阅读说明：本技术 目标物质的分离方法和定量方法 (Method for separating and quantifying target substance ) 是由 佐藤嘉哉 于 2019-10-16 设计创作，主要内容包括：本发明提供一种能够不论目标物质的种类(电荷)地来分离目标物质的方法。本发明的目标物质的分离方法包括：使用含有目标物质的样品和固定有特异性识别上述目标物质的部位的第1受体的磁性粒子,形成目标物质-磁性粒子的复合物,利用磁性和电泳来分离上述目标物质-磁性粒子的复合物。(The present invention provides a method capable of separating a target substance regardless of the kind (charge) of the target substance. The method for separating a target substance of the present invention comprises: a target substance-magnetic particle complex is formed using a sample containing a target substance and magnetic particles to which a1 st receptor that specifically recognizes a site of the target substance is immobilized, and the target substance-magnetic particle complex is separated by magnetism and electrophoresis.)

1. A method of separating a target substance, comprising: a target substance-magnetic particle complex is formed using a sample containing a target substance and magnetic particles to which 1 st receptors that specifically recognize a site of the target substance are immobilized, and the target substance-magnetic particle complex is separated by magnetism and electrophoresis.

2. The method according to claim 1, wherein the mixture containing the target substance-magnetic particle complexes is obtained, and then the target substance-magnetic particle complexes are collected at a predetermined position by magnetism and subjected to electrophoresis, thereby separating the target substance-magnetic particle complexes from the mixture.

3. The method of claim 1 or 2, wherein the electrophoresis is a carrier-free electrophoresis.

4. The method of any one of claims 1 to 3, wherein the sample is selected from blood, interstitial fluid and urine.

5. A method for measuring the amount of a target substance, comprising: a sample containing a target substance, magnetic particles to which a1 st receptor that specifically recognizes a site of the target substance is immobilized, and a labeled substance to which a 2 nd receptor that specifically recognizes a site different from the site of the target substance is immobilized are mixed to obtain a mixture containing a target substance-magnetic particle-labeled substance complex, the target substance-magnetic particle-labeled substance complex is separated by magnetism and electrophoresis, and then the signal intensity of the target substance-magnetic particle-labeled substance complex is detected.

6. The method according to claim 5, wherein the mixture containing the target substance-magnetic particle-labeling substance complex is obtained, and then the target substance-magnetic particle-labeling substance complex is magnetically concentrated at a predetermined position and then subjected to electrophoresis to separate the target substance-magnetic particle-labeling substance complex from the labeling substance to which the unreacted 2 nd receptor is immobilized.

7. The method of claim 5 or 6, wherein the electrophoresis is a carrier-free electrophoresis.

8. The method according to any one of claims 5 to 7, wherein a standard curve of the signal intensity of a target substance-magnetic particle-labeling substance complex with respect to the amount of a target substance is prepared using a target substance of which an amount is already known in advance, and the amount of the target substance contained in the sample is determined based on the standard curve and the detected signal intensity of the target substance-magnetic particle-labeling substance complex.

9. The method according to claim 1 to 5, wherein the labeling substance is at least one selected from the group consisting of a fluorescent substance, a radioisotope, an enzyme and a redox substance.

10. The method of any one of claims 5 to 9, wherein the sample is selected from blood, interstitial fluid and urine.

11. A test kit for a disease related to a target substance, comprising: the magnetic particle comprises a magnetic particle to which a1 st receptor that specifically recognizes a site of a target substance is immobilized, a labeling substance to which a 2 nd receptor that specifically recognizes a site different from the recognition site of the target substance is immobilized, and a magnet.

Technical Field

The present invention relates to a method for separating and quantifying a target substance.

Background

Among peptides present in an organism, there are peptides whose amount varies due to a disease, and these peptides can be used as markers for a specific disease. For example, diabetes is a disease in which the glucose level (blood glucose level) in blood is high due to a decrease in the action and insufficient secretion of insulin. If the blood glucose level is maintained at a high level for a long period of time, damage may occur to the kidney, retina and peripheral nerve, damage may occur to blood vessels, arteriosclerosis may be promoted, and this may be a risk factor for myocardial infarction and cerebral infarction. Therefore, in diagnosis, prevention, and treatment of diabetes, it is important to grasp the blood glucose level and the blood component concentration of factors related to diabetes. For example, Japanese patent application laid-open No. 2014-145680 reports a method of forming a complex of a target substance (e.g., serum C-peptide which is a constituent substance of proinsulin) and a receptor using 2 types of receptors having large molecular weights, and separating the complex from the free receptor by utilizing a difference in molecular weight under electrophoresis.

Disclosure of Invention

The receptor is preferably a protein having a predetermined charge. However, due to the magnitude relationship between the charge and molecular weight of the receptor and the target substance, it may be difficult to clearly separate the complex and the receptor even by the method described in Japanese patent application laid-open No. 2014-145680.

In view of the above circumstances, an object of the present invention is to provide a method capable of separating a target substance.

The present inventors have conducted intensive studies in order to solve the above problems. As a result, it has been found that the above problems can be solved by combining electrophoresis with separation by magnetism. That is, the above object can be achieved by a separation method using a target substance comprising: a target substance-magnetic particle complex is formed using a sample containing a target substance and magnetic particles to which a1 st receptor that specifically recognizes a site of the target substance is immobilized, and the target substance-magnetic particle complex is separated by magnetism and electrophoresis.

Drawings

In fig. 1-1, fig. 1A and B are process diagrams for explaining the method of the present invention. In fig. 1A and 1B, 2 and 3 denote electrodes; 4 represents a reagent; 5 represents an electrophoretic substrate; 6 represents a substrate; 7 represents a sample containing a target substance; and 8 denotes a magnet.

In fig. 1-2, fig. 1C and D are process diagrams for explaining the method of the present invention. In fig. 1C and D, 2, 3 denote electrodes; 5 represents an electrophoretic substrate; 6 represents a substrate; 8 denotes a magnet; and 9 represents a target substance-magnetic particle-labeling substance complex.

In fig. 2, fig. 2A is a photograph of a square glass tube before magnetic recovery in example (production of a square glass tube filled with a migration solution). Fig. 2B is a photograph of a square glass tube after magnetic recovery in example (production of a square glass tube filled with a migration solution).

Fig. 3 is a photograph of an electrophoresis apparatus used in example (electrophoresis).

FIG. 4 is a photograph of a square glass tube before and after electrophoresis in example (electrophoresis) when the measurement is performed by a fluorescence scanner.

FIG. 5 is a graph showing the relationship between the fluorescence intensity and the final concentration of H-FABP in examples.

Detailed Description

Hereinafter, embodiments of the present invention will be described. The present invention is not limited to the following embodiments.

The 1 st aspect of the present invention is a method for separating a target substance, including: a target substance-magnetic particle complex is formed using a sample containing a target substance and magnetic particles to which a1 st receptor that specifically recognizes a site of the target substance is immobilized, and the target substance-magnetic particle complex is separated by magnetism and electrophoresis. According to this mode, the target substance can be separated regardless of the kind (e.g., charge, molecular weight) of the target substance. Further, according to this mode, a target substance which is difficult to detect can be separated.

The 2 nd aspect of the present invention is a method for measuring the amount of a target substance, comprising: a sample containing a target substance, magnetic particles to which a1 st receptor that specifically recognizes a site of the target substance is immobilized, and a labeled substance to which a 2 nd receptor that specifically recognizes a site different from the site of the target substance is immobilized are mixed to obtain a mixture containing a target substance-magnetic particle-labeled substance complex, the target substance-magnetic particle-labeled substance complex is separated by magnetism and electrophoresis, and then the signal intensity of the target substance-magnetic particle-labeled substance complex is detected. According to this embodiment, the amount of the target substance can be measured with high accuracy regardless of the type (e.g., charge or molecular weight) of the target substance. Further, according to this aspect, even a target substance that is difficult to detect can be measured with high accuracy.

In the present specification, the "1 st receptor that specifically recognizes a site of a target substance" is also simply referred to as "1 st receptor". Further, "the magnetic particle to which the 1 st receptor that specifically recognizes the site of the target substance is immobilized" is also simply referred to as "the magnetic particle to which the 1 st receptor is immobilized" or "the magnetic particle of the present invention". The "2 nd receptor that specifically recognizes a site different from the site of the target substance" means that the 2 nd receptor specifically recognizes a site different from the site of the target substance recognized by the 1 st receptor, and is also simply referred to as "2 nd receptor". Further, "the 2 nd receptor-immobilized labeled substance that specifically recognizes a site different from the site of the target substance" is also simply referred to as "the 2 nd receptor-immobilized labeled substance" or "the labeled substance of the present invention".

In the present specification, "X to Y" indicating a range includes X and Y, and means "X to Y. The operation and the measurement of physical properties are carried out under the conditions of room temperature (20 to 25 ℃ C.)/relative humidity of 40 to 50% RH unless otherwise specified, but the conditions are not limited thereto.

< method of separating target substance (1 st embodiment of the present invention) >

The 1 st aspect of the present invention is a method for separating a target substance, including: a target substance-magnetic particle complex is formed using a sample containing a target substance and magnetic particles to which a1 st receptor that specifically recognizes a site of the target substance is immobilized, and the target substance-magnetic particle complex is separated by magnetism and electrophoresis.

If the method of the present invention is used, a complex of a target substance and magnetic particles (target substance-magnetic particle complex) is formed via the 1 st receptor. The magnetic particles contained in the complex are magnetically captured at a predetermined position. Therefore, if the complex is captured magnetically or electrophoresed after the capture, the complex is fixed at a predetermined position by magnetism and does not move. However, components other than the complex (for example, components derived from living bodies such as blood cells) move or do not move. Therefore, the target substance (target substance-magnetic particle complex) can be selectively separated and recovered. The method of the present invention is a method for recovering a target substance as an object using magnetic particles, and therefore, the target substance can be efficiently separated regardless of the kind (for example, charge, isoelectric point, molecular weight) of the target substance.

The following describes the 1 st embodiment of the present invention.

(sample containing target substance)

The target substance may be appropriately selected depending on the purpose (kind of disease to be diagnosed). Therefore, the target substance is not particularly limited. Specifically, there may be mentioned: insulin, insulin precursors (e.g., C-peptide), GLP-1 (glucagon-like peptide-1), GIP (glucose-dependent insulinotropic polypeptide), adiponectin, H-FABP (cardiac fatty acid binding protein), myoglobin, troponin I, troponin T, BNP (brain natriuretic peptide), NT-ProBNP (N-terminal brain natriuretic peptide precursor), fatty acid binding proteins, and other proteins, peptides, sugar chains, nucleic acids, low-molecular compounds, high-molecular compounds, and complexes thereof. Among them, the target substance is preferably a protein or a peptide, and more preferably a protein or a peptide (e.g., insulin, C-peptide, etc.) that can be used for examination of body fluids such as blood and urine, from the viewpoints of benefit, versatility, and the like. The protein or peptide includes a modified form of an isolated form, which has been modified with a phosphate group, a methyl group, an acetyl group, a sugar chain, a lipid, a nitrile group, or the like, a salt with an acid (e.g., an inorganic acid, an organic acid), a base (e.g., an alkali metal salt), or the like, a nucleic acid-binding nucleoprotein, or the like, and a protein or peptide bound to another substance. The protein may be natural or synthetic. In the present specification, "protein" refers to a polypeptide of 10kDa or more, and peptide refers to a polypeptide of less than 10 kDa.

The sample containing the target substance is not particularly limited as long as it contains the desired target substance as described above. Specifically, the test sample (blood, plasma, serum, tissue, synovial fluid, urine, lymph fluid, etc.), cells (cultured cells, cell lines, etc.), culture supernatant thereof, extracts thereof, and partially purified fractions thereof may be mentioned. The source of the test sample is not particularly limited, and may be any of humans or non-human mammals (for example, rodents such as rats, mice, hamsters and guinea pigs, domestic animals such as sheep, pigs, cows and horses, pets such as rabbits, cats and dogs, primates such as macaques, green monkeys, cynomolgus monkeys and chimpanzees). In addition, samples such as soil and water in various environments, or extracts from the above-described samples may be used. Among them, if considering the benefit (e.g., for diagnostic purposes) and the like, the sample containing the target substance is preferably a test sample, more preferably blood (e.g., whole blood, plasma, serum, and the like), interstitial fluid, and urine. That is, from a preferred aspect of the present invention, the sample may be selected from the group consisting of blood, interstitial fluid and urine.

The target substance may be used as it is as a sample without being purified (extracted), or may be used after being purified (extracted). For example, a test sample such as a biological component such as blood, plasma, serum, tissue, synovial fluid, urine, lymph, or the like of a subject can be used. In this case, the test sample may be subjected to a treatment such as dilution or purification. Alternatively, the target substance may be prepared by purifying the target substance by a known protein purification method. Specifically, for example, a tissue or a cell of a mammal may be homogenized in the presence of an appropriate buffer solution, and a crude extract fraction of the obtained tissue may be purified by chromatography such as reverse phase chromatography, ion exchange chromatography, or affinity chromatography. Alternatively, the target substance may be a commercially available product.

(magnetic particles having immobilized thereon a1 st receptor that specifically recognizes a site of a target substance)

The magnetic particle of the present invention (magnetic particle having the 1 st receptor immobilized thereon) is formed by immobilizing (for example, on the surface) the 1 st receptor having a site that specifically recognizes a target substance.

In the present specification, the "receptor" refers to a substance capable of specifically binding to a target substance. The receptor can maintain the binding activity to the target substance during electrophoresis (for example, in an electrophoresis buffer). In the present invention, the 1 st receptor specifically recognizes a certain site (site 1) of a target substance. The 2 nd receptor specifically recognizes a site (site 2) of the target substance different from the site (site 1) of the target substance recognized by the 1 st receptor.

The receptor is not particularly limited, and examples thereof include an antibody, a fragment of an antibody having a binding activity to a target substance (antibody fragment), a nucleic acid such as a DNA aptamer, a protein, a receptor composed of a protein, a binding protein, a peptide, a biological receptor, a chemically synthesized receptor, and a sugar chain. Among them, antibodies or fragments of antibodies are preferably used as receptors, in view of high specific binding to target substances. The 1 st receptor is preferably an antibody or a fragment of an antibody recognizing a structural unit (epitope) composed of a specific sequence of a target substance (epitope 1). Furthermore, the 2 nd receptor is preferably an antibody or a fragment of an antibody recognizing an epitope (epitope 2) different from epitope 1. Alternatively, the 2 nd receptor may be a receptor (including a ligand) that specifically recognizes the 1 st receptor.

Examples of the antibody include a monoclonal antibody, a polyclonal antibody, a single-chain antibody, a modified antibody (for example, "humanized antibody" obtained by humanizing only an antigen recognition site), a chimeric antibody, and a bifunctional antibody capable of recognizing 2 epitopes simultaneously. The antibody may be any of IgA, IgD, IgE, IgG, IgM, etc. From the viewpoint of specific binding to an epitope, a monoclonal antibody is preferably used, and an IgG monoclonal antibody is more preferably used.

Examples of the antibody Fragment (Fragment) include a coupling molecule produced by genetic engineering such as a Fab Fragment, Fab 'Fragment, f (ab)'2 Fragment, single-chain antibody (scFv), scFv-Fc, minibody, or diabody, and a derivative thereof modified with a molecule having a protein stabilizing effect such as polyethylene glycol (PEG). The receptor may be obtained by treating an antibody with various proteases, or by applying an arbitrary label (Tag) according to the purpose.

The receptor to be bound to the target substance can be prepared by a conventionally known method. For example, a monoclonal antibody can be produced by the following procedure. First, an antigen is administered to a site of an animal where an antibody can be produced by antigen administration, either by itself or together with a carrier or diluent. In order to improve the antibody-producing ability at the time of administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be administered. Examples of the animal to be used include mammals such as monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats. The antibody titer in the antiserum can be measured by a conventional method. An individual having confirmed antibody titer is selected from animals immunized with an antigen, the spleen or lymph node is collected 2 to 5 days after the final immunization, and antibody-producing cells contained therein are fused with myeloma cells to prepare hybridoma cells producing a monoclonal antibody. The fusion operation can be carried out by a known method, for example, a method described in Nature 256:495 (1975). Examples of the fusion promoter include polyethylene glycol (PEG). Examples of myeloma cells include NS-1, P3U1, and SP 2/0. The monoclonal antibody can be screened according to a known or corresponding method, but is usually carried out using a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As the medium for screening and breeding, any medium can be used as long as hybridoma cells can be bred. The antibody titer of the hybridoma cell culture supernatant can be measured in the same manner as the measurement of the antibody titer in antiserum. The separation and purification of monoclonal antibodies can be carried out by, for example, a separation and purification method of immunoglobulin by a specific purification method using an antigen-binding solid phase, protein a or protein G, antigen affinity purification, or the like, which is similar to the separation and purification of ordinary polyclonal antibodies, such as a salting-out method, an alcohol precipitation method, an isoelectric point precipitation method, an electrophoresis method, an adsorption/desorption method using an ion exchanger (e.g., DEAE), an ultracentrifugation method, a gel filtration method.

The polyclonal antibody can be prepared, for example, by the following procedure. The polyclonal antibody can be produced, for example, by preparing an antigen from a peptide or the like containing an epitope, preparing a complex with a carrier, immunizing a mammal in the same manner as the above-described method for producing a monoclonal antibody, collecting an antibody-containing substance against active haptoglobin from the immunized animal, and separating and purifying the antibody. In forming the complex of the antigen and the carrier, the type of the carrier and the mixing ratio of the antigen and the carrier may be any ratio as long as an antibody can be efficiently produced with respect to the antigen crosslinked to the carrier. Examples of the carrier include bovine serum albumin, bovine thyroglobulin, and keyhole limpet hemocyanin. In addition, in the coupling of the antigen to the carrier, various condensing agents can be used, and glutaraldehyde, carbodiimide, maleimide active ester, active ester reagent containing a thiol group, dithiopyridyl group, or the like can be used. The complex of the antigen and the carrier may be administered to an immunized animal at a site where an antibody can be produced, by itself, or may be administered together with a carrier or a diluent. In order to improve the antibody-producing ability at the time of administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be administered. The administration can be usually performed 1 time each about 2 to 6 weeks, and about 3 to 10 times in total. The animal used may be a mammal similar to that produced by monoclonal antibody production. The polyclonal antibody can be collected from blood, ascites, and the like of an animal immunized by the above-described method, and is preferably collected from blood. Measurement of polyclonal antibody titer in antiserum was performed in the same manner as the measurement of antibody titer in the above-mentioned serum. The isolation and purification of the polyclonal antibody can be performed by the same procedure as the isolation and purification of the monoclonal antibody described above.

Commercially available antibodies can also be used. For example, when insulin is the target substance, monoclonal mouse anti-human (C7C9), monoclonal mouse anti-human (D4B8), monoclonal mouse anti-human (7F8), monoclonal mouse anti-human (3a6), monoclonal mouse anti-human (8E2), monoclonal mouse anti-human (7F5) (both manufactured by Hytest) and the like can be given. In addition, for example, when a fatty acid binding protein is a target substance, monoclonal mouse anti-human fatty acid binding protein (28), monoclonal mouse anti-human fatty acid binding protein (25), monoclonal mouse anti-human fatty acid binding protein (5B5), monoclonal mouse anti-human fatty acid binding protein (9F3), monoclonal mouse anti-human fatty acid binding protein (10E1), monoclonal mouse anti-human fatty acid binding protein (22), monoclonal mouse anti-human fatty acid binding protein (30) (all manufactured by Hytest) and the like can be given. Further, when C-peptide is a target substance, for example, a combination of any one monoclonal antibody selected from the group consisting of monoclonal mouse anti-human C-peptide (7E10) (manufactured by abcam), anti-C-peptide antibody (5B8) (manufactured by abcam), anti-C-peptide antibody (2B7) (manufactured by abcam), anti-C-peptide antibody (2A11) (manufactured by abnova) and anti-H C-peptide 9101SPTN-5 (manufactured by Medix Biochemica) with anti-H C-peptide 9103 SPRN-5 (manufactured by Medix Biochemica), or a combination of anti-C-peptide antibody (2A11) (manufactured by abnova) and anti-C-peptide antibody (4H8) (manufactured by Medix Biochemica) is given, for example, anti-C-peptide antibody (4H8) (mouse monoclonal antibody (manufactured by abcam)) and anti-C-peptide antibody (5B8) (mouse monoclonal antibody (manufactured by abcam)).

The magnetic particles (magnetic particles before the immobilization of the 1 st receptor) are not particularly limited as long as they are magnetic. For example, the magnetic particles may be formed of the formula MFe₂O₄(M＝Co、Mn、Ni、Mg、Cu、Zn、Li_0.5Fe_0.5Etc.) and maghemite (gamma-Fe)₂O₃) Magnetite (Fe)₃O₄) Nickel zinc ferrite (Ni)_1-XZn_XFe₂O₄) And manganese-zinc ferrite (Mn)_{1- x}Zn_XFe₂O₄) And metals such as iron, manganese, nickel, cobalt, and chromium, and magnetic materials such as alloys of cobalt, nickel, and manganese. Among them, from the viewpoint of high saturation magnetization and low residual magnetization, γ -Fe is preferable₂O₃、Fe₃O₄. The average particle size (primary particle size) of the magnetic particles is not particularly limited, and may be determined according to the electrophoresis conditions (for example, the inside of the electrophoresis substrate)Diameter, compatibility with an electrophoresis buffer), and the like. The average particle size (primary particle diameter) of the magnetic particles is preferably 50nm to 20 μm, more preferably 100nm to 10 μm, and particularly preferably 1 to 5 μm, from the viewpoints of mobility, dispersibility, sufficient magnetic force, and the like in electrophoresis. Note that the "particle size" refers to the largest distance among distances between arbitrary 2 points on the contour line of the magnetic particle. In the present specification, the "average particle size" is a value calculated by extracting 300 or more particles from a photograph obtained using a Scanning Electron Microscope (SEM), a Transmission Electron Microscope (TEM), or an optical microscope, measuring the diameters of the particles, and calculating the arithmetic mean of the diameters.

Further, the magnetic particles may be surface-coated with a polymer or the like. The polymer is not particularly limited, and a known polymer used in the field of biochemistry (for example, a carrier, a polymer bead, or a magnetic particle) can be used in the same manner, or can be used after being modified as appropriate. Specifically, examples thereof include radical polymerizable polymers such as (meth) acrylate polymers and styrene polymers. Among them, polymers having a hydrophobic surface such as polystyrene and polycyclohexylmethacrylate, which can bind to biochemical substances, and polymers having a surface functional group such as carboxyl group and tosyl group can be preferably used. In the present specification, the "(meth) acrylate" includes both "acrylate" and "methacrylate". In addition, in the case where the magnetic particles are surface-coated with a polymer or the like, the coating may have at least 1 polar group selected from amino groups, aldehyde groups, carboxyl groups, tosyl groups, mercapto groups, hydroxyl groups, and epoxy groups. In such a case, the target substance, the receptor such as the antibody, or the like can be easily bound (supported) to the magnetic particles via the polar group.

Instead of the coating, a modifying substance may be fixed to the surface of the magnetic particles, or in addition to the coating, a modifying substance may be fixed to the surface of the magnetic particles. Here, the modifying substance is not particularly limited, and may be appropriately selected depending on the intended use. Specific examples thereof include proteins such as biotin, avidin, streptavidin, neutravidin, protein A, protein G, protein L, antigens, antibodies, and enzymes; nucleic acids such as DNA and RNA; and low molecular compounds such as low molecular drugs, physiologically active substances, oligopeptides, oligonucleotides, and lipids. By modifying with such a modifying substance, binding to a receptor can be facilitated. Here, the method of immobilizing (modifying) the magnetic particles with the modifying substance is not particularly limited, and the method can be performed in the same manner as the methods described in, for example, japanese patent laid-open publication No. 2007-262114, japanese patent laid-open publication No. 2008-32411 (corresponding to US 2008/026222 a1), and japanese patent laid-open publication No. 2012-177691, or can be used after being modified appropriately.

Commercially available magnetic particles may be used. The magnetic particles to be used may be beads in which a plurality of magnetic materials such as magnetic beads and magnetic beads of agarose are coated with a polymer or gel (for example, methacrylate or dextran), or may be magnetic particles modified as appropriate depending on the purpose, such as antibody-bound magnetic beads and labeled antibody-bound beads. Specific examples thereof include Magnosphere^TM MS300/Streptavidin、Magnosphere^TM MS160/Streptavidin、Magnosphere^TM SS150/Streptavidin、Magnosphere^TMStreptavidin-labeled magnetic beads such as SS550/Streptavidin (both JSR Life Sciences Co., Ltd.), Magnosphere^TM MS300/Carboxyl、Magnosphere^TMMS160/Carboxyl、Magnosphere^TM MX200/Carboxyl、Magnosphere^TM SS150/Carboxyl、Magnosphere^TMMagnetic beads, Magnosphere, with Carboxyl groups introduced, such as SS550/Carboxyl (both JSR Life Sciences Co.)^TM MS300/Tosyl、Magnosphere^TMMagnetic beads and the like are introduced with Tosyl groups such as MS160/Tosyl and the like.

The method for immobilizing the 1 st receptor on the surface of the magnetic particle is also not particularly limited. For example, the following method can be used in the same manner as described below, or after appropriate modification: method for labeling biotin to receptor (e.g., antibody), binding biotin-labeled receptor to streptavidin-labeled magnetic bead, method for labeling streptavidin to receptor (e.g., antibody), and binding streptavidin-labeled receptor to biotin-labeled magnetic beadA specific binding pair labeling method of chemically binding a substance exhibiting specific binding properties to a pair of substances; NHS method in which carboxyl groups introduced into magnetic beads are selectively activated with N-hydroxysuccinimide (NHS) to form-COO-NHS groups on the bead surfaces and the resulting groups are bonded to receptors (e.g., antibodies); a maleimide method in which a disulfide bond of an acceptor (e.g., an antibody) is reduced with a reducing agent to form a thiol group (-SH), and a maleimide group is introduced to the surface of a magnetic particle to react with the thiol group of the acceptor; oxidizing sugar chain part of enzyme with periodic acid, introducing aldehyde group, reacting the aldehyde group with amino group of acceptor (such as antibody) to form Schiff base (-CH ═ N-), and reducing the Schiff base to-CH₂Periodic acid method of NH-; treating enzyme with excessive glutaraldehyde, introducing aldehyde group, reacting the aldehyde group with amino group of receptor (such as antibody) to form Schiff base (-CH ═ N-), and reducing the Schiff base to-CH₂NH-glutaraldehyde method and the like.

The amount of the 1 st receptor-immobilized magnetic particles used in the present invention is not particularly limited, and may be appropriately selected depending on the amount of the target substance, and is usually larger than the amount of the target substance. The 1 st receptor-immobilized magnetic particles of the present invention are used in an amount such that 1 mol of the 1 st receptor (e.g., antibody) is present in an amount of preferably 1 to 10000 mol or less, more preferably more than 1 mol and 1000 mol, and particularly preferably 10 to 1000 mol, based on 1 mol of the target substance. In the case where 2 target substances are bound to 1 magnetic particle (for example, in a magnetic particle to which an IgG antibody is immobilized), the amount of the 1 st receptor-immobilized magnetic particle is 2 times the preferable amount by weight. Further, the person skilled in the art can predict the amount of the target substance, such as the amount of insulin contained in blood, to some extent, and can take the upper limit of the prediction range as the amount of the target substance. In the case where the amount of the target substance cannot be predicted by those skilled in the art, an excess amount of the 1 st receptor (for example, 0.1 to 100. mu.g/mL of the 1 st receptor in the reaction solution) may be used.

(formation of target substance-magnetic particle Complex)

The target substance-magnetic particle complex is formed by mixing the target substance-containing sample with the magnetic particles of the present invention (magnetic particles to which the 1 st receptor is immobilized), and binding the target substance to the magnetic particles via the 1 st receptor. The conditions for forming the target substance-magnetic particle complex in this case are not particularly limited as long as the desired complex can be formed. For example, a sample containing a target substance and magnetic particles to which the 1 st receptor is immobilized may be mixed in a buffer solution. Here, the buffer is not particularly limited, and a known buffer can be used. Specific examples thereof include solutions containing organic acids such as citric acid, succinic acid, tartaric acid, and malic acid, and salts thereof; amino acids such as glycine, taurine, and arginine; and solutions containing inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, boric acid, and acetic acid, and salts thereof. Specific examples of the buffer for electrophoresis include Good's buffers (Tris-glycine buffer, Tris-Tricine buffer, Bis-Tris buffer, PIPES buffer, Tris-hydrochloric acid (Tris-HCl) buffer, MOPS buffer, HEPES buffer, PIPES buffer, ACES buffer, MOPSO buffer, BES buffer, TES buffer, DIPSO buffer, TAPSO buffer, POPSO buffer, hepps buffer, EPPS buffer, TAPS buffer, Bicine buffer, CHES buffer, CAPSO buffer, CAPS buffer, etc.), Phosphate Buffer (PBS), acetate buffer, carbonate buffer, glycine buffer, etc. The buffer solution can be used alone in 1, also can be used in 2 or more mixtures of way. The buffer may also contain a stabilizer such as EDTA, Bovine Serum Albumin (BSA), casein, or polyethylene glycol, or a nonionic surfactant such as Tween or Triton-X. The pH of the buffer is not particularly limited as long as it is an environment in which the receptor maintains the binding activity of the target substance. In general, the neutral to weakly basic vicinity is preferable in consideration of the ability of the receptor to maintain the binding activity with the target substance, etc. From such a viewpoint, the pH of the buffer solution is more preferably 6.0 to 9.0, and particularly preferably 6.0 to 8.0. The mixing temperature is preferably 4 to 40 ℃, and more preferably 4 to 25 ℃. In addition, the mixing time is preferably 1 to 72 hours, and more preferably 8 to 24 hours. The mixing time is preferably 1 to 60 minutes, and more preferably 5 to 30 minutes.

By mixing (reacting) a sample containing a target substance with the magnetic particles of the present invention (magnetic particles to which the 1 st receptor is immobilized), a reaction solution containing a target substance-magnetic particle complex in which 1 or 2 target substances are bound to 1 magnetic particle (in the case of using a reducing antibody, a target substance-magnetic particle complex in which 1 target substance is bound to 1 magnetic particle) and unreacted magnetic particles is obtained.

(separation of target substance-magnetic particle Complex)

The target substance-magnetic particle complexes thus formed are separated by magnetic and electrophoretic separation. That is, the magnetic particles contained in the target substance-magnetic particle complex are magnetically captured at a predetermined position. Therefore, if the complex is captured magnetically or electrophoresed after the capture, the complex is fixed at a predetermined position by magnetism, and components other than the complex (for example, components derived from a living body such as blood cells) move or do not move at all. Therefore, according to the method of the present invention, the target substance (target substance-magnetic particle complex) can be efficiently separated and recovered regardless of the type of the target substance, or even a target substance that is difficult to detect can be efficiently separated and recovered. The target substance-magnetic particle complex may be contained in a buffer solution as obtained in the above (formation of the target substance-magnetic particle complex), may be separated from the buffer solution, or may be dispersed in another buffer solution after being separated from the buffer solution.

The electrophoresis is not particularly limited, and known electrophoresis can be used. Specific examples of the Electrophoresis include agarose Gel Electrophoresis, pulse-field Gel Electrophoresis (PFGE), polyacrylamide Gel Electrophoresis (PAGE), and other Gel Electrophoresis, isoelectric point Electrophoresis in which a pH Gradient is formed in an Electrophoresis Gel, two-dimensional Electrophoresis in which isoelectric point Electrophoresis (1-dimensional) and SDS-polyacrylamide Gel Electrophoresis (SDS-PAGE) are combined, and carrier Electrophoresis in which urea or formamide is used as a modifier, such as modifier concentration Gradient Gel Electrophoresis (DGGE); and non-carrier electrophoresis such as non-carrier isoelectric point electrophoresis and microchip electrophoresis. In the above-mentioned carrier electrophoresis, a carrier such as a capillary polymer described below may be used instead of a gel such as dextran in addition to the above. Among them, the carrier-free electrophoresis is preferable for the following reasons. For example, in the case of carrier electrophoresis using blood (whole blood) as a sample, blood cell aggregates may be formed at the interface between the sample and the carrier (e.g., gel). When the target substance can be detected in a normal concentration range (for example, when the target substance is contained in a sample at a level of mM. mu.M), the blood cell aggregation can eliminate the influence on the measurement of the target substance. However, when the fluorescence of the target substance is trace, when the fluorescence is detected, a protein or the like in blood cells contained in a blood cell aggregate at the interface between the sample and the carrier emits fluorescence (autofluorescence), which generates a large noise and further causes a decrease in sensitivity. In addition, in the case of electrophoresis using a carrier, the blood cells may accumulate a part of the target substance-receptor complex, and may cause a signal to decrease. On the other hand, in the case of carrier-free electrophoresis, since there is no interface between the sample and the carrier, blood cells do not aggregate. Blood cells can be electrophoresed (usually on the positive side). That is, autofluorescence by blood cells, which is one cause of sensitivity reduction, can be prevented or suppressed. Therefore, highly sensitive measurement can be realized. Furthermore, since the carrier-free electrophoresis does not contain a carrier, the treatment after the separation is easy. In addition, a large amount of continuous electrophoresis can be performed, and the continuation to other separation methods is also easy. That is, according to a preferred embodiment of the present invention, the electrophoresis is a carrier-free electrophoresis.

In addition, the target substance-magnetic particle complex can be separated from other components by magnetism. Here, the other separable component means a component other than the target substance. Specifically, there are components other than the target substance contained in the sample (for example, blood components), and components of a diluent (for example, buffer solution) if used.

In the present invention, a magnet is provided at a predetermined position in the electrophoresis direction. The magnet is not particularly limited as long as it can trap magnetic particles. Specifically, rare earth magnets (neodymium magnets, bonded magnets, etc.), ferrite magnets, electromagnets, and the like can be mentioned.

Hereinafter, a manner of performing separation of a target substance-magnetic particle complex by carrier-free electrophoresis and magnetism is described with reference to the drawings. The present invention is not limited to the following embodiments.

FIGS. 1A to D are process diagrams for explaining the method of the present invention.

First, as shown in fig. 1A, a reagent 4 containing magnetic particles to which 1 st receptors are immobilized is filled in an electrophoresis substrate (e.g., capillary) 5. The electrophoresis base material 5 is disposed on a substrate 6 of an electrophoresis apparatus. Electrodes (for example, platinum electrodes) 2 and 3 are provided at both ends of the electrophoresis substrate 5. Thereby, an electric field is applied in the longitudinal direction of the electrophoresis substrate 5. The reagent may be composed of only magnetic particles to which the 1 st receptor is immobilized. Alternatively, the reagent may contain, in addition to the magnetic particles to which the 1 st receptor is immobilized, a pH adjuster (e.g., sodium hydroxide, sodium carbonate, sodium bicarbonate, sulfuric acid, hydrochloric acid, phosphoric acid, etc.), a buffer (e.g., sodium hydrogen phosphate, sodium phosphate, Tris-HCL, etc.), a hemolytic agent such as saponin, a surfactant, and the like.

The electrophoretic substrate 5 is not particularly limited, and is preferably transparent from the viewpoint of the flow visibility of the complex. Specifically, the electrophoresis substrate is preferably made of a transparent material such as a transparent resin such as an acrylic resin, quartz glass, or synthetic quartz. From the viewpoint of reducing autofluorescence, quartz glass and synthetic quartz are more preferable, and synthetic quartz is particularly preferable. In addition, an electrophoretic substrate made of a transparent resin is particularly preferable from the viewpoint of cost. The shape of the electrophoretic substrate 5 is not particularly limited, and may be any of a cylinder, a prism, and the like. In consideration of installation stability and the like, the cross-sectional shape of the electrophoresis substrate 5 is preferably a square prism, and particularly the cross-sectional shape of the electrophoresis substrate 5 is preferably a quadrangular prism. The size of the electrophoretic substrate 5 is also not particularly limited. Considering the buffer solution, the ease of Flow of the sample, the prevention and suppression effect of electroosmotic Flow (EOF), and the like, the maximum length of the cross section of the electrophoresis substrate 5 (the length of 1 side (inner side) in the case of a square cross section, and the inner diameter (diameter) in the case of a circular cross section) is preferably 0.1 to 2mm, and more preferably 0.5 to 1 mm. In addition, the length of the electrophoresis substrate 5 is preferably 1 to 10cm, more preferably 2 to 5cm, in consideration of the ease of separation of the complex, the ease of handling, and the like.

The substrate 6 is not particularly limited, and is preferably transparent from the viewpoint of visibility of the composite flow. Specifically, the substrate is preferably made of a transparent material such as a transparent resin such as an acrylic resin, quartz glass, or synthetic quartz. From the viewpoint of less autofluorescence, quartz glass and a synthetic quartz substrate are more preferable, and a synthetic quartz substrate is particularly preferable. Alternatively, the substrate 6 made of a transparent resin is particularly preferable from the viewpoint of cost.

Next, as shown in fig. 1A, a sample 7 containing a target substance is dropped on the platinum electrode 2 side. Thereby, the sample 7 enters the electrophoresis substrate 5 by the capillary phenomenon and is mixed with the reagent 4. Thus, in the electrophoresis substrate 5, the target substance in the sample 7 reacts with the 1 st receptor present in the 1 st receptor-immobilized magnetic particles to form a target substance-magnetic particle complex. The amount of the sample 7 (specimen 1) containing the target substance to be dropped is not particularly limited, and may be appropriately set according to the size of the electrophoresis substrate. The reaction conditions are not particularly limited, and the same conditions as those described above can be used. In this embodiment, the sample 7 is dropped on the electrode 2 side of the positive (+) electrode, but the present invention is not limited to this embodiment. This mode is particularly preferably used when the carboxyl group (-COOH) present in the protein is negatively charged, as described above.

The amount of the 1 st receptor-immobilized magnetic particles in the reagent is not particularly limited, and the same amount as described in the above (1 st receptor-immobilized magnetic particles having a site that specifically recognizes a target substance) may be used.

In the present embodiment, the electrophoresis substrate 5 is filled with a reagent containing the 1 st receptor-immobilized magnetic particles in advance, but the electrophoresis substrate 5 may be filled with the reaction product after the target substance-magnetic particle complex is formed by reacting the 1 st receptor-immobilized magnetic particles with the target substance in advance (embodiment 2). In the case of embodiment 2, it is preferable to separately provide a chamber (not shown) for the above reaction on the substrate 6.

In another embodiment, a reagent solution containing magnetic particles to which the 1 st receptor is immobilized and a buffer is dropped onto both ends of an electrophoresis substrate to prepare a liquid aggregation part, the inside of the electrophoresis substrate is filled with the reagent solution by capillary action, electrodes (for example, platinum electrodes) 2 and 3 are provided on both ends of an electrophoresis substrate 5, and a sample containing a target substance is dropped onto either one of the electrodes (embodiment 3). In embodiment 3, the buffer solution is not particularly limited as long as it is an environment in which the receptor can maintain the target substance binding activity, and the same buffer solution as that used in ordinary electrophoresis may be used or appropriately modified and used. Specifically, the buffer solution is not particularly limited as long as it is a solution containing a conventionally known buffer composition having buffering ability. For example, the same buffer as the buffer exemplified in the above (formation of a complex of a target substance and magnetic particles) can be used. Alternatively, a buffer solution or the like provided in a commercially available electrophoresis kit may be used. The buffer for electrophoresis can be used in a concentration generally usable as a buffer for electrophoresis. The pH of the buffer is not particularly limited as long as it is an environment in which the receptor maintains the binding activity of the target substance. Among them, it is generally preferable that the pH is in the vicinity of neutral to weakly alkaline, considering the ability to maintain the binding activity of the receptor to the target substance, the stability of the reagent, the reactivity, and the like. From such a viewpoint, the pH of the buffer solution is more preferably 6 to 9. In particular, when the target substance is a protein, the pH of the buffer is on the alkaline side, and thus the carboxyl group (-COOH) present in the protein is negatively charged (becomes-COO-), whereby the movement of the complex in electrophoresis can be promoted. The pH of the buffer solution may be appropriately adjusted by using an acidic substance such as hydrochloric acid or a basic substance such as sodium hydroxide.

Next, as shown in fig. 1B, the magnet 8 is provided on the platinum electrode 2 (anode) side of the electrophoresis substrate 5. Thereby, the magnetic particles contained in the target substance-magnetic particle complex can be captured by the magnet 8. Next, as shown in fig. 1C, after the magnet 8 is moved in the longitudinal direction of the electrophoresis substrate 5, electrophoresis is started. Thereby, the target substance-magnetic particle complex can be selectively separated. That is, according to a preferred embodiment of the present invention, after a mixture containing a target substance-magnetic particle complex is obtained, the target substance-magnetic particle complex is magnetically captured at a predetermined position, and then the target substance-magnetic particle complex is separated from the mixture by electrophoresis.

Here, the moving place of the magnet 8 is not particularly limited, and the magnet 8 is preferably provided on the cathode side of the electrophoresis substrate 5 (platinum electrode 3 side in fig. 1C). In this method, components to be separated from the target substance in the sample (for example, a substance that causes noise or increases in background noise at the time of detection (blood cells that emit autofluorescence, a substance that has fluorescence outside the detection target, or the like in embodiment 2 described below)) move toward the anode side. Therefore, at the time of detection, within the electrophoresis substrate 5, the distance between the target substance-magnetic particle complex and a substance that may generate noise can be increased. Therefore, the sensitivity can be more effectively prevented or suppressed from being lowered, and the background noise and the noise can be reduced, thereby further improving the sensitivity. Specifically, the magnet is preferably provided at a position closer to the cathode half during the whole swimming movement from the swimming start point to the swimming end point (position Y/X ≦ 1/2 in fig. 1C). More preferably, the magnets are disposed at the following positions: the distance from the cathode to the magnet (Y in fig. 1C) is a position from the cathode (Y in fig. 1C is 0) to the anode beyond the cathode to the midpoint (a position from Y/X in fig. 1C to Y/X is 0 and 1/2 or less) in the process of the electrophoresis from the cathode (Y in fig. 1C is 0) to the anode. Particularly preferably, the magnets are arranged at the following positions: positions where the ratio (Y/X) of the distance from the cathode to the magnet (Y in fig. 1C) to the total process of migration from the cathode to the anode (X in fig. 1C) is 1/10 to 3/5 (positions where Y/X in fig. 1C is 1/10 to 3/5); most preferably, the magnets are arranged in the following positions: the ratio (Y/X) of the distance from the cathode to the magnet (Y in FIG. 1C) to the total migration process from the cathode to the anode (X in FIG. 1C) is at positions 1/10-2/5 (positions 1/10-2/5Y/X in FIG. 1C). By providing the above-described position, a decrease in sensitivity such as autofluorescence can be more effectively prevented or suppressed. In fig. 1C, "X" represents the length (corresponding to the total length of the electrophoretic substrate 5) between the cathode and the anode (mm). Further, "Y" represents a distance (mm) from the cathode to the midpoint (center) of the width of the magnet. The relationship between the installation distance of the magnet with respect to the cathode and the anode may be the reverse relationship to that in embodiment 2, depending on the type of the detection substance.

After the magnet 8 is disposed at a predetermined position on the electrophoresis substrate 5, electrophoresis is started. The electrophoresis conditions are not particularly limited as long as the conditions are such that components other than the complex (for example, impurities derived from the sample) can be separated, and normal conditions can be used. For example, the voltage applied in electrophoresis may be appropriately selected from the range generally used in this field, and may be applied generally using a direct current voltage in the range of preferably 5 to 200V, more preferably 10 to 100V. The electrophoresis time may be generally 30 to 180 minutes. From the viewpoint of rapid separation, the migration time is preferably about several minutes to 10 minutes. The electrophoresis temperature is not particularly limited, and is usually carried out at room temperature (20 to 25 ℃).

As described above, the sample to be subjected to electrophoresis (hereinafter also referred to as "specimen 1") contains components contained in the sample (for example, components derived from a living body such as blood cells) and components contained in the buffer solution, as well as the target substance-magnetic particle complex and unreacted magnetic particles. In the electrophoresis, a compound of the target substance and the magnetic particles and unreacted magnetic particles can be captured by a magnet. The magnetic particles captured at 1 are typically visible (see, for example, fig. 2B). Therefore, by cutting and taking out only the electrophoretic substrate portion on which the magnet is disposed, the target substance can be efficiently separated from the sample and the components (for example, blood cells) contained in the buffer solution. In order to prevent diffusion of the target (target substance-magnetic particle composite), it is preferable that the operation of taking out only the electrophoretic substrate portion on which the magnet is disposed is performed in a state in which the magnet is disposed.

< method for measuring amount of target substance (2 nd embodiment of the present invention) >)

The 2 nd aspect of the present invention is a method for measuring the amount of a target substance, comprising: a sample containing a target substance, magnetic particles to which a1 st receptor that specifically recognizes a site of the target substance is immobilized, and a labeled substance to which a 2 nd receptor that specifically recognizes a site different from the site of the target substance is immobilized are mixed to obtain a mixture containing a target substance-magnetic particle-labeled substance complex, the target substance-magnetic particle-labeled substance complex is separated by magnetism and electrophoresis, and then the signal intensity of the target substance-magnetic particle-labeled substance complex is detected.

According to the method of the present invention, a target substance is bound to magnetic particles via a1 st receptor, and a target substance is bound to a labeling substance via a 2 nd receptor, thereby forming a complex of magnetic particles, the target substance, and the labeling substance (target substance-magnetic particle-labeling substance complex). The magnetic particles contained in the complex are magnetically captured at a predetermined position. Therefore, if the complex (the complex of the target substance, the magnetic particles, and the labeling substance) is captured magnetically or is subjected to electrophoresis after capture, the complex is fixed at a predetermined position by magnetism, and components other than the complex (for example, components derived from a living body such as blood cells, and proteins) move. In particular, in the case of detection using a receptor in which an object to be measured is fluorescently labeled, impurities (protein, peptide, amino acid, cell membrane, fatty acid, and the like) which are not an object to be detected can be electrophoretically migrated to the cathode side or the anode side. In particular, if an amino acid or peptide having fluorescence by itself, or a protein that is not a detection target is separated by electrophoresis, noise and background noise can be greatly reduced. Therefore, the target substance (target substance-magnetic particle-labeling substance complex) can be selectively separated and recovered. The method of the present invention is a method of recovering a target substance as a target using magnetic particles, and therefore, the target substance can be efficiently separated regardless of the kind (for example, charge, isoelectric point, molecular weight) of the target substance. Further, since the complex of the target substance, the magnetic particle and the labeling substance can be selectively collected, the amount of the target substance can be measured with high accuracy.

The following description of embodiment 2 of the present invention is given, and since the description of the duplicated items (for example, a sample containing a target substance, magnetic particles of the present invention (magnetic particles having 1 st receptors immobilized thereon), magnet, and electrophoresis) is the same as that in embodiment 1 of the present invention, the description thereof will be omitted here.

(labeled substance having immobilized thereon receptor 2 which specifically recognizes a site different from the site of the target substance)

The labeling substance of the present invention (labeling substance having a 2 nd receptor immobilized thereon) is one having a 2 nd receptor specifically recognizing a site different from the 1 st receptor immobilized thereon (for example, on the surface). The 2 nd receptor is not particularly limited, and is the same as that described in the magnetic particle of the present invention (the 1 st receptor-immobilized magnetic particle), and therefore, the description thereof will be omitted here. The 2 nd receptor recognizes a site different from the 1 st receptor, that is, the 1 st receptor and the 2 nd receptor (e.g., antibody) recognize 1 different site of the target substance, respectively. For example, in the case where the 1 st receptor and the 2 nd receptor recognize fatty acid binding proteins, different receptors (e.g., antibodies) are selected such that the monoclonal mouse anti-human fatty acid binding protein (25) is used as the 1 st receptor, the monoclonal mouse anti-human fatty acid binding protein (28) is used as the 2 nd receptor, and the like.

The labeling substance (labeling substance before immobilization of the 2 nd receptor) is not particularly limited as long as it can be used for measuring the amount of a desired target substance in a subsequent step. For example, fluorescent substances, radioisotopes, enzymes, redox substances, and the like are preferably used. The labeling substance can be used alone in 1 kind, or can also be combined with 2 or more kinds. That is, according to a preferred embodiment of the present invention, the labeling substance is selected from the group consisting of fluorescent substances and radioactive substancesAt least one of an isotope, an enzyme, and a redox species. Here, the fluorescent substance is not particularly limited, and a fluorescent substance that can be used to measure the amount of a target substance can be generally used. Specifically, there may be mentioned: alexa flow (Life), Hilyte flow (Ana spec), IRDye (LI-COR Biosciences), IRDye800CW maleimide (LI-COR Biosciences), FITC (fluoroescein Isothiocyanate), PE (phytoerythrin), APC (allophyacin), Cy-3, Cy-5, tetramethylrhodamine isocyanate, semiconductor quantum dots, and the like. Among them, a fluorescent dye excited on the long wavelength side is preferably used. Since a substance that emits fluorescence at a short wavelength is present in a large amount in nature, autofluorescence can be reduced more effectively by using a fluorescent dye that is excited on the long wavelength side. In view of the above, it is preferable to use a fluorescent dye that excites at 700 to 1000nm, particularly at 750 to 900 nm. The fluorescent dye excited in such a wavelength region has an advantage that the detection efficiency is high and the sensitivity can be further improved. The radioisotope is not particularly limited, and a radioisotope that can be used for measuring the amount of a target substance can be generally used. Specific examples thereof include¹²⁵I、³²P、¹⁴C、³⁵S or³Radioactive isotopes such as H. The enzyme is not particularly limited, and an enzyme that can be used for measuring the amount of a target substance can be generally used. Specific examples thereof include alkaline phosphatase, peroxidase (e.g., horseradish peroxidase), β -galactosidase, phycoerythrin, and the like. The redox substance is not particularly limited, and a redox substance that can be used for measuring the amount of a target substance can be generally used. Specifically, there are compounds containing a redox metal ion selected from iron, vanadium, chromium, zinc and mixtures thereof, and more specifically, ferrocene, 1' -dimethylferrocene, ferrocyanide and ferricyanide, ruthenium (III) and ruthenium (II) hexamine, PMS (phenazine methosulfate), m-PMS and the like are cited.

The method for immobilizing the 2 nd receptor to the labeling substance is also not particularly limited, and the same contents as those described in the magnetic particle of the present invention (magnetic particle having the 1 st receptor immobilized thereon) will be omitted here. In the above description of the "method of immobilizing the 1 st receptor by the magnetic particles" (magnetic particles having the 1 st receptor immobilized thereon that specifically recognizes a site of the target substance), "magnetic beads" or "magnetic particles" may be used instead of "labeling substance".

The amount of the 2 nd receptor-immobilized labeling substance used in the present invention is not particularly limited, and may be appropriately selected depending on the amount of the target substance, and is usually larger than the amount of the target substance. For example, in the case where 1 target substance is bound to a labeling substance (for example, in the case of a labeling substance to which a reducing antibody is immobilized), the amount of the 2 nd receptor-immobilized labeling substance of the present invention is preferably more than 1 mole and 100 moles or less, more preferably more than 1 mole and 50 moles or less, and particularly preferably 2 to 50 moles of the 2 nd receptor (e.g., antibody) based on 1 mole of the target substance. In the case where 2 target substances are bound to a labeling substance (for example, in a labeling substance to which an IgG antibody is immobilized), the amount of the 2 nd receptor-immobilized labeling substance is 2 times the preferable amount. Further, the amount of the marker substance, such as the amount of insulin contained in blood, can be predicted to some extent by those skilled in the art, and the upper limit of the prediction range can be set as the amount of the marker substance. In the case where the amount of the labeling substance cannot be predicted by those skilled in the art, an excess amount of the 2 nd receptor-immobilized labeling substance (for example, an amount of 0.1 to 100. mu.g/mL of the 2 nd receptor-immobilized labeling substance or 1 to 100nM of the 2 nd receptor (e.g., antibody)) may be used.

The mixing ratio of the labeling substance of the present invention (labeling substance having 2 nd receptors immobilized thereon) and the magnetic particles of the present invention (magnetic particles having 1 st receptors immobilized thereon) is also not particularly limited, and it is preferable that the number of the magnetic particles having 1 st receptors immobilized thereon is larger than the number of the labeling substance having 2 nd receptors immobilized thereon. This makes it possible to more effectively capture a labeled substance present in a sample while more effectively suppressing a decrease in sensitivity due to an unreacted labeled substance. Specifically, in the case where the labeling substance and the magnetic particles are bound to 1 target substance in common (for example, in the case of magnetic particles or labeling substances to which reducing antibodies are immobilized), the mixing ratio of the labeling substance of the present invention (labeling substance to which 2 nd receptors are immobilized) to the magnetic particles of the present invention (magnetic particles to which 1 st receptors are immobilized) is: the proportion of the 1 st receptor bound to the magnetic particle of the present invention is preferably more than 1 mole and 10000 moles or less, more preferably 10 to 1000 moles, particularly preferably more than 10 moles and 50 moles or less based on 1 mole of the 2 nd receptor bound to the labeling substance of the present invention. With such a mixing ratio, the decrease in sensitivity due to the unreacted labeling substance can be further effectively suppressed, and the labeling substance present in the sample can be further effectively captured.

(formation of a target substance-magnetic particle-labeling substance Complex)

The target substance-containing sample is mixed with the magnetic particles of the present invention (magnetic particles to which the 1 st receptor is immobilized) and the labeling substance of the present invention (labeling substance to which the 2 nd receptor is immobilized), so that the target substance is bound to the magnetic particles via the 1 st receptor and the target substance is bound to the labeling substance via the 2 nd receptor, thereby forming a target substance-magnetic particle-labeling substance complex. The conditions for forming the target substance-magnetic particle-labeling substance complex in this case are not particularly limited as long as the desired complex can be formed. For example, a sample containing a target substance, magnetic particles to which a1 st receptor is immobilized, and a labeling substance to which a 2 nd receptor is immobilized may be mixed in a buffer solution. Here, the buffer is not particularly limited, and a known buffer can be used. Specifically, the same buffer as the buffer exemplified in the above (formation of a complex of a target substance and magnetic particles) can be used. The pH of the buffer is not particularly limited as long as the target substance binding activity is maintained in the environment of the receptor. However, if a decrease in the binding activity of the receptor to the target substance or the like is considered, it is generally preferable to be in the vicinity of neutral to weakly basic. From such a viewpoint, the pH of the buffer solution is more preferably 6.0 to 9.0, and particularly preferably 6.0 to 8.0. The mixing temperature is preferably 4 to 40 ℃, and more preferably 25 to 37 ℃. The mixing time is preferably 1 to 60 minutes, and more preferably 5 to 30 minutes.

By mixing (reacting) a sample containing a target substance, the magnetic particles of the present invention (magnetic particles having the 1 st receptor immobilized thereon), and the labeling substance of the present invention (labeling substance having the 2 nd receptor immobilized thereon), a mixture containing a target substance-magnetic particle-labeling substance complex can be obtained. Specifically, the above mixture comprises: a target substance-magnetic particle-labeling substance complex in which a magnetic particle and a labeling substance are bound via a target substance, a target substance-magnetic particle complex in which a target substance is bound only to a magnetic particle, a target substance-labeling substance complex in which a target substance is bound only to a labeling substance, an unreacted magnetic particle, and an unreacted labeling substance.

(separation of target substance-magnetic particle-labeling substance Complex)

The mixture containing the target substance-magnetic particle-labeling substance complex thus formed is subjected to electrophoresis, in which the target substance-magnetic particle-labeling substance complex is separated magnetically. That is, magnetic particles present in a target substance-magnetic particle-labeling substance complex are magnetically captured during or before electrophoresis, and a target substance (target substance-magnetic particle-labeling substance complex) as a target is collected. The target substance-magnetic particle-labeling substance complex may be contained in a buffer solution as obtained in the above (formation of the target substance-magnetic particle-labeling substance complex), may be separated from the buffer solution, or may be dispersed in another buffer solution after being separated from the buffer solution. Here, the electrophoresis is not particularly limited, and known electrophoresis can be used. Specifically, since the same contents as those described in the above (separation of a target substance-magnetic particle complex) are described, the description thereof will be omitted here.

In the electrophoresis, a target substance-magnetic particle-labeling substance complex as a target is separated magnetically. Specifically, a target substance-magnetic particle-labeling substance complex, a target substance-magnetic particle complex in which a target substance is bound only to magnetic particles, and unreacted magnetic particles can be magnetically captured. Therefore, in the electrophoresis, components other than the target substance contained in the sample, a reagent diluent (e.g., a buffer solution) if used, a substance to which the target substance is non-specifically bound (a target substance-labeled substance complex or the like that is bound only to the labeled substance), and an unreacted labeled substance can be separated.

In the present invention, a magnet is provided in the middle of electrophoresis. Here, the specific description of the magnet is the same as that described in the above (separation of the target substance-magnetic particle complex), and therefore, the description thereof is omitted here.

(detection of Signal Strength of target substance-magnetic particle-labeling substance Complex)

The amount of the target substance to be targeted is determined by detecting the signal intensity of the separated target substance-magnetic particle-labeling substance complex. In the separation step, in addition to the target substance-magnetic particle-labeling substance complex, a target substance-magnetic particle complex in which the target substance is non-specifically bound to the magnetic particles alone, or unreacted magnetic particles are captured by a magnet. In the present embodiment, an antibody that specifically binds to a target substance is used as a labeling substance. Therefore, the target substance is not present in the unreacted magnetic particles, and thus the labeling substance does not bind to the unreacted magnetic particles. That is, the unreacted magnetic particles do not substantially affect the signal intensity (i.e., measurement accuracy) of the target substance-magnetic particle-labeling substance complex. Therefore, in this step, the signal intensity from the target substance can be selectively detected, that is, the amount of the target substance can be measured with high accuracy. Other components (for example, blood cells) in the sample, which cause the decrease in sensitivity, and unreacted labeling substance migrate from the magnet to the cathode or anode side by electrophoresis. The amount of the labeling substance to be mixed is such that the target substance can be sufficiently detected in the range to be measured, taking into consideration the type of the target substance, the amount of other reagent components such as magnetic particles, and the type of the sample. Therefore, according to the method of the present invention, the amount of the target substance present can be measured with high sensitivity (high accuracy).

Hereinafter, a mode of detecting the signal intensity of a complex of a target substance-magnetic particle-labeling substance after separation of the complex by carrier-free electrophoresis and magnetism is described with reference to the drawings. The present invention is not limited to the following embodiments.

FIGS. 1A to D are process diagrams for explaining the method of the present invention.

In the explanation of fig. 1A, B and C, the description thereof will be omitted for the matters overlapping with the explanation of the above-described embodiment 1 of the present invention.

First, as shown in fig. 1A, an electrophoresis substrate (for example, a capillary) 5 is filled with a reagent 4 containing magnetic particles to which a1 st receptor is immobilized and a labeling substance to which a 2 nd receptor is immobilized. The electrophoresis base material 5 is disposed on a substrate 6 of an electrophoresis apparatus. Electrodes (for example, platinum electrodes) 2 and 3 are provided at both ends of the electrophoresis substrate 5. Thereby, an electric field is applied in the longitudinal direction of the electrophoresis substrate 5. The reagent may include any one of a magnetic particle to which a1 st receptor is immobilized and a labeling substance to which a 2 nd receptor is immobilized. Alternatively, the reagent may contain, in addition to the magnetic particles to which the 1 st receptor is immobilized and the labeling substance to which the 2 nd receptor is immobilized, a pH adjuster (for example, sodium hydroxide, sodium carbonate, sodium bicarbonate, sulfuric acid, hydrochloric acid, phosphoric acid, or the like), a buffer (for example, sodium hydrogen phosphate, sodium phosphate, Tris-HCL, or the like), a hemolytic agent such as saponin, a surfactant, or the like.

The amount of the 1 st receptor-immobilized magnetic particles in the reagent is not particularly limited, and the same amount as described in the above (1 st receptor-immobilized magnetic particles having a site that specifically recognizes a target substance) may be used. Similarly, the amount of the 2 nd receptor-immobilized labeling substance and the mixing ratio of the 1 st receptor-immobilized magnetic particles and the 2 nd receptor-immobilized labeling substance are not particularly limited, and the same amounts and ratios as described in the above (the 2 nd receptor-immobilized labeling substance that specifically recognizes a site different from the site of the target substance) can be used.

In this embodiment, the electrophoresis substrate 5 is filled with a reagent containing the 1 st receptor-immobilized magnetic particles and the 2 nd receptor-immobilized labeling substance in advance, but the electrophoresis substrate 5 may be filled with the reaction product after the target substance-magnetic particle-labeling substance complex is formed by reacting the 1 st receptor-immobilized magnetic particles and the 2 nd receptor-immobilized labeling substance with the target substance (sample) in advance (embodiment 2'). In the case of embodiment 2', it is preferable to separately provide a chamber (not shown) for the above reaction on the substrate 6.

Alternatively, in another embodiment, a reagent solution containing magnetic particles to which a1 st receptor is immobilized, a labeling substance to which a 2 nd receptor is immobilized, and a buffer is dropped onto both ends of an electrophoresis substrate to prepare a liquid aggregation part, the inside of the electrophoresis substrate is filled with the reagent solution by capillarity, electrodes (for example, platinum electrodes) 2 and 3 are provided on both ends of an electrophoresis substrate 5, and a sample containing a target substance is dropped onto either one of the electrodes (embodiment 3').

Next, as shown in fig. 1B, the magnet 8 is provided on the platinum electrode 2 (anode) side of the electrophoresis substrate 5. Thereby, the magnetic particles contained in the target substance-magnetic particle complex can be captured by the magnet 8. Next, as shown in fig. 1C, after the magnet 8 is moved in the longitudinal direction of the electrophoresis substrate 5, electrophoresis is started. Thereby, the target substance-magnetic particle-labeling substance complex can be selectively separated. Therefore, in the detection of the signal intensity described in detail below, the amount of the target substance-magnetic particle-labeled substance complex (i.e., the labeled substance contained in the complex) can be measured with high accuracy. That is, according to a preferred embodiment of the present invention, after a mixture including a target substance-magnetic particle-labeling substance complex is obtained, the target substance-magnetic particle-labeling substance complex is magnetically concentrated at a predetermined position, and then subjected to electrophoresis, thereby separating the target substance-magnetic particle-labeling substance complex from a labeling substance to which an unreacted 2 nd receptor is immobilized (a 2 nd receptor-immobilized labeling substance that does not bind to the target substance). Here, the moving place of the magnet 8 is not particularly limited, and the magnet is preferably provided at the same position as described in fig. 1C in the above (separation of the target substance-magnetic particle complex).

The magnet 8 is moved between the cathode and the anode at least 1 time along the electrophoretic substrate 5 at a position on the outer surface of the electrophoretic substrate 5 or in the vicinity of the outer surface of the electrophoretic substrate 5. Then, the magnet 8 is arranged at a predetermined position, and electrophoresis is started. The position of the magnet 8 is not particularly limited as long as it is a position capable of giving an appropriate magnetic force to the electrophoresis substrate 5, and the magnet may be arranged at the same position as defined in the above (separation of the target substance-magnetic particle complex).

As described above, the sample to be electrophoresed (hereinafter also referred to as "specimen 2") contains a component contained in the sample (for example, a component derived from a living body such as a blood cell) and a component contained in the buffer solution, and also contains a target substance-magnetic particle-labeled substance complex, a target substance-magnetic particle complex, a target substance-labeled substance complex, an unreacted magnetic particle, and an unreacted labeled substance. In the electrophoresis, a target substance-magnetic particle-labeling substance complex, a target substance-magnetic particle complex, and unreacted magnetic particles can be captured by a magnet. However, by setting the mixing ratio of the labeling substance and the magnetic particles to an appropriate ratio, the mixing amount of the target substance-magnetic particle composite can be minimized. Therefore, the target substance-magnetic particle-labeling substance complex and unreacted magnetic particles can be selectively captured by the magnet. However, the labeling substance does not specifically bind to the unreacted magnetic particles (is not detected in the following detection of signal intensity). Therefore, by measuring the signal intensity of the separated product after electrophoresis, the amount of the target substance-magnetic particle-labeling substance complex (i.e., the target substance) present can be measured with high sensitivity (high accuracy).

Next, as shown in fig. 1D, the signal intensity of the target substance-magnetic particle-labeling substance complex 9 present in the portion of the electrophoresis substrate 5 where the magnet 8 is disposed is detected. Here, the signal intensity of the labeling substance present in the complex corresponds to the amount of the target substance. That is, a calibration curve of the signal intensity of the measured band of the target substance-magnetic particle-labeled substance and the amount of the target substance is prepared in advance, and the signal intensity of the detected band of the target substance-magnetic particle-labeled substance complex is compared based on the calibration curve, whereby the amount of the target substance in the sample can be accurately measured. That is, according to a preferred embodiment of the present invention, a standard curve of the signal intensity of the target substance-magnetic particle-labeled substance complex with respect to the amount of the target substance is prepared using a target substance of which an amount is known in advance, and the amount of the target substance contained in the sample is measured based on the standard curve and the detected signal intensity of the target substance-magnetic particle-labeled substance complex.

In the present invention, it is preferable that: using a sample whose amount of the target substance has been known in advance, a relationship (standard curve) of the signal intensity of the band of the target substance-magnetic particle-labeling substance complex with respect to the amount of the target substance is prepared. That is, the same procedure as described above is performed on a sample in which the amount of the target substance is known in advance, the signal intensity of the band of the target substance-magnetic particle-labeling substance complex is calculated, and a calibration curve of the amount of the standard substance and the signal intensity of the band is prepared. Here, the signal intensity can be measured by a known method depending on the type of the labeling substance. For example, when the labeling substance is a fluorescent substance and is quantified, the fluorescence intensity is measured using a fluorescence measuring instrument (fluorescence scanner) or an image processing system. In the case where the labeling substance is a radioisotope, the radiation dose is measured by a radiation counting device. In the case where the labeling substance is an enzyme, detection and/or quantification of the product produced by the enzyme can be carried out by measuring the absorbance of the product. For example, when 3,3',5,5' -tetramethylbenzidine is used as an enzyme substrate, the absorbance at 655nm may be measured. In addition, when the labeling substance is a redox substance, the labeling substance can be detected by measuring an electrochemical signal generated.

In the quantification of a target substance, for example, a standard sample containing a target substance at a known concentration is used, and the amount of the target substance contained in the sample is quantified simply by preparing a standard curve using the obtained fluorescence intensity.

The calibration curve obtained by the present invention is constant and is not dependent on external factors such as swimming conditions, and therefore, it is effective as a calibration curve. Therefore, by using the standard curve, the amount of the target substance in the sample can be accurately measured.

In particular, when the target substance is a marker related to a certain disease, the above-described detection method is a very useful detection means in clinical examination.

In fig. 1D, although the magnet is omitted, it is preferable to appropriately detect the signal intensity (for example, from below the substrate) in a state where the magnet is disposed on the electrophoresis substrate 5 in order to prevent diffusion of the target (target substance-magnetic particle-labeling substance complex). From this point of view, it is also preferable that the substrate and the electrophoretic substrate are transparent.

[ diagnostic method ]

The 3 rd embodiment of the present invention is a method for diagnosing a disease related to a target substance using the separation/measurement method according to the 1 st or 2 nd embodiment. Specifically, a method for diagnosing a disease associated with a target substance, comprising: in a stage of collecting a sample from a subject, a target substance in the sample, magnetic particles having a1 st receptor immobilized thereon and a labeled substance having a 2 nd receptor immobilized thereon, the first receptor being capable of specifically recognizing a site different from the site of the target substance, are mixed to obtain a mixture containing a target substance-magnetic particle-labeled substance complex, the mixture is subjected to electrophoresis, the target substance-magnetic particle-labeled substance complex is separated magnetically and electrophoretically, the signal intensity of the target substance-magnetic particle-labeled substance complex is detected, and the signal intensity is compared with the value of the target substance in a healthy subject to determine whether or not the subject has a disease related to the target substance. Accordingly, the present invention also provides a method for diagnosing a disease associated with a target substance, which comprises: collecting a sample from a subject, mixing a target substance in the sample, the magnetic particles having the 1 st receptor immobilized thereon and the labeled substance having the 2 nd receptor immobilized thereon, the magnetic particles having a site specifically recognizing the target substance immobilized thereon, and the labeled substance having a site specifically recognizing the site different from the site recognized by the target substance to obtain a mixture containing a target substance-magnetic particle-labeled substance complex, separating the target substance-magnetic particle-labeled substance complex by magnetism and electrophoresis, and then detecting the signal intensity (signal intensity 1) of the target substance-magnetic particle-labeled substance complex; collecting a sample from a healthy subject, mixing a target substance in the sample, the magnetic particles having the 1 st receptor immobilized thereon and the labeled substance having the 2 nd receptor immobilized thereon, the magnetic particles having a site capable of specifically recognizing the target substance, and the labeled substance having a site capable of specifically recognizing a site different from the site of the target substance, to obtain a mixture containing a target substance-magnetic particle-labeled substance complex, separating the target substance-magnetic particle-labeled substance complex by magnetism and electrophoresis, and then detecting a signal intensity (signal intensity 2) of the target substance-magnetic particle-labeled substance complex; comparing the signal intensity 1 with the signal intensity 2 to determine whether the subject suffers from a disease related to the target substance. Here, the subject or healthy person is preferably a human or a mammal other than a human. Examples of the above diseases include insulinoma, obesity, liver diseases, cushing's syndrome, terminal hypertrophy, dysinsulinemia, insulin autoimmune syndrome, diabetes, hypoglycemia, a low nutrition state, brown cell tumor, and hypophyseal adrenal function reduction, in the case where the target substance is insulin. When the signal intensity of the subject is high as compared with that of a healthy subject, the subject may suffer from such diseases as insulinoma, obesity, liver disease, cushing's syndrome, terminal hypertrophy, dysinsulinemia, insulin autoimmune syndrome, and the like. On the other hand, if the signal intensity of the subject is low as compared with the value of a healthy subject, diabetes, hypoglycemia, a low nutrition state, brown cell tumor, pituitary adrenal gland hypofunction, and the like may be suffered. That is, when the target substance is insulin and the signal intensity 1 is lower than the signal intensity 2, it is determined that the subject is at risk of developing diabetes, hypoglycemia, a low nutrition state, brown cell tumor, or hypophyseal adrenal hypofunction.

[ examination kit or examination system ]

The 4 th aspect of the present invention is a test kit for a disease related to a target substance, comprising: the magnetic particle to which the 1 st receptor that specifically recognizes a site of the target substance is immobilized (magnetic particle to which the 1 st receptor is immobilized), the labeling substance to which the 2 nd receptor that specifically recognizes a site different from a site of the target substance is immobilized (labeling substance to which the 2 nd receptor is immobilized), and the magnet. Accordingly, the present invention also provides a test kit for a disease related to a target substance, comprising: the magnetic particle comprises a magnetic particle to which a1 st receptor that specifically recognizes a site of a target substance is immobilized, a labeling substance to which a 2 nd receptor that specifically recognizes a site different from the site recognized by the target substance is immobilized, and a magnet.

A 5 th aspect of the present invention is a disease detection system for a target substance, including: a magnetic particle having a1 st receptor immobilized thereon, a site that specifically recognizes a target substance, a labeling substance having a 2 nd receptor immobilized thereon, a site that specifically recognizes a site different from the site of the target substance, a magnet, and an electrophoresis device (preferably a carrier-free electrophoresis device). Therefore, the present invention also provides a test system for a disease related to a target substance, including: the magnetic particle comprises a magnetic particle to which a1 st receptor that specifically recognizes a site of a target substance is immobilized, a labeling substance to which a 2 nd receptor that specifically recognizes a site different from the site recognized by the target substance is immobilized, a magnet, and an electrophoresis device. In this embodiment, the electrophoresis device is preferably a carrier-free electrophoresis device.

The magnetic particles to which the 1 st receptor is immobilized and the labeling substance to which the 2 nd receptor is immobilized, which are contained in the test kit, may be placed in 1 container or may be placed in different containers. The magnetic particles to which the 1 st receptor is immobilized and the labeling substance to which the 2 nd receptor is immobilized may be placed in a container together with a buffer solution. The test kit may be an electrophoresis kit containing an assay buffer solution, a concentrated solution thereof, or the like. That is, the 6 th aspect of the present invention is a disease detection system for a target substance, including: a magnetic particle having a1 st receptor immobilized thereon, a site that specifically recognizes a target substance, a labeling substance having a 2 nd receptor immobilized thereon, a site that specifically recognizes a site different from the site of the target substance, a magnet, and an electrophoresis set (preferably a carrier-free electrophoresis set). Therefore, the present invention also provides a disease detection system for a target substance, including: the kit comprises magnetic particles having a1 st receptor immobilized thereon which specifically recognizes a site of a target substance, a labeling substance having a 2 nd receptor immobilized thereon which specifically recognizes a site different from the site recognized by the target substance, a magnet, and an electrophoresis set. In this embodiment, the electrophoresis set is preferably a carrier-free electrophoresis set. Here, the electrophoresis set may include other general electrophoresis instruments such as an electrophoresis tank, an electrophoresis glass plate, a buffer tank, a spacer, a comb, a clip, a power supply, and a petri tower pump; a reagent for electrophoresis; detection reagents, and the like.

In the test kit or test system of the present embodiment, a known amount (concentration) of a standard sample and a container containing the standard sample may be further contained in the kit or system. Each reagent contained in the test kit or the test system may be dispensed into a container in an amount corresponding to 1 sample, or may be provided by combining reagents in an amount corresponding to the amount of a plurality of samples, each of which is contained in a container for each reagent type. In the latter case, each reagent is dispensed into a predetermined measurement vessel and used at the time of use. When the reagents are supplied in a measured amount of 1 sample, the containers containing the reagents may be integrally formed as a cassette, or the reagents may be accommodated in different regions of the cassette. When the magnetic particles to which the 1 st receptor is immobilized and the labeling substance to which the 2 nd receptor is immobilized are contained in a solid form in the kit, the test kit or the test system may further contain a buffer solution as described above for forming a complex. The container that can be included in the test kit or the test system may be made of any material as long as it does not interact with the magnetic particles to which the 1 st receptors are immobilized or the labeling substance to which the 2 nd receptors are immobilized, and does not interfere with reactions used in the assay, for example, enzymatic reactions or chemiluminescent reactions. If necessary, the surface may be previously treated and then provided so that no interaction occurs. In general, an instruction manual is attached to the test kit or the test system.

Examples

The following examples and comparative examples are used to illustrate the effects of the present invention. However, the technical scope of the present invention is not limited to the following examples. In the following examples, the operation was carried out at room temperature (25 ℃ C.) unless otherwise specified. In addition, "%" and "part" represent "% by mass" and "part by mass", respectively, unless otherwise specified.

Example 1

(preparation of magnetic particles having 1 st receptor immobilized thereon)

0.032mL (0.2mg) of mouse anti-human fatty acid binding protein (H-FABP) IgG monoclonal antibody 28 (monoclonal mouse anti-human Fatty Acid Binding Protein (FABP)28, manufactured by Hytest Ltd.) was biotinylated using biotin labeling kit-SH (manufactured by Kabushiki Kaisha chemical Co., Ltd.) according to the manufacturer's protocol, and a PBS solution (pH 7.4) (solution A-1) containing biotin-labeled anti-H-FABP (28) (biotinylated H-FABP antibody) was obtained in an amount of 0.2mL (1 mg/mL).

To 0.2mL of this solution A-1, 0.2mL (magnetic particle amount: 2mg) of MagnosphereMS300/Streptavidin (average particle size: 3.0 μm, manufactured by JSR Life sciences Co., Ltd.) (magnetic particles) was added, and the biotin moiety of the biotinylated FABP antibody and the Streptavidin moiety of the magnetic particles were reacted at 4 ℃ overnight to obtain a reaction solution (solution A-2) containing a biotin-labeled anti-H-FABP (28)/Streptavidin magnetic bead complex (magnetic particles having a receptor 1 immobilized). After the incubation for a predetermined period of time, the magnetic particles to which the 1 st receptor was immobilized were separated from the reaction solution (solution A-2) using a permanent magnet, and washed with PBS (pH 7.4) containing 0.1% BSA. After the final washing, the 1 st receptor-immobilized magnetic particles were separated by decantation, and then 0.2mL of PBS (pH 7.4) containing 0.1% BSA was added to prepare a1 st receptor-immobilized magnetic particle solution (solution a-3). The concentrations of the magnetic particles, antibodies and magnetic particles to which the 1 st receptor was immobilized in solution A-3 were 11mg/mL, 1mg/mL and 10mg/mL, respectively.

(preparation of labeled substance having receptor 2 immobilized thereon)

0.055mL (0.3mg) of mouse anti-human Fatty Acid Binding Protein (FABP) IgG monoclonal antibody 25 (monoclonal mouse anti-human Fatty Acid Binding Protein (FABP)25, manufactured by Hytest Ltd.) was added to 0.3mL of 0.1M phosphate buffer (pH6.0) containing 50mM EDTA to prepare an antibody solution (solution B-1). To the antibody solution (solution B-1), 0.05mL of 0.1M phosphate buffer (pH6.0) containing 0.1M mercaptoethylamine hydrochloride and 50mM EDTA was added. This solution was incubated at 37 ℃ for 2 hours to reduce the disulfide bond of the antibody to form a thiol group (-SH), resulting in a solution containing a reduced antibody (solution B-2). This solution (solution B-2) was desalted to obtain 0.4mL of a PBS solution (solution B-3) containing a reducing antibody.

Further, 0.03mL of N, N-Dimethylformamide (DMF) was added to 0.5mL of IRDye (registered trademark) 800CW maleimide (manufactured by LI-COR Biosciences) (labeling substance) (excitation wavelength: 800nm) to dissolve the substance, thereby preparing a labeling substance solution (solution B-4). 0.01mL of the labeling substance solution (solution B-4) was added to 0.4mL of the PBS solution (solution B-3) containing the reducing antibody, and the mixture was incubated at 37 ℃ for 30 minutes to react the thiol group (-SH) of the reducing antibody with the maleimide group of the labeling substance, thereby obtaining a reaction solution (solution B-5) containing an IRDye800 CW-labeled anti-H-FABP (25) complex (labeling substance having the 2 nd receptor immobilized). After incubation for a prescribed time, the reaction solution thus obtained (solution B-5) was subjected to a Zeba spin desalting column (Zeba)^TMSpin desaling Columns,7K MWCO,0.5, manufactured by seimer feishell scientific), buffer exchange was performed with PBS to obtain 0.4mL of PBS solution (solution B-6) containing the labeling substance having the 2 nd receptor immobilized thereon. The buffer exchange was performed using 2 rotary desalting columns with the solution divided every 0.2mL, and then the column treatment solutions were combined (solution B-6).

Finally, the thus-obtained solution B-6 was diluted with PBS (pH 7.4) containing 0.1% BSA to an antibody concentration of 0.1mg/mL to prepare a 2 nd receptor-immobilized labeling substance solution (solution B-7).

(preparation of H-FABP solution)

A H-FABP solution (H-FABP concentration: 0.05mg/mL) was prepared by adding 2mL of a PBS solution containing 0.1% BSA to 0.1mg of human Fatty acid binding protein (FABP, manufactured by human, Hytest Ltd.) and dissolving the mixture.

(preparation of swimming pools)

The 1 st receptor-immobilized magnetic particle solution (solution A-3) prepared above, the 2 nd receptor-immobilized labeling substance solution (solution B-7), the H-FABP solution, PBS (pH 7.4) containing 0.1% BSA (hereinafter "0.1% BSAPBS") and 0.1M Tris-HCl (pH8.5) (hereinafter "Tris-HCl" in Table 1) were mixed so as to have the composition (pH8.5) shown in Table 1, and reacted at 25 ℃ for 5 minutes to prepare electrophoretic solutions 1 to 5 (samples 1 to 5) containing complexes 1 to 5 (complexes 1 to 5) of fatty acid-binding protein-biotin labeled anti-H-FABP (28)/Streptavidin magnetic bead-IRDye 800CW labeled anti-H-FABP (25). In table 1, the "dilution ratio of the H-FABP solution" indicates the dilution ratio of the prepared H-FABP solution, and the "diluted H-FABP solution" indicates the amount of the H-FABP solution added after dilution at the predetermined dilution ratio. Thus, for example, sample 1 is a solution obtained by diluting the H-FABP solution prepared as described above by 2 times with 1. mu.L. In table 1 below, "antibody/H-FABP (molar ratio) in solution a-3" refers to the molar ratio of anti-H-FABP (28) (1 st receptor) to fatty acid binding protein (H-FABP, target substance) in each sample. Similarly, "antibody/H-FABP (molar ratio) in solution B-7)" means the molar ratio of anti-H-FABP (25) (receptor No. 2) to the fatty acid-binding protein (H-FABP, target substance) in each sample. The binding amount of biotin to the magnetic particles can be calculated based on the amount described in the instruction manual of the magnetic particles to be used. In addition, the calculation can be made assuming that the antibody and biotin are completely bound at a molar ratio of 1: 1.

[ Table 1]

(production of glass Square tube filled with swimming solution)

0.04mL of each of the samples 1 to 5 prepared in the above (preparation of a migration solution) was packed in a square tube made of glass (outer side: 1.5mm, inner side: 1.0mm, thickness: 0.25mm, length: 40mm, material: quartz) (FIG. 2A). The neodymium permanent magnet was slid on the tubes filled with the respective samples along the extending direction of the tubes, and the compounds 1 to 5 and unreacted magnetic particles to which the 1 st receptor was fixed were concentrated at one place (position a), thereby obtaining glass square tubes 1 to 5, respectively (fig. 2B). The magnet is provided at a position where the ratio (Y/X) of the distance (Y) from the cathode to the magnet to the distance (X) from the cathode to the anode is 1/5.

(electrophoresis)

The square glass tubes 1 to 5 prepared above (preparation of electrophoretic solution) were set in the electrophoresis apparatus shown in FIG. 3, and then subjected to electrophoresis at a voltage of 100V for 5 minutes.

After electrophoresis, the neodymium permanent magnet was fixed at position a so that the compound in each tube did not move, and at the same time, the glass square tubes 1 to 5 were removed from the electrophoresis apparatus, respectively. The fluorescence intensities at positions A of the square glass tubes 1 to 5 were measured by a fluorescence scanner (Odyssey CLx imaging System, LI-COR Biosciences). Fig. 4 shows photographs of the glass square tube filled with the sample 1 before and after electrophoresis, measured by a fluorescence scanner. In fig. 4, the portion enclosed by the white square represents the place where the complex exists. As is clear from fig. 4, after electrophoresis, the complex 1 can be separated well from the unreacted (fatty acid-binding protein-unbound) labeled substance to which the 2 nd receptor is immobilized.

In addition, the fluorescence intensity was plotted against the final concentration of H-FABP. The results are shown in FIG. 5. As can be seen from fig. 5, in the range of H-FABP concentration of 0 to 9.2nM (138ng/mL), there was a good positive correlation (linearity) between the signal intensity (fluorescence intensity) of the target substance-magnetic particle-labeling substance complex and the H-FABP concentration (y 21384x +7190 (here, y represents the signal intensity (fluorescence intensity), x represents the H-FABP concentration (nM)), and R represents²0.9867). Therefore, if the standard curve is prepared using a predetermined amount of the target substance, it is expected that the amount of the fatty acid binding protein in the sample can be measured with high accuracy. It should be noted that, although fatty acid bonding is used in this exampleThe protein was used for the experiments, but it is considered that the same results can be obtained for other receptors such as insulin antibody and insulin receptor. In the present example, the fatty acid-binding protein was used, but even when a biological sample such as blood was used, components derived from a living body other than the complex were moved to the anode side by electrophoresis. Therefore, even in such a case, it is considered that the complex can be favorably separated from other biological components.

The present application is based on japanese laid-open application No. 2018-201908, filed on 26.10.2018, the disclosure of which is incorporated in its entirety by reference into this specification.

Description of the symbols

2. 3 … electrodes;

4 … reagent;

5 … an electrophoretic substrate;

6 … a substrate;

7 … sample containing a target substance;

8 … a magnet;

9 … target substance-magnetic particle-labeling substance complex