With the measurement method of the present invention, a time-dependent change such as a transient response may be measured and a kinetic analysis may be performed.

Hereinafter, when the molecule binding substance is a capture molecule binding protein or an enzyme, the measurement method of the measurement object and the measurement kit will be described in detail for each case.
When the molecule binding substance is a capture molecule binding protein When the molecule binding substance is the aforementioned capture molecule binding protein, the measurement of the measurement target is a change in physical properties caused by the binding between the measurement target and the carrier. This can be done by detecting. The measuring method is appropriately selected according to the physical properties. Here, the physical properties include spectroscopic characteristics, light emission and fluorescence, radiation emission, physical shape, dielectric constant, optical characteristics, weight, and the like. Any known measuring means is selected according to the change in physical properties to be measured.

  In one aspect of the present invention, the measurement object is labeled with an antibody to which a labeling substance is bound, or a complex containing the labeling substance and the antibody (hereinafter collectively referred to as an antibody labeling complex). The label can be appropriately selected from enzymes, fluorescent substances, radioisotopes, colloids and the like. In another embodiment, carrier aggregation is measured. Suitable supports for this embodiment include fine particles such as latex or gold colloid.

  When the label is an enzyme (generally referred to as an ELISA method), a dye can be produced by an enzymatic reaction and detected by a known spectroscopic device. In the case where light is emitted by an enzyme reaction, a known chemiluminescence detection device can be used. When the label is a fluorescent substance, a known fluorescence measuring device can be used. When the label is a radioisotope (generally called a radioimmunoassay method), a known radiation detection apparatus can be used. When the label is a colloid, it may be observed directly by a scanning probe microscope such as an atomic force microscope or a scanning tunneling microscope or an electron microscope. However, in the case of a measurement object labeled with a colored substance such as gold colloid, it can also be detected spectroscopically. In the case of aggregation of fine particles such as latex (generally referred to as immunoturbidimetry), spectroscopic techniques such as change in absorbance and light dispersion are used.

  The crystal oscillation method measures changes in vibration frequency, the surface plasmon resonance method (SPR) measures the change in reflection angle caused by the change in dielectric constant, and the micro differential thermal measurement method measures the amount of heat. A measurement object that is not labeled may be directly observed with a high-resolution electron microscope, a scanning probe microscope, a scanning near-field microscope, or the like. In addition, for measurement, any measurement device according to the labeling mode, specifically, a spectroscopic device, a fluorescence detection device, a chemiluminescence detection device, a radiation detection device, or the like can be used. The spectroscopic methods used include infrared absorption, ultraviolet visible absorption, Raman scattering, and the like.

  It is also possible to simultaneously measure a plurality of measurement objects in a measurement sample using the carrier of the present invention. In this case, by using the carrier of the present invention in combination, that is, by immobilizing different types of capture molecules depending on the measurement object, a plurality of types of measurement objects can be measured simultaneously. This simultaneous multi-item measurement method can speed up examination and diagnosis.

  For the simultaneous item measurement method, a plurality of capture molecule binding regions may be provided on one carrier, and different capture molecules may be bound to each region. By binding a plurality of types of capture molecules in one carrier, a large number of samples can be measured at once, and the screening can be speeded up.

  In the case of the above-described embodiment, it is preferable to immobilize a capture molecule in advance on a carrier such as a 96 or 384 plate, a bead, or a chip. In the plate and the chip, a means is required for knowing which measurement object the capture molecule binding region is used for measurement. With beads, means for associating the beads with the measurement object is required. As a preferred embodiment, as disclosed in US Pat. No. 5,981,180, it is possible to identify beads by irradiating laser light and classifying in detail the signals emitted from the fluorescent dyes possessed by the beads. . Alternatively, as disclosed in WO 01/59454, the beads can be identified by detecting the barcode attached to the surface of the capture molecule binding region in the most suitable manner.

  By measuring the physical property change of each capture molecule binding region and making the antibody binding region correspond to the measurement target simultaneously or after a certain time, it is possible to measure each target at once.

  Specific measurement apparatuses capable of identifying the capture molecule binding region include the following. In the case of a plate, it is a plate reader equipped with a detector corresponding to the changing physical properties, and in the case of a chip, a measuring device including an image analysis device equipped with a detector corresponding to the changing physical properties (for example, Perkin Elmer) Scan array series), in the case of beads, a measuring device (for example, Luminex 100 Luminex, Inc.) provided with a detector that reads an identification symbol given to the bead and a detector according to changing physical properties.

  The present invention also relates to a measurement kit comprising the aforementioned carrier. As the constituent elements other than the carrier in the measurement kit, any known element can be selected according to the measurement technique.

  Examples of the ELISA method include enzyme-labeled antibody, enzyme-labeled antibody diluent, enzyme reaction substrate solution, enzyme reaction stop solution, and washing solution. Further, a blocking agent, a standard substance, a measurement sample diluent, etc. can be arbitrarily added.

  Examples of the fluorescence immunoassay include fluorescently labeled antibodies, fluorescently labeled antibody dilutions, and washing solutions. Further, a blocking agent, a standard substance, a measurement sample diluent, etc. can be arbitrarily added.

  In the radioimmunoassay method, a radiolabeled antibody, a radiolabeled antibody diluent, a washing solution and the like can be mentioned. Further, a blocking agent, a standard substance, a measurement sample diluent, etc. can be arbitrarily added.

  In methods such as SPR, quartz oscillator, micro differential heat, scanning electron microscope, and atomic force microscope, an antibody for enhancing the signal, a cleaning solution, a standard substance, a measurement sample diluent, and the like can be arbitrarily added.

  In the immunochromatography method, the measurement kit includes a test strip in which a membrane including an antibody binding region is used as a carrier and a sample pad, a conjugate pad, an absorption pad, and the like are arranged in a housing. Constituent elements other than the test strip in the measurement kit include labeled antibodies having a label arbitrarily selected from enzymes, fluorescence, radioactive substances, colloidal gold, and the like, labeled antibody diluents, washing solutions, and the like. Further, a blocking agent, a standard substance, a measurement sample diluent, etc. can be arbitrarily added.

  In the immunoturbidimetric method and the colloidal gold agglutination method, fine particles such as latex and colloidal gold are used as a carrier, and a carrier diluent, a washing solution, and the like are listed as components of the measurement kit. Further, a blocking agent, a standard substance, a measurement sample diluent, etc. can be arbitrarily added.

The measurement object of the present invention includes human serum albumin (HSA), bovine serum albumin (B
SA), IgG, IgM, IgA, IgE, apoprotein AI, apoprotein AII, apoprotein B, apoprotein E, rheumatoid factor, D-dimer, oxidized LDL, glycoalbumin, triiodothyronine (T3), total thyronin (T4), drugs (such as anti-tencan agents), C-reactive protein (CRP), cytokines, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA19-9 (carbohydrate antigen 19-9), CA15-3 (carbohydrate antigen 15-3), CA-125 (carbohydrate antigen 125), PIVKA-II (Protein induced by vitamin K absence-II), parathyroid hormone (PTH), human chorionic gonadotropin (hCG), thyroid gland Stimulating hormone (TSH), insulin, C-peptide, estrogen, anti-glutamate decarboxylase (GAD) antibody, pepsinogen, HBV antigen, anti-hepatitis B virus (HBV) antibody, hepatitis C virus (HCV) antigen, anti-HCV antibody, adult T-cell leukemia virus type 1 (HTLV-I) antigen, anti-HTLV-I antibody, human immunodeficiency Virus (HIV) antibody, tuberculosis antibody, tuberculosis antigen (TBGL), mycoplasma antibody, hemoglobin A1c, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), troponin T, troponin I, creatinine kinase-MB ( CK-MB), myoglobin, H-FABP (human heart-derived fatty acid binding protein), deoxynivalenol (DON), nivalenol (NIV), mold toxins such as T-2 toxin (T2), bisphenol A, nonylphenol, phthalic acid Dibutyl, polychlorinated biphenyls (PCBs), Dio Endocrine disruptors such as syn, p, p'-dichlorodiphenyltrichloroethane, tributyltin, fungi such as E. coli, food allergens such as eggs, milk, wheat, buckwheat, and peanuts, and allergies such as mites, such as mite and mite Substance, antiallergic substance antibody and the like.

FIG. 1 shows one embodiment of the formation of the carrier of the present invention and the capture molecule-binding carrier and the reaction between the carrier and the measurement target when a capture molecule-binding protein is used as the molecule-binding substance.
When the molecular binding substance is an enzyme When the molecular binding substance is an enzyme, the measurement of the measurement object is performed by measuring the amount of reaction product that increases due to the reaction between the measurement object and the enzyme or the amount of reaction substrate that decreases This can be done by measuring. In the measurement, the reaction product or the reaction substrate can be directly measured, or it can be converted into an appropriate substance and measured. The measurement method and the conversion method are appropriately selected. Here, the measurement includes spectroscopic measurement, luminescence and fluorescence measurement, dielectric constant measurement, and the like, and any known measurement means is selected according to the substance to be measured.

  For example, when the reaction product is a dye or a substance that absorbs light of a specific wavelength, the measurement is performed by a known spectroscopic device. When the reaction product emits light, a known chemiluminescence detection device is used. When the reaction product is a fluorescent substance, it can be carried out using a known fluorescence measuring device.

  For the purpose of performing measurement at high throughput, the embodiment of a sensor equipped with an electrode is more suitable. Examples of the electrode include a hydrogen peroxide electrode, a dissolved oxygen electrode, a conductivity electrode, an ion electrode, a pH electrode, and a redox electrode. The electrode can be properly used depending on the type of reaction product. For example, if the reaction product is hydrogen peroxide, it is a hydrogen peroxide electrode, if it is an electron or a substance or ion having a charge, it is a conductivity electrode, an ion electrode or a pH electrode, and if the reaction involves oxygen consumption, it will be dissolved. An oxygen electrode or the like can be used as long as the reaction causes a change in redox potential. Since the sensor provided with the electrode can constitute a small and simple device, it is suitable for making the device easy to carry.

  Two or more kinds of enzymes may be immobilized on the carrier (enzyme immobilization carrier) of the present invention. When converting an object to be measured into a detectable substance (dye, light, fluorescence, etc.) using two or more kinds of enzymes, all or part of the two or more kinds of enzymes are used in the aggregate region of the support. Can be fixed.

  It is also possible to simultaneously measure a plurality of measurement objects in a measurement sample using the carrier (enzyme-immobilized carrier) of the present invention. In this case, by using the carrier of the present invention in combination, that is, by immobilizing different types of enzymes according to the measurement object, it is possible to measure a plurality of types of measurement objects simultaneously using one sample. it can. This simultaneous multi-item measurement method can speed up examination and diagnosis. For simultaneous item measurement, a plurality of enzyme binding regions may be provided on one carrier, and different enzymes may be bound to each region. By binding a plurality of types of enzymes in one carrier, a large number of samples can be measured at a time, and the screening speed can be increased.

  In the case of the above embodiment, it is preferable to immobilize the enzyme in advance on a carrier such as 96 or 384 plate or chip. In the plate and chip, a means for knowing which measurement object the enzyme binding region is used for is required.

  It is possible to measure each object at a time by measuring a change in the amount of substance in each enzyme binding region and associating the enzyme region with the object to be measured simultaneously or after a certain time.

  Specific measuring apparatuses capable of identifying the enzyme binding region include the following. In the case of a plate, it is a plate reader equipped with a detector corresponding to the amount of substance that changes, and in the case of a chip, a measuring device including an image analysis device equipped with a detector according to the amount of substance that changes (eg, Parkin) Elmer scan array series).

  The carrier of the present invention (enzyme-immobilized carrier) is also suitable for the case where a plurality of samples are continuously measured. For example, this aspect is enabled by putting a plurality of samples into another container and sequentially contacting the carrier with the samples in the container. Alternatively, the carrier and the sample can be brought into contact with each other by sequentially dropping or ejecting the sample onto the carrier by a dropping device or a jetting device. Alternatively, even when a carrier is present in a partial region of the channel or in the channel, it is possible to flow the sample so that a plurality of samples do not cross each other in the channel.

  The diagnostic bioreactor using the carrier (enzyme-immobilized carrier) of the present invention is suitable for measuring a plurality of samples continuously. The diagnostic enzyme is not disposable but is fixed in the reactor (reactor) and used repeatedly. As a preferred embodiment in a diagnostic bioreactor, for example, a porous alkylamine glass (particle size: 125 to 177 μm, pore size: 50 nm) is coated as a support with the polymer of the present invention, and glucose oxidase, cholesterol oxidase, etc. Fix it. Enclose glass particles with enzyme molecules in a plastic tube with an inner diameter of 0.5 to 1.5 nm and a length of 50 to 300 mm, cover both ends with a nylon net, and stop with a nipple of an appropriate length. Can be made.

  A flow type diagnostic bioreactor is provided by providing a sample inlet in one of the diagnostic bioreactors, a reaction product outlet in the other, and a sensor (detector of the detector) for measuring the reaction product at the outlet. You can make a reactor. In the flow type bioreactor, it is not necessary to put a sensor into a sample for each specimen, and the sample can be sent in one direction, so that multi-directional diffusion of the substrate and the reaction product is difficult to occur. This is particularly important when two or more enzyme reactions are carried out continuously, and by connecting the reactors of different enzymes in a straight line, the next reaction is performed after the first reaction is incomplete. Can be minimized.

  When measuring continuously, the change in the amount of substance caused by the measurement object in the sample previously contacted may affect the change in the amount of substance caused by the measurement object in the sample contacted later. In such a case, it is desirable to appropriately clean the sample before adhering to the carrier with a cleaning solution.

  The present invention also relates to a measurement kit including a carrier using the aforementioned enzyme. Arbitrary well-known elements can be selected as constituent elements other than the carrier in the measurement kit. That is, an enzyme reaction stop solution, a blocking agent, a standard substance, a measurement sample diluent, a washing solution, and the like can be arbitrarily added.

  In the immunochromatography method, a test strip in which a membrane including an enzyme immobilization region is used as a carrier and a sample pad, a conjugate pad, an absorption pad and the like are arranged in a housing is included in the measurement kit. As components other than the test strip in the measurement kit, an enzyme reaction stop solution, a blocking agent, a standard substance, a measurement sample diluent, a washing solution, and the like can be arbitrarily added.

  Examples of the measurement object that can be measured by the carrier of the present invention (enzyme-immobilized carrier) include LDL cholesterol, HDL cholesterol, total cholesterol, free cholesterol, triglyceride, phospholipid, free fatty acid, lipid peroxide, creatinine, urea nitrogen, uric acid Ammonia, glucose, hemoglobin A1c, lactic acid, pyruvic acid, sialic acid, L-glutamic acid, L-lactic acid, vitamin C, inorganic phosphorus, alcohol and the like.

  The carrier of the present invention (enzyme-immobilized carrier) can also be applied to the production of substances. In this case, after the substrate is brought into contact with the enzyme bound to the carrier, the reaction solution containing the reaction product is immediately separated from the enzyme, thereby reducing the inhibition of the enzyme reaction by the reaction product. Embodiments made possible by the present invention include continuously removing the reaction product produced by the enzyme. Further, the convenience is further improved by combining a mode in which the substrate can be continuously contacted with the carrier. The aspect in which the substrate is continuously contacted and the reaction product can be continuously taken out makes it possible to simplify the apparatus for continuously producing the reaction product.

  Production of a substance using the carrier of the present invention (enzyme-immobilized carrier) can be performed, for example, by continuously flowing a reaction solution containing a substrate on the surface of the carrier. The carrier can also function as a production means for continuously removing the reaction product and a means for bringing the substrate into continuous contact with the carrier. For example, when the carrier of the present invention is formed in a part of the channel or when the carrier is present in the channel, the substrate and the carrier can be brought into contact by flowing a reaction solution containing the substrate through the channel. . Examples of the case where the carrier of the present invention is formed in a part of the flow path include micro-TAS (Micro-Total Analysis System), MEMS (Micro-Electro Mechanical Systems), or lab-on-a-chip (Lab-on -a-chip) in which the carrier is installed in the microchannel. As an example of the case where a carrier is present in the flow path, there can be mentioned a mode in which a column as a flow path is packed with beads as a support, such as an enzyme reactor or a bioreactor.

  In addition, production of a substance using a carrier (enzyme-immobilized carrier) can also be performed by introducing the carrier of the present invention into a reaction solution containing a substrate. A means for separating the reaction solution and the carrier by putting a carrier such as a bead into the container containing the reaction solution and capturing the carrier with a membrane having a hole that does not pass through the carrier after performing the enzyme reaction is suitable. It is.

  It is preferable to allow the enzyme reaction to proceed while simultaneously monitoring the enzyme reaction as a means for enabling the reaction product to be taken out after the enzyme reaction has sufficiently progressed. In this case, it is possible to use an apparatus provided with means for measuring the measurement object.

  Examples of the apparatus for continuously producing the reaction product include an enzyme reaction column, an enzyme reactor, and a bioreactor. Examples of the apparatus for continuously producing the reaction product include a continuous stirring reactor, a packed bed reactor, a fluidized bed reactor, a membrane reactor, a hollow fiber reactor, and a rotating disk reactor. Examples include a reactor, a magnetic field reactor, and a two-phase reactor.

  The reaction product that can be produced according to the present invention may be any product as long as it can be produced by an enzymatic reaction. For example, alcohols such as ethanol, polyphenols, trans-4-hydroxyproline, glycopeptides, glycolipids, monosaccharides, Polysaccharides such as cyclodextrins, oligosaccharides such as trehalose, amino acids such as L-aspartic acid, L-alanine and L-tryptophan, oligopeptides such as dipeptides and aspartame, sweeteners such as lipids, nucleic acids and isomerized sugars, inosinic acid Umami substances such as food additives such as diacylglycerol, food raw materials such as lactose-decomposed milk and casein degradation products, cosmetic raw materials, raw materials for synthetic polymers such as acrylamide, 6-aminopenicillane, 7-aminocephalosporin, etc. Antibiotic raw material, 1,4-andros Diene, 4-androsten like material for steroid hormones, human insulin, polypeptide pharmaceuticals such as human growth hormone, L- dopa, (6S) - pharmaceuticals or raw materials such as tetrahydrofolic acid and the like.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, these do not limit the scope of the present invention at all. In the examples and comparative examples, the following reagents were used.

PMMA (manufactured by Polymer Source. Inc. or Polyscience, Inc.).
These physical properties are as follows.
it-PMMA: Mn = 19,000, Mw = 21,000, Mw / Mn = 1.10, mm / mr / rr = 95/2/3
st-PMMA: Mn = 18,600, Mw = 23,000, Mw / Mn = 1.23, mm / mr / rr = 10/12/78
at-PMMA: Mn = 22,200, Mw = 23,200, Mw / Mn = 1.03
(In the above abbreviations, Mn represents the number average molecular weight, Mw represents the weight average molecular weight, mm / mr / rr is a triplet symbol representing stereoregularity, mm is isotacticity, rr is syndicated. (It-PMMA represents isotactic PMMA, st-PMMA represents syndiotactic PMMA, and at-PMMA represents atactic PMMA.)
Protein A (manufactured by Cosmo Bio). The protein A solution was prepared as follows.

Protein A (Mw 42,000) stored at 4 ° C is returned to room temperature and then dissolved in 0.02 wt% sodium azide (NaN ₃ ) / PBS (phosphate buffered saline) solution (2.38 mL) to give 20 µmol / L. Protein A solution was prepared and allowed to stand at 4 ° C. for 2 hours. 150 μL of this protein A solution was taken, spin-downed, frozen in liquid nitrogen, and stored at −20 ° C. In use, it was thawed and diluted with PBS to 1 μmol / L.

  Anti-human serum albumin (abbreviated as HSA) rabbit polyclonal antibody (Mw = 160,000; manufactured by SIGMA). Used without purification. The frozen antibody solution was quickly thawed at 37 ° C., diluted with PBS to 1 μmol / L, and stored at 4 ° C. At the time of use, 1 mL was collected in a polypropylene sample tube.

PBS (Ca ²⁺ , Mg ²⁺ free) was prepared by dissolving NaCl 4.00 g, KCl 0.10 g, Na ₂ HPO ₄ 12H ₂ O 1.45 g, and KH ₂ PO ₄ 0.10 g in 50 mL of ultrapure water. A double concentrate was made for stock and stored at 4 ° C. (10 × PBS). In use, it was diluted 10 times with ultrapure water.

  HSA (HSA fatty acid free; Mw = 69,000; manufactured by SIGMA) and bovine serum albumin (abbreviated as BSA; Mw = 67,000; manufactured by SIGMA). Used without purification. HSA is an antigen specific for anti-human HSA antibody, and BSA is a non-specific antigen used as a control. Both HSA and BSA solutions were prepared as follows. First, after allowing albumin to stand in a desiccator for 30 minutes at room temperature, weigh out a predetermined amount, add PBS to an albumin concentration of 10 μmol / L when completely dissolved, and leave it still at 4 ° C. until the next day. Dissolved in. Furthermore, it diluted with PBS and was set to 1 micromol / L. These solutions were used within one week of preparation.

  β-galactosidase [β-D-galactosidase; derived from E. coli, 5-30 units / mL; manufactured by Wako Pure Chemical Industries, Ltd.].

p-Nitrophenylgalactopyranoside (PNPG; manufactured by Wako Pure Chemical Industries, Ltd.).
Example 1
Production of protein A (molecule binding substance) fixed carrier Polymer membrane / protein A comprising a support / stereocomplex was prepared as follows.

  A carrier having an AT-cut quartz crystal (QCM) chip with a fundamental frequency of 9 MHz as a support and a polymer film of a stereo complex of it-PMMA and st-PMMA on the support was prepared as follows. . Note that a gold thin film was formed on the QCM chip, and the polymer film was formed on the gold thin film.

  First, the QCM chip was washed with Piranha solution (sulfuric acid: 30% by weight hydrogen peroxide solution = 3: 1) for 2 minutes twice on each side, and the frequency was measured.

  This QCM chip is immersed in a 1.7 mg / mL it-PMMA solution (solvent: acetonitrile) at 25 ° C for 5 minutes, pulled up, washed with acetonitrile (special grade), then washed with ultrapure water, and dried with nitrogen gas. It was. Subsequently, it was immersed in a 1.7 mg / mL st-PMMA solution (solvent: acetonitrile) at 25 ° C. for 5 minutes, pulled up, washed with acetonitrile (special grade), then washed with ultrapure water, and dried with nitrogen gas. . This operation was defined as one cycle, and this cycle was repeated for a total of 14 times. In this way, a polymer film was formed.

Protein A was immobilized on this polymer film by spin coating. The support on which the above polymer film was formed was immersed in a 1 μmol / L protein A solution at 37 ° C. for 60 minutes, washed with ultrapure water, and dried to prepare a carrier.
[Comparative Example 1] Polymer membrane consisting of support / stereo complex (without protein A)
A measurement carrier was prepared in the same manner as in Example 1 except that protein A fixation was omitted.
[Comparative Examples 2 to 4] Support / single polymer membrane (without protein A)
The same QCM chip as in Example 1 was used as a support, and each of the supports was composed of it-PMMA alone (Comparative Example 2), st-PMMA alone (Comparative Example 3), and at-PMMA (Comparative Example 4). A carrier for measurement was prepared using a support on which a polymer film was formed.

  For it-PMMA, st-PMMA, and at-PMMA solutions, weigh 1.7 mg of PMMA into each sample tube, and add chloroform (reagent for ultraviolet absorption spectrum, manufactured by Nacalai) to a concentration of 1.7 mg / mL. It was prepared by allowing it to stand at room temperature for 1 hour and completely dissolving it. These solutions were stored at −20 ° C. until use. At the time of preparing the polymer film, it was used after returning to room temperature and allowing to stand for 1 hour.

To prepare the polymer film, mount one drop of the above-mentioned it-PMMA, st-PMMA, and at-PMMA solution (solvent: chloroform) with a Pasteur pipette on the support and spin coat (2000 rpm, 60 s). Was done by. After spin coating, washing with ultrapure water was repeated, and after confirming that the frequency was stable, drying was performed.
[Comparative Example 5] Support only (no polymer membrane and protein A)
The QCM chip was washed with Piranha solution, dried and used as a measurement carrier.
[Comparative Examples 6 to 8] Support / single polymer membrane / protein A
Protein A was further immobilized on the measurement carriers of Comparative Examples 2-4. In Comparative Examples 6 to 8, the polymer films are respectively it-PMMA alone (Comparative Example 6), st-PMMA alone (Comparative Example 7), and at-PMMA (Comparative Example 8). Protein A was immobilized in the same manner as in Example 1. To determine the amount of protein A immobilized, the frequency was measured before and after protein A immobilization.
[Comparative Example 9] Support / Protein A
Protein A was immobilized on the measurement carrier of Comparative Example 5. Protein A was immobilized in the same manner as in Example 1 and dried to prepare a carrier. To determine the amount of protein A immobilized, the frequency was measured before and after protein A immobilization.

  The structures of the carriers prepared in Example 1 and Comparative Examples 1 to 9 are shown in Table 1.

[Test Example 1] Amount of protein immobilized on each measurement carrier The carrier prepared in Example 1 and Comparative Examples 6 to 9 was measured for frequency before and after protein A was immobilized, and was immobilized with the difference in frequency. The amount of protein A (fixed amount of protein A) was determined. Table 2 below shows the amount of protein A fixed (protein A fixed amount) as the amount of change in vibration frequency.

As shown in Table 2, the stereocomplex film as in Example 1 was compared with the single film of it-PMMA (Comparative Example 6) and st-PMMA (Comparative Example 7) and at-PMMA (Comparative Example 8). By using it, the amount of protein A fixed increased. The amount of protein A immobilized was almost the same when the stereocomplex membrane was used as in Example 1 and when the polymer membrane was not present as in Comparative Example 9.
Example 2
Preparation of anti-human HSA antibody-binding carrier The carrier prepared in Example 1 was immersed in a 1 µmol / L anti-human HSA antibody solution at 37 ° C for 60 minutes to bind the antibody. Subsequently, it was taken out from the solution, washed with ultrapure water at 37 ° C., immersed in a PBS solution for 1 hour without being dried, further washed with ultrapure water at 37 ° C., and dried to produce an anti-human HSA antibody-binding carrier.
[Test Example 2] Effect of protein A immobilization on antibody and antigen binding amount The measurement carrier prepared in Example 1 (protein A immobilization treatment completed), Comparative Examples 1 to 3 and 5 (no protein A immobilization treatment) were used. The antibody was bound and its binding amount was evaluated. Furthermore, antigens were further bound and the amount of binding was evaluated, and the effect of protein A fixation on the antibody and antigen binding amount was tested.

  The amount of antibody binding was evaluated as follows.

  In Example 1 and Comparative Examples 1 to 3 and 5 to 9, the frequency was measured in advance and used as initial values. Antibody binding was performed as follows. First, each measurement carrier was immersed in a 1 μmol / L anti-human HSA antibody solution at 37 ° C. for 60 minutes to bind the antibody, and then taken out from the solution and washed with ultrapure water at 37 ° C. The measurement carrier was immersed in a PBS solution for 1 hour without being dried, washed with ultrapure water at 37 ° C., and dried. Thereafter, the frequency was measured, and the amount of antibody binding was evaluated based on the difference from the initial value. The amount of antigen binding was evaluated as follows.

  The measurement carrier was immersed in a 1 μmol / L antibody solution at 37 ° C. for 60 minutes to bind the antibody, and then removed from the solution and washed with ultrapure water at 37 ° C. The carrier for measurement was immersed in a 1 μmol / L HSA solution or BSA solution for 1 hour at 37 ° C. without drying. After binding the antigen, it was taken out from the solution, washed with ultrapure water at 37 ° C., and dried. Thereafter, the frequency was measured, and the antibody + antigen binding amount was determined based on the difference from the initial value. The antigen binding amount was determined from the difference from the above-mentioned antibody binding amount. HSA is the antigen of the antibody used this time and corresponds to the measurement object to be measured. On the other hand, BSA is a non-specific antigen that does not bind to the antibody, and an experiment was conducted for comparison.

  The results of antibody binding amount and antigen binding amount are shown in Table 3. The amount of coupling is expressed in terms of frequency change (Hz). The average number of moles of HSA bound per mole of antibody (denoted as HSA / antibody in the table) was calculated from the amount of antibody binding, the amount of HSA binding, and the molecular weight of each.

As shown in Table 3, it can be seen that immobilizing protein A not only increases the amount of antibody binding, but also increases the binding ratio of specific antigen per antibody (HSA / antibody). This result suggests that the denaturation of the antibody can be suppressed by intervening protein A, and / or that the binding state of the antibody is promoted by controlling the adsorption state of the antibody.
[Test Example 3] Effect of Polymer Membrane on Antibody and Antigen Binding Amount Using the measurement carrier prepared with the measurement carrier of Example 1 and Comparative Examples 6 to 9 (all of which protein A is immobilized) Antibodies were bound and the amount of antibody binding was evaluated. Furthermore, antigens were further bound to evaluate the amount of antigen binding, and the influence of the polymer membrane was compared. Table 4 shows the results of the antibody binding amount and the antigen binding amount. The amount of coupling is expressed in terms of frequency change (Hz). The average number of moles of HSA bound per mole of antibody (denoted as HSA / antibody in the table) was calculated from the amount of antibody binding, the amount of HSA binding, and the molecular weight of each.

As shown in Table 4, by using a stereocomplex film in comparison with an it-PMMA single film (Comparative Example 6) and a st-PMMA single film (Comparative Example 7) and at-PMMA (Comparative Example 8) It can be seen that the amount of antibody binding and the amount of HSA binding, which is an antigen specific to the antibody, increase, and the HSA / antibody ratio also increases. Therefore, it is considered that the measurement carrier of the present invention exhibits an excellent performance of being able to efficiently measure a trace amount of measurement object.
[Test Example 4] Storage stability of carrier for HSA measurement Regarding the carrier for measurement of Example 1, Comparative Example 6 and Comparative Example 9, the amount of antibody binding and The amount of antigen binding was measured and the change in performance over time was examined. The storage condition was that the sample was stored in a container containing silica gel that was shielded from light at room temperature. The measurement was carried out 1 day, 3 days, 5 days, and 10 days after the start of storage. The results after 10 days are shown in Table 5.

When a stereocomplex membrane is used, the amount of HSA binding after storage for 10 days is 87% of the original, whereas when it-PMMA single membrane (Comparative Example 6) is used and when a polymer membrane is not used (comparison In Example 9), they decreased to 12% and 55%, respectively. The storage conditions this time are harsh compared to the storage conditions of ordinary carriers, and since the deterioration is suppressed even under such storage conditions, the carrier of the present invention has excellent storage stability. You can see that
Example 3
Quantification of HSA with carrier for HSA measurement The carrier prepared in Example 1 was measured for the frequency in advance and used as an initial value. Antibody binding was performed as follows. First, each measurement carrier was immersed in a 1 μmol / L anti-human HSA antibody solution at 37 ° C. for 60 minutes to bind the antibody, and then taken out from the solution and washed with ultrapure water at 37 ° C. The measurement carrier was immersed in a PBS solution for 1 hour without being dried, washed with ultrapure water at 37 ° C., and dried. Thereafter, the frequency was measured, and the amount of antibody binding was evaluated based on the difference from the initial value.

Antigen binding was performed as follows. The measurement carrier was immersed in a 1 μmol / L antibody solution at 37 ° C. for 60 minutes to bind the antibody, and then removed from the solution and washed with ultrapure water at 37 ° C. This measurement carrier is not dried, but is immersed in a sample solution containing HSA antigen adjusted to a concentration of 0.1, 0.5, 1.0, 5.0 μmol / L for 1 hour at 37 ° C. Then, it was taken out from the solution, washed with ultrapure water at 37 ° C., and dried. A product which was washed with ultrapure water at 37 ° C. without binding HSA antigen and dried was also prepared. Thereafter, the frequency was measured, and the binding amount of the anti-human HSA antibody and the HSA antigen was determined based on the difference from the initial value. The amount of HSA antigen binding was determined from the difference from the amount of anti-human HSA antibody binding described above. A calibration curve representing the relationship between the HSA antigen binding amount and the HSA antigen concentration is shown in FIG. The amount of coupling is expressed in terms of frequency change (Hz).
[Comparative Example 10]
A calibration curve representing the relationship between the amount of HSA antigen binding and the concentration of HSA antigen was prepared in the same manner as in Example 3 using the carrier of Comparative Example 6 instead of the carrier of Example 1. However, the HSA antigen concentration was 0.5, 1.0, and 5.0 μmol / L. The results are shown in FIG.
[Comparative Example 11]
A calibration curve representing the relationship between the amount of HSA antigen binding and the concentration of HSA antigen was prepared in the same manner as in Example 3 using the carrier of Comparative Example 9 instead of the carrier of Example 1. However, the HSA antigen concentration was 0.5, 1.0, and 5.0 μmol / L. The results are shown in FIG.

  As a result of Example 3, Comparative Example 10 and Comparative Example 11, it was confirmed that the carrier using the stereocomplex membrane of Example 1 increased the amount of HSA antigen binding depending on the HSA antigen concentration. Therefore, by preparing a calibration curve in advance, the frequency change (Hz) obtained from the sample solution of unknown concentration can be converted into the HSA antigen concentration, and the HSA antigen concentration can be quantified.

The carrier using the stereocomplex membrane has the same concentration of HSA antigen as compared to the case where it-PMMA single membrane (Comparative Example 6) is used and the case where the polymer membrane is not used (Comparative Example 9). On the other hand, the amount of HSA antigen binding is increased. This indicates that a carrier using a stereocomplex membrane is excellent as a carrier used for quantification of the HSA antigen.
Example 4
Preparation of β-galactosidase binding carrier Preparation of β-galactosidase binding carrier
Β-galactosidase was dissolved in a 50 mmol / L phosphate buffer containing 1 mmol / L magnesium chloride so as to be 0.0025 to 1 μmol / L to prepare a β-galactosidase solution. In each β-galactosidase solution of each concentration, a QCM chip on which a polymer film composed of it-PMMA alone of Comparative Example 1 was formed was immersed for 60 minutes at room temperature, and washed with 50 mmol / L phosphate buffer three times. It was dried to prepare a β-galactosidase binding carrier.
[Comparative Example 12]
Preparation of β-galactosidase binding carrier
Β-galactosidase was dissolved in a 50 mmol / L phosphate buffer containing 1 mmol / L magnesium chloride so as to be 0.0025 to 1 μmol / L to prepare a β-galactosidase solution. In each β-galactosidase solution of each concentration, a QCM chip on which a polymer film composed of it-PMMA alone of Comparative Example 2 was formed was immersed for 60 minutes at room temperature and washed three times with 50 mmol / L phosphate buffer. It was dried to prepare a β-galactosidase binding carrier.
[Test Example 5] Effect on β-galactosidase binding amount The frequencies of Comparative Example 1 and Comparative Example 2 before binding β-galactosidase were measured in advance. Subsequently, the frequency of Example 4 and Comparative Example 12 after binding β-galactosidase was measured to determine the amount of change in frequency (−ΔF, unit Hz). Using the Sauerbrey equation (Equation 1), the amount of β-galactosidase bound to the crystal resonator electrode was calculated from the amount of change in frequency.

Formula 1

Where ΔF is the frequency change amount, F ₀ is the fundamental frequency, Δm is the β-galactosidase binding amount, A is the area of the electrode, ρ _q is the density of the crystal, and μ _q is the shear stress of the crystal.

The amount of β-galactosidase bound per 1 cm ² was calculated by dividing the amount of β-galactosidase bound to the crystal resonator electrode (Δm, unit ng) by the area of the electrode. Table 6 summarizes the amount of change in frequency and the amount of β-galactosidase binding per cm ² for each concentration of β-galactosidase solution used for immersion.

FIG. 4 shows the results of plotting data with the concentration of β-galactosidase solution on the horizontal axis and the amount of β-galactosidase binding per cm ² on the vertical axis.

  In both Example 4 and Comparative Example 12, the amount of β-galactosidase bound to the QCM chip increased depending on the concentration of the β-galactosidase solution, but when the concentration of the β-galactosidase solution was 0.25 μmol / L or more, β -Galactosidase binding was saturated and did not increase any further. From this result, it can be seen that in order to saturate the amount of β-galactosidase to be bound to the QCM chip, it should be immersed in a β-galactosidase solution having a concentration of 0.25 μmol / L or more.

In each case of Example 4 and Comparative Example 12, an apparent binding constant (K _app ) was obtained by approximating the Langmuir adsorption isotherm (Formula 2).

Formula 2

However, A is the binding amount of β-galactosidase, A _max is the maximum binding amount of β-galactosidase, K _app is an apparent binding constant, and [β-Gal] is the concentration of β-galactosidase.

Table 7 summarizes the maximum binding amount (A _max ) and binding constant (K _app ) of β-galactosidase. R ² represents a correlation coefficient.

In Example 4 using a polymer film of a stereocomplex, the maximum binding amount of β-galactosidase is large and the binding constant is small, compared with Comparative Example 12 using a polymer film of it-PMMA alone. Was slight. The maximum binding amount of β-galactosidase of Example 4 was only 1.3 times that of Comparative Example 12.
Example 5 Preparation of β-Galactosidase Binding Plate After adding 200 μL of a 1.7 mg / mL it-PMMA solution (solvent: acetonitrile) to each well of a NUNC 96-well maxisorp plate, and incubating at 25 ° C. for 5 minutes, The it-PMMA solution was removed, washed with acetonitrile (special grade), then washed with ultrapure water, and dried with nitrogen gas. Subsequently, after adding 200 μL of 1.7 mg / mL st-PMMA solution (solvent: acetonitrile) and incubating at 25 ° C. for 5 minutes, the st-PMMA solution is removed, washed with acetonitrile (special grade), and then ultrapure water. And dried with nitrogen gas. This operation was defined as one cycle, and this cycle was repeated for a total of seven times. Thus, the polymer film which consists of a stereocomplex was formed in each well of 96 well maxi soap plate. 150 μL of the β-galactosidase solution prepared at 0.125 to 1 μmol / L was added to the well in which the polymer film composed of stereocomplex was formed, and incubated at room temperature for 1 hour. The β-galactosidase solution was removed, washed 3 times with 50 mmol / L phosphate buffer, and dried to prepare a measurement carrier.
[Comparative Examples 13-14]
Preparation of β-galactosidase-binding plate Using a Nunk 96-well maxi soap plate as a support, add 50 mg to 100 μL of 1.7 mg / mL it-PMMA solution (solvent: acetonitrile) and incubate at 25 ° C. overnight, then it -The PMMA solution was removed and dried with nitrogen gas. In the same manner as in Example 5, 150 μL of the β-galactosidase solution prepared at 0.125 to 1 μmol / L was added to the well in which the polymer film of it-PMMA was formed, and incubated at room temperature for 1 hour. The β-galactosidase solution was removed, washed 3 times with 50 mmol / L phosphate buffer, and dried to prepare a measurement carrier. This was designated as Comparative Example 13. Comparative Example 14 was prepared by fixing β-galactosidase in the same manner as in Example 5 to the well of a NUN 96-well maxisorp plate in which no polymer film was formed.
[Test Example 6]
In order to measure the activity of β-galactosidase, p-nitrophenylgalactopyranoside (hereinafter abbreviated as PNPG) which is a substrate of β-galactosidase was added to the wells prepared in Example 5, Comparative Example 13 and Comparative Example 14. The solution was added, and the amount of color-developing material produced by the decomposition of PNPG was determined colorimetrically. PNPG was added to 0.1 mol / L phosphate buffer (containing 3.36 mmol / L mercaptoethanol and 30 mmol / L magnesium chloride) to prepare a 2 mmol / L PNPG solution. The absorbance at a wavelength of 405 nm immediately after the addition of PNPG was measured, and thereafter, the absorbance at a wavelength of 405 nm was measured every minute after the addition of PNPG. The experiment was performed on 3 wells, and the average value was calculated. FIG. 5 shows a plot of absorbance at a wavelength of 405 nm versus time after addition of PNPG for each case where the concentration of the β-galactosidase solution used for fixation was 0.125, 0.25, 0.5, and 1 μmol / L.

In any case of Example 5 and Comparative Examples 13 and 14, the enzymatic reaction progressed with time, but the activity value was the highest in the case of the polymer film comprising the stereocomplex of Example 5. Dividing the absorbance change of FIG. 5 for 5-20 minutes by the amount of β-galactosidase bound to the wells of the 96-well maxisorp plate at each concentration of β-galactosidase, the unit amount (mg) and per unit time The (min) β-galactosidase activity was determined and summarized in Table 8. The binding amount of β-galactosidase immobilized on the well of a 96-well maxisorp plate was obtained by calculating the contact area of the β-galactosidase solution in the well, and β-galactosidase binding per 1 cm ² of Table 6 was calculated as the area value. It can be calculated by multiplying the quantity.

  The results of Table 8 show that β-galactosidase immobilized on the polymer membrane comprising the stereocomplex of Example 5 is β-galactosidase immobilized on the polymer membrane of it-PMMA of Comparative Example 13 and the polymer membrane of Comparative Example 14 It is shown that the activity is maintained more than β-galactosidase immobilized in wells without any. This maintenance of β-galactosidase activity also indicates that it is independent of the concentration of β-galactosidase solution when immobilizing β-galactosidase.

[Test Example 7] Storage stability of β-galactosidase-immobilized carrier The carriers of Example 5 and Comparative Examples 13 and 14, which are maxisorps immobilized with β-galactosidase, were stored for a certain period after preparation, and were the same as in Test Example 6. The β-galactosidase activity was measured by various methods, and the change in performance over time was examined. The storage condition was to store in a container containing silica gel shielded from light at room temperature, and the measurement was performed 7 days after the start of storage. FIG. 6 shows a plot of absorbance at a wavelength of 405 nm versus time after addition of PNPG when the concentration of the β-galactosidase solution used for immobilization is 1 μmol / L.

  In any case of Example 5 and Comparative Examples 13 and 14, the activity value increased with time, but the activity value was highest in the case of the polymer film made of the stereocomplex of Example 5. The amount of β-galactosidase per unit amount (mg) and per unit time (min) was determined from the change in absorbance for 5 to 20 minutes in FIG.

  In all cases of Example 5 and Comparative Examples 13 and 14, it was found that the activity before storage was reduced by storage for 7 days. In the case of Comparative Example 13, the activity value before storage was If 100%, the activity value after 7 days is 8.5%, whereas in Example 5, if the activity value before storage is 100%, the activity value after 7 days is 14.6%, Example 5 has a smaller reduction rate. From this, it is considered that β-galactosidase immobilized on the stereocomplex of Example 5 is more active.

In the measurement carrier of the present invention, an aggregate of two or more polymers having different stereoregularity is formed on a support, so that denaturation of protein A fixed on the polymer aggregate is suppressed, and the carrier is excellent. Storage stability. Furthermore, since the antibody binding amount and the antigen binding amount per unit area increase, it is possible to improve measurement accuracy and sensitivity, and to quickly measure a small amount of sample. Therefore, the present invention provides a measurement object measurement carrier, a measurement method, and a measurement kit that are useful for diagnosis of diseases.

Claims (23)

Measures an object to be measured in a sample, including a support having an aggregate region of two or more polymers having different stereoregularities on the surface, and a molecular binding substance fixed to the aggregate region of the support. Carrier to do. The carrier according to claim 1, wherein the aggregate includes an aggregate that forms a sterically complementary association. The carrier according to claim 1 or 2, wherein the two or more polymers having stereoregularity include a syndiotactic polymer and an isotactic polymer. The carrier according to any one of claims 1 to 3, wherein the aggregate is an aggregate including a helical structure or a van der Waals structure. The carrier according to any one of claims 1 to 4, wherein at least one of the polymers is polymethacrylate. The carrier according to claim 5, wherein the polymethacrylate is polymethyl methacrylate, polyethyl methacrylate, polyisopropyl methacrylate or poly n-propyl methacrylate. The carrier according to any one of claims 1 to 6, wherein the support is in the form of a plate, a chip, a bead, a fiber or a membrane. The carrier according to any one of claims 1 to 7, wherein the carrier is subjected to a blocking treatment with a blocking agent. The carrier according to any one of claims 1 to 8, wherein the molecular binding substance is an enzyme. The carrier according to any one of claims 1 to 8, wherein the molecule binding substance is a capture molecule binding protein. The carrier according to claim 10, wherein the capture molecule binding protein is protein A, protein G, or protein L. A capture molecule-binding carrier for measuring a measurement object in a sample, wherein a capture molecule is bound to the carrier according to claim 10 or 11 via the capture molecule-binding protein. A reagent for measuring a measurement object, comprising the carrier according to claim 1. A measurement object measuring kit comprising the carrier according to claim 1. A kit for measuring an object to be measured in a sample, comprising a reagent comprising a support having on its surface an aggregate region of two or more polymers having different stereoregularities, and a reagent containing a substance for molecular binding. The measurement kit according to claim 15, further comprising a reagent containing a capture molecule. A step of bringing the carrier according to claim 10 or 11 into contact with a capture molecule to form a capture molecule-bound carrier, contacting the capture molecule-bound carrier with a measurement object, and bringing the measurement object into contact with the capture molecule A method for measuring a measurement object in a sample, comprising the steps of binding to a carrier and measuring the measurement object bound to the carrier. A step of bringing a capture molecule into contact with a measurement object to form a measurement object-bound capture molecule, wherein the carrier according to claim 10 or 11 is brought into contact with the measurement object-bound capture molecule to bind the measurement object to the carrier. And a method for measuring a measurement object in a sample, the method comprising: a step of measuring the measurement object bound to the carrier. A step of bringing the carrier according to claim 9 into contact with a measurement target, causing the measurement target to react with an enzyme immobilized on the carrier, and a step of measuring a change amount of a substance that increases or decreases due to the reaction. A method for measuring an object to be measured in a sample. A sample comprising: a step of bringing the carrier according to claim 12 into contact with a measurement target, binding the measurement target to the carrier via a capture molecule; and measuring the measurement target bound to the carrier. Method of measuring objects. A carrier for measuring an object to be measured in a sample, characterized in that a substance for molecular binding is immobilized on an aggregate region of a support having two or more polymer aggregate regions having different stereoregularities on the surface. Manufacturing method. Immobilizing a molecular binding substance on an aggregate area of a support having two or more polymer aggregate areas having different stereoregularities on the surface, and binding a capture molecule to the molecular binding substance. A method for producing a capture molecule-binding carrier for measuring an object to be measured in a sample. Different stereoregulations used as a carrier for immobilizing a molecular binding substance on an aggregate region of two or more polymers having different stereoregularities on a support surface and measuring a measurement object in a sample A polymer-attached support having an aggregate region of two or more polymers having properties on the surface.