Detection sensitivity improvement method, detection method, and composition

Abstract

Provided is a method for improving detection sensitivity of a test substance in a sandwich immunoassay. The sandwich immunoassay includes a labeling step of forming a complex of the test substance and a labeled body containing a first binder binding to the test substance and a labeling substance. The method includes a step of introducing a histidine-rich segment into the labeled body.

Description

Method for improving detection sensitivity, detection method, and composition

[0001] The present invention relates to a method for improving detection sensitivity, a detection method, and a composition, and more specifically, to a method for improving detection sensitivity in a sandwich immunoassay, a method for detecting a test substance by a sandwich immunoassay, and a labeled substance-containing composition used in these.

[0002] As a method for detecting a test substance in a sample, immunoassays, which utilize the specific immune response between an antigen and an antibody, have been widely used as conventional methods. Among these, sandwich immunoassays, which detect the test substance by forming a complex (capturer-test substance-labeled substance) with a capturer (a binder that binds to the target test substance, such as an antibody or peptide) immobilized on a solid phase such as particles or a plate, a labeled substance (the binder labeled with a labeling substance), and the test substance, and detecting the signal derived from the labeling substance, are generally known as a method with excellent detection sensitivity.

[0003] As an example of such a sandwich immunoassay, International Publication No. 2014 / 024853 (Patent Document 1) describes a sandwich method in which a mixture of an anti-F1 antibody or its antigen-binding fragment that specifically binds to prothrombin fragment 1 and an anti-F2 antibody or its antigen-binding fragment that specifically binds to fragment 2 is used as a labeled antibody (labeled substance), and an anti-PIVKA-II antibody or its antigen-binding fragment that specifically binds to PIVKA-II is used as a solid-phase antibody (capture substance).

[0004] International Publication No. 2014 / 024853

[0005] Regarding sandwich immunoassays, various studies have been conducted to date on detection sensitivity, detection accuracy, and reduction of nonspecific reactions. For example, when the aforementioned test substance is used as a biomarker for various diseases, its detection accuracy affects the treatment strategy for those diseases, and therefore further improvement in detection sensitivity is still required. In particular, the inventors have found that with the diversification of test substances, further improvement in detection sensitivity is desirable when detecting compounds with small molecular weights as test substances or when the sample is small in quantity.

[0006] The present invention has been made in view of the above problems, and aims to provide a method for improving the detection sensitivity of a test substance in a sandwich immunoassay, a detection method that enables highly sensitive detection of a test substance by a sandwich immunoassay, and a labeled substance-containing composition for use in these methods.

[0007] The present inventors have diligently studied methods for detecting a test substance by sandwich immunoassay and have discovered that by introducing a histidine-rich segment, which is a region containing many histidine residues, into the labeling body for labeling the test substance, the amount of signal derived from the labeling substance in the labeling body is increased. This makes it possible to detect the test substance with high sensitivity, even if the test substance is a compound with a small molecular weight or at a low concentration, thus completing the present invention.

[0008] The embodiments of the present invention obtained from these findings are as follows: [1] A method for improving the detection sensitivity of a test substance in a sandwich immunoassay, wherein the sandwich immunoassay includes a labeling step of forming a complex of the test substance with a labeled product comprising a first binder bound to the test substance and a labeling substance, the method comprising a step of introducing a histidine-rich segment into the labeled product. [2] The method for improving detection sensitivity according to [1], wherein the first binder is a VHH antibody. [3] The method for improving detection sensitivity according to [1] or [2], wherein the first binder is a polymer of VHH antibodies in which two or more VHH antibodies are linked by a linker, and the linker is a linker comprising at least one selected from the group consisting of glycine and serine. [4] The method for improving detection sensitivity according to any one of [1] to [3], wherein the molecular weight of the test substance is 2500 or less. [5] The method for improving detection sensitivity according to any one of [1] to [4], wherein the sandwich immunoassay further comprises a capture step of capturing the test substance with a capture body comprising a second binder bound to the test substance and an insoluble carrier. [6] A method for detecting a test substance in a sample by a sandwich immunoassay, comprising a labeling step of forming a complex of the test substance with a labeled body comprising a first binder bound to the test substance and a labeling substance, wherein the labeled body comprises a histidine-rich segment. [7] The detection method according to [6], wherein the first binder is a VHH antibody. [8] The detection method according to [6] or [7], wherein the first binder is a polymer of VHH antibodies in which two or more VHH antibodies are linked by a linker, and the linker is a linker comprising at least one selected from the group consisting of glycine and serine. [9] The detection method according to any one of [6] to [8], wherein the molecular weight of the test substance is 2500 or less.

[10] The detection method according to any one of [6] to [9], further comprising a capture step of capturing the test substance with a capture body comprising a second binder bound to the test substance and an insoluble carrier.

[11] A composition containing a labeled substance, comprising a first binder bound to the test substance and a labeling substance, and further comprising a histidine-rich segment, for improving the detection sensitivity of the test substance in a sandwich immunoassay.

[0009] According to the present invention, it is possible to provide a method for improving the detection sensitivity of a test substance in a sandwich immunoassay, a detection method that enables highly sensitive detection of a test substance by a sandwich immunoassay, and a labeled composition used in these methods.

[0010] The graph in <Test Example 1> shows the signal value (count) at each everolimus concentration [ng / mL] when polyhistidine-introduced labeled solution 1 (Example, No. 1) and polyhistidine-non-introduced labeled solution 3 (Comparative Example, No. 3) were used as the labeled solution. The graph in <Test Example 2> shows the signal value (count) at each everolimus concentration [ng / mL] when polyhistidine-introduced labeled solution 5 (Example, No. 5) and polyhistidine-non-introduced labeled solution 6 (Comparative Example, No. 6) were used as the labeled solution.

[0011] The present invention will be described in detail below with reference to its preferred embodiments.

[0012] <Method for improving detection sensitivity, detection method> The method for improving detection sensitivity of the present invention is a method for improving the detection sensitivity of a test substance in a sandwich immunoassay, wherein the sandwich immunoassay includes a labeling step of forming a complex of the test substance with a labeled product containing a first binder bound to the test substance and a labeling substance, and the method includes a step of introducing a histidine-rich segment into the labeled product. The detection method of the present invention is a method for detecting a test substance in a sample by a sandwich immunoassay, wherein the method includes a labeling step of forming a complex of the test substance with a labeled product containing a first binder bound to the test substance and a labeling substance, and the labeled product contains a histidine-rich segment. In this specification, these methods may be collectively referred to as "the method of the present invention."

[0013] [Test Substance] The "test substance" according to the present invention is not particularly limited as long as it is capable of binding, preferably specifically binding, to the first binder and, optionally, a second binder (hereinafter, these are sometimes collectively referred to simply as "binders"). Examples of such combinations of test substance and binder (or combination of binder and test substance) include combinations that can achieve specific binding, such as combinations of antibody and antigen, combinations of lectin and a glycan that can bind to it (lectin-binding glycan), combinations of receptor and ligand, combinations of antibody containing an Fc region and an Fc-binding protein, and combinations of protein and a peptide that binds to it (peptide binder). In the present invention, "sandwich immunoassay" includes not only a method of detecting a test substance using an immune reaction between an antigen and an antibody, but also a method of detecting a test substance using a reaction by a combination that can achieve the above-mentioned specific binding, similar to an immune reaction between an antigen and an antibody.

[0014] Among these, in the present invention, it is preferable that the binder is an antibody and the test substance is a substance that can act as an antigen against the antibody, or that the binder is a peptide and the test substance is a protein to which the peptide binds. It is also preferable that one of the first binder and the second binder is an antibody and the other is the peptide, and the test substance is an antigen protein that binds to the antibody and the peptide.

[0015] Examples of test substances according to the present invention include, but are not limited to, low-molecular-weight compounds such as pharmaceuticals or their metabolites, including, substances that can act as antigens to the aforementioned antibodies, such as antigen peptides, antibodies, receptor proteins, transport proteins, transcription factors, haptens, enzymes, viral antigens / antibodies, and other proteins and peptides; sugars (polysaccharides, monosaccharides); glycoproteins; nucleic acids; lipids; glycolipids; vitamins, hormones (especially sex hormones, hormones having a steroid skeleton (e.g., estradiol, progesterone, testosterone, etc.)), coenzymes, toxins, antibiotics, immunosuppressants (e.g., macrolide immunosuppressants having a macrolide structure), and antiepileptic drugs.

[0016] For example, as the test substance according to the present invention, from the viewpoint of particularly contributing to the increased sensitivity of detection by the method of the present invention, it is preferable that it be a low molecular weight, for example, more preferably 2500 or less in molecular weight, even more preferably 50 to 2500, even more preferably 100 to 2000, and particularly preferably 200 to 1500. Examples of such low molecular weight test substances include macrolide immunosuppressants (e.g., tacrolimus (FK506), cyclosporine A, sirolimus (rapamycin) and its derivatives (e.g., everolimus, temsirolimus), zotarolimus, biolimus, novolimus, pimecrolimus, myolimus, myolimus, defololimus), and their metabolites; and hormones such as estradiol, progesterone, and testosterone (preferably hormones having a steroid skeleton).

[0017] In this invention, "antibody" includes not only complete antibodies but also antibody fragments (e.g., Fab, Fab', F(ab')). ２ This includes Fv, single-chain antibodies (such as VHH antibodies), diabodies, etc., and low-molecular-weight antibodies to which the variable region of an antibody has been conjugated. Furthermore, the "antibody" according to the present invention may be either a polyclonal antibody or a monoclonal antibody.

[0018] Furthermore, in the present invention, "protein-binding peptide" refers to a polypeptide capable of binding to the protein in question, also known as a "peptide binder," and is typically a polypeptide obtained by designing a protein structure based on the amino acid sequence of the protein. Such peptides can be obtained by conventionally known methods, such as the phage library method. The length of such peptides is typically 5 to 30 amino acid residues, and more preferably 8 to 20 amino acid residues.

[0019] [Sample] The "sample" used in the sandwich immunoassay according to the present invention is not particularly limited as long as it is a sample in which the test substance may be present. For example, various organisms (including cells, tissues, organs, and individuals) and their extracts; 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, amniotic fluid, etc.; feces; tissue); plant biosaturates; biological culture solutions; suspensions of water in the environment (rivers, lakes, harbors, waterways, groundwater, purified water, sewage, wastewater, etc.), solid matter (soil, etc.); food, beverages, etc. can be used as appropriate depending on the purpose. Examples of non-human animals include primates such as chimpanzees and monkeys; domesticated animals such as cattle, pigs, horses, sheep, rabbits, chickens, cats, and dogs; and wild animals and birds such as deer, wild boars, raccoons, weasels, mice, and pigeons.

[0020] Among these, for example, when detecting biomarkers, etc., as indicators for disease diagnosis in the medical field or clinical testing field, the sample generally includes specimens taken from the subject of diagnosis (preferably a human) from which the target biomarker, etc., is to be detected, such as serum, plasma, whole blood, urine, feces, oral mucosa, pharyngeal mucosa, intestinal mucosa, and various biopsy tissues.

[0021] The aforementioned sample may be one that has been processed by grinding or freezing, one that has been appropriately diluted or suspended in a diluent, one that has been appropriately pH-adjusted, or one that has been pre-treated to dissociate the test substance from the substance to which it is bound. Examples of the diluent include water, physiological saline, known buffers (phosphate buffer (sodium phosphate buffer, potassium phosphate buffer), MES buffer, Tris buffer, CFB buffer, MOPS buffer, PIPES buffer, HEPES buffer, trichine buffer, bicine buffer, glycine buffer, citrate buffer, etc.), and organic solvents (dimethyl sulfoxide, etc.). Depending on the type of sample, stabilizing proteins such as BSA or serum may be added.

[0022] The sample to be used in the sandwich immunoassay according to the present invention is preferably an aqueous sample, and is preferably appropriately diluted or suspended with the diluent as needed. Furthermore, if the test substance is a nucleic acid or a substance derived from microorganisms contained in the sample, these nucleic acids or microorganisms may be appropriately isolated. A known method can be appropriately used to isolate such nucleic acids or microorganisms from the sample.

[0023] [Labeled substance] In the present invention, "labeled substance" is a complex comprising a labeling substance and a first binder that binds to the test substance, wherein the labeling substance and the first binder are directly or indirectly bound. In the detection sensitivity improvement method of the present invention, a histidine-rich segment is introduced into the labeled substance. Furthermore, in the detection method of the present invention, a labeled substance into which a histidine-rich segment has been introduced (hereinafter referred to as "histidine-rich segment-introduced labeled antibody") is used.

[0024] (Labeling Substance) The "labeling substance" contained in the labeled product according to the present invention functions primarily as a label for detecting the test substance, and any labeling substance used in known immunological detection methods can be appropriately adopted. The labeling substance according to the present invention is not particularly limited, but it is preferably an enzyme from the viewpoint of particularly contributing to increasing the detection sensitivity of the method of the present invention (in this specification, a histidine-rich segment-transformed labeling antibody in which the labeling substance is an enzyme is referred to as a "histidine-rich segment-transformed enzyme-labeled antibody").

[0025] Examples of the enzymes include alkaline phosphatase (ALP), horseradish peroxidase (HRP), β-galactosidase (β-gal), glucose oxidase, and luciferase, and may be used individually or in combination of two or more of these. When an enzyme is used as the labeling substance, the following signals corresponding to the substrate can be detected by adding a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, etc. as a substrate. Conventional known labeling substances and substrates can be used as appropriate, and commercially available products can also be used as appropriate.

[0026] (First Binder) In the present invention, "first binder" refers to a molecule that can bind to the test substance, preferably specifically. When the following capture step is performed before or simultaneously with the labeling step using the following capture body, "first binder that binds to the test substance" includes a binder that can bind to the complex of the test substance and the second binder, preferably specifically. An example of the mode of binding to the complex of the test substance and the second binder is a mode in which the binding site between the test substance and the second binder is recognized and bound.

[0027] The first binder according to the present invention is any binder that can bind to the test substance, preferably specifically, and may be one type or a combination of two or more types. However, it is preferably an antibody or peptide that binds to the test substance, preferably specifically, and is more preferably a low molecular weight binder, which can particularly contribute to increasing the detection sensitivity of the method of the present invention. It is even more preferably a VHH antibody (Variable domain of Heavy chain of Heavy chain antibody) or a peptide, and even more preferably a VHH antibody. Such a first binder can be prepared by known and established methods depending on the test substance, or commercially available binders may be used as appropriate.

[0028] Furthermore, when a VHH antibody is used as the first binder according to the present invention, such VHH antibody may be a monomer or a polymer containing two or more antibodies. From the viewpoint of further improving detection sensitivity, a polymer is preferable, and the polymer is more preferably a dimer, trimer, or tetramer, and even more preferably a dimer. Furthermore, the polymer is preferably such that two or more VHH antibodies are linked together in the same orientation in the longitudinal direction (front-to-back direction) by a linker, and more preferably two VHH antibodies are linked together in tandem by a linker (i.e., linked together in the same orientation in the longitudinal direction (front-to-back direction)).

[0029] The linker can be any conventionally known linker without particular limitation, but it is preferably a flexible linker, more preferably an amino linker containing at least one selected from the group consisting of glycine (G) and serine (S), and even more preferably a linker consisting of glycine and serine. The length of the linker is preferably, for example, 3 to 25 amino acid residues, more preferably 5 to 20 amino acid residues, and even more preferably 10 to 14 amino acid residues. Specific examples of such linkers include, for example, linkers composed of (GS)n (n = an integer from 1 to 10, preferably 5 to 7), (GGS)n (n = an integer from 1 to 7, preferably 3 to 5), (GGGS)n (n = an integer from 1 to 5, preferably 2 to 4, more preferably 3; GGGS: SEQ ID NO: 1), or (GGGGGS)n (n = an integer from 1 to 4, preferably 2 to 3; GGGGS: SEQ ID NO: 2). Such a multimer of VHH antibodies can be obtained, for example, by expressing a plasmid containing an expressible nucleotide sequence encoding an amino acid sequence in which the amino acid sequences of two or more VHH antibodies are linked via the linker, in a suitable host (e.g., E. coli) using a known method.

[0030] The inventors have found that the detection sensitivity of a sandwich immunoassay can be improved by using a polymer of the VHH antibody described above. Therefore, the present invention provides a method for improving the detection sensitivity of a test substance in a sandwich immunoassay, wherein the sandwich immunoassay includes a labeling step of forming a complex of the test substance with a labeled substance comprising a first binder that binds to the test substance and a labeling substance, and the method also provides a method for improving detection sensitivity, wherein the first binder is a polymer of VHH antibodies in which two or more VHH antibodies are linked by a linker, and the linker is a linker comprising at least one selected from the group consisting of glycine and serine. In this method for improving detection sensitivity, it is preferable to further include a step of introducing a histidine-rich segment into the labeled substance in order to further improve detection sensitivity.

[0031] (Histidine-Rich Segment) In the present invention, a "histidine-rich segment" refers to a region consisting of four or more amino acid residues and containing a large amount of histidine. The length of such a histidine-rich segment (for example, if the first binder is an antibody, the length other than the amino acid sequence constituting the antibody; if the binder is the peptide, the length other than the amino acid sequence involved in the binding of the peptide to the test substance; the length other than the amino acid sequence of the labeling substance or linker; the length of the sequence containing the following percentage of histidine) is preferably 4 to 30 amino acid residues, more preferably 5 to 20 amino acid residues, and even more preferably 6 to 10 amino acid residues. Furthermore, in such a region, "histidine-rich" means that 30% or more, preferably 50% or more, more preferably 80% or more, and even more preferably 100% of the total amino acid residues constituting the region (for example, per 4 to 30, 5 to 20, or 6 to 10 amino acid residues) are histidine residues. Such histidine-rich segments preferably contain polyhistidines consisting of four or more consecutive histidine residues, more preferably contain polyhistidines consisting of four to ten or five to eight consecutive histidine residues, and even more preferably consist of four to ten or five to eight consecutive histidine residues. In the present invention, a region other than the amino acid sequence involved in the binding of the first binder (in the case of an antibody or peptide) to the test substance, and the amino acid sequence involved in the labeling function of the labeling substance (in the case of an enzyme, etc.), where 30% or more, preferably 50% or more, more preferably 80% or more per 4 to 30, 5 to 20, or 6 to 10 amino acid residues are histidine residues (for example, 4 or more amino acid residues per 4 to 30 amino acid residues; 4 or more amino acid residues or 5 or more amino acid residues per 5 to 20 amino acid residues; 3 or more amino acid residues or 5 or more amino acid residues or 6 or more amino acid residues per 6 to 10 amino acid residues), and more preferably 100% of 4 to 30, 5 to 20, or 6 to 10 amino acid residues are histidine residues, and / or a polyhistidine consisting of 4 or more, 4 to 10, or 5 to 8 histidine residues or a region containing the same may be defined as a "histidine-rich segment".

[0032] The histidine-rich segment according to the present invention may be introduced at any position in the labeled product. It may be added to the labeled substance, incorporated into the labeled substance as long as its function as a labeled substance is not impaired, added to the first binder, incorporated into the first binder as long as its function as a first binder is not impaired, or introduced as a linker or part thereof between the labeled substance and the first binder. Furthermore, there may be two or more histidine-rich segments per labeled product, and in this case, the amino acid sequences of the histidine-rich segments may be the same or different. Among these, the histidine-rich segment according to the present invention is preferably introduced into the labeled product by adding it to the first binder. In this case, it is preferable that the first binder be bound to a position other than the binding site with the labeling substance. Furthermore, if the first binder is an antibody, it is not particularly limited as long as the specificity and affinity of the antibody to the test substance are not inhibited, but it is preferable, for example, to bind it to the framework region or constant region of the heavy chain or light chain of the antibody, and more preferably to bind it to the framework region (e.g., the C-terminus) of a single-chain antibody.

[0033] (Composition and manufacturing method of the labeled product (method for introducing histidine-rich segments)) In the labeled product according to the present invention, the molar ratio of the labeled substance to the first binder is not particularly limited and can be appropriately adjusted according to the combination of these types and the ease of binding to the test substance, but for example, the amount of the first binder (the total amount if the first binder is a combination of two or more types, or the number of moles of the macromer in the case of the VHH antibody macromer) is preferably 0.01 to 10,000 moles, and more preferably 0.05 to 10 moles, per mole of the labeled substance (the total amount if the labeled substance is a combination of two or more types).

[0034] Furthermore, in the labeled product (histidine-rich segment-labeled antibody) according to the present invention, the molar ratio of the first binder to the histidine-rich segment is preferably 1 to 3 moles, more preferably 1 to 2 moles, and more preferably 1 mole, of the histidine-rich segment (total of the histidine-rich segment if it is a combination of two or more types) per mole of the first binder (the total of the first binder if it is a combination of two or more types, or the number of moles of the multimer if it is a multimer of the VHH antibody). Furthermore, in the case where the first binder is a multimer of the VHH antibody, preferably, when the total number of individual VHH antibodies constituting the multimer is 1 mole, the total number of individual histidine-rich segments is preferably 1 to 3 moles, more preferably 1 to 2 moles, and more preferably 1 mole.

[0035] In the production of a labeled body according to the present invention, the method for bonding the labeled substance and the first binder can be a conventionally known method or a method similar thereto, depending on the type of labeled substance and the first binder, and the labeled substance and the first binder may be bonded directly to each other or indirectly.

[0036] Examples of the method for said direct binding include a method wherein an active group (e.g., a thiol group, a maleimide group, a succinimide group, a carboxy group, an amino group, an ester group) is imparted to said labeling substance and / or the first binder, or a substance having such an active group is used as said labeling substance and / or the first binder, and binding is achieved via covalent bonding formed by said active group. As the labeling substance provided with the active group and the first binder, commercially available products may be used as they are, or they may be prepared by introducing said active group onto the surface of the labeling substance and / or the first binder under appropriate reaction conditions. Examples of the method for indirectly binding said labeling substance to the first binder include a method for binding via polyethylene glycol, oligopeptides, linker molecules, or the like. Alternatively, binding may be achieved via the following steps: modifying one side with a certain modification, adding a substance that captures the modified moiety to the other side, and binding them via these components. For example, one side may be biotinylated and the other side may be avidinylated, and a binding method based on avidin-biotin binding may be employed.

[0037] Examples of the method for introducing said histidine-rich segment into the labeled substance according to the present invention include a method wherein said histidine-rich segment is used as the oligopeptide and introduced between the labeling substance and the first binder. Further, when the first binder is an antibody or said peptide, a method may be used: using a plasmid that expressibly comprises a nucleotide sequence encoding an amino acid sequence in which a histidine-rich segment sequence is added to the N-terminus or C-terminus (preferably the C-terminus) of these amino acid sequences, and expressing the sequence in an appropriate host (e.g., E. coli) by a known method, whereby the first binder having the histidine-rich segment added thereto can be obtained. By binding the first binder obtained in this way to the labeling substance by the above method, a labeled substance into which a histidine-rich segment has been introduced (a labeled antibody with an introduced histidine-rich segment) can be obtained.

[0038] The ratio of the labeling substance, the first binder, and the histidine-rich segment to be subjected to the above method can be appropriately selected so as to achieve the preferred range for the above labeled substance.

[0039] [Capturing Body] In the sandwich immunoassay according to the present invention, it is preferable to use a capturing body. In the present invention, the "capturing body" refers to a complex including an insoluble carrier and a second binder that binds to the test substance, and is a conjugate in which the second binder is directly or indirectly bound to and supported by the insoluble carrier.

[0040] (Insoluble Carrier) The "insoluble carrier" included in the capturing body according to the present invention is water-insoluble, and functions as a carrier that mainly supports the second binder and immobilizes it as a solid phase. In the present invention, "water-insoluble" means that it is insoluble in water at normal temperature and normal pressure (the solubility in water is 0.001 g / mL or less, preferably 0.0001 g / mL or less, the same applies hereinafter).

[0041] As the material of such an insoluble carrier, those used as insoluble carriers in known immunological detection methods can be appropriately used. Examples include at least one selected from the group consisting of polymer compounds (organic compounds such as polystyrene, (meth)acrylic acid esters, polymethyl methacrylate, polyimide, nylon, etc.; gelatin, cellulose, nitrocellulose, etc.; resins such as gum arabic; latex; etc.), glass, silica, metals (gold, platinum, etc.), and metal compounds (iron oxide, ferrite, cobalt oxide, nickel ferrite, etc.). The material of the insoluble carrier may also be a composite material thereof. For example, it may be an organic-inorganic composite material including at least one selected from the group consisting of the above polymer compounds (gelatin, cellulose, gum arabic, latex, etc.) and at least one selected from the group consisting of the above metals or metal compounds (iron oxide (spinel ferrite, etc.), cobalt oxide, nickel ferrite, etc.). In the present invention, the shape of the insoluble carrier is not particularly limited, and may be, for example, any of a plate, fiber, membrane, particle, or the like. As such an insoluble carrier, conventionally known ones can be appropriately used, and commercially available products can also be appropriately used.

[0042] Among these, the insoluble carrier according to the present invention is preferably a particle from the viewpoint of reaction efficiency, and more preferably a particle with an average particle diameter of 0.1 to 10 μm, or 0.5 to 8 μm. Furthermore, from the viewpoint of automation and time reduction, magnetic particles are even more preferable. Examples of the magnetic particles include particles made of the metal compound; particles in which the metal compound is used as a core and is coated with the polymer compound (polymer compound coated particles); and particles in which the polymer compound is used as a core and is coated with the metal compound (metal compound coated particles). There are no particular limitations, but polymer compound coated particles are preferred from the viewpoint of further reducing the detection background.

[0043] (Second Binder) In the present invention, "second binder" refers to a molecule that can bind to the test substance, preferably specifically. When the following capture step is performed after or simultaneously with the labeling step, "second binder that can bind to the test substance" includes a binder that can bind to the complex of the test substance and the first binder, preferably specifically. An example of the mode of binding to the complex of the test substance and the first binder is a mode in which the binding site between the test substance and the first binder is recognized.

[0044] The second binder according to the present invention is any binder that can bind to the test substance, preferably specifically, and may be one type or a combination of two or more types. However, it is preferably an antibody or peptide that binds to the test substance, preferably specifically, and is more preferably a low molecular weight, even more preferably a VHH antibody or peptide, and even more preferably a VHH antibody. It may also be a polymer of the above VHH antibody. The second binder according to the present invention may be different from the first binder, or it may be the same as the first binder as long as it does not hinder the effects of the present invention. Such a second binder can be prepared by known established methods depending on the test substance, or a commercially available one may be used as appropriate.

[0045] (Composition and Manufacturing Method of the Capture Body) In the capture body according to the present invention, the content of the second binder is not particularly limited, but in order to further improve the detectability of the substance to be tested, it is preferable to set the number of molecules of the second binder bound to one molecule of the insoluble carrier to be as large as possible. For example, the mass of the second binder (the total of two or more types of second binders if there 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.

[0046] The capture body according to the present invention can be manufactured by binding and fixing a second binder to the insoluble carrier. Depending on the type of insoluble carrier and second binder, a conventionally known method or a method similar thereto may be used for this manufacturing method, and the second binder may be directly or indirectly bound to the insoluble carrier. For example, such a binding method is the method mentioned for binding the labeling substance to the first binder. The ratio of the insoluble carrier to the second binder used in this manufacturing method can be appropriately selected to achieve a preferred range for the content of the second binder in the capture body. Furthermore, commercially available capture bodies such as antibody-bound particles may be used as appropriate.

[0047] [Labeling Step] In the sandwich immunoassay according to the present invention, in the labeling step, the sample and the label are brought into contact, and if the test substance is present in the sample, a complex of the label and the test substance, i.e., a label-test substance complex (hereinafter referred to as the "first complex") is formed by binding of the test substance to the first binder. Alternatively, if the detection method of the present invention includes the following capture step before or simultaneously with the labeling step, in the labeling step, a complex of the label and the test substance captured by the capture body (third complex), i.e., a label-test substance-capture body complex (hereinafter referred to as the "second complex") is formed as the complex.

[0048] The method for bringing the sample (or third composite) into contact with the labeling agent is not particularly limited, and any conventionally known method or a method similar thereto can be used as appropriate. For example, one method is to add the labeling agent or the labeling agent-containing composition described below to the aqueous sample (or third composite).

[0049] In the method of the present invention, from the viewpoint of further improving detection sensitivity, it is preferable that, in the labeling step, at least one selected from imidazole and its derivatives (collectively referred to as "imidazole, etc." in this specification) and a polyvalent metal ion be present in the reaction system between the test substance and the label. Furthermore, it is even more preferable that a pH buffer be further present in the reaction system. In the present invention, the "reaction system between the test substance and the label" is an aqueous system, and it is preferable that it is an aqueous solution containing these for reacting the label with the test substance.

[0050] (Imidazole, etc.) In the present invention, "imidazole and its derivatives" refers to compounds having an imidazole skeleton, for example, imidazole, 1-methylimidazole, 1-ethylimidazole, 1-propylimidazole, 1-butylimidazole, 1-phenylimidazole, 1-vinylimidazole, 1-allyliimidazole, 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-vinyl Examples include midazole, 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-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, and any one of these or a combination of two or more may be used.

[0051] The aforementioned imidazoles, etc., also include chemically acceptable salts of the imidazole or its derivatives, such as potassium salts, magnesium salts, lithium salts, calcium salts, zinc salts, adipine salts, alginates, citrates, aspartates, benzoates, benzenesulfonates, bisulfates, butyrates, camphorates, camphor sulfonates, digluconates, glycerophosphates, hemisulfates, heptanoates, hexanoates, fumarates, sesqui(fumarates), hydrochlorides, hydrochlorides, hydrochlorides, hydrobroms, hydroiodides, 2-hydroxyethanesulfonates (isethionates), lactates, maleates, methanesulfonates, nicotine salts. Examples include tinate, 2-naphthalene sulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, sulfate, bis(tartrate), tartrate, (L)tartrate, bis((L)tartrate), (D)tartrate, bis((D)tartrate), (DL)tartrate, bis((DL)tartrate), mesotartrate, bis(mesotartrate), thiocyanate, phosphate, glutamate, bicarbonate, bis(bromide), bis(sulfate), bis(phosphate), tris(hydrochloride), p-toluenesulfonate, and undecanoate, and any one of these or a combination of two or more may be used. Of these, in terms of controlling the concentrations of the imidazole and the polyvalent metal ions listed below in the reaction system, it is preferable that the imidazole added to the reaction system be a salt that does not produce the polyvalent metal ions, and more preferably not a salt. However, this does not negate the possibility that the imidazole and the polyvalent metal ions listed below may form a salt in the reaction system.

[0052] In the labeling step according to the present invention, the concentration of imidazole, etc. in the reaction system (the total of two or more types of imidazole, etc., if there are two or more types of imidazole, etc., 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, in terms of imidazole equivalent, i.e., in terms of imidazole skeleton amount. If the concentration of imidazole, etc. is below the lower limit, the effect of further improving the detection sensitivity tends not to be fully realized, on the other hand, if it exceeds the upper limit, the detection sensitivity may conversely decrease.

[0053] (Polyvalent Metal Ions) In the present invention, "polyvalent metal ions" refers to metal ions with a valency of two or more, and examples include divalent metal ions such as zinc ions, calcium ions, magnesium ions, copper ions, iron(II) ions, nickel ions, manganese ions, and cobalt ions; and trivalent metal ions such as iron(III) ions, chromium ions, and aluminum ions. It may be one of these or a combination of two or more. 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 ions, magnesium ions, and nickel ions, and even more preferably at least one selected from the group consisting of zinc ions and magnesium ions.

[0054] These polyvalent metal ions may be added to the reaction system in the form of these chemically acceptable ionic compounds. In this case, examples of the ionic compounds include chlorides, sulfides, iodides, and hydroxides, and it may be one of these or a combination of two or more. However, if the labeled substance is a histidine-rich segment-transfer enzyme-labeled antibody, it is preferable that it be a chloride from the viewpoint of not inhibiting the enzyme activity. Furthermore, if the imidazole or the like added to the reaction system is in the form of a salt that generates the polyvalent metal ions, it may also serve as the ionic compound, but it is preferable that the added imidazole or the like does not generate the polyvalent metal ions.

[0055] In the labeling step according to the present invention, the concentration of polyvalent metal ions in the reaction system (the sum of the two or more polyvalent metal ions if there are two or more types, the same applies hereinafter) is preferably 0.01 mM or higher, 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 polyvalent metal ions is below the lower limit, the effect of further improving detection sensitivity tends not to be fully realized, while if it exceeds the upper limit, the metal ions tend to precipitate.

[0056] (pH buffering agent) In the present invention, the "pH buffering agent" can be any agent that has pH buffering capacity for aqueous solutions (a function that suppresses rapid changes in pH), but the pH buffering 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 6.5 to 7.5.

[0057] 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(acetamide)iminodiacetic acid), PIPES (piperazine-1,4-bis(2-ethanesulfonic acid)), ACES (N-(2-acetamide)-2-aminoethanesulfonic acid), BES (N, Examples 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 any one of these or a combination of two or more may be used. Among these, if the labeled product is a histidine-rich segment-transfer enzyme-labeled antibody, Tris and / or MES are preferred from the viewpoint of not inhibiting the enzyme activity.

[0058] In the labeling step according to the present invention, the concentration of the pH buffer in the reaction system can be appropriately selected depending on the type of pH buffer used, but it is preferable that the pH of the reaction solution be 6.0 to 8.0, preferably 6.5 to 7.5. Specifically, for example, if Tris or MES is used, the concentration is preferably 10 to 500 mM, and more preferably 50 to 200 mM.

[0059] (Labeled substance) In the labeling step according to the present invention, the concentration of the labeled substance in the reaction system is not particularly limited as it can be adjusted appropriately depending on the type and concentration of the sample and labeled substance. For example, the concentration of the labeled substance, which is the histidine-rich segment-labeled antibody, is preferably 0.1 to 5 μg / mL, and more preferably 0.5 to 2 μg / mL.

[0060] (Other) In the labeling step according to the present invention, the pH of the reaction system is preferably 6.0 to 8.0, and more preferably 6.5 to 7.5, as described above. The temperature and reaction time of the reaction system are not particularly limited and can be adjusted as appropriate, but for example, it can be carried out at room temperature to 45°C, preferably 20 to 37°C, for about 5 seconds to 10 minutes, preferably 30 seconds to 8 minutes.

[0061] In the labeling step according to the present invention, the reaction system may further contain, in addition to the test substance, the labeled product, the imidazole, the polyvalent metal ions, and the pH buffer, other components such as salts, sugars, proteins, surfactants, and preservatives.

[0062] The aforementioned salts include salts other than those derived from imidazoles, pH buffers, and ionic compounds of polyvalent metal ions, which are added to the reaction system. Examples include sodium chloride and potassium chloride, and it may be one of these or a combination of two or more. When the reaction system contains the aforementioned salts, the content (total of two or more types, the same applies hereinafter) is preferably 50 to 1000 mM, and more preferably 50 to 300 mM.

[0063] Examples of the aforementioned sugars include glucose, sucrose, maltose, sorbitol, mannitol, xylitol, trehalose, and cyclodextrin, and may be one of these or a combination of two or more. When the reaction system contains the aforementioned sugars, the content (total of two or more if there are two or more, the same applies hereinafter) is preferably, for example, 0.5 to 50 w / v%, and more preferably 1 to 40 w / v%.

[0064] Examples of the aforementioned proteins include casein, sodium caseinate, bovine serum albumin (BSA), and fetal bovine serum (FBS), and may be one of these or a combination of two or more. When the reaction system contains the aforementioned proteins, the content (total of two or more if there are two or more, the same applies hereinafter) is preferably, for example, 0.01 to 10 w / v%, and more preferably 0.1 to 5 w / v%.

[0065] Examples of the surfactant include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants. One of these or a combination of two or more may be used, but nonionic surfactants (e.g., Tween 20, Tween 80 (manufactured by Merck)) are preferred. When the reaction system contains the surfactant, its content (the total of two or more if there are two or more, the same applies hereinafter) is preferably 0.01 to 5 w / v%, and more preferably 0.1 to 1 w / v%.

[0066] Examples of the preservatives include sodium azide, antibiotics, and proclin (manufactured by Merck), and may be one of these or a combination of two or more. If the reaction system contains the preservatives, the amount can be adjusted as appropriate depending on the type of preservative.

[0067] Furthermore, other components include, for example, if the labeled substance is an enzyme-labeled antibody, an inactivated enzyme obtained by inactivating the same enzyme; for example, if the labeled substance is an ALP-labeled antibody, the inactivated enzyme is inactivated ALP. This makes it possible to suppress nonspecific reactions (see Japanese Patent Publication No. 2008-281489). If the reaction system contains the inactivated enzyme, the content is preferably 10 to 5000 parts by mass per 1 part by mass of the labeled substance. Other components include, for example, polymers such as PEG, Dextran, and Lipidure (manufactured by NOF Corporation); and amino acids.

[0068] In the labeling step according to the present invention, the reaction system may further contain components derived from the sample (for example, impurities other than the test substance contained in the sample), but in the sandwich immunoassay according to the present invention, it is preferable to remove such components by performing the capture step and washing step described below before the labeling step in the reaction system. Furthermore, in the labeling step according to the present invention, the reaction system may further contain components derived from the labeled substance (for example, a storage buffer for the labeled substance (for example, the one listed as the diluent for the sample)), but it is preferable to add the labeled substance to the reaction system alone or in the form of the labeled substance-containing composition described below.

[0069] [Capture Step] In the sandwich immunoassay according to the present invention, it is preferable to include a capture step before the labeling step in which the sample and the capture body are brought into contact, and if the test substance is present in the sample, the capture body captures the test substance via binding between the test substance and the second binder, thereby forming a complex of the capture body and the test substance, i.e., a capture body-test substance complex (hereinafter referred to as the "third complex"). Alternatively, it is also preferable to include a capture step after or simultaneously with the labeling step in which the first complex obtained in the labeling step is brought into contact with the capture body, thereby forming a second complex of label body-test substance-capture body as the complex. Such a capture step is more preferably included before the labeling step from the viewpoint of removing impurities other than the test substance contained in the sample and from the viewpoint of further improving detection accuracy by performing the washing step multiple times.

[0070] The method for bringing the sample (or the first composite) into contact with the capture body is not particularly limited, and conventionally known methods or similar methods can be used as appropriate. For example, if the insoluble carrier is a plate, the sample (or the first composite liquid) can be injected into it, or if the insoluble carrier is particles, the particle liquid containing them can be mixed with the sample (or the first composite). Examples of dispersion media for the particle liquid include those listed as diluents for the sample, and salts, proteins, chelating agents, sugars, surfactants, preservatives, etc., may be added as appropriate if necessary.

[0071] In the reaction between the capture agent and the test substance, the content (final concentration) of the capture agent in the reaction solution containing the capture agent is not particularly limited and can be adjusted as appropriate depending on the type and concentration of the sample and capture agent. However, for example, the amount of the second binder is preferably 0.75 to 75 μg / mL, and more preferably 5 to 30 μg / mL. Furthermore, the conditions for the capture step are not particularly limited and can be adjusted as appropriate. For example, it can be carried out at room temperature to 45°C, preferably 20 to 37°C, pH 6 to 9, preferably pH 7 to 8, for about 5 seconds to 10 minutes, preferably 30 seconds to 8 minutes, but is not limited to these conditions.

[0072] [Washing Step] In the sandwich immunoassay according to the present invention, it is preferable to further include a washing step to remove impurities that were not captured by the capture body. If 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 impurities that were not captured by the capture body, i.e., components other than the third complex. In this case as well, it is more preferable to include a washing step after the labeling step to remove impurities that were not captured by the capture body, i.e., components other than the second complex contained in the reaction system.

[0073] The method for removing the aforementioned impurities is not particularly limited, and conventionally known methods or similar methods can be used as appropriate. For example, if the insoluble carrier is a plate, a method of removing the liquid phase (supernatant) from the plate can be used, or if the insoluble carrier is particles, 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. In the washing step, the injection and removal of the washing solution may be repeated as needed. Examples of the washing solution include known neutral (preferably pH 6 to 9) buffers (phosphate buffer (sodium phosphate buffer, potassium phosphate buffer), MES buffer, Tris buffer, CFB buffer, MOPS buffer, PIPES buffer, HEPES buffer, tricine buffer, bicine buffer, glycine buffer, etc.), and may also contain stabilizer proteins such as BSA or surfactants.

[0074] [Detection Step] In the sandwich immunoassay according to the present invention, the test substance is detected indirectly by detecting a signal derived from the labeling substance of a label bound to the test substance (detection step). The "signal" may include, for example, fluorescence, luminescence, or color development, depending on the type of labeling substance, and may be visible to the naked eye as well as visible by fluorescence microscopy or electrical analysis. According to the method of the present invention, by using a labeling substance into which the above-mentioned histidine-rich segment has been introduced, for example, even if the molecular weight of the test substance is small or the concentration is low, the sensitivity of detection can be greatly increased, and the amount of signal generated from the reaction between the test substance and the labeling substance can be increased.

[0075] When an enzyme is used as the labeling substance, preferably, the capture step and washing step are performed before the labeling step, and the washing step is performed after the labeling step to remove impurities that were not captured by the capture material. Then, a chromogenic substrate, fluorescent substrate, chemiluminescent substrate, etc., depending on the type of enzyme, is added as a substrate and reacted to detect a signal (fluorescence, emission, color development) corresponding to the substrate. Such substrates and reaction conditions can be appropriately adjusted depending on the type of enzyme, etc. In the sandwich immunoassay according to the present invention, the detected signal amount (count) may be used directly as the value corresponding to the amount of the test substance, or, if necessary, the test substance may be quantified by comparing it with the signal amount value in a standard sample of known concentration of the test substance.

[0076] <Labeled Substance-Containing Composition, Kit> The present invention provides a labeled substance-containing composition for improving the detection sensitivity of a test substance in a sandwich immunoassay, and a labeled substance-containing composition for use in the detection method of the present invention described above, comprising a first binder that binds to the test substance and a labeling substance, and further containing a labeled substance (histidine-rich segment-introduced labeled antibody). Such a labeled substance-containing composition preferably further contains the imidazole and the polyvalent metal ions, and more preferably further contains the pH buffering agent.

[0077] In the labeling agent-containing composition, the labeling agent is, as described above, a labeling agent into which the above-described histidine-rich segment is introduced, including its preferred embodiment. Furthermore, as a preferred embodiment of the labeling agent-containing composition, the above-described embodiment can be cited as the reaction system for the labeling step, and the composition of the reaction system and the content of each component can be replaced with the composition of the labeling agent-containing composition and the content of each component, respectively. The labeling agent-containing composition of the present invention is preferably an aqueous solution containing each of the above-described components. Furthermore, the labeling agent-containing composition of the present invention can be, for example, a concentrated solution in which the content of each component is concentrated to 1 to 10 times so that it can be used after dilution. The labeling agent-containing composition of the present invention can be distributed and stored as is, and furthermore, for example, the labeling step according to the method of the present invention can be carried out by adding it as is or diluted as necessary after performing the capture step and washing step on the sample.

[0078] Furthermore, the present invention provides a kit for improving the detection sensitivity of a test substance in a sandwich immunoassay, and a kit for use in the detection method of the present invention described above, which includes a first binder that binds to the test substance and a labeling substance, and further includes a labeling agent or a labeling agent-containing composition comprising a histidine-rich segment. The kit of the present invention preferably further includes a reaction buffer containing the imidazole and the polyvalent metal ions, and the reaction buffer more preferably further includes the pH buffering agent. In this case, the reaction buffer included in the kit may further include the labeling agent-containing composition described above. The kit of the present invention also preferably further includes the capture agent. Such a capture agent is also as described above, including its preferred embodiments. These labeling agents, the components of the reaction buffer, and the capture agent may each be independently in solid (powder) form or in liquid form dissolved or suspended in a storage buffer (for example, the one listed as the sample diluent).

[0079] The kit of the present invention may further include, for example, at least one selected from the group consisting of standard samples (at various concentrations), control samples, the diluent, the washing solution, the substrate, an enzyme-substrate reaction buffer, and an enzyme-substrate reaction termination buffer. The kit of the present invention may also further include instructions for use of the kit.

[0080] The present invention will be described more specifically below based on examples and comparative examples, but the present invention is not limited to the following examples. In each of the following test examples, unless otherwise specified, the "%" indicates weight / volume (w / v) percent (w (g) / v (mL) × 100).

[0081] [Detection Test 1] (1) Preparation of Captured Body Fluid (1-1) In an MES buffer, gelatin particles (particles containing iron oxide in a coacervate of gelatin and gum arabic: manufactured by Fujirebio Inc., average particle size 2-3 μm) were used as magnetic particles, to which capture anti-everolimus antibody (antibody obtained from hybridomas obtained by fusing spleen cells obtained from mice immunized with everolimus immunogen with myeloma cells) was added, and the mixture was incubated at 25°C for 1 hour with gentle stirring. After the reaction, the magnetic particles were magnetized with a magnet, and the magnetized magnetic particles were washed to obtain capture anti-everolimus antibody-immobilized particles. These capture anti-everolimus antibody-immobilized particles were suspended in a particle diluent containing Tris as a pH buffer to obtain capture body fluid 1 (particle concentration: 0.015%).

[0082] (1-2) Except that carboxylated particles (particles having carboxyl groups on the surface of particles with ferrite plating on a latex core: manufactured by Fujirebio Inc., average particle diameter 2-7 μm) were used as magnetic particles instead of gelatin particles, the same procedure as in [Detection Test 1] (1-1) above was used to obtain captured body liquid 2 (particle concentration: 0.015%).

[0083] (2) Preparation of labeled body fluid (2-1) Preparation of polyhistidine-introduced anti-everolimus antibody First, a VHH antibody that recognizes the complex of everolimus and the above-mentioned anti-everolimus antibody for capture was obtained by phage display. A plasmid was created so that six consecutive histidine residues were added to the C-terminus of the amino acid sequence of the obtained VHH antibody, and it was introduced into E. coli. E. coli introduced with the plasmid was cultured to induce protein expression and cultured at 37°C for 3 to 5 hours. After culturing, the culture supernatant was removed by centrifugation, and PBS was added to the recovered bacterial cells (precipitate) and subjected to sonication. The fraction solubilized by PBS was removed, and the insoluble fraction was dissolved in 8M Urea and recovered. Affinity purification was performed using a Ni column with the added sequential histidine residues. The column eluate obtained by purification was dialyzed, and finally, an antibody in which polyhistidine was added to the VHH antibody (polyhistidine-modified anti-everolimus antibody) was obtained in a solution of 50 mM Tris buffer, 150 mM NaCl, pH 8.0.

[0084] (2-2) Preparation of polyhistidine-free anti-everolimus antibody The VHH antibody obtained in the same manner as in [Detection Test 1] (2-1) above was cleaved with HRV3C Protein, and the polyhistidine moiety was removed by purification using a Ni column again, thereby obtaining an antibody in which polyhistidine was not attached to the VHH antibody (polyhistidine-free anti-everolimus antibody).

[0085] (2-3) Preparation of Polyhistidine-Introduced Labeled Body Solutions 1 and 2 First, desalted alkaline phosphatase (ALP) and N-(4-maleimidobutyryloxy)-succinimide (GMBS) were mixed and allowed to stand at 30°C for 1 hour to obtain maleimidized ALP. Next, the polyhistidine-introduced anti-everolimus antibody obtained in [Detection Test 1] (2-1) above and the maleimidized ALP were mixed in a molar ratio of 1:10 and reacted at 37°C for 1 hour. The mixture of the reacted antibody and ALP was purified using Superdex 200 10 / 300 (trade name, manufactured by GE) column chromatography to obtain polyhistidine-introduced anti-everolimus antibody (labeled product) labeled on ALP. The obtained labeled compound was suspended in labeled compound dilution A containing MES as a pH buffer to prepare polyhistidine-introduced labeled compound solution 1 (labeled compound concentration 0.5 μg / mL).

[0086] Alternatively, instead of labeled diluent A, labeled diluent B (labeled diluent A with an additional 0.3 mM ZnCl) can be used. ２ and 1 mM MgCl ２ Polyhistidine-labeled body solution 2 was obtained in the same manner as described above, except that a composition containing [the specified substance] was used.

[0087] (2-4) Preparation of Polyhistidine-Free Labeled Body Solutions 3 and 4 Polyhistidine-Free Labeled Body Solution 3 (using Label Diluent A) and Polyhistidine-Free Labeled Body Solution 4 (using Label Diluent B) were obtained in the same manner as in [Detection Test 1] (2-3) above, except that the polyhistidine-free anti-everolimus antibody obtained in [Detection Test 1] (2-2) above was used instead of the polyhistidine-introduced anti-everolimus antibody.

[0088] (2-5) Preparation of Polyhistidine-Introduced Labeled Body Solution 5 The polyhistidine-introduced labeled body solution was prepared using Alkaline Phosphate Labeling Kit-NH2 (Dojindo, product code LK12) according to the attached manual, by an alternative method to [Detection Test 1] (2-3) above. Specifically, first, a solution containing 200 μg of the polyhistidine-introduced anti-everolimus antibody obtained in [Detection Test 1] (2-1) above and 100 μL of Washing Buffer were added to a Filter Tube. After light mixing by pipetting, the mixture was centrifuged at 8,000 × g for 10 minutes. Furthermore, 100 μL of Washing Buffer was added to the Filter Tube and centrifuged at 8,000 × g for 10 minutes. Next, 10 μL of Reaction Buffer was added to NH2-Reactive Alkaline Phosphate, dissolved by pipetting, and added to the membrane of the Filter Tube. After thoroughly mixing with the polyhistidine-introduced anti-everolimus antibody on the membrane by pipetting, the mixture was reacted at 37°C for 2 hours to obtain the labeled product. Next, 190 μL of Storage Buffer was added and pipetted about 10 times to collect the obtained labeled product. The collected labeled product was suspended in the above-mentioned labeled product dilution A to prepare polyhistidine-introduced labeled product solution 5 (labeled product concentration 0.1 μg / mL).

[0089] (2-6) Preparation of polyhistidine-free labeled body solution 6 Polyhistidine-free labeled body solution 6 was obtained in the same manner as in [Detection Test 1] (2-5) above, except that the polyhistidine-free anti-everolimus antibody obtained in [Detection Test 1] (2-2) above was used instead of the polyhistidine-introduced anti-everolimus antibody.

[0090] (2-7) Preparation of Polyhistidine-Introduced Labeled Body Solution 7 First, a plasmid was prepared such that a linker was inserted between two amino acid sequences in which six consecutive histidine residues were added to the C-terminus of the amino acid sequence of the VHH antibody obtained in [Detection Test 1] (2-1) above, thereby linking them in tandem. Using this plasmid, it was introduced into E. coli in the same manner as in [Detection Test 1] (2-1) above, cultured, and purified to obtain a dimer (a dimer of polyhistidine-introduced anti-everolimus antibody) in which two polyhistidine-added VHH antibodies were linked via a single linker and arranged in tandem. The linker was "(GGGS) ２ Linker or (GGGS) ３ A "linker" was used.

[0091] For the obtained polyhistidine-introduced anti-everolimus antibody dimers, the same procedure as in [Detection Test 1] (2-3) above was used, except that a labeling diluent containing Tris was used instead of labeling diluents A and B, and the polyhistidine-introduced labeled solution 7 (linker: (GGGS)) was used. ２ or (GGGS) ３ They obtained the following results.

[0092] (3) Preparation of everolimus solution Everolimus was dissolved in dimethyl sulfoxide to prepare a solution of 10 mg / mL, which was used as the primary stock solution. The primary stock solution was dissolved in everolimus diluent (phosphate buffer-based diluent) to prepare a solution of 500 ng / mL, which was used as the secondary stock solution. The secondary stock solution was added to the everolimus diluent to prepare an everolimus solution at various everolimus concentrations (0.4 ng / mL to 25.0 ng / mL, 0.06 ng / mL to 31.0 ng / mL, or 0.1 to 0.4 ng / mL).

[0093] <Test Example 1> Measurement of Everolimus 1 Everolimus was detected from the everolimus solution prepared in [Detection Test 1] (3) above using the captured body fluid prepared in [Detection Test 1] (1) above and the labeled body fluid prepared in [Detection Test 1] (2) above. Specifically, first, 50 μL of the everolimus dilution or everolimus solution prepared in [Detection Test 1] (3) above (everolimus concentration: 0 to 25.0 [ng / mL]) was mixed with 50 μL of the captured body fluid 1 prepared in [Detection Test 1] (1-1) above, and the mixture was reacted at 37°C for 8 minutes. Magnetic particles were collected and washed to remove unbound impurities from the magnetic particles. Next, one of the polyhistidine-labeled solutions 1-2 (Examples, No. 1-2) prepared in [Detection Test 1] (2-3) above, or the polyhistidine-unlabeled solutions 3-4 (Comparative Examples, No. 3-4) prepared in [Detection Test 1] (2-4) above, was added and reacted at 37°C for 8 minutes. After the reaction, the magnetic particles were magnetized and washed to remove any unbound impurities, and 200 μL of substrate solution containing AMPPD (Lumipulse® substrate solution, manufactured by Fujirebio Inc.) was added. The amount of luminescence produced by the enzymatic reaction was counted, and the average of the two measurements was taken as the signal value [count]. This measurement was performed using the automated analyzer Lumipulse L2400 (registered trademark, manufactured by Fujirebio Inc.).

[0094] Figure 1 shows the signal values ​​(counts) at various everolimus concentrations [ng / mL] when polyhistidine-introduced labeled solution 1 (Example, No. 1) and polyhistidine-free labeled solution 3 (Comparative Example, No. 3) were used as the labeled solution. Table 1 below shows the signal values ​​(counts) at various everolimus concentrations [ng / mL] when polyhistidine-introduced labeled solutions 1-2 (Examples, No. 1-2) and polyhistidine-free labeled solutions 3-4 (Comparative Examples, No. 3-4) were used as the labeled solution. Table 1 also shows the composition of metal ions in the diluted labeled solution (i.e., the reaction system between the labeled solution and everolimus) (upper row) and the ratio of the signal values ​​when the everolimus concentration is 0.4 ng / mL to when it is 0 ng / mL (0.4 / 0, lower row).

[0095]

[0096] As shown in Figure 1 and Table 1, it was confirmed that when polyhistidine was introduced as the labeling agent (Nos. 1-2), the detection sensitivity was significantly increased compared to when polyhistidine was not introduced (Nos. 3-4). In particular, as shown in Table 1, it was confirmed that sufficient detection sensitivity could be achieved even at a low everolimus concentration of 0.4 ng / mL.

[0097] <Test Example 2> Measurement of Everolimus 2 First, 50 μL of the everolimus dilution or everolimus solution prepared in [Detection Test 1] (3) above (everolimus concentration: 0-31.0 [ng / mL]) and 50 μL of the captured body solution 2 prepared in [Detection Test 1] (1-2) above were mixed and reacted at 37°C for 8 minutes. The magnetic particles were magnetized and washed to remove unbound impurities from the magnetic particles. Next, either the polyhistidine-labeled body solution 5 (Example, No. 5) prepared in [Detection Test 1] (2-5) above (polyhistidine-unlabeled body solution 6 (Comparative Example, No. 6) prepared in [Detection Test 1] (2-6) above (polyhistidine-unlabeled body solution) was added and reacted at 37°C for 8 minutes. After the reaction, the magnetic particles were collected and washed to remove any unbound impurities. 200 μL of substrate solution containing AMPPD (Lumipulse® substrate solution, manufactured by Fujirebio Inc.) was added, and the amount of luminescence produced by the enzymatic reaction was counted. The average of the two measurements was taken as the signal value [count]. This measurement was performed using the automated analyzer Lumipulse L2400 (registered trademark, manufactured by Fujirebio Inc.).

[0098] Figure 2 shows the signal values ​​(counts) at various everolimus concentrations [ng / mL] when polyhistidine-introduced labeled solution 5 (Example, No. 5) was used as the labeled solution and when polyhistidine-free labeled solution 6 (Comparative Example, No. 6) was used. As shown in Figure 2, in this embodiment as well, it was confirmed that when polyhistidine-introduced labeled solution was used (No. 5), the detection sensitivity was significantly increased (approximately 5 times increased at an everolimus concentration of 16.0 ng / mL) compared to when polyhistidine was not introduced (No. 6).

[0099] <Test Example 3> Measurement of everolimus using dimer of polyhistidine-introduced anti-everolimus antibody First, 50 µL of the everolimus diluent or everolimus solution prepared in the above [Detection Test 1] (3) (everolimus concentration: 0 to 0.4 [ng / mL]) was mixed with 50 µL of the capturing body fluid 1 prepared in the above [Detection Test 1] (1-1), and the mixture was reacted at 37°C for 8 minutes. Magnetic particles were collected by magnetism and washed to remove unbound impurities from the magnetic particles. Next, each polyhistidine-introduced labeled body fluid 7 prepared in the above [Detection Test 1] (2-7) (Example, No. 7; linker: (GGGS) ２ or (GGGS) ３ ) was added, and the mixture was reacted at 37°C for 8 minutes. After the reaction, magnetic particles were collected by magnetism and washed to remove unbound impurities from the magnetic particles, 200 µL of a substrate solution containing AMPPD (Lumipulse (registered trademark) substrate solution, manufactured by Fujirebio Corporation) was added, the amount of luminescence generated by the enzymatic reaction was counted, and the average value of two measurements was taken as the signal value [count]. This measurement was performed using an automatic analyzer Lumipulse L2400 (registered trademark, manufactured by Fujirebio Corporation).

[0100] <Test Example 3> As the labeled body fluid, each polyhistidine-introduced labeled body fluid 7 (Example, No. 7; linker: (GGGS) ２ or (GGGS) ３ ) the signal values (counts) at each everolimus concentration [ng / mL] when using the above are shown in Table 2 below. Table 2 also shows the ratio of signal values between when the everolimus concentration was 0.4 ng / mL and when it was 0 ng / mL (0.4 / 0, lower row).

[0101]

[0102] As shown in Table 2, also in this embodiment, excellent detection sensitivity was achieved by using a labeled body into which polyhistidine was introduced as the labeled body, in particular (GGGS) ３ It was confirmed that the detection sensitivity is further improved when a VHH antibody is dimerized with a linker for use.

[0103] <Test Example 4> Examination of the reaction system of the test substance and the labeled body In place of the labeled body diluent A, labeled body diluent C (further 0.3 mM ZnCl ２, 1 mM MgCl ２ Polyhistidine-labeled body solution 8 was obtained in the same manner as polyhistidine-labeled body solution 1 in [Detection Test 1] (2-3) above, except that a composition containing 15 mM imidazole was used. Everolimus was measured in the same manner as in <Test Example 1> above, except that polyhistidine-labeled body solution 8 (Example, No. 8) was used in place of polyhistidine-labeled body solution 1 (Example, No. 1).

[0104] Table 3 below shows the signal values ​​(counts) at various everolimus concentrations [ng / mL] when polyhistidine-labeled body solution 8 (Example, No. 8) was used as the labeled body solution. Table 3 also shows the ratio of the signal values ​​(0.4 / 0) when the everolimus concentration was 0.4 ng / mL to when it was 0 ng / mL. Furthermore, for reference, Table 3 also shows the results for No. 1 in Table 1.

[0105]

[0106] As shown in Table 3, the reaction between the test substance (everolimus) and the labeled product was observed to involve imidazole and polyvalent metal ions (Zn). ２＋ Mg ２＋ It was confirmed that the detection sensitivity was particularly increased by performing the test in the presence of both (No. 8).

[0107] [Detection Test 2] (1) Preparation of Captured Body Fluid Captured body fluid 3 (particle concentration: 0.015%) was obtained in the same manner as in [Detection Test 1] (1-1) above, except that an anti-progesterone antibody for capture (an antibody obtained from a hybridoma obtained by fusing spleen cells obtained from mice immunized with a progesterone immunogen with myeloma cells) was used instead of the anti-everolimus antibody for capture.

[0108] (2) Preparation of labeled body fluid (2-1) Preparation of polyhistidine-modified anti-progesterone antibody A polyhistidine-modified anti-progesterone antibody was obtained in the same manner as in [Detection Test 1] (2-1) above, except that a VHH antibody that recognizes the complex of progesterone and the above-mentioned anti-progesterone antibody for capture was used as the VHH antibody.

[0109] (2-2) Preparation of polyhistidine-free anti-progesterone antibody An antibody (polyhistidine-free anti-progesterone antibody) was obtained in the same manner as in [Detection Test 2] (2-1) above, except that a Myc sequence (EQKLISEEDL: SEQ ID NO: 3) was added to the C-terminus of the amino acid sequence of the VHH antibody in place of six consecutive histidine residues, and affinity purification was performed using an anti-Myc antibody instead of affinity purification using a Ni column.

[0110] (2-3) Preparation of polyhistidine-labeled solutions 9 and 10 Polyhistidine-labeled solution 9 (using labeling solution A) and polyhistidine-labeled solution 10 (using labeling solution B) were obtained in the same manner as in [Detection Test 1] (2-3), except that the polyhistidine-labeled anti-progesterone antibody obtained in [Detection Test 2] (2-1) above was used instead of the polyhistidine-labeled anti-everolimus antibody.

[0111] (2-4) Preparation of polyhistidine-free labeled solutions 11 and 12 Polyhistidine-free labeled solution 11 (using labeled solution diluent A) and polyhistidine-introduced labeled solution 12 (using labeled solution diluent B) were obtained in the same manner as in [Detection Test 1] (2-3), except that the polyhistidine-free anti-progesterone antibody obtained in [Detection Test 2] (2-2) above was used instead of the polyhistidine-introduced anti-everolimus antibody.

[0112] (3) Preparation of progesterone solutions Progesterone solutions were prepared by dissolving progesterone in progesterone diluents (phosphate buffer-based diluents) to achieve the following concentrations (0.2 ng / mL, 2.0 ng / mL, 20 ng / mL).

[0113] <Test Example 5> Measurement of Progesterone Progesterone was measured in the same manner as in <Test Example 1>, except that the progesterone dilution or progesterone solution prepared in [Detection Test 2] (3) above was used as the capture fluid, and one of the polyhistidine-introduced labeled fluids 9-10 (Examples, No. 9-10) prepared in [Detection Test 2] (2-3) above or the polyhistidine-unintroduced labeled fluids 11-12 (Comparative Examples, No. 11-12) prepared in [Detection Test 2] (2-4) above was used as the labeling fluid.

[0114] Table 4 below shows the signal values ​​(counts) at various progesterone concentrations [ng / mL] when polyhistidine-introduced labeled solutions 9-10 (Examples, No. 9-10) were used as the labeled solution, and when non-polyhistidine-introduced labeled solutions 11-12 (Comparative Examples, No. 11-12) were used. Table 4 also shows the metal ion composition in the labeled solution dilution (i.e., the reaction system between the labeled solution and everolimus) (upper row) and the ratio of the signal values ​​at progesterone concentrations of 2.0 ng / mL to 0 ng / mL (2.0 / 0, lower row).

[0115]

[0116] As shown in Table 4, in this embodiment as well, it was confirmed that when polyhistidine was introduced as the labeling agent (Nos. 9-10), the detection sensitivity was significantly increased compared to when polyhistidine was not introduced (Nos. 11-12).

[0117] According to the present invention, it is possible to provide a method for improving the detection sensitivity of a test substance in a sandwich immunoassay, a detection method that enables highly sensitive detection of a test substance by a sandwich immunoassay, and a labeled composition used in these methods.

Claims

This is a method to improve the detection sensitivity of a test substance in a sandwich immunoassay. The sandwich immunoassay includes a labeling step of forming a complex of a test substance and a labeled substance comprising a first binder that binds to the test substance and a labeling substance, The method includes the step of introducing a histidine-rich segment into the labeled material. Methods for improving detection sensitivity. The method for improving detection sensitivity according to claim 1, wherein the first binder is a VHH antibody. 　 The method for improving detection sensitivity according to claim 1 or 2, wherein the first binder is a polymer of VHH antibodies in which two or more VHH antibodies are linked by a linker, and the linker is a linker comprising at least one selected from the group consisting of glycine and serine. 　 The method for improving detection sensitivity according to claim 1 or 2, wherein the molecular weight of the substance to be tested is 2500 or less. 　 The method for improving detection sensitivity according to claim 1 or 2, wherein the sandwich immunoassay further comprises a capture step of capturing the test substance with a capture body comprising a second binder bound to the test substance and an insoluble carrier. 　 This method detects the test substance in a sample using a sandwich immunoassay. The process includes a labeling step of forming a composite of a test substance and a labeled substance comprising a first binder that binds to the test substance and a labeling substance, The labeled substance includes a histidine-rich segment. Detection method. 　 The detection method according to claim 6, wherein the first binder is a VHH antibody. 　 The detection method according to claim 6 or 7, wherein the first binder is a polymer of VHH antibodies in which two or more VHH antibodies are linked by a linker, and the linker is a linker comprising at least one selected from the group consisting of glycine and serine. 　 The detection method according to claim 6 or 7, wherein the molecular weight of the substance to be tested is 2500 or less. 　 The detection method according to claim 6 or 7, further comprising a capture step of capturing the test substance with a capture body comprising a second binder bound to the test substance and an insoluble carrier. 　 A composition containing a labeled substance, comprising a first binder that binds to the test substance and a labeling substance, and further comprising a histidine-rich segment, for improving the detection sensitivity of the test substance in a sandwich immunoassay.

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