Method for improving photocleavage efficiency of photocleavable linker, photocleavable linker-bound magnetic particles, and method for measuring target substance and kit for use therein

Abstract

This method for improving the photocleavage efficiency of a photocleavable linker comprises: preparing a photocleavable linker-bound magnetic particle including a magnetic particle and a photocleavable linker; and inserting a spacer between the magnetic particle and the photocleavable linker.

Description

[Technical field]

[0001] The present invention relates to a method for improving the photocleavage efficiency of a photocleavable linker, photocleavable linker-bound magnetic particles, and a measurement method for measuring a target substance using the photocleavable linker-bound magnetic particles, and a kit for use in the method. [Background technology]

[0002] Conventionally, immunological measurement methods such as sandwich method, competitive method, and immunoturbidimetric method, and measurement methods based on these principles, are known as measurement methods for detecting the presence or absence of biomolecules and the like contained in a sample or quantifying them. Examples of the sandwich method include a method in which a probe molecule capable of specifically binding to a target substance such as the biomolecule is immobilized on a solid phase such as a plate or particles, and the target substance is captured and detected by the probe molecule. Examples of the competitive method include a method in which the target substance is indirectly measured by making the probe molecule immobilized on the solid phase compete with a competitor of the target substance, capturing the competitor, or immobilizing the competitor on the solid phase, making the target substance compete with the competitor, capturing the probe molecule for detection with the competitor, and detecting it.

[0003] It is known that the detection of the target substance or competing substance is carried out, for example, via detection of a labeled substance such as an enzyme or a luminescent substance. Furthermore, for example, International Publication No. 2005 / 075997 (Patent Document 1) describes the use of a labeled specific binding substance, which is a substance that specifically binds to the test substance and is bound to magnetic beads via a spacer, as a probe molecule immobilized on the solid phase, and detection of the test substance (target substance) is carried out via detection of a magnetic signal.

[0004] In addition, it is known that a photocleavable linker is used for binding the solid phase to the probe molecule or the competitor, because it can easily recover (re-release) the captured detection target while suppressing damage to the target. The photocleavable linker is mainly used in a method for recovering a biomolecule or the like from a sample or specifically releasing a target substance. For example, Bioconjug Chem. 2010 October 20; 21(10): 1917-1924 (Non-Patent Document 1) describes that, for the purpose of evaluating protein kinase, magnetic beads to which a spacer, a photocleavable linker, and a substrate peptide are bound are used, and the substrate peptide after the reaction is released by cleavage of the photocleavable linker, and this is measured using MALDI-TOF MS. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2005 / 075997 [Non-patent literature]

[0006] [Non-Patent Document 1] Bioconjug Chem.2010 October 20;21(10):1917-1924 Summary of the Invention [Problem to be solved by the invention]

[0007] As the solid phase for immobilizing the probe molecule, the competitor, etc., a microwell plate, a membrane, particles, etc. can be used. In particular, it is preferable to use particles from the viewpoint of reaction efficiency. On the other hand, in recent years, in the detection of the labeled substance in the above-mentioned immunological measurement method, etc., the use of a microchemical analysis method using a fine flow path (microchannel) provided on a glass or plastic substrate has been considered due to advantages such as reaction efficiency, measurement sensitivity, and shortened measurement time. However, in order to use such a microchemical analysis method, it is necessary to separate the detection target from the microwell plate, membrane, etc. In addition, the particle size of the above-mentioned particles is large compared to the captured detection target (target substance, competitor substance, etc.) and the labeled substance for detection, so it is difficult to directly subject them to the microchemical analysis method, and it is necessary to separate the particles from the detection target.

[0008] As the particle, it is more preferable to use magnetic particles from the viewpoint of automation and shortening the time. Therefore, the present inventors have investigated the use of the photocleavable linker for binding the magnetic particles to the probe molecules, with the aim of easily separating the captured detection target from the magnetic particles while suppressing damage to the target. However, the present inventors have found that when the photocleavable linker is combined with the magnetic particles, the photocleavage efficiency is significantly reduced compared to the case of using other particles or plates, and when the amount of light irradiation or time is increased to increase the reduced photocleavage efficiency, some detection targets are denatured or decomposed by light or heat, resulting in a new problem of reduced measurement accuracy.

[0009] The present invention has been made in consideration of the problems newly discovered by the inventors described above, and aims to provide a method for improving the photocleavage efficiency of a photocleavable linker in a photocleavable linker-bound magnetic particle comprising a magnetic particle and a photocleavable linker, photocleavable linker-bound magnetic particles with excellent photocleavage efficiency, and a measurement method for measuring a target substance using these and a kit for use in said measurement. [Means for solving the problem]

[0010] As a result of intensive research to solve the above problems, the present inventors have found that in a photocleavable linker-bound magnetic particle comprising a magnetic particle and a photocleavable linker, by inserting a spacer between the magnetic particle and the photocleavable linker, the photocleavage efficiency of the photocleavable linker can be significantly improved. In addition, by binding a probe molecule capable of capturing a detection target to the photocleavable linker and using it in the above immunological measurement method, the detection target can be easily recovered (re-released) while suppressing damage to the detection target, and therefore the present invention has been completed.

[0011] The present invention, based on these findings, has the following features. [1] A photocleavable linker-bound magnetic particle comprising a magnetic particle and a photocleavable linker, Inserting a spacer between the magnetic particle and the photocleavable linker; A method for improving the photocleavage efficiency of a photocleavable linker. [2] The method according to [1], wherein the photocleavable linker is photocleaved in solution. [3] The method according to [1] or [2], wherein the distance between the magnetic particle and the photo-cleavable linker is set to 4 to 170 nm by inserting the spacer. [4] The method according to any one of [1] to [3], wherein the spacer is at least one selected from the group consisting of proteins, polyethylene glycol, and dextran. [5] A photocleavable linker-bound magnetic particle comprising a magnetic particle and a photocleavable linker, A spacer is provided between the magnetic particle and the photocleavable linker, and A probe molecule is provided at the end of the photocleavable linker opposite the spacer. Photocleavable linker-bound magnetic particles. [6] The photo-cleavable linker-bound magnetic particle according to [5], wherein the distance between the magnetic particle and the photo-cleavable linker is 4 to 170 nm. [7] The photo-cleavable linker-bound magnetic particle described in [5] or [6], wherein the spacer is at least one selected from the group consisting of proteins, polyethylene glycol, and dextran. [8] The photo-cleavable linker-bound magnetic particle described in any one of [5] to [7], wherein the probe molecule is at least one selected from the group consisting of polynucleotides, polypeptides, and low molecular weight compounds. [9] A measurement method for measuring a target substance in a sample, a capture step of contacting the photo-cleavable linker-bound magnetic particle according to any one of [5] to [8], the probe molecule being a molecule capable of specifically binding to the target substance, with the sample, and causing the photo-cleavable linker-bound magnetic particle to capture the target substance; a release step of photocleaving the photocleavable linker to release a complex containing the target substance and the probe molecule from the magnetic particle to obtain a prepared sample; a measuring step of measuring the target substance by detecting the complex in the prepared sample; A measurement method including:

[10] The measurement method described in [9], further comprising a labeling step of capturing the target substance with a labeled detection molecule in which a detection molecule capable of specifically binding to the target substance is labeled with a labeling substance, the complex is a complex containing the target substance, the probe molecule, and the labeled detection molecule, and the complex is detected by the labeling substance.

[11] A kit for use in the method for measuring a target substance in a sample according to [9] or

[10] , [5] to [8], the photo-cleavable linker-bound magnetic particle according to any one of the above [5] to [8], wherein the probe molecule is a molecule capable of specifically binding to the target substance. Including the kit.

[12] The kit described in

[11] , further comprising a labeled detection molecule in which the detection molecule capable of specifically binding to the target substance is labeled with a labeling substance.

[13] A measurement method for measuring a target substance in a sample, a capture step of contacting the photo-cleavable linker-bound magnetic particle according to any one of [5] to [8], the probe molecule being a molecule capable of specifically binding to the target substance and a competitor of the target substance, with the sample and the competitor, and allowing the target substance to compete with the competitor, thereby capturing the competitor on the photo-cleavable linker-bound magnetic particle; a releasing step of photocleaving the photocleavable linker to release a complex containing the competitor and the probe molecule from the magnetic particles to obtain a prepared sample; a measuring step of measuring the target substance by detecting the complex in the prepared sample; A measurement method including:

[14] The measurement method according to

[13] , wherein the competitor is a labeled competitor labeled with a labeling substance, and the detection of the complex is detection using the labeling substance.

[15] A kit for use in the method for measuring a target substance in a sample according to

[13] or

[14] , [5] to [8], the photo-cleavable linker-bound magnetic particle according to any one of the above [5] to [8], wherein the probe molecule is a molecule capable of specifically binding to the target substance and a competitive substance of the target substance. Including the kit.

[16] The kit described in

[15] , further comprising a labeled competitor in which the competitor to the target substance is labeled with a labeling substance.

[17] A measurement method for measuring a target substance in a sample, a capture step of contacting the photo-cleavable linker-bound magnetic particle according to any one of [5] to [8], the probe molecule being a competitor of the target substance, with the sample and a detection molecule capable of specifically binding to the target substance and the competitor, and allowing the detection molecule to compete with the target substance and the probe molecule, thereby capturing the detection molecule on the photo-cleavable linker-bound magnetic particle; a release step of photocleaving the photocleavable linker to release a complex containing the detection molecule and the probe molecule from the magnetic particle to obtain a prepared sample; a measuring step of measuring the target substance by detecting the complex in the prepared sample; A measurement method including:

[18] The measurement method according to

[17] , wherein the detection molecule is a labeled detection molecule labeled with a labeling substance, and the detection of the complex is detection using the labeling substance.

[19] A kit for use in the method for measuring a target substance in a sample according to

[17] or

[18] , [5] to [8], wherein the photo-cleavable linker-bound magnetic particle is the photo-cleavable linker-bound magnetic particle according to any one of [5] to [8], and the probe molecule is a competitive substance for the target substance. Including the kit.

[20] The kit described in

[19] , further comprising a labeled detection molecule in which the detection molecule capable of specifically binding to the target substance and the competitor is labeled with a labeling substance.

[0012] Although the reason why the above object is achieved by the configuration of the present invention is not necessarily clear, the present inventors speculate as follows. That is, when a photocleavable linker is combined with a magnetic particle, the light absorption rate of the magnetic particle is high, so that when the photocleavable linker is irradiated with light to cleave it, the irradiated light is absorbed by the magnetic particle, and the amount of light that reaches the photocleavable linker is reduced. Therefore, the present inventors speculate that the cleavage of the photocleavable linker is inhibited and the photocleavage efficiency is reduced. In contrast, in the present invention, the present inventors speculate that the distance between the magnetic particle and the photocleavable linker is maintained by inserting a spacer between the magnetic particle and the photocleavable linker, so that the absorption of light by the magnetic particle in the vicinity of the photocleavable linker by the magnetic particle is suppressed, and the amount of light that reaches the photocleavable linker can be sufficiently secured, thereby achieving excellent photocleavage efficiency.

[0013] In addition, even if the detection target captured by the magnetic particles is a substance that is easily decomposed by light or heat, the improved photocleavage efficiency eliminates the need to apply excessive light or heat, and damage to the detection target can be sufficiently suppressed. Therefore, according to the measurement method for measuring a target substance using the photocleavable linker-bound magnetic particles of the present invention, it is also possible to improve the measurement accuracy. Furthermore, since the magnetic particles and the detection target can be easily separated by light while enjoying the advantages of automation and shortening the time by the magnetic particles, it is easily possible to use more accurate measurement means such as microchemical analysis and the measurement method described in International Publication No. 2018 / 079761. Effect of the Invention

[0014] According to the present invention, it is possible to provide a method for improving the photocleavage efficiency of a photocleavable linker-bound magnetic particle comprising a magnetic particle and a photocleavable linker, a photocleavable linker-bound magnetic particle having excellent photocleavage efficiency, and a measurement method for measuring a target substance using these and a kit for use therein. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram showing an example of a first embodiment of a target substance measurement method of the present invention. [Diagram 2] FIG. 2 is a schematic diagram showing an example of a second embodiment of the target substance measurement method of the present invention. [Diagram 3] FIG. 2 is a schematic diagram showing an example of a third embodiment of the target substance measurement method of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention will be described in detail below based on preferred embodiments thereof.

[0017] <Method for improving the photocleavage efficiency of photocleavable linkers> The method for improving the photocleavage efficiency of a photocleavage linker of the present invention (hereinafter sometimes simply referred to as the "method for improving the photocleavage efficiency") is characterized in that in a photocleavage linker-bound magnetic particle comprising a magnetic particle and a photocleavage linker, a spacer is inserted between the magnetic particle and the photocleavage linker.

[0018] In the present invention, the term "photo-cleavable linker-bound magnetic particles" refers to those comprising at least magnetic particles and a photo-cleavable linker.

[0019] [Magnetic particles] In the present invention, the term "magnetic particles" refers to particles that contain at least a magnetic material and are magnetized at least while a magnet is applied from the outside. Examples of the magnetic material include ferromagnetic metals such as iron, cobalt, and nickel (e.g., spinel ferrite, cobalt oxide, and nickel ferrite) and alloys containing at least one of them, and one of them may be used alone or in combination of two or more. The magnetic particles according to the present invention may be formed by molding the magnetic material into a particle shape alone, or may be formed by using the magnetic material as a core and coating the surface of the core with a non-magnetic material, or may be formed by using the non-magnetic material as a core and coating the magnetic material.

[0020] The non-magnetic material is not particularly limited, but examples thereof include high molecular weight polymers (e.g., polystyrene, (meth)acrylic acid esters, polymethyl methacrylate, polyimide, nylon), gelatin, cellulose, nitrocellulose, glass, latex, silica, and non-magnetic metals (e.g., gold, platinum), and any one of these may be used alone or two or more may be used in combination.

[0021] The magnetic particles according to the present invention may be surface-modified with at least one active group selected from the group consisting of carboxyl, epoxy, tosyl, amino, hydroxyl, isothiocyanate, isocyanate, azide, aldehyde, carbonate, allyl, aminooxy, maleimide, thiol, NHS ester (N-hydroxyester), and pyridyl disulfide groups, or may have a bond residue between the active group and the spacer described below. The magnetic particles may also be modified with biotin or the like.

[0022] The particle diameter of the magnetic particles according to the present invention is preferably within the range of 0.1 to 10 μm, more preferably within the range of 0.5 to 8 μm. If the particle diameter of the magnetic particles is less than the lower limit, more magnetism and time will be required for magnetic collection, while if the particle diameter exceeds the upper limit, the specific surface area of ​​the particles will be small, and when the probe molecule described below is immobilized and a target substance is measured using a labeling substance, the amount of the labeling substance bound to the magnetic particles will tend to be small.

[0023] As such magnetic particles, conventionally known ones can be appropriately used, and for example, commercially available magnetic particles such as the magnetic particles described in JP-A-3-115862 (ferrite-coated particles, carboxylated ferrite particles), Dynabeads (manufactured by Thermo Fisher Scientific), Magnosphere (manufactured by JSR Corporation), Magrapid (manufactured by Sanyo Chemical Industries, Ltd.) and the like can also be appropriately used. Furthermore, the magnetic particles according to the present invention may be one of these alone or a combination of two or more of them.

[0024] [Photocleavable linker] In the present invention, the term "photocleavable linker" refers to a compound containing a structure (photocleavable unit) that is decomposed by irradiation with light. Examples of the photocleavable unit include 3-amino-3-(2-nitrophenyl)propionic acid (ANP) and coumarin. The photocleavable linker according to the present invention may have a structure such as an alkyl chain or an ethylene glycol chain in addition to the photocleavable unit. In addition, the photocleavable linker may have at least one active group added thereto, such as a carboxy group, an epoxy group, a tosyl group, an amino group, a hydroxy group, an isothiocyanate group, an isocyanate group, an azide group, an aldehyde group, a carbonate group, an allyl group, an aminooxy group, a maleimide group, a thiol group, an NHS ester (N-hydroxyester) group, a pyridyl disulfide group, or the like, or may have a binding residue between the active group and the spacer and / or probe molecule described below. In addition, the photocleavable linker may be modified with biotin or the like.

[0025] As such a photocleavable linker, a conventionally known one can be appropriately used, and for example, commercially available photocleavable linkers such as N-hydroxysuccinimide (NHS)-photocleavable linker (PC)-biotin (trade name: PC-Biotin-NHS, manufactured by Ambergen), Fmoc-aminoethyl photolinker (manufactured by Novabiochem), PC DBCO-PEG4-NHS ester (manufactured by BroadPharm), PC Mal-NHS carbonate ester (manufactured by BroadPharm, etc.) can also be appropriately used. Furthermore, the photocleavable linker according to the present invention may be one of these alone or a combination of two or more of them.

[0026] [Spacer] In the present invention, the "spacer" may be any molecule that can be inserted between the magnetic particle and the photocleavable linker and can maintain the distance between the magnetic particle and the photocleavable linker by its length and / or size based on the molecular weight or mass per molecule. The molecule constituting the spacer according to the present invention may be at least one selected from the group consisting of proteins, polyethylene glycol, and dextran.

[0027] Examples of proteins constituting the spacer according to the present invention include avidins such as avidin D and streptavidin; albumin, casein, and collagen, and one of these may be used alone or two or more may be used in combination. Among these, the protein is preferably avidins, and more preferably streptavidin, from the viewpoint of maintaining the distance between the magnetic particle and the photocleavage linker within a more suitable range and being more effective in improving the photocleavage efficiency.

[0028] The polyethylene glycol constituting the spacer according to the present invention has a weight average molecular weight (Mw) measured by using polymethyl methacrylate as a standard polymer using gel permeation chromatography (GPC) in order to maintain the distance between the magnetic particles and the photocleavable linker within a more suitable range and to have a more excellent effect of improving the photocleavage efficiency. The weight average molecular weight (Mw) is preferably 0.5K to 21K, more preferably 0.8K to 20K, even more preferably 0.8K to 10K, even more preferably 0.8K to 8K, and particularly preferably 1K to 6K. If the weight average molecular weight of the polyethylene glycol is less than the lower limit, the distance between the magnetic particles and the photocleavable linker tends to be short, and the effect of improving the photocleavage efficiency by the spacer tends to be insufficient. On the other hand, if it exceeds the upper limit, when the following probe molecule is immobilized and the target substance is measured using a labeling substance, the amount of the labeling substance bound to the magnetic particles tends to be reduced.

[0029] From the viewpoint of a more excellent effect of improving the photocleavage efficiency, the dextran constituting the spacer according to the present invention preferably has a mass per molecule of 5K to 75KDa, more preferably 10K to 70KDa, and even more preferably 20K to 70KDa. If the mass of the dextran is less than the lower limit, the effect of improving the photocleavage efficiency by the spacer tends to be insufficient, while if it exceeds the upper limit, when the probe molecule described below is immobilized and the target substance is measured using the labeling substance, the amount of the labeling substance bound to the magnetic particles tends to be reduced.

[0030] The spacer according to the present invention may be one to which at least one active group selected from the group consisting of a carboxy group, an epoxy group, a tosyl group, an amino group, a hydroxy group, an isothiocyanate group, an isocyanate group, an azide group, an aldehyde group, a carbonate group, an allyl group, an aminooxy group, a maleimide group, a thiol group, an NHS ester (N-hydroxyester) group, a pyridyl disulfide group, etc. is added, or one having a binding residue between the active group and the magnetic particle and / or the photocleavable linker. Also, the spacer may be one modified with biotin, etc.

[0031] The spacer according to the present invention may be one or more of the above proteins, polyethylene glycol, and dextran. Among these, the spacer according to the present invention is preferably one made of the above polyethylene glycol, one made of the above polyethylene glycol and avidins (more preferably streptavidin), or one made of dextran, and more preferably one made of the above polyethylene glycol, or one made of the above polyethylene glycol and avidins (more preferably streptavidin).

[0032] (Method for improving light cutting efficiency) In the method for improving photocleavage efficiency of the present invention, in the photocleavable linker-bound magnetic particle, the spacer is inserted between the magnetic particle and the photocleavable linker.

[0033] Regarding the insertion of the spacer, the distance between the magnetic particle and the photocleavable linker after the insertion of the spacer is the longest distance in an aqueous solution from the surface of the magnetic particle to the end of the photocleavable linker on the magnetic particle side in the vertical direction, and is preferably within the range of 4 to 170 nm, more preferably within the range of 5 to 160 nm, even more preferably within the range of 6 to 80 nm, and even more preferably within the range of 6 to 50 nm. If the distance between the magnetic particle and the photocleavable linker is less than the lower limit, the effect of improving the photocleavage efficiency tends to be insufficient, while if it exceeds the upper limit, when the probe molecule described below is immobilized and the target substance is measured using a labeling substance, the amount of the labeling substance bound to the magnetic particle tends to be small. Such a longest distance indicates the longest diameter of the structure that the molecule constituting the spacer can have in an aqueous solution, and can be calculated, for example, by a known technique from the molecular weight and structure of the molecule constituting the spacer.

[0034] In addition, when the spacer is inserted, if the spacer is made of the protein and / or polyethylene glycol, the distance between the magnetic particle and the photocleavable linker can be maintained within a more suitable range, and from the viewpoint of excellent effect of improving the photocleavage efficiency, it is preferable that the protein is the avidin (more preferably streptavidin), and the magnetic particle and the photocleavable linker are bonded via a bond with a spacer made of at least one molecule (more preferably one molecule) of the avidin. From the same viewpoint, in this case, the polyethylene glycol preferably has a weight average molecular weight (Mw) of 0.5K to 21K, more preferably 0.8K to 20K, even more preferably 0.8K to 10K, even more preferably 0.8K to 8K, and particularly preferably 1 to 6K. Furthermore, in this case, it is preferable that the magnetic particle and the photocleavable linker are bonded via a bond with a spacer made of at least one molecule (more preferably one molecule) of polyethylene glycol. Alternatively, the magnetic particle and the photocleavable linker are preferably bonded to a spacer consisting of at least one molecule (more preferably one molecule) of the avidin and at least one molecule (more preferably one molecule) of polyethylene glycol, although two or more photocleavable linkers may be bonded to one molecule of the protein and / or polyethylene glycol.

[0035] In addition, when the spacer is inserted and the spacer is made of dextran, the mass of the dextran per molecule is preferably 5K to 75KDa, more preferably 10K to 70KDa, and even more preferably 20K to 70KDa, from the viewpoint of a more excellent effect of improving the photocleavage efficiency. Furthermore, it is preferable that the magnetic particle and the photocleavable linker are bonded via a bond with a spacer made of at least one molecule (more preferably one molecule) of dextran. However, two or more photocleavable linkers may be bonded to one molecule of dextran.

[0036] In the method for improving photocleavage efficiency of the present invention, the method for inserting the spacer between the magnetic particle and the photocleavage linker is not particularly limited, and any known method can be adopted as appropriate. For example, such a method includes a method of binding (fixing) the spacer and the photocleavage linker to the magnetic particle in this order, and such binding method is not particularly limited, and any known method or a method similar thereto can be adopted as appropriate. In this case, each bond between the magnetic particle and the spacer and between the spacer and the photocleavage linker may be direct or indirect, independently of each other (hereinafter, the former of "-" will be referred to as "bound substance" and the latter as "bound substance").

[0037] Methods for directly binding the magnetic particles, the spacer, and the photocleavable linker, respectively, include, for example, a method in which at least one of the components constituting at least one of the above-mentioned conjugated substance and conjugated substance is provided with at least one active group selected from the group consisting of a carboxy group, an epoxy group, a tosyl group, an amino group, a hydroxy group, an isothiocyanate group, an isocyanate group, an azide group, an aldehyde group, a carbonate group, an allyl group, an aminooxy group, a maleimide group, a thiol group, an NHS ester (N-hydroxyester) group, a pyridyl disulfide group, etc., or a method in which a component having such an active group is used to bind the conjugated substance and the conjugated substance through a covalent bond via the active group.

[0038] The components to which the active groups have been added may be commercially available as they are, or may be prepared by introducing the active groups under appropriate reaction conditions. As an example, the introduction of a thiol group can be carried out using commercially available reagents such as S-acetylmercaptosuccinic anhydride, N-succinimidyl 3-(2-pyridyldithio)propionate, and 2-iminothiolane, and the introduction of a maleimide group into an amino group can be carried out using commercially available reagents such as N-(6-maleimidocaproyloxy)succinimide and N-(4-maleimidobutyryloxy)succinimide. In addition, the introduction of a pyridyl disulfide group can be carried out using commercially available reagents such as N-{6-[3-(2-Pyridyldithio)propionamido]hexanoyloxy}sulfosuccinimide, sodium salt (Sulfo-AC5-SPDP), and the like.

[0039] As a method for indirectly binding the magnetic particle, the spacer, and the photocleavable linker, for example, a component constituting one of the above-mentioned bound substance and the binding substance may be modified in some way, a substance that captures the modified portion may be immobilized on the other, and binding may be performed via such capture. For example, a representative example of the modified portion is biotin, and a representative example of the substance that captures the modified portion is streptavidin, but the methods are not limited thereto.

[0040] The ratio of the above-mentioned conjugate and conjugate to be subjected to these bindings can be appropriately selected, for example, so as to achieve the preferred range of ratios in the photocleavable linker-bound magnetic particles described below.

[0041] In the method for improving photocleavage efficiency of the present invention, the magnetic particles, the spacer, and the photocleavage linker may be bound at once or may be bound separately and sequentially. However, from the viewpoint of ease of operation and ease of control of the amount of each part, it is preferable to bind them separately and sequentially. Furthermore, it is also preferable to bind some of the conjugates and binders in advance and then combine and bind them. In addition, as such pre-bound conjugates and binders, commercially available conjugates such as SA-bound Dynabeads (trade name: Dynabeads M-280 Streptavidin, manufactured by Thermo Fisher Scientific) and magnetic streptavidin beads (trade name: Magnosphere MS300 / Streptavidin, manufactured by JSR Corporation) may be appropriately used.

[0042] Such a method for improving photocleavage efficiency of the present invention can sufficiently improve the photocleavage efficiency of the photocleavage linker, more preferably the photocleavage efficiency of the photocleavage linker cleaved in solution (even more preferably in an aqueous solution) (preferably represented by the following formula: photocleavage efficiency (cleavage rate, %) = {cleaved photocleavable linker / (cleaved photocleavable linker + uncleaved photocleavable linker)} x 100), compared to when the photocleavage linker is bound to the magnetic particle without the spacer. More specifically, it is possible to improve the photocleavage efficiency by about 6 to 30 times or more, compared to when the photocleavage linker is bound to the magnetic particle without the spacer.

[0043] <Photocleavable linker-bound magnetic particles> The photo-cleavable linker-bound magnetic particles of the present invention are, among the photo-cleavable linker-bound magnetic particles, The spacer is provided between the magnetic particle and the photocleavable linker, and The probe molecule is provided at the end of the photocleavable linker opposite to the spacer. That is, the photo-cleavable linker-bound magnetic particle of the present invention is composed of a magnetic particle, a spacer, a photo-cleavable linker, and a probe molecule bound in this order. Preferably, the magnetic particle is a core, and a layer of the spacer, a layer of the photo-cleavable linker, and a layer of the probe molecule are laminated on the surface of the core in this order.

[0044] [Probe molecule] In the present invention, the term "probe molecule" refers to a molecule capable of specifically binding to a target to be captured by a photocleavable linker-bound magnetic particle (sometimes referred to as a "detection target" in this specification). Examples of the detection target include the target substance, competitor, and detection molecule described in the measurement method for measuring the target substance below.

[0045] Probe molecules according to the present invention are not particularly limited, but include, for example, polynucleotides, polypeptides, low molecular weight compounds, composite molecules containing two or more of these molecules, and molecules having the same or similar structure as these.

[0046] In the present invention, the term "polynucleotide" refers to a compound consisting of two or more nucleotides, and the polynucleotide according to the present invention includes DNA, RNA, and fragments thereof.

[0047] In the present invention, the term "polypeptide" refers to a substance consisting of two or more amino acids, and the polypeptide according to the present invention includes not only full-length polypeptides encoded by genes, but also fragments thereof and synthetic polypeptides. The polypeptide also includes glycopeptides modified with sugar chains. Examples of the polypeptide as the probe molecule include antibodies; antigen peptides; binding proteins such as protein A, protein G, and protein L; avidins such as avidin D and streptavidin; various lectins such as concanavalin A, lentil lectin, and kidney bean lectin; glycopeptides having lectin-binding sugar chains (lectin-binding glycopeptides); and receptor proteins, transport proteins, and transcriptional regulators. The origin of the polypeptide is not particularly limited, and may be any of mammals, birds, reptiles, amphibians, fish, plants, insects, microorganisms, and viruses.

[0048] The antibody may be a polyclonal antibody or a monoclonal antibody. In the present invention, the term "antibody" includes complete antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, single-chain antibodies, diabodies, etc.) and minibodies in which variable regions of antibodies are bound. Among these, antibody fragments are more preferable, since the photocleavage efficiency of the photocleavable linker tends to be improved.

[0049] Examples of low molecular weight compounds as probe molecules according to the present invention include vitamins, coenzymes, hormones, toxins, antibiotics, antibacterial drugs, antiviral drugs, anticancer drugs, carcinogens, narcotics, psychotropic drugs, and immunosuppressants.

[0050] Furthermore, in the present invention, a "molecule having the same or similar structure" as the above-mentioned polynucleotide, polypeptide, low molecular weight compound, or complex molecule refers to a substance that can bind to the detection target, similar to the polynucleotide, polypeptide, low molecular weight compound, or complex molecule, and more specifically, includes a substance that cross-reacts with the polynucleotide, polypeptide, low molecular weight compound, or complex molecule when an antigen-antibody reaction is carried out with the polynucleotide, polypeptide, low molecular weight compound, or complex molecule.

[0051] The probe molecule according to the present invention may be one to which at least one active group selected from the group consisting of a carboxy group, an epoxy group, a tosyl group, an amino group, a hydroxy group, an isothiocyanate group, an isocyanate group, an azide group, an aldehyde group, a carbonate group, an allyl group, an aminooxy group, a maleimide group, a thiol group, an NHS ester (N-hydroxyester) group, a pyridyl disulfide group, etc. has been added, or one having a binding residue between the active group and the photocleavable linker. Also, the probe molecule may be one modified with biotin, etc.

[0052] The probe molecule of the present invention may be one of the above alone or a combination of two or more of them, and is not particularly limited, and can be appropriately selected depending on the principle adopted in the measurement method using the photo-cleavable linker-bound magnetic particles of the present invention and the substance to be measured. For example, from the viewpoint of reaction specificity, it is preferable that the probe molecule is at least one type selected from the group consisting of polynucleotides, polypeptides, hormones, and molecules having the same or similar structure as these, and it is more preferable that the probe molecule is at least one type selected from the group consisting of antibody fragments, DNA fragments, and RNA fragments.

[0053] (Configuration and manufacturing method of photocleavable linker-bound magnetic particles) The photo-cleavable linker-bound magnetic particles of the present invention may have multiple spacers bound to one magnetic particle, multiple photo-cleavable linkers bound to one spacer, or multiple photo-cleavable linkers bound to one probe molecule.

[0054] In the photocleavable linker-bound magnetic particles of the present invention, the content of the spacer is preferably set so that the number of spacers bound to one magnetic particle is as large as possible, and the mass of the spacer (the total amount of a combination of two or more types of magnetic particles, the same below) per part by mass of the magnetic particles (preferably with an average particle diameter of 8 μm or less, the same below) is preferably 0.1 to 5 parts by mass, more preferably 0.2 to 2 parts by mass.

[0055] When the spacer is the protein (preferably an avidin; the same applies below), the mass of the protein (if the protein is a combination of two or more types, the total amount thereof; the same applies below) per part by mass of the magnetic particle, when the spacer is polyethylene glycol (if the polyethylene glycol is a combination of two or more types, the total amount thereof; the same applies below), the total mass of the protein and polyethylene glycol per part by mass of the magnetic particle when the spacer is a combination of the protein and polyethylene glycol, and the mass of dextran (if the dextran is a combination of two or more types, the total amount thereof; the same applies below) per part by mass of the magnetic particle are also preferably 0.1 to 5 parts by mass, and more preferably 0.2 to 2 parts by mass.

[0056] In the photo-cleavable linker-bound magnetic particles of the present invention, the content of the photo-cleavable linker is preferably set so that a sufficient amount of probe molecules can be bound, and the mass of the photo-cleavable linker per 1 part by mass of the spacer (when the photo-cleavable linker is a combination of two or more types, the total amount of the photo-cleavable linkers, the same applies below) is preferably 0.001 to 1 part by mass, more preferably 0.02 to 1 part by mass.

[0057] For example, when the spacer is the protein, the mass of the photo-cleavable linker relative to 1 part by mass of the spacer is preferably 0.001 to 1 part by mass, more preferably 0.02 to 1 part by mass. For example, when the spacer is polyethylene glycol, the mass of the photo-cleavable linker relative to 1 part by mass of the spacer is preferably 0.01 to 1 part by mass, more preferably 0.02 to 1 part by mass. For example, when the spacer is a combination of the protein and polyethylene glycol, the mass of the photo-cleavable linker relative to 1 part by mass of the spacer is preferably 0.01 to 1 part by mass, more preferably 0.02 to 1 part by mass. For example, when the spacer is dextran, the mass of the photo-cleavable linker relative to 1 part by mass of the spacer is preferably 0.01 to 0.5 parts by mass.

[0058] In the photo-cleavable linker-bound magnetic particles of the present invention, the content of the probe molecules is preferably set so that the density of the probe molecules in one particle is as low as possible within a range that does not cause a decrease in sensitivity, from the viewpoint that the photocleavage efficiency of the photo-cleavable linker tends to be further improved.

[0059] The suitable mass ratio between the magnetic particles, spacers, photocleavable linkers and probe molecules varies greatly depending on the materials used, molecular weights, etc., and may therefore differ significantly from the above-mentioned range of mass ratios.

[0060] The photocleavable linker-bound magnetic particles of the present invention can be produced by binding (fixing) the spacer, the photocleavable linker, and the probe molecule to the magnetic particles in this order. There are no particular limitations on the production method, and any conventionally known method or a method similar thereto can be appropriately adopted. In this case, each bond between the magnetic particle and the spacer, between the spacer and the photocleavable linker, and between the photocleavable linker and the probe molecule may be direct or indirect, independently of each other (hereinafter, the former of "-" will be referred to as "bound substance" and the latter as "bound substance").

[0061] Methods for directly binding the magnetic particles, the spacer, the photocleavable linker, and the probe molecule, respectively, and methods for indirectly binding them, respectively, include methods similar to those listed as the method for inserting a spacer in the above-mentioned method for improving photocleavage efficiency.

[0062] The ratio of the above-mentioned conjugate and conjugate to be subjected to these bindings can be appropriately selected so as to achieve the preferred range of ratios in the above-mentioned photocleavable linker-bound magnetic particles.

[0063] In addition, in the production of the photocleavable linker-bound magnetic particles, the magnetic particles, the spacer, the photocleavable linker, and the probe molecule may be bound at once, or may be bound separately and sequentially, but from the viewpoints of ease of production and ease of control of the amount of each part, it is preferable to bind them separately and sequentially. Furthermore, it is also preferable to bind some of the conjugates and binders in advance and then combine and bind them. In addition, as such pre-bound conjugates and binders, commercially available conjugates similar to those listed in the above method for improving photocleavage efficiency may be used appropriately.

[0064] <Method for measuring target substances and kit for use therein> In the measurement method of the present invention for measuring a target substance (sometimes referred to as a "target substance measurement method" in this specification), the target substance in a sample is measured using the above-mentioned photocleavable linker-bound magnetic particles of the present invention.

[0065] 〔sample〕 The "sample" used in the method for measuring a target substance of the present invention is not particularly limited as long as it is a sample in which the target substance may be present. In general, examples of the sample include serum, plasma, whole blood, urine, stool, saliva, cerebrospinal fluid, oral mucosa, pharyngeal mucosa, intestinal mucosa, cell culture fluid, and various biopsy samples collected from a subject (human or animal, preferably human) for measuring the target substance of interest. Aqueous samples are preferred as samples according to the present invention. These samples may be diluted or suspended as necessary.

[0066] [Target substances] In the present invention, the term "target substance" refers to a substance that is desired to be measured. The target substance according to the present invention is not particularly limited, but may be, for example, a biomolecule, more specifically, a polynucleotide, a polypeptide, a low molecular weight compound, and a complex molecule containing two or more of these molecules, and one of these may be used alone or two or more may be used in combination. The polynucleotide, the polypeptide, the low molecular weight compound, and the complex molecule may each independently be the same as those listed in the above-mentioned probe molecule, including their preferred embodiments.

[0067] The target substance according to the present invention is not particularly limited, and can be selected by appropriately selecting the principle adopted in the measurement method using the photocleavable linker-bound magnetic particles of the present invention, the type of the probe molecule described above, and the type of the competitor substance described below, but for example, from the standpoint of specificity, it is preferable that the target substance is at least one type selected from the group consisting of polynucleotides, polypeptides, and hormones, and it is more preferable that the target substance is at least one type selected from the group consisting of antigenic substances, DNA having a complementary sequence, and RNA having a complementary sequence.

[0068] 〔measurement〕 In the present invention, "measurement" includes detection of the presence or absence and amount of a target substance in a sample as a signal, as well as quantification or semi-quantification of the amount of the target substance. In the present invention, the measurement of the target substance is preferably performed by detecting a signal generated by a labeling substance and quantifying it as necessary. The "signal" includes coloration (color development), reflected light, luminescence, fluorescence, radiation from a radioisotope, etc., and includes those that can be confirmed by the naked eye as well as those that can be confirmed by a measurement method or device according to the type of signal. According to the target substance measurement method of the present invention, while enjoying the advantages of automation and shortening the time by the magnetic particles, the magnetic particles and the detection target can be easily and efficiently separated by light, so that it is possible to easily use high-precision signal measurement means such as microchemical analysis and the measurement method described in International Publication No. 2018 / 079761.

[0069] The method of measuring the target substance using the photocleavable linker-bound magnetic particles of the present invention includes immunological measurement methods such as the sandwich method, competitive method, and immunoturbidimetric method, and measurement methods based on these principles, and is not particularly limited. In the target substance measurement method of the present invention, the target substance in the sample can be quantified by performing detection and quantification according to the type of the labeling substance and comparing it with the measured value in a standard sample. In addition, from the viewpoint of detecting the target substance more simply and quickly, a method such as immunochromatography can be adopted.

[0070] The method for measuring a target substance of the present invention will be described below in more specific embodiments. A preferred embodiment of the present invention will be described in more detail with reference to the drawings, but the present invention is not limited thereto. In the following description and drawings, the same or corresponding elements are denoted by the same reference numerals, and duplicated descriptions will be omitted.

[0071] (First aspect) As the method for measuring a target substance of the present invention, a sandwich method in which a target substance is detected using a capture body capable of capturing the target substance is preferred from the viewpoint of being able to construct a detection system with higher sensitivity and specificity. As the sandwich method, the following first aspect: a capture step of contacting the photo-cleavable linker-bound magnetic particle of the present invention, the probe molecule of which is a molecule capable of specifically binding to the target substance, with the sample, and causing the photo-cleavable linker-bound magnetic particle to capture the target substance; a release step of photocleaving the photocleavable linker to release a complex containing the target substance and the probe molecule from the magnetic particle to obtain a prepared sample; a measuring step of measuring the target substance by detecting the complex in the prepared sample; Measurement methods including is preferred.

[0072] In a first aspect, the photo-cleavable linker-bound magnetic particle of the present invention uses a photo-cleavable linker-bound magnetic particle in which the probe molecule is a molecule capable of specifically binding to the target substance. Examples of such a combination of a target substance and a molecule (probe molecule) capable of specifically binding to the target substance, or a combination of the probe molecule and the target substance, include combinations of an antigen (antigen peptide, low molecular weight compound, etc.) and an antibody, a lectin-binding glycopeptide and a lectin, and a nucleic acid and a nucleic acid having a complementary sequence to the nucleic acid.

[0073] [Capturing process] In the capture step of the first embodiment, the photocleavable linker-bound magnetic particle of the present invention (hereinafter sometimes referred to as "capture body") is brought into contact with the sample, and the target substance is captured by the capture body via the bond between the target substance and the probe molecule. The method of bringing the capture body into contact with the sample is not particularly limited, and a conventionally known method or a method similar thereto can be appropriately adopted, for example, a method of adding the capture body to the sample or a method of adding the sample to the capture body fluid.

[0074] The sample may be used after diluting with a sample diluent, or may be used after suspending in a particle suspension medium of the capture body, and further, other reaction buffers may be appropriately added to the reaction system (e.g., antigen-antibody reaction system) between the capture body and the sample. The sample diluent, particle suspension medium, and reaction buffer are not particularly limited, and examples thereof include known buffer solutions (sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, tricine buffer, bicine buffer, glycine buffer, etc.), and the particle suspension medium and reaction buffer may each independently contain a stabilizing protein such as BSA, serum, etc.

[0075] In the reaction between the capture body and the sample in the capture step of the first embodiment, the content (final concentration) of the capture body in the reaction system (when the capture body is a combination of two or more types, the total amount of the capture bodies, the same applies below) is not particularly limited as it is appropriately adjusted depending on the type, concentration, detection method, etc. of the sample, but from the viewpoint of efficient capture in a short period of time, it is preferably, for example, 0.005 to 0.2 mass%, and more preferably 0.01 to 0.1 mass%.

[0076] The conditions for the capture step of the first embodiment are also not particularly limited and can be adjusted as appropriate. For example, the capture step can be performed at room temperature to 45°C, preferably 20 to 37°C, at a pH of about 6 to 9, preferably 7 to 8, for about 5 seconds to 10 minutes, preferably 30 seconds to 5 minutes, but is not limited to these conditions.

[0077] [Cleaning process] In the method for measuring a target substance of the present invention, it is preferable to further include a washing step in which the target substance bound to the capture body is separated from other impurities not bound to (captured by) the capture body after the capture step and before the release step described below, and the impurities are removed. The method for removing the impurities is not particularly limited, and a conventionally known method or a method similar thereto can be appropriately adopted, for example, a method in which the capture body is recovered by centrifugation or magnetic collection after the capture step and the liquid phase (supernatant) is removed. In addition, in the washing step, injection and removal of a washing solution may be repeated as necessary. Examples of the washing solution include known neutral (preferably pH 6 to 9) buffer solutions (sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, tricine buffer, bicine buffer, glycine buffer, etc.), and may also be those to which a stabilizing protein such as BSA or a surfactant has been added.

[0078] [Release process] In the release step of the first embodiment, the photocleavable linker is cleaved to release the complex of the target substance bound to the capture body and the probe molecule from the magnetic particle, thereby obtaining a prepared sample containing the complex as a sample to be subjected to the measurement step.

[0079] The method of cleaving the photocleavable linker includes a method of cleaving it by light irradiation. In the release step, the light source and the light amount of the light to be irradiated are not particularly limited and can be appropriately selected depending on the type of the photocleavable linker. For example, when PC Mal-NHS carbonate ester is used, the conditions include irradiation with ultraviolet light having a wavelength of 300 to 400 nm, preferably 340 to 370 nm, at 10 to 50°C, preferably 20 to 37°C, for about 10 seconds to 10 minutes, preferably 10 seconds to 5 minutes, but are not limited to these conditions. However, in the present invention, the photocleavage efficiency of the photocleavable linker is superior to that when the photocleavable linker is bound to the magnetic particle without the spacer, so that the light irradiation time can be shortened.

[0080] [Measurement process] In the measurement step of the first embodiment, the target substance is measured by detecting the complex in the prepared sample. The detection of the complex is preferably carried out by capturing the target substance with a labeled detection molecule obtained by labeling a detection molecule capable of specifically binding to the target substance with a labeling substance (labeling step), forming a complex containing the target substance, the probe molecule, and the labeled detection molecule as the complex, and detecting the complex with the labeling substance.

[0081] The detection molecule according to the first aspect may be selected from the molecules similar to those listed as the probe molecule according to the type of the target substance as long as it can specifically bind to the target substance. The detection molecule may be the same type as the probe molecule contained in the photocleavable linker-bound magnetic particle of the present invention.

[0082] In the present invention, the labeling substance may be any of those used as labeling substances in known immunological assays without any particular limitation. Examples of such labeling substances include enzymes, luminescent substances such as acridinium derivatives, fluorescent substances such as europium, fluorescent proteins such as allophycocyanin (APC) and phycoerythrin (R-PE), etc. 125Examples of the labeling substance include radioactive substances such as I; low molecular weight labeling substances such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC); gold particles; avidin; biotin; latex; dinitrophenyl (DNP); and digoxigenin (DIG), and one of these may be used alone or in combination of two or more. For example, when an enzyme is used as the labeling substance, various detection and quantification can be performed according to the substrate by adding a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, or the like as a substrate. Among these, enzymes are preferable as the labeling substance according to the present invention from the viewpoint of tending to enable simple and stable tests and from the viewpoint of being able to perform simple, rapid, and highly sensitive measurements. Examples of the enzyme include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase (β-gal), glucose oxidase, and luciferase.

[0083] The labeled detection molecule according to the first aspect can be produced by binding the detection molecule to the labeling substance. As the binding method, a conventionally known method or a method similar thereto can be appropriately adopted, and the detection molecule and the labeling substance may be directly or indirectly bound. As such a binding method, the same method as the method given as the method for producing the photocleavable linker-bound magnetic particle of the present invention (i.e., the same method as the method given as the method for inserting a spacer in the above-mentioned method for improving photocleavage efficiency) can be mentioned. In addition, as the labeled detection molecule according to the first aspect, a commercially available one may be appropriately used, and for example, when the target substance is an antigenic substance, a commercially available labeled antibody against it can be appropriately used.

[0084] In the labeling step, the amount of the labeled detection molecule added (final concentration) (when the labeled detection molecule is a combination of two or more types, the total amount of the labeled detection molecules; the same applies below) is appropriately adjusted depending on the type, concentration, detection method, etc. of the sample, and is not particularly limited, but is preferably 0.05 to 1 μg / mL, and more preferably 0.1 to 0.5 μg / mL, for example.

[0085] Examples of such sandwich methods include the two-step forward sandwich method (in which a photocleavable linker-bound magnetic particle (capture body) reacts with the target substance in a sample, and then the target substance bound to the capture body reacts with a labeled detection molecule), the reverse sandwich method (in which a labeled detection molecule is reacted with the target substance in a sample in advance, and the resulting complex is reacted with the capture body), and the one-step method (in which the capture body, the target substance in a sample, and the labeled detection molecule react simultaneously in one step), and any of these can be used.

[0086] As a first embodiment, for example, in the forward sandwich method, first, in a capture step, the capturer and the sample are brought into contact with each other, and the target substance is captured by the probe molecule of the capturer (primary reaction). Next, the labeled detection molecule is brought into contact with the target substance captured by the capturer and bound to it (secondary reaction). This reaction forms a complex (e.g., an immune complex) containing the capturer, target substance, and labeled detection molecule. After washing the unbound sample and labeled detection molecule in a washing step as necessary, the photocleavable linker is photocleaved in a release step to release the complex from the magnetic particle, and the labeled substance of the labeled detection molecule is measured by a predetermined method. For example, when the labeled substance of the labeled detection molecule is an enzyme, a chromogenic or luminescent substrate corresponding to the enzyme is added, and a signal is detected by reacting the enzyme with the substrate. This allows the presence or absence and amount of the target substance in the sample to be detected as a signal, and the amount of the target substance in the sample to be quantified by comparing with the measured value in a standard sample.

[0087] More specifically, as an example of the first embodiment, for example, as shown in FIG. 1, a magnetic particle 11 and a photocleavable linker 13 are bonded via a spacer 12, and a photocleavable linker-bonded magnetic particle 101 is used in which a probe molecule 14 (preferably an antibody against the target substance 15) capable of specifically binding to a target substance 15 in a sample is bonded to the photocleavable linker 13. First, in a capture step, the target substance 15 is captured by the photocleavable linker-bonded magnetic particle 101 present in a reaction system (reaction solution) via the probe molecule 14. Next, in a washing step, the target substance 15 that has not bonded to the collected photocleavable linker-bonded magnetic particle 101, impurities, etc. are removed, and then a solution containing a labeled detection molecule 102 in which a detection molecule 16 (preferably an antibody against the target substance 15) capable of specifically binding to the target substance 15 is labeled with a labeling substance 17 such as an enzyme is added, and the labeled detection molecule 102 is bonded to the target substance 15 (a). Next, in another washing step, the unreacted labeled detection molecule 102 is removed, and the labeled detection molecule 102 bound to the photocleavable linker-bound magnetic particle 101 via the target substance 15 is left. Next, the collected photocleavable linker-bound magnetic particle 101 is suspended in ion-exchanged water or the like, and then irradiated with UV light to photocleave the photocleavable linker 13, thereby releasing the complex 103 containing the probe molecule 14, the target substance 15, and the labeled detection molecule 102 (b). Next, the magnetic particles are collected, and only the supernatant is taken out, thereby obtaining a complex 103 solution not containing the magnetic particles 11 (c). By detecting the labeling substance 17 in the complex 103 solution, it is possible to measure the concentration of the target substance 15. Note that this specific example shows an example of the first embodiment, and the conditions may be appropriately changed, for example, by performing the washing step only once, or by selecting a substance as the labeling substance 17 or the probe molecule 14.

[0088] Depending on the type of photo-cleavable linker used and the cleavage conditions, there may be cases where no photo-cleavable linker fragments remain in the complex 103 after cleavage, as in the illustrated example. In addition, the illustrated example is a schematic diagram, and as described above, the numbers of magnetic particles, spacers, photo-cleavable linkers, and probe molecules do not have to be in a 1:1:1:1 relationship. These are the same in other embodiments.

[0089] (Kit for use in the first aspect) The kit for use in the first aspect of the present invention includes, as a constituent reagent, at least the photocleavable linker-bound magnetic particle of the present invention, and the probe molecule is a molecule capable of specifically binding to the target substance. The kit preferably further includes the detection molecule according to the first aspect, more preferably the labeled detection molecule.

[0090] These may each be in a solid (powder) form or in a liquid form dissolved in a buffer solution. When in a liquid form, the concentration of the photocleavable linker-bound magnetic particles in each solution (preparation) is not particularly limited, but is preferably 0.005 to 0.2% by mass, more preferably 0.01 to 0.1% by mass, from the viewpoint that excessive addition may tend to increase non-specific signals in some cases. The concentration of the detection molecule is also not particularly limited, but is preferably 0.05 to 1 μg / mL, more preferably 0.1 to 0.5 μg / mL, from the same viewpoint.

[0091] The kit for use in the first aspect of the present invention may further comprise components to be included in normal immunological measurements such as ELISA, CLEIA, immunochromatography, etc. For example, it may further comprise at least one selected from the group consisting of a standard sample (standard specimen reagent, each concentration), a control reagent, the sample dilution solution, the particle suspension medium, the reaction buffer, the washing solution, and a dilution cartridge. It may also further comprise a labeling substance for labeling the detection molecule according to the first aspect.

[0092] For example, when the labeling substance is an enzyme, the kit may further include a substrate necessary for detecting and quantifying the labeling substance, a reaction stop solution, etc. Furthermore, the kit may further include a pretreatment solution for pretreatment of the sample and an instruction manual for use of the kit, as necessary.

[0093] (Second aspect) As the method for measuring a target substance of the present invention, in cases where the size of the antigen is small and the epitope portion is limited, a competitive method is also preferred in which the target substance is indirectly measured by causing a competition between the target substance and a competing substance and detecting the competing substance. As the competitive method, the following second embodiment is also preferred: a capture step of contacting the photo-cleavable linker-bound magnetic particle of the present invention, the probe molecule of which is a molecule capable of specifically binding to the target substance and a competitor of the target substance, with the sample and the competitor, and allowing the target substance to compete with the competitor, thereby capturing the competitor on the photo-cleavable linker-bound magnetic particle; a releasing step of photocleaving the photocleavable linker to release a complex containing the competitor and the probe molecule from the magnetic particles to obtain a prepared sample; a measuring step of measuring the target substance by detecting the complex in the prepared sample; Measurement methods including is preferred.

[0094] In a second embodiment, the photo-cleavable linker-bound magnetic particles of the present invention are photo-cleavable linker-bound magnetic particles in which the probe molecules are molecules capable of specifically binding to the target substance and a competitor of the target substance.

[0095] In the present invention, a "competitor of a target substance" refers to a substance that competes with the target substance in binding to a molecule capable of specifically binding to the target substance, and refers to a substance of the same type as the target substance, or a molecule having the same or similar structure as the target substance (a structure that is the same or similar to the site of the target substance that is recognized by the molecule capable of specifically binding to the target substance), and can be appropriately selected from molecules similar to those exemplified as the probe molecules depending on the type of the target substance.

[0096] In the second aspect, combinations of the target substance / the competitor / a molecule (probe molecule) capable of specifically binding to the target substance and the competitor include, for example, combinations of antigen (antigen peptide, low molecular weight compound, etc.) / the antigen or a molecule having the same or a similar antibody recognition site / antibody, antibody / the antibody or a molecule having the same or a similar antigen recognition site / antigen, and lectin-binding glycopeptide / the glycopeptide or a molecule having the same or a similar lectin recognition site / lectin.

[0097] [Capturing process] In the capture step of the second embodiment, the competitor is added to the sample, the photocleavable linker-bound magnetic particles (capture body) of the present invention are brought into contact with the sample and the competitor, and the competitor is captured by the capture body via the binding between the competitor and the probe molecule. At this time, the binding between the target substance and the binding between the probe molecule and the competitor competes with each other. The method of contacting the capture body, the sample, and the competitor is not particularly limited, and a conventionally known method or a method similar thereto can be appropriately adopted, for example, a method of adding the capture body and the competitor to the sample, or a method of adding two other liquids to the capture body liquid or the competitor liquid.

[0098] The sample may be used after diluting with a sample diluent, or may be used after suspending in a particle suspension medium of the capture body, and further, other reaction buffers may be appropriately added to the reaction system (e.g., antigen-antibody reaction system) between the capture body, the sample, and the competitor. Examples of the sample diluent, particle suspension medium, and reaction buffer include the same ones as those listed in the first embodiment above.

[0099] In the reaction between the capture body, the sample, and the competitor in the capture step of the second embodiment, the content (final concentration) of the capture body in the reaction system is not particularly limited since it is appropriately adjusted depending on the type, concentration, detection method, etc. of the sample, but from the viewpoint of efficient capture in a short time, it is preferably, for example, 0.005 to 0.2 mass%, more preferably 0.01 to 0.1 mass%. The amount of the competitor added (final concentration) can be appropriately adjusted mainly depending on the concentration of the sample, etc., and can also be appropriately adjusted to a stepped concentration depending on the calculation method of the measured value of the target substance.

[0100] The conditions for the capture step of the second embodiment are also not particularly limited and can be adjusted as appropriate. For example, the capture step can be performed at room temperature to 45°C, preferably 20 to 37°C, at a pH of about 6 to 9, preferably 7 to 8, for about 5 seconds to 10 minutes, preferably 30 seconds to 5 minutes, but is not limited to these conditions.

[0101] [Cleaning process] In the second embodiment of the method for measuring a target substance of the present invention, it is preferable to further include a washing step, after the capture step and before the release step described below, in which the competitor bound to the capture body is separated from other impurities not bound to (not captured by) the capture body, and the impurities are removed. Such a washing step is not particularly limited, and a conventionally known method or a method similar thereto can be appropriately adopted, and examples thereof include the same method as the washing step of the first embodiment described above.

[0102] [Release process] In the release step of the second embodiment, the photocleavable linker is cleaved to release the complex of the competitor bound to the capture body and the probe molecule from the magnetic particles, thereby obtaining a prepared sample containing the complex as a sample to be subjected to the measurement step.

[0103] The method of cleaving the photocleavable linker includes a method of cleaving it by light irradiation. In the release step, the light source and the amount of light to be irradiated are not particularly limited and can be appropriately selected according to the type of the photocleavable linker. For example, the same conditions as those listed in the release step of the first embodiment, including the preferred conditions, can be included. However, in the present invention, the photocleavage efficiency of the photocleavable linker is superior compared to the case where the photocleavable linker is bound to the magnetic particle without the spacer, so the light irradiation time can be shortened.

[0104] [Measurement process] In the measurement step of the second embodiment, the target substance is measured by detecting the complex in the prepared sample. The detection of the complex is preferably performed by using a labeled competitor that is labeled with a labeling substance as the competitor.

[0105] The labeled competitor is the competitor labeled with a labeling substance. As the labeling substance, any labeling substance used in known immunological measurements can be used without particular limitation, and examples thereof include the same as those exemplified as the labeling substances of the labeled detection molecule of the first embodiment, including preferred embodiments.

[0106] The labeled competitor can be produced by binding the competitor and the label. As the binding method, a conventionally known method or a method similar thereto can be appropriately adopted, and the competitor and the label may be directly or indirectly bound. As such a binding method, the same method as the method exemplified as the method for producing the photocleavable linker-bound magnetic particle of the present invention (i.e., the same method as the method exemplified as the method for inserting a spacer in the above-mentioned method for improving photocleavage efficiency) can be mentioned. In addition, as the labeled competitor, a commercially available product may be appropriately used. For example, when the target substance is an antigen, the antigen or a molecule having an antibody recognition site identical or similar to that of the antigen may be appropriately labeled.

[0107] In the second embodiment, for example, in the capture step, the capture body, the sample, and the labeled competitor are brought into contact with each other, and the labeled competitor is captured by the probe molecule of the capture body. This reaction forms a complex (e.g., an immune complex) containing the capture body and the labeled competitor. After the unbound sample and the labeled competitor are washed in a washing step as necessary, the photocleavable linker is photocleaved in a release step to release the complex from the magnetic particle, and the labeled substance of the labeled competitor is measured by a predetermined method. For example, when the labeled substance of the labeled competitor is an enzyme, a colorimetric substrate or a luminescent substrate corresponding to the enzyme is added, and the enzyme is reacted with the substrate to detect a signal. At this time, when a large amount of the target substance is present in the sample, the binding between the labeled competitor and the probe molecule is inhibited by the competing binding between the target substance and the probe molecule, and the signal is reduced accordingly. Therefore, by detecting the signal of the labeled competitor, the presence or absence and the amount of the target substance in the sample can be indirectly detected, and the amount of the target substance in the sample can be quantified by comparing with the measured value in a standard sample.

[0108] More specifically, as an example of the second embodiment, for example, as shown in FIG. 2, a magnetic particle 21 and a photocleavable linker 23 are bound via a spacer 22, and a photocleavable linker-bound magnetic particle 201 is used, in which a probe molecule 24 (preferably an antibody against the target substance 25 and the competitor 26) capable of specifically binding to a target substance 25 and its competitor 26 in a sample is bound to the photocleavable linker 23. A labeled competitor 202 of known concentration, in which the competitor 26 is labeled with a labeling substance 27, coexists in the reaction system. First, in a capture step, the target substance 25 and the competitor 26 are made to compete with each other, and are captured by the photocleavable linker-bound magnetic particle 201 present in the reaction system (reaction solution) via the probe molecule 24 (a). Here, the higher the concentration of the target substance 25, the more difficult it is for the competitor 26 to be captured by the probe molecule 24. Next, in a washing step, the target substance 25, the labeled competitor 202, impurities, etc. that have not bound to the collected photocleavable linker-bound magnetic particle 201 are removed. Next, the collected photocleavable linker-bound magnetic particles 201 are suspended in ion-exchanged water or the like, and then irradiated with UV light to photocleave the photocleavable linker 23, thereby releasing a complex 203 containing the probe molecule 24 and the labeled competitor 202, and a complex containing the probe molecule 24 and the target substance 25 (b). Next, the magnetic particles are collected, and only the supernatant is taken out to obtain a solution containing the complex 203 without containing the magnetic particles 21, and the labeling substance 27 in the solution of the complex 203 is detected (c). The higher the concentration of the target substance 25 in the sample, the lower the signal of the detected labeling substance 27, so that the concentration of the target substance 25 can be calculated using a calibration curve or the like. Note that this specific example shows one example of the second aspect, and the conditions may be changed as appropriate, for example, by selecting substances as the labeling substance 27, the probe molecule 24, and the competitor 26.

[0109] (Kit for use in the second embodiment) The kit for use in the second aspect of the present invention contains, as a constituent reagent, at least the photocleavable linker-bound magnetic particle of the present invention, and the probe molecule is a molecule capable of specifically binding to the target substance and a competitor of the target substance. In addition, the kit preferably further contains the competitor, more preferably the labeled competitor.

[0110] These may each be in the form of a solid (powder) or a liquid dissolved in a buffer solution. When in the form of a liquid, the concentration of the photocleavable linker-bound magnetic particles in each solution (preparation) is not particularly limited, but is preferably 0.005 to 0.2% by mass, more preferably 0.01 to 0.1% by mass, from the viewpoint that excessive addition may tend to increase non-specific signals. The concentration of the competitor is not particularly limited, but is preferably 0.05 to 1 μg / mL, more preferably 0.1 to 0.5 μg / mL, from the same viewpoint.

[0111] The kit for use in the second aspect of the present invention may further comprise components to be included in normal immunological measurements such as ELISA, CLEIA, immunochromatography, etc. For example, the kit may further comprise at least one selected from the group consisting of a standard sample (standard specimen reagent, each concentration), a control reagent, the sample dilution solution, the particle suspension medium, the reaction buffer, the washing solution, and a dilution cartridge. The kit may further comprise a labeling substance for labeling the competitor.

[0112] Furthermore, for example, when the labeling substance is an enzyme, the kit may further contain a substrate necessary for detection and quantification of the labeling substance, a reaction stop solution, etc. Furthermore, if necessary, the kit may further contain a pretreatment solution for pretreatment of the sample and instructions for use of the kit.

[0113] (Third aspect) As the method for measuring a target substance of the present invention, the competitive method may be the following third embodiment: a capture step of contacting the photo-cleavable linker-bound magnetic particle of the present invention, the probe molecule of which is a competitor of the target substance, with the sample and a detection molecule capable of specifically binding to the target substance and the competitor, and allowing the detection molecule to compete with the target substance and the probe molecule, thereby capturing the detection molecule on the photo-cleavable linker-bound magnetic particle; a release step of photocleaving the photocleavable linker to release a complex containing the detection molecule and the probe molecule from the magnetic particle to obtain a prepared sample; a measuring step of measuring the target substance by detecting the complex in the prepared sample; Measurement methods including is also preferred.

[0114] In a third embodiment, the photo-cleavable linker-bound magnetic particles of the present invention are photo-cleavable linker-bound magnetic particles in which the probe molecule is a competitive substance for the target substance.

[0115] In the third embodiment, a detection molecule capable of specifically binding to the target substance and the competing substance is added to the sample, and the photocleavable linker-bound magnetic particle (capture body) of the present invention is brought into contact with the sample and the detection molecule. The detection molecule according to the third embodiment may be any molecule capable of specifically binding to both the target substance and the competing substance, and may be appropriately selected from the same molecules as those listed as the probe molecule according to the type of the target substance.

[0116] In the third aspect, combinations of the target substance / the competitor (probe molecule) / a molecule (detection molecule) capable of specifically binding to the target substance and the competitor of the target substance include, for example, combinations of antigen (antigen peptide, low molecular weight compound, etc.) / the antigen or a molecule having the same or similar antibody recognition site / antibody, antibody / the antibody or a molecule having the same or similar antigen recognition site / antigen, and lectin-binding glycopeptide / the glycopeptide or a molecule having the same or similar lectin recognition site / lectin.

[0117] [Capturing process] In the capture step of the third embodiment, the detection molecule is captured by the capture body through the binding between the competitor and the detection molecule by the contact. At this time, the binding between the detection molecule and the target substance competes with the binding between the target substance and the competitor (probe molecule) contained in the capture body. The method of contacting the capture body, the sample, and the detection molecule is not particularly limited, and a conventionally known method or a method similar thereto can be appropriately adopted, for example, a method of adding the capture body and the detection molecule to the sample, or a method of adding two other liquids to the capture body liquid or the detection molecule liquid.

[0118] The sample may be used after diluting with a sample dilution solution, or may be used after suspending in a particle suspension medium of the capture body, and further, other reaction buffers may be appropriately added to the reaction system (e.g., antigen-antibody reaction system) of the capture body, the sample, and the detection molecule. Examples of the sample dilution solution, particle suspension medium, and reaction buffer include the same ones as those listed in the first embodiment above.

[0119] In the reaction between the capture body, the sample, and the detection molecule in the capture step of the third embodiment, the content (final concentration) of the capture body in the reaction system is not particularly limited since it is appropriately adjusted depending on the type, concentration, detection method, etc. of the sample, but is preferably 0.005 to 0.2% by mass, more preferably 0.001 to 0.1% by mass, from the viewpoint of efficient capture in a short time. In addition, the amount (final concentration) of the detection molecule to be added (when two or more types of detection molecules are combined, the total amount thereof, the same applies below) is not particularly limited since it is appropriately adjusted depending on the type, concentration, detection method, etc. of the sample, but is preferably 0.05 to 1 μg / mL, more preferably 0.1 to 0.5 μg / mL, for example.

[0120] The conditions for the capture step of the third embodiment are also not particularly limited and can be adjusted as appropriate. For example, the capture step can be performed at room temperature to 45°C, preferably 20 to 37°C, at a pH of about 6 to 9, preferably 7 to 8, for about 5 seconds to 10 minutes, preferably 30 seconds to 5 minutes, but is not limited to these conditions.

[0121] [Cleaning process] In the third embodiment of the method for measuring a target substance of the present invention, it is preferable to further include a washing step, after the capture step and before the release step described below, in which the detection molecule bound to the capturer is separated from other impurities not bound to (not captured by) the capturer, and the impurities are removed. Such a washing step is not particularly limited, and a conventionally known method or a method similar thereto can be appropriately adopted, and examples thereof include the same method as the method given as the washing step of the first embodiment above.

[0122] [Release process] In the release step of the third embodiment, the photocleavable linker is cleaved to release the complex of the detection molecule and the probe molecule bound to the capture body from the magnetic particles, thereby obtaining a prepared sample containing the complex as a sample to be subjected to the measurement step.

[0123] The method of cleaving the photocleavable linker includes a method of cleaving it by light irradiation. In the release step, the light source and the amount of light to be irradiated are not particularly limited and can be appropriately selected according to the type of the photocleavable linker. For example, the same conditions as those listed in the release step of the first embodiment, including the preferred conditions, can be included. However, in the present invention, the photocleavage efficiency of the photocleavable linker is superior compared to the case where the photocleavable linker is bound to the magnetic particle without the spacer, so the light irradiation time can be shortened.

[0124] [Measurement process] In the measurement step of the third embodiment, the target substance is measured by detecting the complex in the prepared sample. The detection of the complex is preferably performed by using a labeled detection molecule labeled with a labeling substance as the detection molecule according to the third embodiment and detecting the complex with the labeling substance.

[0125] The labeled detection molecule according to the third embodiment is a detection molecule labeled with a labeling substance. As the labeling substance, any labeling substance used in known immunological measurements can be used without particular limitation, and examples thereof include the same as those exemplified as the labeling substances for the labeled detection molecule according to the first embodiment, including preferred embodiments.

[0126] The labeled detection molecule according to the third aspect can be produced by binding the detection molecule to the labeling substance. As the binding method, a conventionally known method or a method similar thereto can be appropriately adopted, and the detection molecule and the labeling substance may be directly or indirectly bound. As such a binding method, the same method as that given as the method for producing the photocleavable linker-bound magnetic particle of the present invention (i.e., the same method as the method given as the method for inserting a spacer in the above-mentioned method for improving photocleavage efficiency) can be mentioned. In addition, as the labeled detection molecule according to the third aspect, a commercially available one may be appropriately used. For example, when the target substance is an antigen and the competitive substance is the antigen or a molecule having an antibody recognition site identical or similar to the antigen, a labeled antibody against the antigen can be appropriately used.

[0127] In the third embodiment, for example, in the capture step, the capturer, the sample, and the labeled detection molecule are brought into contact with each other, and the capturer captures the labeled detection molecule via the probe molecule of the capturer. This reaction forms a complex (e.g., an immune complex) containing the capturer and the labeled detection molecule. After the unbound sample and the labeled detection molecule are washed in a washing step as necessary, the photocleavable linker is photocleaved in a release step to release the complex containing the labeled detection molecule and the probe molecule from the magnetic particle, and the labeling substance of the labeled detection molecule is measured by a predetermined method. For example, when the labeling substance of the labeled detection molecule is an enzyme, a colorimetric substrate or a luminescent substrate corresponding to the enzyme is added, and the enzyme is reacted with the substrate to detect a signal. At this time, when a large amount of the target substance is present in the sample, the binding between the probe molecule and the labeled detection molecule is inhibited by the competing binding between the target substance and the labeled detection molecule, and the signal is reduced accordingly. Therefore, by detecting the signal of the labeled detection molecule, it is possible to indirectly detect the presence or absence and amount of the target substance in a sample, and the amount of the target substance in a sample can be quantified by comparing it with the measured value in a standard sample.

[0128] More specifically, as an example of the third embodiment, for example, as shown in FIG. 3, a magnetic particle 31 and a photocleavable linker 33 are bound via a spacer 32, and a photocleavable linker-bound magnetic particle 301 is used in which a competitor (probe molecule 34) of a target substance 35 in a sample is bound to the photocleavable linker 33. In the reaction system, a labeled detection molecule 302 in which a detection molecule 36 (preferably an antibody against the target substance 35 and the competitor 34) capable of specifically binding to the target substance 35 and its competitor 34 in the sample is labeled with a labeling substance 37 coexists. First, in a capture step, the target substance 35 and the competitor 34 are made to compete with each other, and the labeled detection molecule 302 is captured by the photocleavable linker-bound magnetic particle 301 present in the reaction system (reaction solution) via the competitor 34 and the detection molecule 36 (a). Here, the higher the concentration of the target substance 35, the more difficult it becomes for the competitor 34 to capture the labeled detection molecule 302. Next, in a washing step, the target substance 35, the labeled detection molecule 302, and impurities that have not bound to the collected photocleavable linker-bound magnetic particles 301 are removed. Next, the collected photocleavable linker-bound magnetic particles 301 are suspended in ion-exchanged water or the like (b), and then irradiated with UV light to photocleave the photocleavable linker 33, thereby releasing the complex 303 containing the probe molecule 34 (competitor) and the labeled detection molecule 302 (c). Next, the magnetic particles are collected, and only the supernatant is taken out to obtain a solution containing the complex 303 without containing the magnetic particles 31, and the labeled substance 37 in the complex 303 solution is detected (d). The higher the concentration of the target substance 35 in the sample, the lower the signal of the detected labeled substance 37, so the concentration of the target substance 35 can be calculated using a calibration curve or the like. Note that this specific example shows one example of the third embodiment, and the conditions may be changed as appropriate, such as by selecting substances as the labeled substance 37, the probe molecule 34, and the detection molecule 36.

[0129] (Kit for use in the third aspect) The kit for use in the third aspect of the present invention contains, as a constituent reagent, at least the photocleavable linker-bound magnetic particle of the present invention, and the probe molecule is a competitive substance for the target substance. The kit preferably further contains the detection molecule according to the third aspect, more preferably the labeled detection molecule.

[0130] These may be in the form of a solid (powder) or a liquid dissolved in a buffer solution. When in the form of a liquid, the concentration of the photocleavable linker-bound magnetic particles in each solution (preparation) is not particularly limited, but is preferably 0.005 to 0.2% by mass, more preferably 0.01 to 0.1% by mass, from the viewpoint that excessive addition may increase non-specific signals. The concentration of the detection molecule is not particularly limited, but is preferably 0.05 to 1 μg / mL, more preferably 0.1 to 0.5 μg / mL, from the same viewpoint.

[0131] The kit for use in the third aspect of the present invention may further comprise components to be included in normal immunological measurements such as ELISA, CLEIA, immunochromatography, etc. For example, it may further comprise at least one selected from the group consisting of a standard sample (standard specimen reagent, each concentration), a control reagent, the sample dilution solution, the particle suspension medium, the reaction buffer, the washing solution, and a dilution cartridge. It may also further comprise a labeling substance for labeling the detection molecule according to the third aspect.

[0132] Furthermore, for example, when the labeling substance is an enzyme, the kit may further contain a substrate necessary for detection and quantification of the labeling substance, a reaction stop solution, etc. Furthermore, if necessary, the kit may further contain a pretreatment solution for pretreatment of the sample and instructions for use of the kit. EXAMPLES

[0133] The present invention will be described in more detail below based on Reference Examples, Examples, and Comparative Examples, but the present invention is not limited to the following Examples. In each Reference Example, Example, and Comparative Example, the indication of "%" indicates weight / volume (w / v: g / mL) percentage unless otherwise specified. In each Example and Comparative Example, the equipment, reagents, etc. used are the same as those used in Reference Example 1 unless otherwise specified.

[0134] (Reference Example 1) Cleavage rate test of photocleavable linker immobilized on a microplate (1) Preparation of alkaline phosphatase-photocleavable linker-biotin conjugate 2 mg of alkaline phosphatase (ALP) (ALP-24, Oriental Yeast) was loaded onto a gel filtration spin column (PD-10, GE) and eluted with buffer 1 (20 mM PB (pH 7.2)). Next, 2.86 μL of dimethylformamide (DMF) containing 50 mM N-hydroxysuccinimide (NHS)-photocleavable linker (PC)-biotin (trade name: PC-Biotin-NHS, Ambergen) was added to the eluate, and the mixture was shaken at 25° C. for 1 hour to obtain an alkaline phosphatase-photocleavable linker-biotin conjugate (ALP-PC-biotin conjugate). The mixture was then loaded onto the gel filtration spin column again and eluted with buffer 1.

[0135] (2) Preparation of ALP-PC-immobilized plates 100 μL of buffer 1 containing 5 μg / mL of streptavidin (SA) was added to each well of a 96-well microwell plate (made of polystyrene, manufactured by Nunc Co., Ltd.) and mixed by shaking at 37 ° C. for 1 hour. After washing three times with Lumipulse (registered trademark) washing solution (manufactured by Fujirebio Co., Ltd.), 100 μL of buffer 2 (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 2% BSA (pH 7.2)) was added and left to stand for 30 minutes at 37 ° C. After washing three times with Lumipulse washing solution, 100 μL of buffer 3 (50 mM MES, 150 mM NaCl, 1 mM MgCl2, 0.1 mM ZnCl2, 0.5% BSA (pH 6.8)) containing ALP-PC-biotin conjugate was added and reacted at 37 ° C. for 30 minutes. Here, the buffer solution 3 was adjusted so that the ALP concentration was 0.1 μg / mL or 0.01 μg / mL. The plate was washed three times with Lumipulse washing solution to obtain an ALP-PC-immobilized plate.

[0136] (3) Cleavage of the photocleavable linker 100 μL of ultrapure water was added to the ALP-PC solid-phase plate, and UV irradiation was performed for 10 minutes. UV irradiation was performed using a UV-LED (Optocode Co., Ltd.) with 365 nm light (UV) at 15 mW / cm 2 The plate was irradiated with UV light at an intensity of 100 μL (unless otherwise specified, the same applies below). The supernatant of the plate after UV irradiation was collected, and 20 μL was added to a 96-well microwell plate, followed by addition of 100 μL of substrate solution (p-nitrophenyl phosphate sodium (pNPP), Fujifilm Wako Pure Chemical Industries, Ltd.). After reaction at 37° C. for 15 minutes, 80 μL of reaction stop solution (0.2 mmol / L sodium hydroxide) was added and shaken at 37° C. for 1 minute. On the other hand, the ALP-PC solid-phase plate (remaining plate) after supernatant recovery was washed three times with Lumipulse washing solution, and then 20 μL of ultrapure water and 100 μL of substrate solution were added. After reaction at 37° C. for 15 minutes, 80 μL of reaction stop solution was added and shaken at 37° C. for 1 minute.

[0137] Next, the absorbance at 405 nm of each well of the supernatant and the remaining plate was measured (using a Varioskan flash (Thermo Fisher Scientific) (hereinafter the same). As a control, the absorbance at 405 nm of each well of the supernatant and the remaining plate was measured in the same manner as above, except that the ALP-PC solid-phase plate was not irradiated with UV light.

[0138] The plate absorbance and cleavage rate ({supernatant absorbance / (supernatant absorbance+remaining plate absorbance)}×100(%)) under each condition are shown in Table 1. The absorbance in Table 1 is the value after subtracting the blank (ultrapure water+microwell plate).

[0139] [Table 1]

[0140] As shown in Table 1, in the case of the photocleavable linker immobilized on a polystyrene microwell plate, ALP was efficiently transferred from the plate to the supernatant upon UV irradiation, confirming high photocleavage efficiency.

[0141] Comparative Example 1: Cleavage rate test of photocleavable linker directly bound to magnetic particles (1) Preparation of ALP-PC-bound magnetic particles Dynabeads M-270 Amine (manufactured by Thermo Fisher Scientific) with amino groups were used as magnetic particles. 10 mg of magnetic particles were washed three times with DMF and suspended in 485 μL of DMF and 15 μL of 50 mM NHS-PC-biotin / DMF solution. After shaking for 2 hours at 25° C., the particles were washed three times with DMF. The particles were suspended in 1 mL of DMF to obtain a biotin-PC-bound particle suspension.

[0142] Next, the biotin-PC-bound particles were washed three times with Buffer 4 (20 mM PB, 0.1% Tween 20 (pH 7.2)) and then suspended in 100 μL of Buffer 4 containing 20 μg / mL SA-bound ALP (Mabtech AB). The particles were shaken at 37° C. for 30 minutes to obtain ALP-PC-bound magnetic particles, which were then washed three times with Buffer 4.

[0143] (2) Cleavage of the photocleavable linker The ALP-PC-bound magnetic particles were suspended in buffer 3 and shaken at 37°C for 5 minutes, and then washed three times with Lumipulse washing solution. The magnetic particles were suspended in 100 μL of ultrapure water, and the entire amount was transferred to an 8 mL test tube and irradiated with UV light for 0, 5, 10, and 15 minutes.

[0144] The supernatant was then collected and transferred to a 96-well microwell plate at 20 μL. 100 μL of substrate solution was added and reacted at 37°C for 15 minutes, after which 80 μL of reaction stop solution was added and shaken at 37°C for 1 minute. The absorbance at 405 nm of each well was measured. Meanwhile, the remaining particles after the supernatant was collected were washed three times with Lumipulse washing solution, and then suspended in 20 μL of ultrapure water and 100 μL of substrate solution. After reacting at 37°C for 15 minutes, 80 μL of reaction stop solution was added and shaken at 37°C for 1 minute. 150 μL of the supernatant after the reaction was transferred to a microwell plate, and the absorbance at 405 nm of each well was measured.

[0145] The plate absorbance and cleavage rate ({supernatant absorbance / (supernatant absorbance+residual particle absorbance)}×100(%), the same in Tables 3 to 5 and 7 below) under each condition are shown in Table 2 below. The absorbance in Table 2 is the value after subtracting the blank (ultrapure water + microwell plate).

[0146] [Table 2]

[0147] As shown in Table 2, it was confirmed that when the photocleavable linker was bound to the magnetic particles without a spacer, the photocleavable linker was hardly cleaved even when irradiated with UV light. Furthermore, even 10 minutes after UV irradiation, the sum of the supernatant absorbance and the residual particle absorbance remained almost constant, confirming that ALP was not decomposed by UV irradiation in this test.

[0148] (Example 1) Cleavage rate test of a photocleavable linker bound to magnetic particles via streptavidin (1) Preparation of ALP-PC-SA-bound magnetic particles SA-bound Dynabeads (trade name: Dynabeads M-280 Streptavidin, manufactured by Thermo Fisher Scientific) were used as magnetic particles. 1 mg of magnetic particles was washed three times with buffer 4 in an 8 mL test tube. Next, 100 μL of buffer 3 containing 20 μL / mL of ALP-PC-biotin conjugate obtained in the same manner as in Reference Example 1 (1) was added, and the mixture was shaken at 37° C. for 30 minutes to obtain ALP-PC-SA-bound magnetic particles. After washing three times with buffer 4, the mixture was washed six times with buffer 1, and then washed three times with buffer 2. 400 μL of buffer 2 was added, and the mixture was shaken at 37° C. for 1 hour, after which the mixture was washed three times with buffer 2, and 100 μL of buffer 2 was further added to obtain an ALP-PC-SA-bound magnetic particle liquid.

[0149] (2) Cleavage of the photocleavable linker 1100μL of Buffer 3 containing 0.15μg / mL of ALP-PC-SA-bound magnetic particles was placed in an 8mL test tube and left to stand at 37℃ for 5 minutes. After washing three times with Lumipulse washing solution, 440μL of ultrapure water was added and suspended, and 100μL was dispensed into test tubes. Next, UV irradiation was performed for 10 minutes, and the supernatant was collected and transferred to a 96-well microwell plate in 20μL. After adding 100μL of substrate solution and reacting for 15 minutes at 37℃, 80μL of reaction stop solution was added and shaken at 37℃ for 1 minute. The absorbance at 405nm of each well was measured.

[0150] On the other hand, the remaining particles after collecting the supernatant were washed three times with Lumipulse washing solution, and then suspended in 20 μL of ultrapure water and 100 μL of substrate solution. After reacting for 15 minutes at 37°C, 80 μL of reaction stop solution was added and the mixture was shaken for 1 minute at 37°C. After the reaction, 150 μL of the supernatant was transferred to a microwell plate, and the absorbance at 405 nm of each well was measured.

[0151] As a control, the absorbance at 405 nm of each well was measured for the supernatant and the remaining particles in the same manner as above, except that the ALP-PC-SA-bound magnetic particles were not irradiated with UV light. The measurements were performed in duplicate (sample 1 and sample 2) for each condition with and without UV light irradiation.

[0152] The absorbance and cleavage rate of the plate under each condition are shown in the following Table 3. Note that the absorbance in Table 3 is the value after subtracting the blank (ultrapure water + microwell plate).

[0153] [Table 3]

[0154] As shown in Table 3, when the photocleavable linker was bound to the magnetic particles via avidin, the cleavage rate of the photocleavable linker by UV irradiation was 20-30%, which was improved compared to Comparative Example 1.

[0155] (Example 2) Cleavage rate test of photocleavable linker bound to magnetic particles via polyethylene glycol (1) Preparation of ALP-PC-polyethylene glycol-bound magnetic particles Dynabeads carboxylated particles (Dynabeads M-270 Carboxylic acid, manufactured by Thermo Fisher Scientific) were used as magnetic particles. 10 mg of magnetic particles were washed three times with 1 mL of ultrapure water in an 8 mL test tube, and then 70 μL of ultrapure water, 505 μL of 19 mg / mL EDC (1-Ethyl-3-[3-dimethyl amino propyl]carbodiimide hydrochloride) solution, and 425 μL of 20 mg / mL NH2-polyethylene glycol (PEG)-NH2 (weight average molecular weight of PEG: 1K, 2K, 3.4K, 5K, 10K, or 20K; product name: SUNBRIGHT DE-010PA (1K), DE-020PA (2K), DE-034PA (3.4K), DE-050PA (5K), DE-100PA (10K), DE-200PA (20K); NOF Corp.) were added and shaken at 25° C. for 2 hours. After washing three times with 1 mL of ultrapure water, the particles were washed three times with 1 mL of DMF.

[0156] Next, 485 μL of DMF and 15 μL of a DMF solution of 50 mM NHS-PC-biotin (manufactured by Ambergen) were added, and the mixture was shaken at 25° C. for 2 hours. The mixture was washed three times with 1 mL of DMF and suspended in 1 mL of DMF. Next, 0.1 mL of the above suspension was washed three times with buffer 4 in a test tube, and then washed six times with buffer 1. After further washing three times with buffer 2, the mixture was suspended in buffer 2 and shaken at 37° C. for 1 hour. After washing three times with buffer 2, the mixture was suspended in 100 μL of buffer 2 to obtain a biotin-PC-PEG-bound magnetic particle liquid. Next, the supernatant was removed from 25 μL of biotin-PC-PEG-bound magnetic particle solution, and the solution was suspended in 1,667 μL of a solution of SA-bound ALP (Mabtech) diluted 10,000 times with buffer 2, and the solution was shaken at 37°C for 1 hour to obtain an ALP-PC-polyethylene glycol-bound magnetic particle (ALP-PC-PEG-bound magnetic particle) solution.

[0157] (2) Cleavage of the photocleavable linker The ALP-PC-PEG-bound magnetic particles were washed three times with Lumipulse washing solution. The particles were suspended in 667 μL of ultrapure water, and 100 μL of each was dispensed into 8 mL test tubes, and UV irradiation was performed for each particle. The UV irradiation time was 5 minutes, 12.5 minutes, 15 minutes, and 17.5 minutes for particles using PEG with a weight-average molecular weight of 3.4K, and 15 minutes for particles using other PEGs. After UV irradiation, the supernatant was collected and transferred to a 96-well microwell plate in 20 μL portions. 100 μL of substrate solution was added to each well and reacted at 37°C for 15 minutes, after which 80 μL of reaction stop solution was added and shaken at 37°C for 1 minute. The absorbance at 405 nm of each well was measured.

[0158] On the other hand, the remaining particles after collecting the supernatant were washed three times with Lumipulse washing solution, and then suspended in 20 μL of ultrapure water and 100 μL of substrate solution. After reacting for 15 minutes at 37°C, 80 μL of reaction stop solution was added and the mixture was shaken for 1 minute at 37°C. After the reaction, 150 μL of the supernatant was transferred to a microwell plate, and the absorbance at 405 nm of each well was measured.

[0159] As a control, the absorbance at 405 nm of each well was measured for the supernatant and the remaining particles in the same manner as above, except that the ALP-PC-PEG-bound magnetic particles were not irradiated with UV light.

[0160] The absorbance and cleavage rate of the plate under conditions in which various PEGs were used (UV irradiation time: 15 minutes) are shown in Table 4 below. In addition, the absorbance and cleavage rate measured under each UV irradiation time condition for particles using PEG with a weight-average molecular weight of 3.4K are shown in Table 5 below. Note that the absorbance in Tables 4 and 5 is the value after subtracting the blank (ultrapure water + microwell plate).

[0161] [Table 4]

[0162] [Table 5]

[0163] As shown in Table 4, it was confirmed that when a photocleavable linker was bound to a magnetic particle via polyethylene glycol, the cleavage rate of the photocleavable linker by UV irradiation was high, and it was also confirmed that the cleavage rate tended to be higher by using polyethylene glycol with a weight-average molecular weight of 3.4 K. Furthermore, as shown in Table 5, it was confirmed that the cleavage rate tended to be higher by extending the UV irradiation time.

[0164] (Example 3) Cleavage rate test of photocleavable linker bound to magnetic particles via dextran (1) Preparation of ALP-PC-dextran-bound magnetic particles Dynabeads carboxylated particles (Dynabeads M-270Carboxylic acid, Thermo Fisher) were used as magnetic particles. 10 mg of magnetic particles were washed three times with 1 mL of ultrapure water in an 8 mL test tube, and 300 μL of ultrapure water, 500 μL of 19 mg / mL EDC (1-Ethyl-3-[3-dimethyl amino propyl]carbodiimide hydrochloride) solution, and 200 μL of 10 mg / mL NH2-dextran (Dex) (mass per molecule of Dex: 10K, 40K, or 70K; Thermo Fisher) were added and shaken overnight at 25°C. After washing three times with 1 mL of ultrapure water, the particles were suspended in 1 mL of ultrapure water.

[0165] Next, 0.2 mL of the above suspension was washed three times with DMF in a test tube, and then 194 μL of DMF and 6 μL of a DMF solution of 50 mM NHS-PC-biotin (manufactured by Ambergen) were added, and the mixture was shaken at 25 ° C. for 2 hours. The mixture was washed three times with DMF and three times with buffer 1. After further washing three times with buffer 2, the mixture was suspended in 400 μL of buffer 2 and shaken overnight at 37 ° C. to obtain a biotin-PC-Dex-bound magnetic particle liquid. Next, the supernatant of the biotin-PC-Dex-bound magnetic particle liquid was removed, and SA-bound ALP (manufactured by Mabtech) was suspended in 667 μL of a solution diluted 10,000 times with buffer 2, and the mixture was shaken at 37 ° C. for 1 hour to obtain an ALP-PC-dextran-bound magnetic particle (ALP-PC-Dex-bound magnetic particle) liquid. (2) Cleavage of the photocleavable linker The ALP-PC-Dex-bound magnetic particles were washed three times with Lumipulse washing solution. The particles were suspended in Buffer 5 (50 mM HEPES (pH 7.0)), dispensed in 100 μL portions into 8 mL test tubes, and irradiated with UV for 15 minutes. After UV irradiation, each sample was transferred to a cuvette for Lumipulse Presto (registered trademark), washed six times with Lumipulse washing solution, and reacted by adding 200 μL of Lumipulse substrate solution. The amount of luminescence (count) of each sample was measured using a luminescence reader BLR-201 (manufactured by Aloka). In addition, the count was measured for each sample in the same manner as above, except that the ALP-PC-Dex-bound magnetic particles were not irradiated with UV.

[0166] The luminescence intensity and cleavage rate ({(luminescence intensity without UV irradiation - luminescence intensity with UV irradiation) / luminescence intensity without UV irradiation} x 100 (%)) under the conditions using various Dex are shown in Table 6. The counts in Table 6 are values ​​after subtracting the blank (ultrapure water).

[0167] [Table 6]

[0168] As shown in Table 6, it was confirmed that when a photo-cleavable linker was bound to a magnetic particle via dextran, the cleavage rate of the photo-cleavable linker by UV irradiation was high, and it was also confirmed that the cleavage rate tended to be higher when a dextran with a larger mass per molecule was used.

[0169] (Example 4) Immunoassay using photocleavable linker-bound magnetic particles (1) Preparation of IgG-PC-SA-PEG-bound magnetic particles Dynabeads (trade name: Dynabeads M-270 Amine, manufactured by Thermo Fisher Scientific) having an amino group were used as magnetic particles. 600 μL of DMF containing 2 μmol / mL of NHS-PEG (weight average molecular weight of PEG: 5K)-biotin (trade name: SUNBRIGHT BI-050TS, manufactured by NOF Corp.) was added to 6 mg of magnetic particles, and the particles were allowed to infiltrate for 2 hours at 25° C. After washing three times with 600 μL of DMF, the particles were washed three times with buffer 1. After further washing three times with buffer 4, the particles were suspended in buffer 4 to prepare a biotin-PEG-bound magnetic particle liquid.

[0170] In addition, 2 mg of anti-alpha fetoprotein (AFP) mouse IgG antibody (IgG, Fujirebio) was loaded onto a gel filtration spin column (PD-10, GE) and eluted with buffer 1. Next, 2.86 μL of DMF containing 50 mM NHS-PC-biotin (Ambergen) was added, and the mixture was shaken at 25° C. for 1 hour, after which the mixture was loaded onto the gel filtration spin column again and eluted with buffer 1 to obtain an IgG-PC-biotin conjugate. 2 mL of buffer 4 containing 250 μg / mL of IgG-PC-biotin conjugate was added to 2 mL of buffer 4 containing 100 μg / mL of streptavidin (SA), and the mixture was shaken at 25° C. for 2 hours. The IgG-PC-SA conjugate was fractionated using HPLC using Superdex200.

[0171] The supernatant was removed from the biotin-PEG-bound magnetic particle solution, and 1600 μL of Buffer 4 containing 1 μg / mL of IgG-PC-SA conjugate was added. After shaking at 25° C. for 1 hour, the solution was washed three times with Lumipulse washing solution, and then 1600 μL of Buffer 4 was added and suspended to obtain a suspension of IgG-PC-SA-PEG-bound magnetic particle conjugate.

[0172] (2) Detection of AFP The suspension of IgG-PC-SA-PEG-bound magnetic particles was divided into 250 μL portions in 8 mL test tubes. 20 μL of Lumipulse Presto AFP calibrator (0 ng / mL or 2000 ng / mL, Fujirebio) was added to each test tube and reacted at 37°C for 10 minutes. After washing six times with Lumipulse washing solution, 250 μL of ALP-labeled anti-AFP antibody (anti-AFP mouse IgG antibody) solution (Fujirebio, 0.1 μg / mL) was added. After reacting at 37°C for 10 minutes, the tube was washed six times with Lumipulse washing solution, and 100 μL of buffer 4 was added and suspended. The entire suspension was transferred to an 8 mL test tube and UV irradiation was performed for 15 minutes.

[0173] After UV irradiation, the supernatant was collected and transferred to a 96-well microwell plate in an amount of 20 μL. 100 μL of substrate solution was added to each well and reacted at 37°C for 15 minutes, after which 80 μL of reaction stop solution was added and shaken at 37°C for 1 minute. The absorbance of each well was measured at 405 nm.

[0174] On the other hand, the remaining particles after collecting the supernatant were washed three times with Lumipulse washing solution, and then suspended in 20 μL of ultrapure water and 100 μL of substrate solution. After reacting for 15 minutes at 37°C, 80 μL of reaction stop solution was added and the mixture was shaken for 1 minute at 37°C. After the reaction, 150 μL of the supernatant was transferred to a microwell plate, and the absorbance at 405 nm of each well was measured.

[0175] As a control, the absorbance at 405 nm of each well was measured for the supernatant and the remaining particles in the same manner as above, except that the IgG-PC-SA-PEG-bound magnetic particles were not irradiated with UV light.

[0176] The absorbance and cleavage rate of the plate under each condition are shown in Table 7. The absorbance in Table 7 is the value after subtracting the blank (ultrapure water + microwell plate).

[0177] [Table 7]

[0178] As shown in Table 7, in an immunoassay using the photo-cleavable linker-conjugated magnetic particles of the present invention, it was confirmed that the cleavage rate of the photo-cleavable linker by UV irradiation could be increased by binding the photo-cleavable linker to the magnetic particles via streptavidin and polyethylene glycol.

[0179] (Example 5) Immunoassay using photocleavable linker-bound magnetic particles containing antibody fragments (Fab') (1) Preparation of SA-PEG-bound magnetic particles 1.28 mg of streptavidin (SA) was loaded onto a gel filtration spin column (PD-10, GE) together with 0.1 M sodium bicarbonate buffer (pH 8.5). Iminothiolane hydrochloride was added and the mixture was shaken at 25°C for 1 hour, after which it was purified using buffer 6 (0.1 M PB, 1 mM EDTA2Na, 0.1% Tween20 (pH 6.3)) to obtain SH group-modified streptavidin (SA-SH).

[0180] Dynabeads M-270 Amine (manufactured by Thermo Fisher Scientific) having an amino group was used as the magnetic particles. After 2 mg of magnetic particles were washed three times with DMF, 200 μL of NHS-PEG (weight average molecular weight of PEG: 5K)-maleimide group (Mal) (2 μmol / mL DMF, product name: SUNBRIGHT MA-050TS, manufactured by NOF Corp.) was added and reacted overnight at 25° C. After washing three times with DMF, the particles were washed three times with buffer 6, and then 200 μL of SA-SH solution (400 μg / mL) was added. After shaking overnight at 25° C., the particles were washed three times with buffer 6. A 5 mM cysteine / buffer 6 solution was added and the particles were shaken at 25° C. for 3 hours to obtain SA-PEG-bound magnetic particles. The particles were washed six times with buffer solution 7 (0.1 M PB, 1 mM EDTA2Na (pH 6.3)) and three times with buffer solution 6, and the washed SA-PEG-bound magnetic particles were suspended in buffer solution 2.

[0181] (2) Preparation of IgG-PC-SA-PEG-bound magnetic particles and Fab'-PC-SA-PEG-bound magnetic particles 2 mg of anti-IL-6 mouse IgG antibody (Fujirebio) was loaded onto a gel filtration spin column (PD-10, GE) and eluted with 0.1 M citrate buffer (pH 3.5) to obtain a 5 mg / mL antibody solution. 1.0 mg / mL pepsin solution (0.1 M citrate buffer (pH 3.5)) was added at a volume ratio of 1 / 100, reacted at 37°C for 1 hour, and 2 M Tris buffer (pH 10.0) was added at a volume ratio of 1 / 10. Gel filtration purification was performed using a Superdex200 column and buffer 6 (0.1 M PB, 1 mM EDTA2Na (pH 6.3)) to obtain a 5 mg / mL F(ab')2 fragment solution.

[0182] To the F(ab')2 fragment solution, 0.2M 2-mercaptoethylamine (2-MEA) was added at a volume ratio of 1 / 20, and the mixture was allowed to react at 37°C for 1 hour. The mixture was then loaded onto a gel filtration spin column and washed with buffer 7 to obtain a Fab' solution. 0.3μL of 25mM Mal-PC-biotin conjugate solution (prepared from PC-biotin-NHS and 2-aminoethylmaleimide) was added thereto, and the mixture was shaken overnight at 25°C. The mixture was then loaded onto a centrifugal filter (Amicon Ultra (3K)), and 500μL of PBS was added and centrifuged. After repeating the centrifugation three times, the mixture was dissolved in buffer 2 to obtain a Fab'-PC-biotin conjugate solution.

[0183] Furthermore, an IgG-PC-biotin conjugate solution was obtained in the same manner as above, except that a complete antibody (IgG) solution of anti-IL-6 mouse IgG antibody was used instead of the above Fab' solution.

[0184] 1,500 μL of Fab'-PC-biotin conjugate solution or IgG-PC-biotin conjugate solution (both 400 ng / mL) was added to 225 μg of SA-PEG-bound magnetic particles and reacted at 37°C for 30 minutes to obtain IgG-PC-SA-PEG-bound magnetic particle solution and Fab'-PC-SA-PEG-bound magnetic particle solution, respectively. (3) Measurement of IL-6 After washing the IgG-PC-SA-PEG-bound magnetic particles and Fab'-PC-SA-PEG-bound magnetic particles three times with Lumipulse washing solution (Fujirebio), 1500 μL of buffer 2 was added and dispensed into Lumipulse cartridges (Fujirebio). 50 μL of Lumipulse IL-6 calibrator (0 pg / mL or 400 pg / mL, Fujirebio) was added to each cartridge and reacted at 37°C for 10 minutes. After washing six times with Lumipulse washing solution, 250 μL of Lumipulse-labeled body fluid (ALP-labeled anti-IL-6 mouse IgG antibody) was added and reacted at 37°C for 10 minutes. After washing six times with Lumipulse washing solution, the particles were suspended in 100 μL of buffer 3 and irradiated with UV for 1 minute. UV irradiation was performed using a LIGHTNINGCURE LC-L1V3 (manufactured by Hamamatsu Photonics) with a wavelength of 365 nm and a light intensity of 10,000 mW / cm 2The magnetic particles were irradiated from one direction under the conditions of UV irradiation. After UV irradiation, the magnetic particles were further washed six times with Lumipulse washing solution, and 200 μL of Lumipulse substrate solution was added and reacted at 37° C. for 5 minutes. The amount of luminescence (count) of the magnetic particle solution under each condition was measured using a luminescence reader BLR-201 (manufactured by Aloka). In addition, the count was measured for each sample in the same manner as above, except that the IgG-PC-SA-PEG-bound magnetic particles and the Fab'-PC-SA-PEG-bound magnetic particles were not irradiated with UV.

[0185] The amount of luminescence under each condition is shown in Table 8. The "cleavage rate" in Table 8 is calculated based on the difference between the count value at an IL-6 concentration of 400 pg / mL and the blank, assuming that the cleavage rate without UV irradiation under the same conditions is 0% (residual rate 100%).

[0186] [Table 8]

[0187] As shown in Table 8, even when a complete antibody (IgG) was used, the cleavage rate of the photo-cleavable linker was excellent at about 80%, but when an antibody fragment (Fab') was used, the cleavage rate of the photo-cleavable linker was further improved to 90%. [Industrial Applicability]

[0188] According to the present invention, it is possible to provide a method for improving the photocleavage efficiency of a photocleavable linker-bound magnetic particle comprising a magnetic particle and a photocleavable linker, a photocleavable linker-bound magnetic particle having excellent photocleavage efficiency, and a measurement method for measuring a target substance using these and a kit for use therein. [Explanation of symbols]

[0189] 11, 21, 31...magnetic particles, 12, 22, 32...spacer, 13, 23, 33...photo-cleavable linker, 14, 24...probe molecule, 34...probe molecule (competitor), 15, 25, 35...target substance, 16, 36...detection molecule, 26...competitor, 17, 27, 37...labeled substance, 101, 201, 301...photo-cleavable linker-bound magnetic particles, 102, 302...labeled detection molecule, 202...labeled competitor, 103, 203, 303...complex.

Claims

1. In the photocleavable linker-bound magnetic particle, the magnetic particle and the photocleavable linker are provided, Inserting a spacer between the magnetic particle and the photocleavable linker; A method for improving the photocleavage efficiency of a photocleavable linker, comprising: The spacer is made of polyethylene glycol, or made of polyethylene glycol and avidins, or made of dextran, wherein the weight-average molecular weight of the polyethylene glycol is 1K to 6K, and the mass of the dextran is 5K to 75KDa. method.

2. The method of claim 1 , wherein the photocleavable linker is photocleavable in solution.

3. A photocleavable linker-bound magnetic particle comprising a magnetic particle and a photocleavable linker, A spacer is provided between the magnetic particle and the photocleavable linker, and A probe molecule is provided at the end of the photocleavable linker opposite to the spacer, The spacer is made of polyethylene glycol, or made of polyethylene glycol and avidins, or made of dextran, wherein the weight-average molecular weight of the polyethylene glycol is 1K to 6K, and the mass of the dextran is 5K to 75KDa. Photocleavable linker-bound magnetic particles.

4. The photocleavable linker-bound magnetic particle according to claim 3 , wherein the probe molecule is at least one selected from the group consisting of polynucleotides and polypeptides.

5. A measurement method for measuring a target substance in a sample, a capture step of contacting the photo-cleavable linker-bound magnetic particle according to claim 3 or 4, the probe molecule being a molecule capable of specifically binding to the target substance, with the sample, and causing the photo-cleavable linker-bound magnetic particle to capture the target substance; a release step of photocleaving the photocleavable linker to release a complex containing the target substance and the probe molecule from the magnetic particle to obtain a prepared sample; a measuring step of measuring the target substance by detecting the complex in the prepared sample; A measurement method including:

6. 6. The measurement method according to claim 5, further comprising a labeling step of capturing the target substance with a labeled detection molecule in which a detection molecule capable of specifically binding to the target substance is labeled with a labeling substance, the complex is a complex containing the target substance, the probe molecule, and the labeled detection molecule, and the detection of the complex is detection by the labeling substance.

7. A kit for use in the method for measuring a target substance in a sample according to claim 5 or 6, 5. The photocleavable linker-bound magnetic particle according to claim 3 or 4, wherein the probe molecule is a molecule capable of specifically binding to the target substance. Including the kit.

8. The kit according to claim 7 , further comprising a labeled detection molecule in which the detection molecule capable of specifically binding to the target substance is labeled with a labeling substance.

9. A measurement method for measuring a target substance in a sample, a capture step of contacting the photo-cleavable linker-bound magnetic particle according to claim 3 or 4, the photo-cleavable linker-bound magnetic particle being a molecule capable of specifically binding to the target substance and a competitor of the target substance, with the sample and the competitor, and allowing the target substance to compete with the competitor, thereby capturing the competitor on the photo-cleavable linker-bound magnetic particle; a releasing step of photocleaving the photocleavable linker to release a complex containing the competitor and the probe molecule from the magnetic particles to obtain a prepared sample; a measuring step of measuring the target substance by detecting the complex in the prepared sample; A measurement method including:

10. The method according to claim 9 , wherein the competitor is a labeled competitor labeled with a labeling substance, and the detection of the complex is detection using the labeling substance.

11. A kit for use in the method for measuring a target substance in a sample according to claim 9 or 10, 5. The photocleavable linker-bound magnetic particle according to claim 3 or 4, wherein the probe molecule is a molecule capable of specifically binding to the target substance and a competitor of the target substance. Including the kit.

12. The kit according to claim 11 , further comprising a labeled competitor in which the competitor for the target substance is labeled with a labeling substance.

13. A measurement method for measuring a target substance in a sample, a capture step of contacting the photo-cleavable linker-bound magnetic particle according to claim 3 or 4, the photo-cleavable linker-bound magnetic particle having the probe molecule as a competitor of the target substance, the sample, and a detection molecule capable of specifically binding to the target substance and the competitor, and allowing the detection molecule to compete with the target substance and the probe molecule, thereby capturing the detection molecule on the photo-cleavable linker-bound magnetic particle; a release step of photocleaving the photocleavable linker to release a complex containing the detection molecule and the probe molecule from the magnetic particle to obtain a prepared sample; a measuring step of measuring the target substance by detecting the complex in the prepared sample; A measurement method including:

14. The method according to claim 13 , wherein the detection molecule is a labeled detection molecule labeled with a labeling substance, and the detection of the complex is detection by the labeling substance.

15. A kit for use in the method for measuring a target substance in a sample according to claim 13 or 14, 5. The photocleavable linker-bound magnetic particle according to claim 3 or 4, wherein the probe molecule is a competitive substance for the target substance. Including the kit.

16. The kit according to claim 15 , further comprising a labeled detection molecule in which the detection molecule capable of specifically binding to the target substance and the competitor is labeled with a labeling substance.

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