Method for detecting substance being tested for, and labeling buffer and kit used therein

Abstract

This method for immunologically detecting a substance being tested for in a sample includes a labeling step for forming a complex of the substance being tested for and a label containing a first probe molecule capable of binding to the substance being tested for and a labeling substance, in the presence of: at least one selected from imidazole and derivatives thereof; a pH buffering agent; and a polyvalent metal ion.

Description

Method for detecting a test substance, and labeling buffer and kit used therefor

[0001] The present invention relates to a method for detecting a test substance, and a labeling buffer and kit used therein.

[0002] Immunological detection methods (immunoassays) that utilize a specific immune reaction between an antigen and an antibody have been widely used as methods for detecting a analyte in a sample. In currently mainly used immunological detection methods, for example, a complex (antigen-antibody complex) formed by binding an antigen or antibody (probe molecule) that can specifically bind to the analyte of interest is labeled with a labeling substance, and the analyte is detected by detecting a signal derived from the labeling substance.

[0003] Such immunological detection methods include non-competitive methods in which a complex of a test substance and an antibody or antigen capable of specifically binding to the test substance is bound to an antibody or antigen labeled with a labeling substance (labeled product), and competitive methods in which a test substance is detected by competitively binding a label obtained by labeling the antigen or antibody to a fixed amount of antibody or antigen. Immunological detection methods are also broadly divided into homogeneous detection methods performed in a liquid state and heterogeneous detection methods performed using a solid-phase antibody or the like.

[0004] For example, non-competitive methods include a sandwich method in which the analyte is captured using a capturer in which an antibody or antigen is immobilized on an insoluble carrier, and then an antibody or antigen (labeled) labeled with a labeling substance is bound to the capturer, and the analyte is detected by detecting a signal such as luminescence corresponding to the labeling substance. One example of such a sandwich method is described in International Publication No. 2014 / 024853 (Patent Document 1), which describes a sandwich method in which a mixture of an anti-F1 antibody or an antigen-binding fragment thereof that specifically binds to prothrombin fragment 1 and an anti-F2 antibody or an antigen-binding fragment thereof that specifically binds to fragment 2 is used as the labeled antibody (labeled), and an anti-PIVKA-II antibody or an antigen-binding fragment thereof that specifically binds to PIVKA-II is used as the immobilized antibody (capturer). Various studies have been conducted on immunological detection methods, including those aimed at improving detection sensitivity, detection accuracy, and reducing non-specific reactions. However, further improvements are required due to the need for smaller samples and the increasing variety of test substances, and in particular, further improvements in detection sensitivity are desired.

[0005] International Publication No. 2014 / 024853

[0006] The present invention has been made in view of the above-mentioned problems, and aims to provide a detection method capable of detecting a test substance with high sensitivity by an immunological detection method, as well as a labeling buffer to be used in the detection method and a kit containing the same.

[0007] The present inventors have conducted extensive research into methods for detecting a test substance by immunological detection, and have found that by adding at least one selected from imidazole and its derivatives, a pH buffer, and a polyvalent metal ion to a reaction system in which a complex between the test substance and a label is formed, the signal amount derived from the labeling substance of the label increases, and the test substance can be detected with high sensitivity even at a low concentration, thereby completing the present invention.

[0008] The present invention, which was made possible by these findings, is as follows: [1] A method for immunologically detecting a analyte in a sample, comprising a labeling step of forming a complex between the analyte and a label comprising a label and a first probe molecule capable of binding to the analyte in the presence of at least one selected from imidazole and its derivatives, a pH buffer, and a polyvalent metal ion. [2] The detection method according to [1], wherein the concentration of the at least one selected from imidazole and its derivatives is 1 mM or more in terms of imidazole in the reaction system of the labeling step. [3] The detection method according to [1] or [2], wherein the pH of the reaction system of the labeling step is 6.0 to 8.0. [4] The detection method according to any one of [1] to [3], wherein the concentration of the polyvalent metal ion is 0.01 mM or more in the reaction system of the labeling step. [5] The detection method according to any one of [1] to [4], further comprising a capture step of capturing the analyte with a capture body comprising a second probe molecule capable of binding to the analyte and an insoluble carrier. [6] The detection method according to [5], further comprising a washing step of removing impurities not captured by the capture body after the capture step. [7] The detection method according to any one of [1] to [6], wherein the labeling substance is an enzyme and the first probe molecule is an antibody capable of binding to the analyte. [8] A labeling buffer for use in the detection method according to any one of [1] to [7], comprising at least one selected from imidazole and its derivatives, a pH buffer, and a polyvalent metal ion. [9] The buffer according to [8], further comprising a label comprising a first probe molecule capable of binding to the analyte and a labeling substance.

[10] A kit for use in the detection method according to any one of [1] to [7], comprising a labeling buffer containing at least one selected from imidazole and its derivatives, a pH buffer, and a polyvalent metal ion.

[11] The kit according to

[10] , further comprising a label containing a labeling substance and a first probe molecule capable of binding to the analyte.

[0009] According to the present invention, it is possible to provide a detection method capable of detecting a test substance with high sensitivity by an immunological detection method, as well as a labeling buffer to be used in the detection method and a kit containing the same.

[0010] The present invention will be described in detail below based on preferred embodiments thereof.

[0011] <Detection Method> The detection method of the present invention is a method for immunologically detecting a test substance in a sample, and includes a labeling step of forming a complex between the test substance and a label containing a labeling substance and a first probe molecule capable of binding to the test substance in the presence of at least one selected from imidazole and its derivatives, a pH buffer, and a polyvalent metal ion.

[0012] [Analyte] The "analyte" according to the present invention is not particularly limited, as long as it is capable of binding, preferably specifically binding, to the first probe molecule and, optionally, the second probe molecule (hereinafter sometimes collectively referred to as "probe molecules"). Examples of such combinations of analyte and probe molecule (or combinations of probe molecule and analyte) include combinations that can achieve specific binding, such as a combination of an antibody and an antigen, a combination of a lectin and a sugar chain capable of binding to the lectin (lectin-binding sugar chain), a combination of a receptor and a ligand, a combination of avidin and biotin, and a combination of an antibody containing an Fc region and an Fc-binding protein. Among these, in the present invention, it is preferable that the probe molecule is an antibody and the analyte is a substance that can serve as an antigen for the antibody.

[0013] Examples of test substances according to the present invention include substances that can serve as antigens for the antibodies, such as antigen peptides, antibodies, receptor proteins, transport proteins, transcriptional regulatory factors, haptens, enzymes, viral antigens and antibodies, and other proteins and peptides; sugars (polysaccharides, monosaccharides); glycoproteins; nucleic acids; lipids; glycolipids; vitamins, hormones, coenzymes, toxins, antibiotics, immunosuppressants (e.g., cyclosporine, tacrolimus, everolimus, sirolimus, methotrexate, etc.), and low-molecular-weight compounds such as pharmaceuticals such as antiepileptic drugs, but are not limited to these.

[0014] In the present invention, the term "antibody" includes not only a complete antibody but also an antibody fragment (e.g., Fab, Fab', F(ab')). ２ The "antibody" according to the present invention may be either a polyclonal antibody or a monoclonal antibody.

[0015] [Sample] The "sample" used in the detection method of the present invention is not particularly limited as long as it is a sample in which the test substance can be present, and examples thereof include various organisms (including cells, tissues, organs, and individuals) and extracts thereof; specimens collected from humans and non-human animals (body fluids such as saliva, oral mucosa, pharyngeal mucosa, tears, sweat, urine, sputum, bronchoalveolar lavage fluid, intestinal mucosa, serum, plasma, whole blood, cerebrospinal fluid, lymph, semen, ascites, and amniotic fluid; feces; tissues); plant biological fluids; biological culture solutions; environmental water (rivers, lakes, harbors, waterways, groundwater, purified water, sewage, wastewater, etc.), and suspensions of solids (soil, etc.), which can be used as appropriate depending on the purpose. Examples of the non-human animals include primates such as chimpanzees and monkeys; livestock, poultry, and pets such as cows, pigs, horses, sheep, rabbits, chickens, cats, and dogs; and wild animals and birds such as deer, wild boars, raccoons, weasels, rats, and pigeons.

[0016] Among these, for example, in the medical field or clinical testing field, when a biomarker or the like serving as a standard for diagnosing a disease is detected as a test substance, the sample generally includes specimens collected from a subject (preferably a human) to be diagnosed or the like, in which the target biomarker or the like is to be detected, such as serum, plasma, whole blood, urine, feces, oral mucosa, pharyngeal mucosa, intestinal mucosa, and various biopsy tissues.

[0017] The sample may be one that has been subjected to processing such as pulverization or freezing, one that has been appropriately diluted or suspended in a diluent, or one that has been appropriately pH-adjusted. Examples of the diluent include water, physiological saline, known buffer solutions (sodium phosphate buffer, MES buffer, Tris buffer, CFB buffer, MOPS buffer, PIPES buffer, HEPES buffer, tricine buffer, bicine buffer, glycine buffer, citrate buffer, phosphate buffer, etc.), and organic solvents (dimethyl sulfoxide, etc.), and may also contain a stabilizing protein such as BSA, serum, etc.

[0018] The sample used in the detection method of the present invention is preferably an aqueous sample, and is preferably diluted or suspended as needed. Furthermore, when the test substance is a nucleic acid or a substance derived from a microorganism contained in the sample, the nucleic acid or microorganism may be appropriately isolated. As a method for isolating such nucleic acids or microorganisms from the sample, any known method can be used.

[0019] [Labeled Product] In the present invention, the term "labeled product" refers to a complex comprising a labeling substance and a first probe molecule capable of binding to the analyte, and is a conjugate in which the labeling substance and the first probe molecule are directly or indirectly bound. The labeling substance may further comprise a water-soluble carrier or the like that supports the labeling substance and the first probe molecule.

[0020] (Labeling substance) The "labeling substance" contained in the labeled body according to the present invention functions mainly as a label for detecting the test substance, and any labeling substance used in known immunological detection methods can be appropriately adopted. However, from the viewpoint of improving the detection sensitivity particularly by the detection method of the present invention, an enzyme is preferred in the present invention.

[0021] Examples of the enzyme include alkaline phosphatase (ALP), horseradish peroxidase (HRP), β-galactosidase (β-gal), glucose oxidase, and luciferase, and these may be used alone or in combination of two or more. When an enzyme is used as the labeling substance, a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, or the like may be added as a substrate to detect the following signals corresponding to the substrate. As such labeling substances and substrates, conventionally known substances can be used as appropriate, and commercially available substances can also be used as appropriate.

[0022] (First probe molecule) In the present invention, the term "first probe molecule" refers to a molecule capable of binding, preferably specifically binding, to the analyte. When a capturer described below is used and the capture step described below is performed before or simultaneously with the labeling step, the "first probe molecule capable of binding to the analyte" includes a probe molecule capable of binding, preferably specifically binding, to a complex of the analyte and a second probe molecule. Examples of the mode of binding to the complex of the analyte and the second probe molecule include a mode in which the analyte recognizes and binds to the binding site of the analyte and the second probe molecule.

[0023] The first probe molecule according to the present invention may be any molecule capable of binding, preferably specifically binding, to the analyte, and may be one type or a combination of two or more types, but is preferably an antibody corresponding to the analyte. Furthermore, the label according to the present invention is preferably an enzyme-labeled antibody, in which the label is an enzyme and the first probe molecule is an antibody. Such a first probe molecule can be prepared by a known, established method depending on the analyte, or a commercially available one may be used as appropriate.

[0024] (Constitution of Labeled Body and Production Method) In the labeled body according to the present invention, the molar ratio of the labeling substance to the first probe molecule is not particularly limited and can be adjusted as appropriate depending on the combination of these types, the ease of binding to the test substance, and the like. For example, the ratio of the first probe molecule (the total amount of the first probe molecule when two or more types of first probe molecules are combined) to 1 mole of the labeled substance (the total amount of the first probe molecule when two or more types of first probe molecules are combined) is preferably 0.01 to 10,000 moles, and more preferably 0.05 to 10 moles.

[0025] The label according to the present invention can be produced by binding the labeling substance to a first probe molecule. As a production method, a conventionally known method or a method based thereon can be appropriately adopted depending on the types of the labeling substance and the first probe molecule, and the labeling substance and the first probe molecule may be directly or indirectly bound to each other.

[0026] Examples of the direct binding method include a method in which an active group (e.g., a thiol group, a maleimide group, or a succinimide group) is added to the labeling substance and / or the first probe molecule, or a labeling substance and / or a first probe molecule having such an active group is used, and the labeling substance and / or the first probe molecule are bound by a covalent bond via the active group. The labeling substance and the first probe molecule to which the active group has been added may be commercially available products, or may be prepared by introducing the active group onto the surface of the labeling substance and / or the first probe molecule under appropriate reaction conditions. Examples of a method in which the labeling substance is indirectly bound to the first probe molecule include a method in which the labeling substance is bound via polyhistidine, polyethylene glycol, an oligopeptide, a linker molecule, or the like. Alternatively, one of the molecules may be modified in some way, and a substance that captures the modified portion may be added to the other, and the two may be bound via these. For example, one molecule may be biotinylated and the other molecule may be avidinylated, and a binding method using avidin-biotin bonding may be employed. The ratio of the labeling substance to the first probe molecule used in this production method can be appropriately selected so as to achieve the preferred range for the label. In addition, as such a label, for example, a commercially available product such as an enzyme-labeled antibody may be appropriately used.

[0027] [Capture Body] In the production method of the present invention, it is preferable to use a capture body. In the present invention, the term "capture body" refers to a complex comprising an insoluble carrier and a second probe molecule capable of binding to the analyte, in which the second probe molecule is directly or indirectly bound to and supported on the insoluble carrier.

[0028] (Insoluble Carrier) The "insoluble carrier" contained in the labeled body according to the present invention is water-insoluble and functions as a carrier that mainly supports and immobilizes the second probe molecule. In the present invention, "water-insoluble" means that the carrier is insoluble in water (the solubility in water is 0.001 g / mL or less, preferably 0.0001 g / mL or less, the same applies hereinafter) at room temperature and normal pressure.

[0029] The material of such an insoluble carrier can be any material used as an insoluble carrier in known immunological detection methods, and is not particularly limited. For example, at least one selected from the group consisting of high molecular weight polymers (polystyrene, (meth)acrylic acid esters, polymethyl methacrylate, polyimide, nylon, etc.), gelatin, cellulose, nitrocellulose, glass, latex, silica, metals (gold, platinum, etc.), and metal compounds (iron oxide, ferrite, cobalt oxide, nickel ferrite, etc.) can be mentioned. The material of the insoluble carrier may also be a composite of these materials, for example, an organic-inorganic composite composed of at least one organic polymer selected from the group consisting of high molecular weight polymers, gelatin, cellulose, and latex, and at least one metal compound selected from the group consisting of iron oxide (spinel ferrite, etc.), cobalt oxide, and nickel ferrite.

[0030] In the present invention, the shape of the insoluble carrier is not particularly limited, and may be, for example, a plate, a fiber, a membrane, a particle, etc., but from the viewpoint of reaction efficiency, particles are preferable, and from the viewpoint of automation and shortening the reaction time, magnetic particles are more preferable. As such an insoluble carrier, conventionally known carriers can be used as appropriate, and commercially available carriers can also be used as appropriate.

[0031] (Second probe molecule) In the present invention, the term "second probe molecule" refers to a molecule capable of binding, preferably specifically binding, to the analyte. When the capture step described below is performed after or simultaneously with the labeling step, the "second probe molecule capable of binding to the analyte" includes a probe molecule capable of binding, preferably specifically binding, to a complex of the analyte and the first probe molecule. Examples of the mode of binding to the complex of the analyte and the first probe molecule include a mode in which the second probe molecule recognizes the binding site between the analyte and the first probe molecule.

[0032] The second probe molecule according to the present invention may be any molecule capable of binding, preferably specifically binding, to the analyte, and may be one type or a combination of two or more types. Preferably, the second probe molecule is an antibody against the analyte. The second probe molecule may be different from the first probe molecule, or may be the same as the first probe molecule as long as it does not inhibit the effects of the present invention. Such second probe molecules can be prepared by known, established methods depending on the analyte, or commercially available ones may be used as appropriate.

[0033] (Configuration and manufacturing method of capture body) In the capture body of the present invention, the content of the second probe molecule is not particularly limited, but in order to further improve the detectability of the analyte, it is preferable to set the number of second probe molecules bound to one molecule of the insoluble carrier so that it is as large as possible. For example, the mass of the second probe molecule (the total mass when the second probe molecule is a combination of two or more types) per 100 parts by mass of the insoluble carrier is preferably 0.005 to 0.05 parts by mass, and more preferably 0.01 to 0.04 parts by mass.

[0034] The capture body of the present invention can be produced by binding and immobilizing a second probe molecule to the insoluble carrier. Such a production method can be a conventionally known method or a method similar thereto, depending on the types of insoluble carrier and second probe molecule. The second probe molecule may be directly or indirectly bound to the insoluble carrier. Examples of such a binding method include the methods listed above for binding the labeling substance to the first probe molecule. The ratio of the insoluble carrier and the second probe molecule used in such a production method can be appropriately selected so as to achieve a preferred range for each content in the capture body. Furthermore, commercially available capture bodies, such as antibody-bound particles, may also be used.

[0035] [Labeling Step] In the detection method of the present invention, in the labeling step, the sample is contacted with the label, and if an analyte is present in the sample, a complex between the label and the analyte (sometimes referred to herein as a "first complex"), i.e., a label-analyte complex, is formed via binding between the analyte and a first probe molecule. Alternatively, when the detection method of the present invention includes the capture step described below before or simultaneously with the labeling step, a complex between the label and the analyte captured by the capturer, i.e., a label-analyte-capturer complex (sometimes referred to herein as a "second complex") is formed in the labeling step.

[0036] The method for contacting the sample with the labeled substance is not particularly limited, and any conventionally known method or a method based thereon can be appropriately adopted, for example, a method in which the labeled substance is added to the aqueous sample.

[0037] In the labeling step according to the present invention, the reaction system between the test substance and the label contains at least one selected from imidazole and its derivatives (sometimes collectively referred to as "imidazole, etc." in this specification), a pH buffer, and a polyvalent metal ion. In the present invention, the "reaction system between the test substance and the label" is preferably an aqueous system containing these compounds for reacting the label with the test substance.

[0038] (Imidazole, etc.) In the present invention, "imidazole and its derivatives" refers to compounds having an imidazole skeleton, and examples thereof include imidazole, 1-methylimidazole, 1-ethylimidazole, 1-propylimidazole, 1-butylimidazole, 1-phenylimidazole, 1-vinylimidazole, 1-allylimidazole, 1-benzyl-2-methylimidazole, 1-benzyl-2-formylimidazole, 1-benzyl-4-hydroxymethylimidazole, 1-benzyl-5-hydroxymethylimidazole, 1-(2-hydroxyethyl)-imidazole, 1-(2-hydroxyethyl)-2-methylimidazole, 2-methylimidazole, 2-ethylimidazole, 2-propylimidazole, 2-butylimidazole, 2-phenylimidazole, 2-formylimidazole, 2-hydroxymethylimidazole, 2-methyl-1-vinylimidazole, imidazole, 2-butyl-4-formylimidazole, 2-butyl-4-hydroxymethylimidazole, 2-butyl-4-chloro-5-formylimidazole, 2-hydroxymethyl-1-benzylimidazole, 2-hydroxymethyl-2-methylimidazole, 2-ethyl-4-methylimidazole, 4-butylimidazole, 4-formylimidazole, 4-formyl-1-methylimidazole, 4-formyl-1-trisylimidazole, 5-formyl-1-methylimidazole, 4-formyl-5-methylimidazole, 4-hydroxymethylimidazole hydrochloride, methylimidazole-4-carboxylate, ethylimidazole-4-carboxylate, 1,2-dimethylimidazole, and 1,2,4-trimethylimidazole may be used alone or in combination of two or more.

[0039] The imidazole and the like also include chemically acceptable salts of the imidazole or a derivative thereof. Examples of such salts include potassium salt, magnesium salt, lithium salt, calcium salt, zinc salt, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, sesqui(fumarate), hydrochloride, dihydrochloride, trihydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, methanesulfonate, nicotinate, hydroxypropyl methyl acrylate ...

[0039] Examples of the salt include tinates, 2-naphthalenesulfonates, oxalates, pamoates, pectinates, persulfates, 3-phenylpropionates, picrates, pivalates, propionates, succinates, sulfates, bis(tartrates), tartrates, (L)tartrates, bis((L)tartrates), (D)tartrates, (DL)tartrates, bis((DL)tartrates), meso-tartrates, bis(meso-tartrates), thiocyanates, phosphates, glutamates, bicarbonates, bis((D)tartrates), bis(bromides), bis(sulfates), bis(phosphates), tris(hydrochlorides), p-toluenesulfonates, and undecanoates, and these may be used alone or in combination of two or more. Among these, from the viewpoint of controlling the concentrations of the imidazole etc. and the polyvalent metal ions described below in the reaction system, the imidazole etc. added to the reaction system is preferably a salt that does not generate the polyvalent metal ions, and more preferably is not a salt, although this does not deny the possibility that the imidazole etc. and the polyvalent metal ions described below form salts in the reaction system.

[0040] In the labeling step according to the present invention, the concentration of imidazole or the like in the reaction system (when two or more types of imidazole or the like are used, the total of those imidazoles or the like; the same applies hereinafter) is preferably 1 mM or more, more preferably 1 to 30 mM, even more preferably 2 to 28 mM, even more preferably 3 to 25 mM, and particularly preferably 5 to 15 mM, calculated as imidazole, i.e., calculated as the amount of imidazole skeleton. If the concentration of imidazole or the like is below the lower limit, the effect of improving detection sensitivity tends to be insufficient, whereas if it exceeds the upper limit, the detection sensitivity may actually decrease.

[0041] (pH Buffer Agent) In the present invention, the "pH buffer agent" may be any agent that has a pH buffering ability for an aqueous solution (a function of suppressing a sudden change in pH), but the pH buffer agent according to the present invention is preferably one that can maintain the pH of the reaction system, which is an aqueous solution, at 6.0 to 8.0, preferably 7.0 to 7.5.

[0042] Examples of such pH buffering agents include lactic acid, acetic acid, hydrochloric acid, succinic acid, phthalic acid, phosphoric acid, boric acid, citric acid, maleic acid, Tris (tris(hydroxymethyl)aminomethane), Bis-Tris (bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane), MES (2-morpholinoethanesulfonic acid), ADA (N-2(acetamido)iminodiacetic acid), PIPES (piperazine-1,4-bis(2-ethanesulfonic acid)), ACES (N-(2-acetamido)-2-aminoethanesulfonic acid), BES (N, Examples of such glycerides include N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid), MOPS (3-morpholinopropane-1-sulfonic acid), Tricine (N-[tris(hydroxymethyl)methyl]glycine), and Bicine (N,N-bis(2-hydroxyethyl)glycine), and these may be used alone or in combination of two or more. Among these, when the label is an enzyme-labeled antibody, Tris is preferred from the viewpoint of not inhibiting the enzyme activity.

[0043] In the labeling step according to the present invention, the concentration of the pH buffer in the reaction system may be appropriately selected depending on the type of pH buffer used, but is preferably a concentration that makes the pH of the reaction solution 6.0 to 8.0, preferably 7.0 to 7.5. Specifically, for example, in the case of Tris, the concentration is preferably 10 to 500 mM, more preferably 50 to 200 mM.

[0044] (Polyvalent Metal Ion) In the present invention, the term "polyvalent metal ion" refers to a metal ion having a valence of divalent or higher, and examples thereof include divalent metal ions such as zinc ion, calcium ion, magnesium ion, copper ion, iron (II) ion, nickel ion, manganese ion, and cobalt ion; and trivalent metal ions such as iron (III) ion, chromium ion, and aluminum ion, and may be one of these or a combination of two or more of these. Among these, the polyvalent metal ion according to the present invention is preferably a divalent metal ion, more preferably at least one selected from the group consisting of zinc ion, magnesium ion, and nickel ion, even more preferably zinc ion and / or nickel ion, and even more preferably zinc ion.

[0045] These polyvalent metal ions may be added to the reaction system in the form of a chemically acceptable ionic compound. In this case, examples of the ionic compound include chloride, sulfide, iodide, and hydroxide. One or a combination of two or more of these compounds may be used. However, when the label is an enzyme-labeled antibody, chloride is preferred in order not to inhibit the enzyme activity. Furthermore, when the imidazole or the like added to the reaction system is in the form of a salt that generates the polyvalent metal ions, this may also serve as the ionic compound, but it is preferred that the imidazole or the like added does not generate the polyvalent metal ions.

[0046] In the labeling step according to the present invention, the concentration of the polyvalent metal ion in the reaction system (when there are two or more types of polyvalent metal ions, the total of all the polyvalent metal ions; the same applies hereinafter) is preferably 0.01 mM or more, more preferably 0.1 to 10 mM, even more preferably 0.2 to 8 mM, even more preferably 0.3 to 5 mM, and particularly preferably 0.3 to 3 mM. If the concentration of the polyvalent metal ion is below the lower limit, the effect of improving detection sensitivity tends to be insufficient, while if it exceeds the upper limit, the detection sensitivity tends to decrease or the metal ions tend to precipitate.

[0047] (Label) In the labeling step according to the present invention, the concentration of the label in the reaction system is not particularly limited as it is adjusted appropriately depending on the type and concentration of the sample and label. For example, when the label is an enzyme-labeled antibody, the concentration is preferably 0.1 to 5 μg / mL, and more preferably 0.5 to 2 μg / mL.

[0048] (Others) In the labeling step according to the present invention, as described above, the pH of the reaction system is preferably 6.0 to 8.0, more preferably 7.0 to 7.5. The temperature and reaction time of the reaction system are not particularly limited and can be adjusted appropriately, but can be, for example, performed at room temperature to 45°C, preferably 20 to 37°C, for about 5 seconds to 10 minutes, preferably 30 seconds to 8 minutes.

[0049] In the labeling step according to the present invention, the reaction system may further contain other components such as salts, sugars, proteins, surfactants, preservatives, etc., in addition to the test substance, the label, the imidazole or the like, the pH buffer, and the polyvalent metal ion.

[0050] The salts refer to salts added to the reaction system other than the above-mentioned salts of imidazole and the like, pH buffers, and salts derived from ionic compounds of polyvalent metal ions, and include, for example, sodium chloride and potassium chloride, and may be one of these or a combination of two or more of these. When the reaction system contains the salts, the content thereof (when two or more types are present, the total content thereof; the same applies hereinafter) is, for example, preferably 50 to 1000 mM, and more preferably 50 to 300 mM.

[0051] Examples of the sugars include glucose, sucrose, maltose, sorbitol, mannitol, xylitol, trehalose, and cyclodextrin, and the reaction system may contain one of these or a combination of two or more of them. When the reaction system contains the sugars, the content (when two or more types are contained, the total content thereof; the same applies hereinafter) is, for example, preferably 0.5 to 50 w / v %, and more preferably 1 to 40 w / v %.

[0052] Examples of the protein include casein, sodium caseinate, bovine serum albumin (BSA), and fetal bovine serum (FBS), and the protein may be one of these or a combination of two or more of these. When the reaction system contains the protein, the content (when two or more proteins are present, the total content of the proteins; the same applies hereinafter) is, for example, preferably 0.01 to 10 w / v %, and more preferably 0.1 to 5 w / v %.

[0053] Examples of the surfactant include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants, and these may be used alone or in combination of two or more, but nonionic surfactants (e.g., Tween 20, Tween 80 (manufactured by Merck)) are preferred. When the reaction system contains the surfactant, the content thereof (when two or more types are used, the total content thereof; the same applies hereinafter) is, for example, preferably 0.01 to 5 w / v %, and more preferably 0.1 to 1 w / v %.

[0054] Examples of the preservative include sodium azide, antibiotics, and Proclin (manufactured by Merck & Co.), and one of these may be used alone or in combination of two or more. When the reaction system contains the preservative, the content thereof can be adjusted appropriately depending on the type of the preservative.

[0055] Furthermore, other examples of the other components include, for example, when the label is an enzyme-labeled antibody, an inactivated enzyme obtained by inactivating the same enzyme as the enzyme. For example, when the label is an ALP-labeled antibody, the inactivated enzyme can be inactivated ALP. This makes it possible to suppress nonspecific reactions (see JP 2008-281489 A). When the reaction system contains the inactivated enzyme, the content thereof is preferably 10 to 5,000 parts by mass per part by mass of the label. Other examples of the other components include polymers such as PEG, Dextran, and Lipidure (manufactured by NOF Corporation); and amino acids.

[0056] In the labeling step according to the present invention, the reaction system may further contain components derived from the sample (e.g., contaminants contained in the sample other than the analyte), but in the detection method of the present invention, it is preferable to carry out the labeling step in the above reaction system after carrying out the capture step and washing step described below prior to the labeling step to remove such components. Also, in the labeling step according to the present invention, the reaction system may further contain components derived from the label (e.g., if the storage buffer for the label is other than the labeling buffer described below, the storage buffer (e.g., one of the diluents listed above)), but it is preferable to add the label alone or in the form of the labeling buffer described below to the reaction system.

[0057] [Capture Step] The detection method of the present invention preferably includes a capture step, prior to the labeling step, in which the sample is contacted with the capture body, and if an analyte is present in the sample, the capture body captures the analyte via binding between the analyte and a second probe molecule, thereby forming a complex between the capture body and the analyte, i.e., a capture body-analyte complex (sometimes referred to as a "third complex" herein). Alternatively, it is also preferable to include a capture step, subsequent to or simultaneously with the labeling step, in which the first complex obtained in the labeling step is contacted with the capture body to form a second complex of label-analyte-capture body. From the viewpoint of removing contaminants other than the analyte contained in the sample and further improving detection accuracy by performing a washing step multiple times, such a capture step is more preferably included prior to the labeling step.

[0058] The method for contacting the sample with the capture body is not particularly limited, and any conventionally known method or a method based thereon can be appropriately adopted, for example, a method in which the sample is injected into a plate when the insoluble carrier is a plate, or a method in which the sample is mixed with a particle liquid containing the insoluble carrier when the insoluble carrier is particles. Examples of the dispersion medium for the particle liquid include those listed as the diluent.

[0059] In the reaction between the capturer and the analyte, the content (final concentration) of the capturer in the reaction solution containing the capturer is not particularly limited and can be adjusted appropriately depending on the type and concentration of the sample and capturer, etc., but is not particularly limited, for example, the amount of the second probe molecule is preferably 0.75 to 75 μg / mL, more preferably 5 to 30 μg / mL. The conditions for the capture step are also not particularly limited and can be adjusted appropriately, for example, at room temperature to 45°C, preferably 20 to 37°C, at a pH of about 6 to 9, preferably 7 to 8, for about 5 seconds to 10 minutes, preferably 30 seconds to 8 minutes, but are not limited to these conditions.

[0060] [Washing step] When the detection method of the present invention includes the capture step, it is preferable that the detection method further includes a washing step for removing contaminants not captured by the capture body. When the capture step is included before the labeling step, it is more preferable to include a washing step between the capture step and the labeling step to remove contaminants not captured by the capture body, i.e., components other than the third complex. In this case, it is also more preferable to include a washing step after the labeling step to remove contaminants not captured by the capture body, i.e., components other than the second complex contained in the reaction system.

[0061] The method for removing the impurities is not particularly limited, and any conventionally known method or a method based thereon can be used as appropriate. For example, when the insoluble carrier is a plate, a method of removing the liquid phase (supernatant) from the plate can be used. When the insoluble carrier is a particle, a method of recovering the particles from the reaction buffer by centrifugation or magnetic collection and then removing the liquid phase (supernatant) can be used. Furthermore, after the washing step, injection and removal of a washing solution can be repeated as necessary. Examples of the washing solution include known neutral (preferably pH 6 to 9) buffers (such as sodium phosphate buffer, MES buffer, Tris buffer, CFB buffer, MOPS buffer, PIPES buffer, HEPES buffer, tricine buffer, bicine buffer, and glycine buffer), and these may also contain stabilizing proteins such as BSA, surfactants, and the like.

[0062] [Detection Step] In the detection method of the present invention, the analyte is indirectly detected by detecting a signal derived from the label of the labeled substance bound to the analyte (detection step). Examples of the "signal" include fluorescence, luminescence, and color development (coloration), and include signals that can be confirmed with the naked eye as well as signals that can be confirmed using a fluorescence microscope or electrical analysis. According to the detection method of the present invention, by performing the labeling step in a reaction system under the above conditions, the amount of signal generated from the reaction between one analyte and a label can be increased, and detection sensitivity can be significantly increased even when the concentration of the analyte is low.

[0063] When an enzyme is used as the labeling substance, preferably, the capture step and washing step are performed before the labeling step, and a washing step is performed after the labeling step. After removing impurities not captured by the capturer, a chromogenic substrate, fluorescent substrate, chemiluminescent substrate, or the like depending on the type of enzyme is added as a substrate and reacted to detect a signal (fluorescence, luminescence, color (color development)) depending on the substrate. Such substrates and reaction conditions can be adjusted appropriately depending on the type of enzyme, etc. In the detection method of the present invention, the value of the detected signal amount (count) may be directly used as a value corresponding to the amount of the test substance, or, if necessary, the test substance may be quantified by comparing the signal amount with the value of the signal amount in a standard sample of a known concentration of the test substance.

[0064] <Labeling buffer and kit> The present invention provides a labeling buffer for use in the detection method of the present invention, which contains at least one selected from imidazole and its derivatives, a pH buffer, and a polyvalent metal ion.

[0065] The labeling buffer, including its preferred embodiments, is as described above as the reaction system for the labeling step, and the composition and content of each component of the reaction system can be substituted with the composition and content of each component of the labeling buffer, respectively. The labeling buffer according to the present invention is preferably an aqueous solution containing each of the components. Furthermore, the labeling buffer according to the present invention can be, for example, a concentrated solution in which the content of each component is 1 to 10 times the original concentration so that it can be used after dilution.

[0066] The labeling buffer preferably further contains the label. Such a label, including its preferred embodiments, is as described above. The labeling buffer containing the label can be distributed and stored as a labeled body fluid as is, and can also be added to the sample after the capturing step and washing step, either directly or diluted as necessary, to carry out the labeling step according to the present invention.

[0067] The present invention also provides a kit for use in the detection method of the present invention, comprising the labeling buffer. The kit of the present invention preferably further comprises the label. In this case, the labeling buffer included in the kit may be a labeling buffer containing the label, or the kit may contain a label-free labeling buffer and the label separately. The kit of the present invention may also further comprise the capture body. Such capture bodies, including their preferred embodiments, are as described above. Each of these may be independently in solid (powder) form or in liquid form dissolved or suspended in the storage buffer.

[0068] The kit of the present invention may further include, for example, at least one selected from the group consisting of a standard sample (each concentration), a control sample, the diluent, the washing solution, the substrate, a buffer for the enzyme-substrate reaction, and a buffer for stopping the enzyme-substrate reaction. Furthermore, the kit for the detection method of the present invention may further include instructions for use of the kit.

[0069] The present invention also provides a method for improving the detection sensitivity of a method for immunologically detecting an analyte in a sample, the method comprising a labeling step of forming a complex between the analyte and a label comprising a first probe molecule capable of binding to the analyte and a labeling substance in the presence of at least one selected from imidazole and its derivatives, a pH buffer, and a polyvalent metal ion. The sensitivity improving method of the present invention preferably further comprises a capture step, a washing step, and / or a detection step. The labeling step, capture step, washing step, detection step, sample, analyte, label, imidazole (at least one selected from imidazole and its derivatives), pH buffer, polyvalent metal ion, and other components and conditions involved in the sensitivity improving method can be any of the aspects described above for the detection method of the present invention, including preferred aspects thereof.

[0070] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples. In each example and comparative example, "%" indicates weight / volume (w / v: g / mL) percentage unless otherwise specified.

[0071] (1) Preparation of antibody-bound magnetic particles N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC hydrochloride) were added to magnetic particles (ferrite particles) and allowed to react. After the reaction, the particles were washed, and then the first anti-cyclosporine antibody was added and allowed to react. To stop the reaction, 1 M Tris-HCl was added, followed by pre-masking with a 10% BSA solution, and then a masking buffer (100 mM NaHCO ３ , 2.0% BSA, 1mM EDTA, 0.1% NaN ３ , pH 9.0) was reacted. This was dispersed in a storage buffer (50 mM Tris-HCl, 2.0% BSA, 150 mM NaCl, 10% sucrose, 0.1% ProClin 300, pH 7.2) to prepare a magnetic particle liquid containing antibody-bound magnetic particles. A monoclonal antibody against cyclosporine was used as the first anti-cyclosporine antibody.

[0072] (2) Preparation of alkaline phosphatase (ALP)-labeled antibody. The second anti-cyclosporine antibody, desalted with thiolation buffer, was mixed with iminothiolane and thiolated. The mixture was then desalted using coupling buffer (0.1 M phosphate buffer, 0.1% CHAPS, 1 mM EDTA, pH 7.0) to obtain a thiolated antibody. Meanwhile, desalted alkaline phosphatase was mixed with N-(4-maleimidobutyloxy)-succinimide (GMBS) in 0.1 M phosphate buffer to obtain maleimidated ALP. After desalting using coupling buffer, this was mixed with the thiolated antibody and coupled. After coupling, L-cysteine ​​was added, followed by iodoacetamide to block free thiol groups. The resulting mixture was concentrated and purified by gel filtration to obtain an ALP-labeled antibody. The second anti-cyclosporine antibody was a monoclonal antibody that was purified by expressing a VHH antibody (derived from an alpaca naive library) in Escherichia coli using standard methods, which has an amino acid sequence that binds to the binding site between cyclosporine and the first anti-cyclosporine antibody.

[0073] (3) Preparation of Cyclosporine Solution Cyclosporine was dissolved in dimethyl sulfoxide to a concentration of 10 mg / mL, which was used as a primary stock solution. The primary stock solution was dissolved in a cyclosporine dilution solution (phosphate buffer-based dilution solution) to a concentration of 50 μg / mL, which was used as a secondary stock solution. The secondary stock solution was added to the cyclosporine dilution solution to obtain various cyclosporine concentrations, thereby preparing cyclosporine solutions.

[0074] (Examples 1 to 3, Comparative Example 1) Cyclosporine was detected from the cyclosporine solution prepared in (3) above using the antibody-bound magnetic particles and ALP-labeled antibody prepared in (1) and (2) above. That is, first, 20 μL of the cyclosporine solution prepared in (3) above (cyclosporine concentration: 0 to 2000 [ng / mL]) was mixed with 100 μL of a citrate buffer-based pretreatment solution, and reacted at 37°C for 6.5 minutes. Next, this was mixed with 50 μL of the magnetic particle solution containing the antibody-bound magnetic particles prepared in (1) above, and reacted at 37°C for 8 minutes. The magnetic particles were collected and washed to remove impurities unbound to the magnetic particles. Next, 50 μL of labeled body solution (concentration of labeled antibody in the reaction system: 2 μg / mL) prepared by diluting the ALP-labeled antibody prepared in (2) above with a labeling buffer containing either no imidazole or 5 to 20 mM imidazole was added, and the reaction was allowed to proceed at 37°C for 8 minutes. After the reaction, the magnetic particles were collected and washed to remove any unbound contaminants, and 200 μL of a substrate solution containing AMPPD (Lumipulse (registered trademark) substrate solution, Fujirebio Inc.) was added, and the amount of luminescence generated by the enzyme reaction was counted as the signal level. All detection steps in this example were performed using the automated analyzer Lumipulse L2400 (registered trademark, Fujirebio Inc.). Each labeling buffer was an aqueous solution containing the components shown in Table 1 below. The count values ​​at each cyclosporine concentration (ng / mL) are shown in Table 2 below. Table 2 also shows the ratio of each count value when the cyclosporine concentration was 100 ng / mL to that when the cyclosporine concentration was 0 ng / mL (100 (ng / mL) / 0 (ng / mL)), and the difference of each count value (100 (ng / mL) - 0 (ng / mL)) (the same applies to Tables 4 and 6 below).

[0075]

[0076]

[0077] As shown in Table 2, it was confirmed that, compared to the reaction system of cyclosporine with a labeled substance, i.e., the condition where the labeling buffer does not contain imidazole (e.g., Comparative Example 1), the detection sensitivity was significantly increased in the condition where imidazole was added (e.g., Examples 1 to 3), even when the concentration of the test substance (cyclosporine) was low (e.g., 100 ng / μL).

[0078] (Examples 4-5, Comparative Example 2) 20 μL of the cyclosporine solution prepared in (3) above was mixed with 100 μL of the pretreatment solution and reacted at 37°C for 6.5 minutes. Subsequently, this was mixed with 50 μL of the magnetic particle solution containing the antibody-bound magnetic particles prepared in (1) above and reacted at 37°C for 8 minutes. The magnetic particles were collected and washed to remove impurities unbound to the magnetic particles. Next, 50 μL of a labeled body solution (label concentration in the reaction system: 2 μg / mL) prepared by diluting the ALP-labeled antibody prepared in (2) above with a labeling buffer containing 15 mM imidazole was added and reacted at 37°C for 8 minutes. After the reaction, the magnetic particles were collected and washed to remove components unbound to the magnetic particles, and 200 μL of a substrate solution containing AMPPD (Lumipulse (registered trademark) substrate solution, Fujirebio Inc.) was added. The amount of luminescence generated by the enzyme reaction was counted as the signal amount. All detection steps in this example were carried out using an automatic analyzer, Lumipulse L2400 (registered trademark, manufactured by Fujirebio Inc.). Each labeling buffer was an aqueous solution containing the composition shown in Table 3 below. The count values ​​at each cyclosporine concentration (ng / mL) are shown in Table 4 below.

[0079]

[0080]

[0081] As shown in Table 4, it was confirmed that the detection sensitivity was significantly increased even when the concentration of the test substance (cyclosporine) was low in the reaction system between cyclosporine and a labeled substance, i.e., in the condition where the labeling buffer did not contain a polyvalent metal ion (zinc ion) (e.g., Comparative Example 2), in the condition where a polyvalent metal ion was added (e.g., Examples 4 and 5).

[0082] (Examples 6-7, Comparative Example 3) 20 μL of the cyclosporine solution prepared in (3) above was mixed with 100 μL of the pretreatment solution and reacted at 37°C for 6.5 minutes. Subsequently, this was mixed with 50 μL of the magnetic particle solution containing the antibody-bound magnetic particles prepared in (1) above and reacted at 37°C for 8 minutes. The magnetic particles were collected and washed to remove impurities unbound to the magnetic particles. Next, 50 μL of a labeled body solution (label concentration in the reaction system: 2 μg / mL) prepared by diluting the ALP-labeled antibody prepared in (2) above with a labeling buffer containing 15 mM imidazole was added and reacted at 37°C for 8 minutes. After the reaction, the magnetic particles were collected and washed to remove components unbound to the magnetic particles, and 200 μL of a substrate solution containing AMPPD (Lumipulse (registered trademark) substrate solution, Fujirebio Inc.) was added. The amount of luminescence generated by the enzyme reaction was counted as the signal amount. All detection steps in this example were carried out using an automatic analyzer, Lumipulse L2400 (registered trademark, manufactured by Fujirebio Inc.). Each labeling buffer was an aqueous solution containing the components shown in Table 5 below. The count values ​​at each cyclosporine concentration (ng / mL) are shown in Table 6 below.

[0083]

[0084]

[0085] As shown in Table 6, it was confirmed that the detection sensitivity was increased, particularly at low concentrations of the test substance (cyclosporine), in the reaction system of cyclosporine with a labeled substance, i.e., in the condition where the labeling buffer did not contain a polyvalent metal ion (nickel ion) (e.g., Comparative Example 3), in the condition where a polyvalent metal ion was added (e.g., Examples 6 and 7).

[0086] According to the present invention, it is possible to provide a detection method capable of detecting a test substance with high sensitivity by an immunological detection method, as well as a labeling buffer to be used in the detection method and a kit containing the same.

Claims

1. A method for immunologically detecting a substance to be detected in a sample, comprising a labeling step of forming a complex of the substance to be detected with a labeled body including a first probe molecule capable of binding to the substance to be detected and a labeling substance in the presence of at least one selected from imidazole and its derivatives, a pH buffer, and a polyvalent metal ion.

2. The detection method according to claim 1, wherein the concentration of at least one selected from imidazole and its derivatives is 1 mM or more in terms of imidazole in the reaction system of the labeling step.

3. The detection method according to claim 1, wherein the pH of the reaction system of the labeling step is 6.0 to 8.

0.

4. The detection method according to claim 1, wherein the concentration of the polyvalent metal ion is 0.01 mM or more in the reaction system of the labeling step.

5. The detection method according to claim 1, further comprising a capturing step of capturing the substance to be detected with a capturing body including a second probe molecule capable of binding to the substance to be detected and an insoluble carrier.

6. The detection method according to claim 5, further comprising a washing step of removing impurities not captured by the capturing body after the capturing step.

7. The detection method according to claim 1, wherein the labeling substance is an enzyme and the first probe molecule is an antibody capable of binding to the substance to be detected.

8. A labeling buffer for use in the detection method according to any one of claims 1 to 7, the buffer containing at least one selected from imidazole and its derivatives, a pH buffer, and a polyvalent metal ion.

9. The buffer according to claim 8, further containing a labeled body including a first probe molecule capable of binding to the substance to be detected and a labeling substance.

10. A kit for use in the detection method according to any one of claims 1 to 7, the kit including a labeling buffer containing at least one selected from imidazole and its derivatives, a pH buffer, and a polyvalent metal ion.

11. The kit according to claim 10, further including a labeled body including a first probe molecule capable of binding to the substance to be detected and a labeling substance.

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