NT-probnp detection method, and antibody composition, antibody-immobilized particles, and kit for use in same

Abstract

This method for detecting a B-type natriuretic peptide precursor N-terminal fragment (NT-proBNP) in a sample comprises a step for bringing the sample into contact with antibody-immobilized particles. The antibody-immobilized particles contain insoluble particles and an anti-NT-proBNP antibody supported on the insoluble particles, and the insoluble particles are at least one selected from the group consisting of latex particles and colloidal particles of metal or the like. The anti-NT-proBNP antibody is a combination of a first antibody that recognizes an epitope contained at positions 12 to 30 of the amino acid sequence of NT-proBNP and a second antibody that recognizes an epitope contained at positions 33 to 55 of the amino acid sequence of NT-proBNP. The first antibody and the second antibody may be supported on the same insoluble particle or may be supported on separate insoluble particles.

Description

NT-proBNP detection method, and antibody composition, antibody-immobilized particles, and kit for use therein.

[0001] The present invention relates to a method for detecting N-terminal fragments of type B natriuretic peptide precursors (NT-proBNP), and to an antibody composition, antibody-immobilized particles, and a kit for use in the NT-proBNP detection method.

[0002] Heart failure is a disease that progresses due to heart disease and aging, and its incidence continues to increase against the backdrop of a super-aging society. In addition to physical examination, electrocardiograms, chest X-rays, echocardiograms, and biomarker measurements are used to diagnose and understand the pathophysiology of heart failure. Currently in Japan, the use of B-type natriuretic peptide (BNP) or N-terminal fragment of B-type natriuretic peptide precursor (NT-proBNP) is recommended as a biomarker, especially for chronic heart failure.

[0003] BNP and NT-proBNP are hormones produced when proBNP (precursor B-type natriuretic peptide), a precursor hormone synthesized in the ventricles, is broken down, and their blood concentrations increase in response to ventricular load. Furthermore, since BNP and NT-proBNP are released into the bloodstream in a 1:1 ratio, they are known as equivalent biomarkers in the diagnosis of heart failure. Among them, NT-proBNP is more useful in clinical tests because it has a longer half-life in serum or plasma and is more storable than BNP.

[0004] NT-proBNP is the N-terminal peptide fragment of proBNP and typically consists of a sequence of 76 amino acids. Many methods for detecting NT-proBNP have been developed. For example, Japanese Patent Publication No. 7-507210 (Patent Document 1), Japanese Patent Publication No. 2003-508724 (Patent Document 2), Japanese Unexamined Patent Publication No. 2005-181304 (Patent Document 3), Japanese Patent Publication No. 2007-525427 (Patent Document 4), Japanese Patent Publication No. 2007-535470 (Patent Document 5), Japanese Unexamined Patent Publication No. 2021-156677 (Patent Document 6), and Japanese Patent Publication No. 2022-544394 (Patent Document 7) each describe a method for immunologically detecting NT-proBNP using antibodies that recognize and bind to various regions of NT-proBNP.

[0005] As immunological methods for detecting NT-proBNP, for example, there is a known labeled antibody method in which the formation of a complex between the target substance (NT-proBNP) and an antibody that binds to it is detected using a labeling substance such as a luminescent substance or a fluorescent substance. Such labeled antibody methods include a method in which a signal corresponding to the labeling substance is detected from a complex (target substance-antibody-labeling substance) formed by labeling the target substance and the antibody that binds to it with a labeling substance (for example, a non-competitive detection method such as the sandwich method in which the target substance is captured with an antibody immobilized on a solid phase and the antibody that binds to it is labeled with a labeling substance for detection), and a method in which a complex is formed between the target substance and its competitors and an antibody that binds to them, and a signal corresponding to the labeling substance that labels the competitors is detected (a competitive detection method). Furthermore, other immunological detection methods include, for example, an agglutination method in which a complex (target substance-antibody-microparticle) is formed between the target substance (NT-proBNP) and antibody-immobilized particles (microparticles) on which an antibody that binds to it is supported. This complex formation then causes the microparticles to aggregate, and the increase in turbidity or scattered light due to this aggregation is used as a signal for detection.

[0006] Japanese Patent Publication No. 7-507210, Japanese Patent Publication No. 2003-508724, Japanese Patent Publication No. 2005-181304, Japanese Patent Publication No. 2007-525427, Japanese Patent Publication No. 2007-535470, Japanese Patent Publication No. 2021-156677, Japanese Patent Publication No. 2022-544394

[0007] Among the immunological detection methods described above, the inventors conducted further investigations into the detection of NT-proBNP by agglutination and found that simply using conventional antibodies against NT-proBNP still presents a problem of insufficient detection sensitivity.

[0008] The present invention has been made in view of the problems of the prior art described above, and aims to provide a method for detecting N-terminal fragments of type B natriuretic peptide precursors (NT-proBNP) with high sensitivity, as well as an antibody composition, antibody-immobilized particles, and a kit for use therein.

[0009] To achieve the above objective, the inventors diligently conducted research and first investigated a method of combining two antibodies that recognize different regions of NT-proBNP in an agglutination method. As antibodies that bind to NT-proBNP, for example, in the above-mentioned labeled antibody method, antibodies that recognize positions 41 to 45 of the amino acid sequence of NT-proBNP, which typically consists of all 76 amino acid residues, as described in Patent Document 6, etc., have been widely used. Next, the inventors created an antibody (second antibody) that recognizes an epitope contained in positions 33 to 55 of the amino acid sequence of NT-proBNP as a similar antibody, and conducted verification by creating multiple antibodies to be combined with this antibody in an agglutination method. Among these, the inventors newly created an antibody (first antibody) that recognizes an epitope contained in positions 12 to 30 of the amino acid sequence of NT-proBNP, and found that by using this in combination with the second antibody, the detection sensitivity was significantly increased compared to when antibodies that recognize other regions were combined. Furthermore, surprisingly, this combination of two antibodies (the first antibody and the second antibody) actually resulted in a decrease in detection sensitivity in the labeled antibody method described above. We discovered that this combination of antibodies is particularly suitable for the agglutination method, and thus completed the present invention.

[0010] In other words, the present invention relates to an NT-proBNP detection method, and to an antibody composition, antibody-immobilized particles, and a kit for use in the NT-proBNP detection method, and more specifically to the following. [1] A method for detecting the N-terminal fragment of a B-type natriuretic peptide precursor (NT-proBNP) in a sample, comprising the step of contacting the sample with antibody-immobilized particles, wherein the antibody-immobilized particles comprise insoluble particles and an anti-NT-proBNP antibody supported on the insoluble particles, and the insoluble particles are at least one selected from the group consisting of latex particles and metal or other colloidal particles, and the anti-NT-proBNP antibody is a combination of a first antibody that recognizes an epitope contained in positions 12 to 30 of the amino acid sequence of NT-proBNP and a second antibody that recognizes an epitope contained in positions 33 to 55 of the amino acid sequence of NT-proBNP, wherein the first antibody and the second antibody may be supported on the same insoluble particle or on separate insoluble particles. [2] The NT-proBNP detection method according to [1], wherein the first antibody is reactive to a peptide containing positions 12 to 30 of the amino acid sequence of NT-proBNP, and is substantially inactive to the peptide consisting of positions 5 to 19 of the amino acid sequence of NT-proBNP and the peptide consisting of positions 21 to 35 of the amino acid sequence of NT-proBNP. [3] The NT-proBNP detection method according to [1] or [2], wherein the second antibody is reactive to a peptide containing positions 33 to 55 of the amino acid sequence of NT-proBNP, and is substantially inactive to the peptide consisting of positions 31 to 45 of the amino acid sequence of NT-proBNP and the peptide consisting of positions 43 to 57 of the amino acid sequence of NT-proBNP. [4] The NT-proBNP detection method according to any one of [1] to [3], wherein at least one of the bindings between the insoluble particles and the first antibody, and the binding between the insoluble particles and the second antibody, is a covalent bond. [5] An antibody composition for use in the NT-proBNP detection method according to any one of [1] to [4], comprising a first antibody that recognizes an epitope located at positions 12 to 30 of the amino acid sequence of NT-proBNP.[6] The antibody composition according to [5], wherein the first antibody is reactive to a peptide comprising the amino acid sequence of NT-proBNP from positions 12 to 30, and substantially inactive to a peptide comprising the amino acid sequence of NT-proBNP from positions 5 to 19 and a peptide comprising the amino acid sequence of NT-proBNP from positions 21 to 35. [7] Antibody-immobilized particles for use in the NT-proBNP detection method described in any one of [1] to [4], comprising insoluble particles and an anti-NT-proBNP antibody supported on the insoluble particles, wherein the insoluble particles are at least one selected from the group consisting of latex particles and metal or other colloidal particles, and the anti-NT-proBNP antibody is a combination of a first antibody that recognizes an epitope contained in positions 12 to 30 of the amino acid sequence of NT-proBNP and a second antibody that recognizes an epitope contained in positions 33 to 55 of the amino acid sequence of NT-proBNP, wherein the first antibody and the second antibody may be supported on the same insoluble particle or on separate insoluble particles. [8] The antibody-immobilized particle according to [7], wherein the first antibody is reactive to a peptide containing positions 12 to 30 of the amino acid sequence of NT-proBNP, and substantially inactive to the peptide consisting of positions 5 to 19 of the amino acid sequence of NT-proBNP and the peptide consisting of positions 21 to 35 of the amino acid sequence of NT-proBNP. [9] The antibody-immobilized particle according to [7] or [8], wherein the second antibody is reactive to a peptide containing positions 33 to 55 of the amino acid sequence of NT-proBNP, and substantially inactive to the peptide consisting of positions 31 to 45 of the amino acid sequence of NT-proBNP and the peptide consisting of positions 43 to 57 of the amino acid sequence of NT-proBNP.

[10] The antibody-immobilized particle according to any one of [7] to [9], wherein at least one of the binding between the insoluble particle and the first antibody, and the binding between the insoluble particle and the second antibody, is a covalent bond.

[11] A kit for use in the NT-proBNP detection method described in any one of [1] to [4], comprising a first antibody that recognizes an epitope located at positions 12 to 30 of the amino acid sequence of NT-proBNP, and a second antibody that recognizes an epitope located at positions 33 to 55 of the amino acid sequence of NT-proBNP.

[12] The kit according to

[11] , wherein the first antibody is reactive to a peptide located at positions 12 to 30 of the amino acid sequence of NT-proBNP, and substantially inactive to a peptide located at positions 5 to 19 of the amino acid sequence of NT-proBNP and a peptide located at positions 21 to 35 of the amino acid sequence of NT-proBNP.

[13] The kit according to

[11] or

[12] , wherein the second antibody is reactive to a peptide comprising positions 33 to 55 of the amino acid sequence of NT-proBNP, and substantially inactive to a peptide comprising positions 31 to 45 of the amino acid sequence of NT-proBNP and a peptide comprising positions 43 to 57 of the amino acid sequence of NT-proBNP.

[14] The kit according to any one of

[11] to

[13] , further comprising insoluble particles, wherein the insoluble particles are at least one selected from the group consisting of latex particles and metal colloidal particles.

[15] The kit according to

[14] , wherein the first antibody, the second antibody, and the insoluble particles are antibody-immobilized particles on which the first antibody and the second antibody are supported on the same or separate insoluble particles.

[0011] According to the present invention, it is possible to provide a method for detecting N-terminal fragments of type B natriuretic peptide precursors (NT-proBNP) with high sensitivity, as well as an antibody composition, antibody-immobilized particles, and a kit for use therein.

[0012] This is a conceptual diagram showing type B natriuretic peptide precursor (proBNP), type B natriuretic peptide precursor N-terminal fragment (NT-proBNP), and type B natriuretic peptide (BNP). This graph shows the relationship between NT-proBNP peptide concentration [pg / mL] and luminescence [luminous value] when antibody b is used as the antibody-immobilized magnetic particle and antibody a is used as the luciferase-labeled antibody obtained in <Test Example 2> (antibody a-antibody b: reference example), and when antibody c is used as the magnetic particle-immobilized antibody and antibody a is used as the luciferase-labeled antibody (antibody a-antibody c: reference example). This graph shows the relationship between the dilution ratio of the sample and the calculated NT-proBNP peptide concentration [pg / mL] when sample 1, obtained in <Test Example 2>, is used in combination with antibody-immobilized latex particles carrying antibody a and antibody-immobilized latex particles carrying antibody b. This graph shows the relationship between the dilution ratio of the sample and the calculated NT-proBNP peptide concentration [pg / mL] when sample 2, obtained in <Test Example 2>, is used in combination with antibody-immobilized latex particles carrying antibody a and antibody-immobilized latex particles carrying antibody b. The graphs in <Test Example 3> show the relationship between NT-proBNP peptide concentration [pg / mL] and absorbance change [ΔOD × 10,000] for antibody a-antibody b (Example) and antibody a-antibody c (Comparative Example). The graphs in <Test Example 4> show the relationship between NT-proBNP peptide concentration [pg / mL] and absorbance change [ΔOD × 10,000] for each combination of antibody a(+) and antibody b(+) (antibody a(+) × antibody b(+)), antibody a(-) and antibody b(+) (antibody a(-) × antibody b(+)), and antibody a(-) and antibody b(-) (antibody a(-) × antibody b(-)).

[0013] The present invention will be described in detail below with reference to its preferred embodiments, but the embodiments of the present invention are not limited to the embodiments described below.

[0014] <NT-proBNP Detection Method> The present invention is a method for detecting the N-terminal fragment of a type B natriuretic peptide precursor (NT-proBNP) in a sample, comprising the step of contacting the sample with antibody-immobilized particles, wherein the antibody-immobilized particles comprise insoluble particles and anti-NT-proBNP antibodies supported on the insoluble particles, and the insoluble particles are at least one selected from the group consisting of latex particles and metal or other colloidal particles, and the anti-NT-proBNP antibody is a combination of a first antibody that recognizes an epitope contained in positions 12 to 30 of the amino acid sequence of NT-proBNP and a second antibody that recognizes an epitope contained in positions 33 to 55 of the amino acid sequence of NT-proBNP, wherein the first antibody and the second antibody may be supported on the same insoluble particle or on separate insoluble particles. The present invention provides an NT-proBNP detection method (hereinafter referred to as "the detection method of the present invention" in some cases).

[0015] [NT-proBNP] In the present invention, "NT-proBNP" refers to the N-terminal pro-brain natriuretic peptide, and is also called the "BNP precursor N-terminal fragment," "brain natriuretic peptide precursor N-terminal fragment," etc. As shown in Figure 1, NT-proBNP is a protein consisting of the N-terminal amino acid sequence obtained by separating the C-terminal B-type natriuretic peptide (BNP) from proBNP (pro-B type natriuretic peptide precursor), a precursor hormone synthesized in the ventricle.

[0016] NT-proBNP typically contains the amino acid sequence from positions 1 to 76 of proBNP if it is of human origin. Human-derived NT-proBNP is preferred for the NT-proBNP according to the present invention, and human NT-proBNP is typically a protein containing the amino acid sequence from positions 1 to 76 of the amino acid sequence described in UniPro Access No.: P16860. The amino acid sequence of this typical NT-proBNP is shown in Sequence ID No. 1.

[0017] Furthermore, the DNA sequence of a gene can be mutated unintentionally (in nature) or artificially, and the amino acid sequence of the protein it encodes is also modified accordingly. Therefore, the "NT-proBNP" detected by the detection method of the present invention is not limited to proteins consisting of the typical amino acid sequence described above, but also includes such variants. Moreover, the "NT-proBNP" detected by the detection method of the present invention only needs to contain each of the following epitopes (or amino acid sequences of peptides exhibiting each reactivity). For example, it may be a proBNP, BNP, or degradation products thereof, in which one or more amino acids at the N-terminus and / or C-terminus of the typical NT-proBNP amino acid sequence described above have been degraded, or in which one or more amino acids have been added to the N-terminus and / or C-terminus of NT-proBNP.

[0018] [Anti-NT-proBNP Antibody] The present invention provides an anti-NT-proBNP antibody that specifically binds to NT-proBNP, comprising: a first antibody that recognizes an epitope located at positions 12 to 30 of the amino acid sequence of NT-proBNP; and a second antibody that recognizes an epitope located at positions 33 to 55 of the amino acid sequence of NT-proBNP (in this specification, the first antibody and the second antibody may be collectively referred to as "the antibody of the present invention").

[0019] In the present invention, "antibody" includes not only complete antibodies but also functional fragments thereof. The "functional fragment" refers to a part (partial fragment) of a complete antibody that is reactive with NT-proBNP, and specifically includes Fab, F(ab')2, Fab', variable region fragment (Fv), single-chain Fv (scFv), sc(Fv)2, diabody, VHH antibody, and polymers thereof. "Fab" refers to a monovalent antigen-binding fragment of immunoglobulin consisting of one light chain and part of a heavy chain, and can be obtained, for example, by papain digestion or recombination of the antibody. "F(ab')2" refers to a divalent antigen-binding fragment of immunoglobulin consisting of both light chains and parts of both heavy chains, and can be obtained, for example, by pepsin digestion or recombination of the antibody. "Fab'" can be obtained, for example, by reducing F(ab')2, and differs from Fab in that it does not contain the hinge portion of the antibody. Furthermore, a "variable region fragment (Fv)" refers to the smallest antibody fragment that has complete antigen recognition and binding sites. A "single-chain Fv (scFv)" includes the heavy chain variable region and light chain variable region of the antibody, and these regions are located on a single polypeptide chain. "sc(Fv)2" is formed by linking two heavy chain variable regions and two light chain variable regions together with a linker or the like to create a single chain. A "diabody" is a small antibody fragment with two antigen-binding sites. This fragment contains a heavy chain variable region bound to a light chain variable region within the same polypeptide chain, and each region forms a pair with a complementary region on another chain.

[0020] Furthermore, in the present invention, "antibody" includes all classes and subclasses of immunoglobulins, and also includes polyclonal antibodies and monoclonal antibodies. The antibody of the present invention is at least one selected from the group consisting of polyclonal antibodies and monoclonal antibodies, but is preferably a monoclonal antibody.

[0021] Furthermore, the origin of the antibody of the present invention is not particularly limited, and it may be a human-derived antibody, an antibody derived from a non-human animal (for example, a rabbit antibody, a mouse antibody, a rat antibody, a camel antibody), a chimeric antibody, a humanized antibody, etc.

[0022] (First Antibody) The first antibody of the present invention is an antibody that recognizes an epitope located at positions 12 to 30 of the amino acid sequence of NT-proBNP (preferably the amino acid sequence described in SEQ ID NO: 1, the same applies hereinafter). More preferably, it is an antibody that recognizes an epitope located at positions 14 to 28 of the amino acid sequence of NT-proBNP, and even more preferably, it is an antibody that recognizes an epitope located at positions 16 to 26 of the amino acid sequence of NT-proBNP. In this specification, "epitope" refers to an antigenic determinant present in an antigen, or a site on an antigen to which the antigen-binding domain in an antibody binds. In the present invention, "recognition" of an antibody as an epitope means that the presence of that region on NT-proBNP causes it to exhibit binding activity (affinity) and / or specificity to the antigen NT-proBNP.

[0023] The first antibody of the present invention is preferably an antibody that reacts to a peptide containing positions 12 to 30 of the amino acid sequence of NT-proBNP, and is substantially inactive to a peptide consisting of positions 5 to 19 and a peptide consisting of positions 21 to 35 of the amino acid sequence of NT-proBNP. More preferably, it is an antibody that reacts to a peptide consisting of positions 12 to 30 of the amino acid sequence of NT-proBNP, even more preferably a peptide consisting of positions 14 to 28 of the amino acid sequence of NT-proBNP, and even more preferably an antibody that reacts to a peptide consisting of positions 16 to 26 of the amino acid sequence of NT-proBNP.

[0024] The first antibody of the present invention is preferably an antibody that is reactive to at least one (preferably all) selected from the group consisting of peptides from positions 12 to 26, positions 14 to 28, and positions 16 to 30 of the amino acid sequence of NT-proBNP, and / or an antibody that is reactive to the peptide from positions 12 to 30. Furthermore, it is even more preferable that the antibody is substantially inactive not only to peptides from positions 5 to 19 and positions 21 to 35 of the amino acid sequence of NT-proBNP, but also to at least one (preferably all) selected from the group consisting of peptides from positions 1 to 15, positions 3 to 17, positions 23 to 37, and positions 25 to 39 of the amino acid sequence of NT-proBNP.

[0025] In the present invention, "reactivity" of an antibody to a peptide means that it exhibits binding activity (affinity) to the peptide, and "substantially unreactivity" of an antibody to a peptide means that it does not exhibit binding activity (affinity) to the peptide. The "reactivity" of an antibody to a peptide can be evaluated by immunological methods known to those skilled in the art or by similar methods, for example, by the method shown in the examples described below. That is, first, a target peptide (for example, a peptide consisting of positions 12 to 30 of the amino acid sequence shown in SEQ ID NO: 1) or a peptide consisting of 10 to 15 amino acid residues contained in the target peptide is prepared from the amino acid sequence of NT-proBNP, and for example, this is used as a solid-phase peptide to react with and capture the target antibody. Next, the formed peptide-antibody complex is detected with a labeled antibody, etc. (for example, a secondary antibody to which a labeling substance has been conjugated: if the target antibody is mouse-derived, HRP-labeled anti-mouse IgG, etc.) (for example, the sandwich method). For example, when an HRP-labeled antibody is used as the labeled antibody (more preferably, for example, the method in (6) of <Test Example 1> below), the antibody can be evaluated as "reactive" to the target peptide if the absorbance at a wavelength of 450 nm is 0.100 or higher, more preferably 0.200 or higher. Such evaluation criteria can be appropriately set by those skilled in the art depending on the detection method and its conditions.

[0026] On the other hand, in the present invention, the fact that an antibody is "substantially inactive" towards a peptide can be evaluated, for example, by preparing the target peptide (for example, a peptide consisting of amino acid positions 5 to 19 of the sequence shown in SEQ ID NO: 1) and detecting the peptide-antibody complex using the method described above. For example, when an HRP-labeled antibody is used as the labeled antibody (in the case of the method in (6) of <Test Example 1> below), if the absorbance at a wavelength of 450 nm is less than 0.100, the antibody can be evaluated as "substantially inactive" towards the target peptide. It should be noted that this evaluation criterion can be appropriately set by those skilled in the art depending on the detection method and its conditions.

[0027] (Second Antibody) The second antibody of the present invention is an antibody that recognizes an epitope located at positions 33 to 55 of the amino acid sequence of NT-proBNP. More preferably, it is an antibody that recognizes an epitope located at positions 35 to 53 of the amino acid sequence of NT-proBNP, even more preferably, it is an antibody that recognizes an epitope located at positions 37 to 51 of the amino acid sequence of NT-proBNP, even more preferably, it is an antibody that recognizes an epitope located at positions 39 to 49 of the amino acid sequence of NT-proBNP, and particularly preferably, it is an antibody that recognizes an epitope located at positions 41 to 47 of the amino acid sequence of NT-proBNP.

[0028] More specifically, the second antibody of the present invention is preferably an antibody that reacts to a peptide containing positions 33 to 55 of the amino acid sequence of NT-proBNP, and is substantially inactive to the peptide consisting of positions 31 to 45 of the amino acid sequence of NT-proBNP and the peptide consisting of positions 43 to 57 of the amino acid sequence of NT-proBNP. More preferably, it is an antibody that reacts to the peptide consisting of positions 33 to 55 of the amino acid sequence of NT-proBNP, even more preferably a peptide consisting of positions 37 to 51 of the amino acid sequence of NT-proBNP, and even more preferably an antibody that reacts to the peptide consisting of positions 41 to 47 of the amino acid sequence of NT-proBNP.

[0029] The second antibody of the present invention is preferably an antibody that is reactive to, for example, at least one (preferably all) selected from the group consisting of peptides from positions 33 to 47, 35 to 49, 37 to 51, 39 to 53, and 41 to 55 of the amino acid sequence of NT-proBNP, and / or an antibody that is reactive to the peptide from positions 33 to 55. Furthermore, it is preferable that the antibody is substantially inactive not only to the peptides from positions 31 to 45 and 43 to 57 of the amino acid sequence of NT-proBNP, but also to at least one (preferably all) selected from the group consisting of peptides from positions 29 to 43 and 45 to 59 of the amino acid sequence of NT-proBNP.

[0030] Those skilled in the art can produce antibodies that are reactive to each of the above epitopes or peptides by conventionally known methods or similar methods. For example, antibodies of the present invention can be produced by the hybridoma method. A typical example of the hybridoma method is the Kohler and Milstein method (Kohler & Milstein, Nature, 256:495 (1975)). The antibody-producing cells used in the cell fusion step in the hybridoma method are spleen cells, lymph node cells, peripheral blood leukocytes, etc., of animals (e.g., mice, rats, hamsters, rabbits, monkeys, goats) immunized with an antigen (e.g., NT-proBNP, its partial peptides, proteins in which Fc proteins, etc., are fused, or cells expressing these; preferably, if an antibody is to be produced that is reactive to a peptide consisting of positions 12 to 30 of the amino acid sequence of NT-proBNP, then the peptide) Furthermore, antibody-producing cells obtained by applying the antigen to the above-mentioned cells or lymphocytes, etc., previously isolated from an unimmunized animal, in a culture medium can also be used. Various known cell lines can be used as myeloma cells in the cell fusion step. The antibody-producing cells and myeloma cells may be from different animal species, provided they are fused, but it is preferable that they be from the same animal species. Hybridomas can be produced, for example, by cell fusion between spleen cells obtained from an antigen-immunized mouse and mouse myeloma cells, and subsequent screening can yield hybridomas that produce monoclonal antibodies specific to NT-proBNP. Monoclonal antibodies against NT-proBNP can be obtained in a substantially pure and homogeneous form by culturing the hybridoma, separating and purifying them from within the hybridoma or from the culture medium. They can also be obtained from the ascites fluid of mammals administered the hybridoma. Furthermore, antibody genes can be obtained from the hybridoma and transfected into mammalian cells such as CHO cells. Antibody isolation and purification can be carried out using methods commonly used for antibody purification.Whether the obtained antibodies show reactivity with each of the epitopes or peptides can be evaluated by the method described above.

[0031] (Antibody Composition) The present invention also provides an antibody composition containing at least one anti-NT-proBNP antibody selected from the group consisting of the first antibody and the second antibody described above. The antibody composition of the present invention may contain only one of the first antibody and the second antibody, or it may contain both the first antibody and the second antibody. Furthermore, each anti-NT-proBNP antibody may be contained in the form of the antibody-immobilized particles described below (i.e., at least one form selected from the group consisting of the first antibody-immobilized particle, the second antibody-immobilized particle, and the third antibody-immobilized particle).

[0032] The antibody composition of the present invention may be a dry product or an aqueous solution containing an aqueous solvent. Examples of the aqueous solvent include sterile water, physiological saline; and buffers such as Good's buffer (HEPES buffer, MOPS buffer, MES buffer, etc.), TRIS buffer, glycine buffer, borate buffer, and PBS buffer. The antibody composition of the present invention may also further contain additives such as pH adjusters, stabilizers, preservatives, antiseptics, and surfactants.

[0033] [Antibody-Immobilized Particles] The present invention provides antibody-immobilized particles comprising insoluble particles and an anti-NT-proBNP antibody supported on the insoluble particles. The anti-NT-proBNP antibody is as described above, including its preferred embodiment.

[0034] (Insoluble Particles) In the present invention, "insoluble particles" are fine particles that function as insoluble carriers supporting the anti-NT-proBNP antibody in the immunoaggregation method and are capable of agglutinating with each other via binding between the anti-NT-proBNP antibody and NT-proBNP. The "insoluble particles" according to the present invention are at least one selected from the group consisting of latex particles and metal or other colloidal particles, and if they are latex particles, they are preferably in the form of an emulsion in which they are dispersed, and if they are metal or other colloidal particles, they are preferably in the form of a colloidal solution in which they are dispersed.

[0035] Examples of the "latex particles" according to the present invention include particles composed of polymer latex such as polystyrene, polyethylene, styrene-methacrylic acid copolymer, polyglycidyl methacrylate, and acrolein-ethylene glycol dimethacrylate copolymer. The latex particles are not particularly limited, and any latex particles conventionally used in known immunoagglutination methods or methods equivalent thereto can be used. The latex particles can be obtained by conventional methods such as emulsion polymerization, soap-free emulsion polymerization, seed polymerization, stage feed emulsion polymerization, power feed polymerization, and suspension polymerization, but commercially available products may also be used as appropriate.

[0036] The latex particles according to the present invention are not particularly limited as long as they can be applied to the production of physically adsorbed particles by the physical adsorption method described below or the production of chemically bonded particles by the chemical bonding method described below, and it is preferable to appropriately select a type suitable for each of the physical adsorption method and the chemical bonding method. Preferable examples of latex particles suitable for the physical adsorption method include polystyrene latex particles, extremely low carboxylic acid latex particles, and hydrophilic group-localized latex particles. Further, preferable examples of latex suitable for the chemical bonding method include latex particles having one or more types selected from the group consisting of a carboxy group, a hydroxy group, an amino group, an aldehyde group, a tosyl group, a sulfhydryl group, and an epoxy group on the surface thereof.

[0037] In the "metal or similar colloid particles" according to the present invention, the "metal or similar" includes, in addition to metals, oxidized metalloids such as silica that can form colloids, and the "metal or similar colloid particles" can also be expressed as "colloid particles of a metal or an oxidized metalloid". Examples of the metal or similar colloid particles include colloid particles of silica, gold, silver, iron, aluminum, and platinum. The metal or similar colloid particles are also not particularly limited, and any metal or similar colloid particles conventionally used in known immunoagglutination methods or methods equivalent thereto can be used. The metal or similar colloid particles can be obtained by conventional methods such as an atomization method and a chemical reduction method, but commercially available products may also be used as appropriate.

[0038] Among the above, the "insoluble particles" according to the present invention are preferably latex particles and / or gold colloid particles, and more preferably latex particles.

[0039] The average particle diameter of the "insoluble particles" according to the present invention is preferably 0.01 to 1.0 µm, more preferably 0.05 to 0.40 µm, still more preferably 0.10 to 0.40 µm, and even more preferably 0.20 to 0.40 µm. Such an average particle diameter can be appropriately adjusted by adjusting the types and amounts of monomers, emulsifiers, and initiators, the polymerization temperature, and the like, for example, in the case of an emulsion polymerization method for latex particles.

[0040] (Structure of Antibody-Immobilized Particles) The antibody-immobilized particles of the present invention contain the anti-NT-proBNP antibody and the insoluble particles, and are particles in which the anti-NT-proBNP antibody is supported on the surface of the insoluble particles. Among the anti-NT-proBNP antibodies, the first antibody and the second antibody may each be supported on separate insoluble particles. That is, the embodiment of the antibody-immobilized particles of the present invention may be a combination of insoluble particles having the first antibody supported thereon (hereinafter sometimes referred to as "first antibody-immobilized particles" in the present specification) and insoluble particles having the second antibody supported thereon (hereinafter sometimes referred to as "second antibody-immobilized particles" in the present specification). Alternatively, the first antibody and the second antibody may be supported on the same insoluble particle. That is, the embodiment of the antibody-immobilized particles of the present invention may be insoluble particles having the first antibody and the second antibody supported thereon (hereinafter sometimes referred to as "third antibody-immobilized particles" in the present specification). Among these, the antibody-immobilized particles of the present invention are preferably a combination of the first antibody-immobilized particles and the second antibody-immobilized particles.

[0041] The amount of the anti-NT-proBNP antibody supported on the insoluble particles (the total amount thereof in the case of two types, the same applies hereinafter) is not particularly limited, but is based on the surface area of the insoluble particles (m ２ ) the amount (mg) of anti-NT-proBNP antibody per unit, which is 0.1 to 100 mg / m ２ , and preferably 1 to 10 mg / m ２It is more preferable that this is the case. The amount of antibody to be loaded can also be calculated by subtracting the amount of antibody after loading from the amount of antibody before loading onto the insoluble particles.

[0042] In the insoluble particles, the ratio of the first antibody to the second antibody, that is, the ratio of the first antibody to the second antibody in a combination of the first antibody-immobilized particles and the second antibody-immobilized particles, or in a third antibody-immobilized particle, is preferably 10:1 to 1:10, more preferably 5:1 to 1:5, and even more preferably 1:1 in terms of mass ratio (first antibody:second antibody). Furthermore, when the insoluble particles of the present invention are a combination of the first antibody-immobilized particles and the second antibody-immobilized particles, it is preferable that the amount of the first antibody loaded in the first antibody-immobilized particle and the amount of the second antibody loaded in the second antibody-immobilized particle are the same, and it is more preferable that the mass ratio of the first antibody-immobilized particles to the second antibody-immobilized particles falls within the range of the preferred mass ratio of the first antibody to the second antibody.

[0043] (Method for producing antibody-immobilized particles) A ​​conventionally known method or a method similar thereto can be used for the method of supporting the anti-NT-proBNP antibody on the insoluble particles, that is, for the method of binding the insoluble particles and the anti-NT-proBNP antibody. Known methods such as physical adsorption, chemical bonding, or a combination thereof can be used as appropriate.

[0044] [Physical Adsorption Method] In the physical adsorption method, the anti-NT-proBNP antibody is mainly bound to the surface of the insoluble particles by utilizing hydrophobic interactions. As an example of the physical adsorption method, a particle solution containing insoluble particles in an aqueous dispersion medium such as distilled water or buffer solution at a concentration of about 0.1 to 1% by mass is mixed with the anti-NT-proBNP antibody to be supported (a first antibody, a second antibody, or a mixture of the first and second antibodies) to obtain antibody-immobilized particles in which the anti-NT-proBNP antibody is physically adsorbed onto the surface of the insoluble particles in the aqueous dispersion medium. Furthermore, the method may appropriately include particle recovery and washing steps. For example, the unadsorbed antibody and antibody-immobilized particles can be separated by centrifugation and redispersed in the aqueous dispersion medium (preferably a buffer solution) to obtain a particle solution of the antibody-immobilized particles (physically adsorbed particles) (the particle composition of the present invention). Examples of the buffer include Good's buffer (HEPES buffer, MOPS buffer, MES buffer, etc.), TRIS buffer, glycine buffer, borate buffer, PBS buffer, etc. The pH of the buffer is preferably 6.0 to 8.0, and the pH of the aqueous dispersion medium used in the physical adsorption reaction is more preferably 6.5 to 7.5. The aqueous dispersion medium may also further contain basic amino acids, nonionic surfactants, etc.

[0045] [Chemical Bonding Method] In the chemical bonding method, functional groups are conferred to the surface of insoluble particles by a conventional method, or particles having functional groups on their surface are used as insoluble particles, and the anti-NT-proBNP antibody is covalently bonded to the surface of the insoluble particles by such functional groups. Examples of the functional groups include carboxyl groups, hydroxyl groups, amino groups, aldehyde groups, tosyl groups, sulfhydryl groups, and epoxy groups, and it may be one of these or a combination of two or more. As an example of the chemical bonding method, when the functional group is a carboxyl group, a hydroxyl group, or an amino group, an activator such as a condensing agent, such as carbodiimide, bromocyanide, glutaraldehyde, or DMT-MM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride), is added to a particle solution containing the insoluble particles in an aqueous dispersion medium (preferably a buffer solution) at a concentration of about 0.1 to 1% by mass, and then mixed with the anti-NT-proBNP antibody to be supported (a first antibody, a second antibody, or a mixture of the first and second antibodies) to obtain antibody-immobilized particles (chemically bonded particles) in which the anti-NT-proBNP antibody is covalently bonded to the surface of the insoluble particles in the aqueous dispersion medium. The addition of the activator and the mixing with the anti-NT-proBNP antibody may be simultaneous, or the addition of the activator may be performed first. Furthermore, if the functional group is an aldehyde group or a tosyl group, it is not necessary to use the activator. The binding between the insoluble particles and the anti-NT-proBNP antibody can also be achieved indirectly through spacer molecules such as oligoamino acids or aminocarboxylic acids via covalent bonding.

[0046] Furthermore, the chemical bonding method may include particle recovery and washing steps as appropriate. For example, the unbound antibody and antibody-immobilized particles can be separated by centrifugation and redispersed in the aqueous dispersion medium (preferably a buffer solution) to obtain a particle liquid of the antibody-immobilized particles (covalently bonded particles) (the particle composition of the present invention). Examples of the buffer solution include Good's buffer (HEPES buffer, MOPS buffer, MES buffer, etc.), TRIS buffer, glycine buffer, borate buffer, PBS buffer, etc. The pH of the buffer solution can be appropriately adjusted according to the isoelectric point (pI) of the anti-NT-proBNP antibody to be supported. The aqueous dispersion medium may also further contain preservatives, surfactants, etc.

[0047] The ratio of insoluble particles to anti-NT-proBNP antibody used in these manufacturing methods can be appropriately selected to achieve, for example, a preferred range of loading amounts in the antibody-immobilized particles described above. Among the above manufacturing methods, the chemical bonding method is preferred as the method for binding the insoluble particles to the anti-NT-proBNP antibody, from the viewpoint that the detection sensitivity of the agglutination method using antibody-immobilized particles tends to be superior. That is, in the antibody-immobilized particles of the present invention, it is preferable that at least one of the bindings between the insoluble particles and the first antibody, and the binding between the insoluble particles and the second antibody, is a covalent bond, and it is more preferable that both are covalent bonds.

[0048] The antibody-immobilized particles of the present invention may further include other components, for example, the surface of the insoluble particles may be blocked with a blocking agent. The blocking agent is not particularly limited, and any known blocking agent can be used as long as it does not inhibit the reaction between the antibody-immobilized particles and NT-proBNP. Examples include milk proteins such as skim milk, casein and its salts, and purified milk protein; other proteins such as BSA and gelatin; and water-soluble polymers. One of these or a combination of two or more of these may be used.

[0049] In this case, the blocking method using the blocking agent is not particularly limited, and conventionally known methods or similar methods can be used as appropriate. For example, after producing the antibody-immobilized particles (after binding the insoluble particles to the anti-NT-proBNP antibody), the antibody-immobilized particles are immersed in a blocking solution containing the blocking agent, and then washed. The solvent for the blocking solution is, for example, the buffer solution, and may also contain surfactants, salts (such as NaCl), reducing agents (such as dithiothreitol and 2-mercaptoethanol) as appropriate. The amount of blocking agent used in the blocking method is not particularly limited and can be adjusted as appropriate, but for example, the amount per unit area of ​​the particle surface of the insoluble particles is 1 to 1,000 mg / m². ２ Preferably, it is 1 to 500 mg / m². ２ It is preferable that it be so.

[0050] (Particle Composition) The present invention also provides a particle composition containing at least one of the antibody-immobilized particles selected from the group consisting of first antibody-immobilized particles, second antibody-immobilized particles, and third antibody-immobilized particles. The particle composition of the present invention may contain any one of the above antibody-immobilized particles, but it is preferable to contain the first antibody-immobilized particles and the second antibody-immobilized particles, or the third antibody-immobilized particles, and it is more preferable to contain the first antibody-immobilized particles and the second antibody-immobilized particles.

[0051] The particle composition of the present invention is preferably a dispersion containing an aqueous dispersion medium in addition to the antibody-immobilized particles of the present invention. Examples of the aqueous dispersion medium include buffers such as Good's buffer (HEPES buffer, MOPS buffer, MES buffer, etc.), TRIS buffer, glycine buffer, borate buffer, and PBS buffer. The particle composition of the present invention may also further contain salts (such as NaCl), stabilizers (such as BSA and water-soluble polymers), surfactants, sensitizers (such as PEG and nonionic surfactants that promote aggregation).

[0052] [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 NT-proBNP may be present. Examples include specimens such as body fluids isolated from living organisms (blood (plasma, serum, whole blood), lymph, tissue fluid, body cavity fluid, cerebrospinal fluid, synovial fluid, nasal mucus, etc.), tissues, etc.; cell culture media, etc. Blood is preferred, and plasma or serum is more preferred. These may also be diluted or suspended with a diluent as needed. Examples of the diluent include physiological saline; Good's buffer (HEPES buffer, MOPS buffer, MES buffer, etc.), TRIS buffer, glycine buffer, borate buffer, PBS buffer, etc., and may further contain, for example, salt (NaCl, etc.), stabilizers (BSA, water-soluble polymers, etc.), surfactants, etc.

[0053] [Detection Step] The detection method of the present invention is a method for detecting NT-proBNP in a sample by an agglutination method, more specifically, the detection method of the present invention is an immunological detection method that includes a detection step of contacting the sample with antibody-immobilized particles of the present invention, and if NT-proBNP is present in the sample, detecting NT-proBNP in the sample based on an immune complex (NT-proBNP = (anti-NT-proBNP antibody = insoluble particles)) formed by the antigen-antibody reaction between these, wherein the method is an immunoaggregation method (immunoturbidimetry, immunowax method, etc.) that agglutinates the insoluble particles via the binding of NT-proBNP and anti-NT-proBNP antibody and detects such agglutination.

[0054] Examples of the immunoaggregation methods include slide agglutination, optical measurement, microtiter, and filter separation. Depending on these methods, it is preferable to detect the presence or absence of NT-proBNP in the sample by detecting the aggregation of the insoluble particles, for example, by visual or microscopic detection of aggregation; detection of aggregation based on the magnitude or change in turbidity, scattered light, or absorbance; or detection by mass measurement of the aggregated material.

[0055] The method for bringing the sample into contact with the antibody-immobilized particles is not particularly limited, and any conventionally known method or a similar method can be used as appropriate. For example, one method is to add the sample to a particle composition containing the antibody-immobilized particles.

[0056] In the reaction between NT-proBNP and the antibody-immobilized particles, the content (final concentration) of the antibody-immobilized particles in the reaction system is not particularly limited, as it can be adjusted appropriately depending on the type and concentration of the sample and antibody-immobilized particles. For example, the concentration of antibody-immobilized particles in the reaction system is preferably 0.0001 to 0.1 w / v%, and more preferably 0.001 to 0.01 w / v%. The reaction system is preferably aqueous, and may further contain a diluent of the sample, a dispersion medium for the antibody-immobilized particles, or a reaction buffer (preferably the aqueous solvent or the buffer listed as the aqueous dispersion medium). The reaction conditions are also not particularly limited and can be adjusted as appropriate. For example, the reaction can be carried out at room temperature to 45°C, preferably 30 to 40°C, pH 4 to 10, preferably pH 6 to 9, for about 30 seconds to 30 minutes, preferably 60 seconds to 20 minutes, but is not limited to these conditions.

[0057] [Quantitative Step] In the detection method of the present invention, the measured value of the amount of detected agglutination (for example, the change in absorbance by an optical measurement method) may be used as the detection value, but the method may further include a quantitative step of quantifying the amount of NT-proBNP in the sample based on the measured value of the amount of agglutination. When quantifying the amount of NT-proBNP from the measured value of the detected amount of agglutination, it can generally be done by comparing it with the measured value in a standard sample. In this case, for example, the amount of NT-proBNP in the sample can be determined by finding where the actual measured value is positioned on a standard curve created based on the measured value of a standard sample with a known concentration of NT-proBNP.

[0058] <Diagnostic support method, diagnostic method> NT-proBNP is known as a biomarker for heart failure. Therefore, the present invention also provides a method (hereinafter simply referred to as "the diagnostic support method of the present invention") for supporting the diagnosis of heart failure or related diseases in a subject when using a sample taken from the subject as the sample in the detection method of the present invention described above.

[0059] In the present invention, "diseases related to heart failure" include, for example, myocardial infarction, angina pectoris, cardiomyopathy, arrhythmia, atrial fibrillation, and valvular heart disease.

[0060] Furthermore, in the present invention, "subject" refers to a person from whom the sample for the diagnostic support method and the diagnostic method described below originates, and refers to the person on whom the diagnostic support method and the diagnostic method are performed. The subject is preferably a human. The subject according to the present invention is not particularly limited and may be a healthy person who does not have the disease, or a patient who has the disease. In addition, it may be a patient who is already known to have the disease or a patient who has had the disease in the past, for the purpose of observing the progress of the disease or observing the prognosis of treatment.

[0061] Furthermore, the method for obtaining data (information) regarding the presence or absence of NT-proBNP detection or its amount using the detection method of the present invention can also be used as a method for collecting data (information) regarding the amount of NT-proBNP for the diagnosis of heart failure or related diseases by a physician, a method for presenting such data (information) to a physician, a method for examining the disease, or a method for detecting the disease.

[0062] The diagnostic support method of the present invention preferably further includes a determination step of determining whether the sample is a sample from a subject who has the disease or is highly likely to have the disease, using the presence or absence of detection of NT-proBNP or the amount thereof as an indicator.

[0063] Furthermore, the present invention also provides a method (hereinafter simply referred to as "the diagnostic method of the present invention") which includes a detection step of detecting NT-proBNP from a sample taken from a subject as the sample using the detection method of the present invention described above, and a determination step of determining whether the subject has the disease or is highly likely to have it, using the presence or absence or amount of NT-proBNP detected as an indicator.

[0064] In the diagnostic support method and diagnostic method of the present invention, the detection step for detecting NT-proBNP and the quantitative step for quantifying it as necessary are as described in the detection method of the present invention, including preferred embodiments thereof.

[0065] [Determination Step] In the determination step, the presence or absence of NT-proBNP detection may be directly expressed as "positive / negative," or the amount of NT-proBNP obtained may be compared with a predetermined cutoff value, and if it is equal to or greater than the cutoff value, it may be determined as "positive," and if it is less than or less than the cutoff value, it may be determined as "negative." The "cutoff value" is not fixed in any one way, as it is set appropriately according to the stage and type of disease being determined, each detection method, the subject, etc. For example, a person skilled in the art can set it based on the above detection method and statistical analysis method. Furthermore, the degree to which it is "higher" or "lower" than the cutoff value can also be appropriately determined by a person skilled in the art based on statistical analysis method, depending on the purpose of the determination and the subject, etc. In this invention, "positive" indicates that the person is suffering from the disease being tested for, or has a high probability of suffering from the disease, while "negative" indicates that the person is not suffering from the disease being tested for, or has a low probability of suffering from the disease. However, this can be used not only to distinguish between healthy individuals, but also to distinguish between individuals suffering from other diseases, or to distinguish between different stages of disease progression.

[0066] <Kit> The present invention provides a kit for use in the detection method, diagnostic support method, or diagnostic method of the present invention described above, comprising a first antibody that recognizes an epitope located at positions 12 to 30 of the amino acid sequence of NT-proBNP, and a second antibody that recognizes an epitope located at positions 33 to 55 of the amino acid sequence of NT-proBNP.

[0067] The first antibody and the second antibody, including their preferred embodiments, are as described in the detection method of the present invention described above. The first antibody and the second antibody, individually or together, may be the antibody composition of the present invention, or the antibody-immobilized particles or particle composition of the present invention. Such antibody compositions, antibody-immobilized particles, and particle compositions, including their preferred embodiments, are as described in the detection method of the present invention described above.

[0068] The kit of the present invention may further include, for example, a diluent for the sample, a diluent for the antibody (e.g., the aqueous solvent), a dispersion medium for the antibody-immobilized particles (e.g., the aqueous dispersion medium), standard samples (e.g., NT-proBNP (positive control), negative control), the reaction buffer, quality control samples, a standard of the target antigen, etc. Furthermore, if the first antibody and / or the second antibody are not supported on the insoluble particles, the kit may further include, for example, the insoluble particles, reagents for binding them (e.g., aqueous dispersion medium, activator, blocking agent), various electrolytes, buffers, stabilizers, surfactants, sensitizers, etc. In addition, the kit of the present invention may include instructions for use of the kit.

[0069] The present invention will be described in more detail below based on test examples, but the present invention is not limited to the following test examples. In these test examples, unless otherwise specified, "%" indicating concentration means "weight volume %, weight / volume (w / v) %", i.e., w (g) / v (mL) × 100.

[0070] <Test Example 1> Production of Antibodies Against NT-proBNP (1) Immunotherapy First, a conjugate was prepared by attaching a peptide, in which a spacer and an N-terminal cysteine ​​were introduced to the N-terminus of a peptide fragment consisting of positions 1-19, 12-30, 21-39, 33-55, or 59-72 of the amino acid sequence of NT-proBNP shown in Sequence ID No. 1, to a carrier molecule (maleimide-activated KLH). Specifically, 10 times the amount of the peptide relative to the molar concentration of the carrier molecule was added, and after standing at 25°C for 2 hours, it was purified using a desalting column PD-10 (Cytiva). Next, the prepared conjugate was immunized intraperitoneally into ddY mice (4 weeks old, female) and Balb / c mice (4 weeks old, female), respectively. For the initial immunization, an emulsion of the conjugate and complete Freund's adjuvant (FAC) was prepared and administered at a dose of 50 μg. For subsequent immunizations, an emulsion of the conjugate and incomplete Freund's adjuvant (FIA) was prepared and administered at a dose of 50 μg each time. The interval between immunizations was set at three weeks, and a total of four immunizations were performed.

[0071] (2) Antibody titer measurement A portion of the conjugate prepared in (1) above was dispensed into a 96-well immunoassay plate (manufactured by ThermoFisher) and left to stand overnight at 4°C to prepare an antibody titer confirmation measurement plate with the conjugate immobilized on the phase. In addition, serum was prepared from blood collected from mice two weeks after four immunizations in (1) above, diluted, dispensed into the measurement plate, and left to stand at 25°C for 1 hour to allow the conjugate to capture the serum antibodies. Next, after washing the measurement plate, 100 μL / well of HRP-labeled anti-mouse IgG (200 ng / mL) was added to the measurement plate and left to stand at 25°C for 1 hour. After washing the measurement plate again, a detection reagent (OPD tablet, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the measurement plate, and the absorbance at a wavelength of 450 nm was measured 10 minutes after addition to confirm the formation of a complex between the conjugate and the serum antibodies. Among those in which complex formation with each conjugate was confirmed, mice from which serum with high absorbance was derived were selected as mice with high antibody titers against each peptide fragment corresponding to each conjugate (peptide fragment consisting of positions 1-19, 12-30, 21-39, 33-55, or 59-72), and designated as mouse d, mouse a, mouse e, mouse b, and mouse c, respectively.

[0072] (3) Hybridoma Production The spleen was removed from each mouse individual (a to e) selected in (2) above for having a high antibody titer, and hybridomas were produced by cell fusion of splenic B cells and autologous myeloma cells NS-1 using electroporation. After fusion, hybridoma cell samples with high antibody titers were screened using the limiting dilution method, and the limiting dilution method was repeated until a single clone was obtained.

[0073] (4) Antibody Expression by CHO Cells RNA was extracted from the hybridomas that were single-cloned in (3) above using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from the obtained RNA using the SMARTer RACE 5' / 3' Kit (Takara Bio), and the antibody gene was decoded by sequencing. The nucleotide sequence of the decoded antibody gene was inserted into pcDNA3.4-TOPO vector (ThermoFisher) to construct an expression vector. In addition, ExpiCHO-S cells (ThermoFisher) were subjected to CO2 expression at 37°C. ２ Cells were cultured with shaking at a concentration of 8% and a 125 rpm. The viable cell concentration was 6 × 10⁶. ６ Once the cell count reached cells / mL, the expression vector constructed above was transfected using the ExpiFectamine CHO Transfection Kit (ThermoFisher). 22 hours after transfection, the culture conditions were changed to 32°C and CO2. ２ The concentration was changed to 5% and the rate to 125 rpm. On day 12 after transfection, CHO cell samples with high antibody titers were screened and antibodies were recovered.

[0074] (5) Screening for NT-proBNP In (3) and (4) above, screening of each cell sample with a high antibody titer was performed by the following method. Specifically, 100 μL / well of NT-proBNP (500-2000 ng / mL) was dispensed into a 96-well immunoassay plate (ThermoFisher) and left to stand overnight at 4°C to allow NT-proBNP to solidify. After blocking with BSA, a measurement plate for screening was prepared. In addition, antibodies were purified and diluted from the culture supernatant of each cell sample using a general affinity purification method to prepare antibody samples (200 ng / mL). 100 μL / well of the prepared antibody sample was dispensed into the measurement plate and left to stand for 1 hour at 25°C to allow NT-proBNP to capture the antibodies in the sample. Next, after washing the measurement plate, 100 μL / well of HRP-labeled anti-mouse IgG (200 ng / mL) was added to the measurement plate and left to stand at 25°C for 1 hour. After washing the measurement plate again, a detection reagent (OPD tablet, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the measurement plate, and the absorbance at a wavelength of 450 nm was measured 10 minutes after addition to confirm the formation of a complex between NT-proBNP and the antibody in the sample. Among those in which complex formation was confirmed, cells derived from samples with high absorbance were selected as cell samples with high antibody titers. The monoclonal antibodies derived from CHO cells screened by the above method were designated as "antibody a," "antibody b," "antibody c," "antibody d," and "antibody e," respectively. Here, "a to e" indicate that they are derived from CHO cells obtained in (4) based on the nucleotide sequences of RNA extracted from hybridomas prepared in (3) from mice a to e selected in (2) above.

[0075] (6) Epitope Mapping of Antibodies (6-1) For epitope analysis, 67 peptides located within the range of positions 1 to 76 of the amino acid sequence of NT-proBNP shown in Sequence ID No. 1 were used. Each peptide consists of 10 amino acid residues (antibody c) or 15 amino acid residues (antibody a, b, d, e) with biotin modified at the terminal group. For example, in the case of 15 amino acid residues, the sequence is shifted by one amino acid residue at a time, resulting in amino acid sequences at positions 1 to 15, amino acid sequences at positions 2 to 16, ..., amino acid sequences at positions 61 to 75, and amino acid sequences at positions 62 to 76. These peptides (9.6 M) were dispensed into streptavidin-immobilized 96-well immunoassay plates (manufactured by ThermoFisher), left to stand at 25°C for 1 hour, and then washed to prepare measurement plates for epitope analysis with each peptide immobilized on the plate. Furthermore, antibodies were purified and diluted from the culture supernatant of the CHO cells obtained in (4) above using a general affinity purification method to prepare antibody samples (500 ng / mL). The prepared antibody samples were dispensed at a rate of 100 μL / well into the measurement plate and allowed to stand at 25°C for 1 hour to allow the peptides to capture the antibodies in the samples. Next, the measurement plate was washed, and 100 μL / well of HRP-labeled anti-mouse IgG (200 ng / mL) was added to the plate and allowed to stand at 25°C for 1 hour. After washing the measurement plate again, a detection reagent (OPD tablet, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the absorbance at a wavelength of 450 nm was measured 10 minutes after addition to confirm the formation of complexes between the peptides and the antibodies in the samples. The reactivity of each antibody with each peptide was evaluated according to the following criteria: Judgment "A": Absorbance of 0.200 or higher, indicating high reactivity with the peptide; Judgment "B": Absorbance of 0.100 or higher but less than 0.200, indicating slight reactivity with the peptide; Judgment "C": Absorbance of less than 0.100, indicating virtually no reactivity with the peptide.

[0076] Table 1 below shows the results of the reactivity evaluations of antibody a with the following peptides: biotinylated peptides consisting of the amino acid sequence of positions 1 to 15 of NT-proBNP shown in Sequence ID No. 1 (hereinafter simply referred to as "peptides 1 to 15," the same applies to other peptides), peptides 3 to 17, peptides 5 to 19 (positions 1 to 19); peptides 12 to 26, peptides 14 to 28, peptides 16 to 30 (positions 12 to 30); peptides 21 to 35, peptides 23 to 37, and peptides 25 to 39 (positions 21 to 39). Table 2 below shows the results of the reactivity evaluations of antibody b with the following peptides: peptides 29 to 43, peptides 31 to 45 (positions 29 to 45); peptides 33 to 47, peptides 35 to 49, peptides 37 to 51, peptides 39 to 53, peptides 41 to 55 (positions 33 to 55); peptides 43 to 57, and peptides 45 to 59 (positions 43 to 59).

[0077] Furthermore, the results of the reactivity evaluations of antibody c with peptides 55-64, 57-66 (positions 55-66); 59-68, 61-70, 63-72 (positions 59-72); 65-74, 67-76 (positions 65-76) are shown in Table 3 below. In addition, the results of the reactivity evaluations of antibody d and antibody e with peptides 1-15, 3-17, 5-19 (positions 1-19); 12-26, 14-28, 16-30 (positions 12-30); 21-35, 23-37, 25-39 (positions 21-39) are shown in Table 4 below, along with the results for antibody a.

[0078]

[0079]

[0080]

[0081]

[0082] As shown in Table 1, antibody a was confirmed to be reactive to peptides consisting of the amino acid sequence from positions 12 to 30 of NT-proBNP shown in SEQ ID NO: 1, more specifically peptides 12-26, 14-28, and 16-30, but substantially inactive to peptides 1-15, 3-17, 5-19, 21-35, 23-37, and 25-39. Furthermore, as shown in Table 2, antibody b was confirmed to be reactive to peptides consisting of the amino acid sequence from positions 33 to 55 of NT-proBNP shown in SEQ ID NO: 1, more specifically peptides 33-47, 35-49, 37-51, 39-53, and 41-55, but substantially inactive to peptides 29-43, 31-45, 43-57, and 45-59.

[0083] Furthermore, as shown in Table 3, antibody c was confirmed to be reactive to the peptide consisting of the amino acid sequence from positions 59 to 72 of the NT-proBNP shown in SEQ ID NO: 1, but substantially inactive to the peptide contained in the amino acid sequences from positions 55 to 66 and 65 to 76. Also, as shown in Table 4, antibody d was confirmed to be reactive to the peptide consisting of the amino acid sequence from positions 1 to 19 of the NT-proBNP shown in SEQ ID NO: 1, but substantially inactive to the peptide contained in the amino acid sequence from positions 12 to 39, and antibody e was confirmed to be reactive to the peptide consisting of the amino acid sequence from positions 21 to 39 of the NT-proBNP, but substantially inactive to the peptide contained in the amino acid sequence from positions 1 to 30.

[0084] (6-2) For epitope analysis, six peptides located within the range of positions 1 to 76 of the amino acid sequence of NT-proBNP shown in Sequence ID No. 1 were used. Each peptide is a biotinylated peptide consisting of the amino acid sequence of positions 5 to 19 of NT-proBNP shown in Sequence ID No. 1 (hereinafter simply referred to as "peptide 5-19," the same applies to other peptides), with biotin modified at the terminal group, consisting of the amino acid sequence of positions 12 to 30 (peptide 12-30), the amino acid sequence of positions 21 to 35 (peptide 21-35), the amino acid sequence of positions 31 to 45 (peptide 31-45), the amino acid sequence of positions 33 to 55 (peptide 33-55), and the amino acid sequence of positions 43 to 57 (peptide 43-57). Except for using these peptides, the formation of complexes between each peptide and the antibody in the sample was confirmed and the reactivity between each antibody and each peptide was evaluated, in the same manner as described in (6-1) above.

[0085] The results of the reactivity evaluations between antibody a and peptides 5-19, 12-30, and 21-35 are shown in Table 5 below. The results of the reactivity evaluations between antibody b and peptides 31-45, 33-55, and 43-57 are shown in Table 6 below. Furthermore, the results of the reactivity evaluations between antibody c and peptides 5-19, 12-30, 21-35, 31-45, 33-55, and 43-57 are shown in Table 7 below.

[0086]

[0087]

[0088]

[0089] As shown in Table 5, consistent with the results shown in Table 1, antibody a was confirmed to be reactive to the peptide consisting of the amino acid sequence from positions 12 to 30 of NT-proBNP shown in SEQ ID NO: 1 (peptide 12-30), but substantially inactive to peptides 5-19 and 21-35. Also, as shown in Table 6, consistent with the results shown in Table 2, antibody b was confirmed to be reactive to the peptide consisting of the amino acid sequence from positions 33 to 55 of NT-proBNP shown in SEQ ID NO: 1 (peptide 33-55), but substantially inactive to peptides 31-45 and 43-57. Furthermore, as shown in Table 7, antibody c was confirmed to be substantially inactive to all of peptides 5-19, 12-30, 21-35, 31-45, 33-55, and 43-57.

[0090] (7) Biacore measurement Using a Biacore surface plasmon resonance analyzer, the binding amount (magnitude of affinity) between each antibody and the antigen (NT-proBNP) was measured. As antibodies, each antibody (Antibody a, Antibody b, Antibody c, Antibody d, Antibody e) obtained by purification from the culture supernatant of CHO cells obtained in (4) above by a general affinity purification method was used. As the antigen, serially diluted NT-proBNP peptide (manufactured by eurofins) prepared by Fmoc synthesis at concentrations from 0.625 nM to 10 nM was used. As the sensor chip, Series S sensor Chip CM5 (manufactured by Cytiva) immobilized with anti-mouse-IgG (manufactured by Cytiva) was used. HBS-EP+ Buffer (manufactured by Cytiva) was used for dilution of each antibody and antigen and as the running buffer during measurement. Using Biacore T200 (manufactured by Cytiva), the measurement temperature was set to 25°C and the flow rate was set to 30 μL / min. Each antibody was captured by anti-mouse-IgG on the sensor chip to 300 RU (±5%), then antigen at each concentration was applied for 120 seconds, the binding reaction was observed, and measurement was performed by allowing dissociation for 180 seconds. Biacore T200 Evaluation software (manufactured by Cytiva) was used for analysis, with the reaction model set as 1:1 Binding. From the obtained binding amount between each antibody and the antigen, the association rate constant (ka [1 / Ms]) and dissociation rate constant (kd [1 / s]) were determined, and the dissociation constant (KD [M]) was calculated for each. The results are shown in Table 8 below. In the present specification, the numerical value represented by "A×10+n" has the same meaning as "A×10 ｎ ".

[0091]

[0092] As shown in Table 8, all of Antibodies a to e have a dissociation constant (KD) of 2.0×10 １０ to 6.0×10 １０ [M], and it was confirmed that their affinities for NT-proBNP are equivalent to each other.

[0093] <Test Example 2> Detection of NT-proBNP by Labeled Antibody Method (1) Preparation of Antibody-Immobilized Magnetic Particles 15 mg of magnetic particles (Dynabeads M-280 Tosyactivated, Invitrogen) were washed with 50 mM carbonate buffer (pH 10.0), and 500 μL of 50 mM carbonate buffer (pH 10.0) solution of each antibody prepared in <Test Example 1> above was added and stirred overnight at 37°C to immobilize the antibodies on the surface of the magnetic particles. After immobilization, the supernatant was discarded and the particles were resuspended in 1% BSA PBS solution to block unreacted functional groups. After blocking, the particles were washed three times with PBS and stored in a storage solution (1% BSA, 0.01% NaN ３ The magnetic particles were suspended in PBS (or similar) to a final concentration of 2.0 mg / mL to obtain a particle solution of antibody-immobilized magnetic particles.

[0094] (2) For the detection of NT-proBNP by bioluminescence (BLEIA method), NT-proBNP peptide (Eurofins) prepared by the Fmoc synthesis method was serially diluted in a 1% BSA-containing PBS solution (standard substance solution). For the luciferase-labeled antibody solution, a 1% BSA-containing PBS solution of luciferase-labeled antibody prepared using each antibody prepared in <Test Example 1> above and recombinant luciferase (Promega) was used. To detect NT-proBNP in the sample, first, as a first reaction, reaction buffer (1% BSA-containing PBS, 100 μL) and the sample (10 μL) were mixed, and then the antibody-immobilized magnetic particle solution (50 μL) prepared in (1) above was added and stirred at 37°C for 30 minutes. After magnetic collection and washing, as a second reaction, 100 μL of the luciferase-labeled antibody solution (200 ng / mL) was added and stirred at 37°C for 30 minutes to form a sandwich complex of antibody (antibody-immobilized magnetic particles) - antigen in the sample (NT-proBNP) - antibody (luciferase-labeled antibody). After washing again, 100 μL of luciferin solution (Promega) was added as a luminescent substrate and the luminescence (luminous value) derived from the complex was measured.

[0095] Figure 2 shows a graph illustrating the relationship between NT-proBNP peptide concentration [pg / mL] and luminescence [luminous value] when antibody b is used as the antibody-immobilized magnetic particle and antibody a is used as the luciferase-labeled antibody (antibody a-antibody b: reference example), and when antibody c is used as the magnetic particle-immobilized antibody and antibody a is used as the luciferase-labeled antibody (antibody a-antibody c: reference example).

[0096] <Test Example 3> Detection of NT-proBNP by Agglutination Method (1) Preparation of Antibody-Immobilized Latex Particles (Physical Adsorption) First, each antibody prepared in <Test Example 1> above is added to 10 mM HEPES buffer (pH 7.4) at a concentration of 10 mg / m³ ２ (mg / m ２ : Unit surface area (m²) of latex particles ２ The solution was diluted to the desired antibody mass (mg) per liter (the same applies below). An appropriate amount of latex particles (average particle size: 0.36 μm, polystyrene latex) was added and stirred and mixed. Then, 25 mg / m³ was added as a blocking agent. ２ The required amount of BSA was added, and the mixture was allowed to stand at 50°C for 3 hours to block the particle surface. Next, the particles were collected by centrifugation, washed with 10 mM HEPES buffer (pH 7.4), collected again by centrifugation, and suspended in 50 mM HEPES buffer (pH 7.4) to obtain a particle solution of antibody-immobilized latex particles in which each antibody was physically adsorbed onto the surface of the latex particles.

[0097] (2) For the detection of NT-proBNP by latex immunoturbidimetry, human serum samples containing NT-proBNP (samples 1-2) were serially diluted with a PBS solution containing 1% BSA. For the standard substance solution, NT-proBNP peptide (Eurofins) prepared by the Fmoc synthesis method was serially diluted with a PBS solution containing 1% BSA. First, a reagent containing 50 mM HEPES (pH 7.4), 150 mM NaCl, and 1% water-soluble polymer was prepared as the first reagent. A reagent containing 10 mM HEPES (pH 7.4), 0.03% BSA, and 0.025% antibody-immobilized latex particles (prepared in (1) above) was prepared as the second reagent. Next, 120 μL of the first reagent was mixed with 8 μL of the sample or standard substance solution and incubated at 37°C for 5 minutes. Then, 40 μL of the second reagent was mixed into this mixture and reacted at 37°C. The change in absorbance (ΔOD) at 660 nm was measured approximately 5 minutes after mixing with the second reagent. The series of measurements were performed using a Hitachi 7180 automatic analyzer (Hitachi High-Tech Corporation). A calibration curve was created from the change in absorbance measured using the standard substance concentration, and the NT-proBNP concentration [pg / mL] in each sample was calculated.

[0098] Figures 3 and 4 show graphs illustrating the relationship between the dilution ratio of each sample and the calculated NT-proBNP peptide concentration [pg / mL] when antibody-immobilized latex particles carrying antibody a and antibody-immobilized latex particles carrying antibody b are used in a 1:1 mass ratio for sample 1 and sample 2, respectively. As shown in Figures 3 and 4, good dilution linearity was obtained for both sample 1 and sample 2 across the entire range of NT-proBNP concentrations, from low to high.

[0099] (3) NT-proBNP was detected by latex immunoturbidimetry in the same manner as in (2) of this <Test Example 3>, except that NT-proBNP peptide (manufactured by Eurofins) prepared by the Fmoc synthesis method was serially diluted in a 1% BSA-containing PBS solution (standard substance solution) as a comparative sample between the labeled antibody method and the agglutination method. As antibody-immobilized latex particles, a combination of antibody-immobilized latex particles carrying antibody a and antibody-immobilized latex particles carrying antibody b (mass ratio 1:1, antibody a-antibody b: example) or a combination of antibody-immobilized latex particles carrying antibody a and antibody-immobilized latex particles carrying antibody c (mass ratio 1:1, antibody a-antibody c: comparative example) was used. Figure 5 shows graphs illustrating the relationship between NT-proBNP peptide concentration [pg / mL] and the change in absorbance [ΔOD × 10,000] for antibody a-antibody b (example) and antibody a-antibody c (comparative example).

[0100] As shown in Figure 2 above, in the labeled antibody method (BLEIA method), combinations of two antibodies with separated recognition regions, i.e., combinations of antibodies where the N-terminal region of one antibody that is reactive and the C-terminal region of the other antibody that is reactive are separated (antibody a-antibody c), resulted in greater luminescence and higher detection sensitivity compared to combinations of two antibodies with closer recognition regions (antibody a-antibody b). On the other hand, as shown in Figure 5, in the agglutination method (latex immunoturbidimetric method), surprisingly, the opposite was true: combinations of two antibodies with closer recognition regions, i.e., combinations of antibody a and antibody b (antibody a-antibody b), resulted in a greater change in absorbance and superior detection sensitivity compared to combinations of two antibodies with separated recognition regions, i.e., combinations of antibody a and antibody c (antibody a-antibody c).

[0101] (4) NT-proBNP was detected by latex immunoturbidimetry in the same manner as in (2) of this <Test Example 3>, except that human serum samples containing NT-proBNP (samples 1 to 6) were diluted in a 1% BSA-containing PBS solution as comparative samples for detection sensitivity based on the recognition region of the antibody. The antibody-immobilized latex particles used were a combination of antibody-immobilized latex particles carrying antibody a and antibody-immobilized latex particles carrying antibody b (mass ratio 1:1, antibody a-antibody b: example), a combination of antibody-immobilized latex particles carrying antibody e and antibody-immobilized latex particles carrying antibody b (mass ratio 1:1, antibody e-antibody b: comparative example), or a combination of antibody-immobilized latex particles carrying antibody d and antibody-immobilized latex particles carrying antibody b (mass ratio 1:1, antibody d-antibody b: comparative example).

[0102] Tables 9 to 10 below show the absorbance change [ΔOD × 10,000] for each sample in antibody a-antibody b (Example), antibody e-antibody b (Comparative Example), and antibody d-antibody b (Comparative Example). Table 9 also shows the ratio of the absorbance change in antibody a-antibody b to the absorbance change in antibody e-antibody b (antibody a-antibody b / antibody e-antibody b [%]), and Table 10 shows the ratio of the absorbance change in antibody a-antibody b to the absorbance change in antibody d-antibody b (antibody a-antibody b / antibody d-antibody b [%]).

[0103]

[0104]

[0105] As shown in Tables 9 to 10, combining antibody a and antibody b resulted in a significantly larger change in absorbance, demonstrating particularly excellent detection sensitivity. The change in absorbance for the combination of antibody a and antibody b (antibody a-antibody b) was more than twice as large in all samples, even though the peptides to which these antibodies react partially overlapped with the peptides to which antibody a reacts, compared to cases where antibody a was replaced with antibody e, whose reactive region is closer to the C-terminus (antibody e-antibody b), or where antibody d, whose reactive region is closer to the N-terminus (antibody d-antibody b).

[0106] <Test Example 4> Examination of binding mode in antibody-immobilized particles (1) Preparation of antibody-immobilized latex particles by covalent bond First, a corresponding amount of latex particles (average particle size: 0.36 μm, polystyrene latex having carboxyl groups on the surface) was added to a DMT-MM solution so that the amount of DMT-MM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride) was 10 molar equivalents relative to the functional group (carboxyl group) of the latex particles, and the mixture was stirred and mixed. To this, antibody a or antibody b prepared in <Test Example 1> above was added at a concentration of 3 mg / m³ relative to the latex particles in 10 mM HEPES (pH 7.0). ２ A diluted solution was added to achieve the following result. Next, 25 mg / m² was added as a blocking agent. ２ The amount of BSA was added and allowed to stand at 50°C for 3 hours to block the particle surface. Next, the particles were recovered by centrifugation, washed with 10 mM HEPES (pH 7.4), and recovered again by centrifugation. Then, an additional 20 mg / m³ of BSA was added as a blocking agent. ２ The required amount of BSA was added, and the mixture was allowed to stand at 50°C for 3 hours to block the particle surface, thereby obtaining particle solutions of antibody-immobilized latex particles (antibody a(+), antibody b(+)) in which antibody a or antibody b was covalently bonded to the latex particle surface.

[0107] As a control, particle solutions of antibody-immobilized latex particles (antibody a(-), antibody b(-)) were obtained by physically adsorbing antibody a or antibody b onto the surface of the latex particles in the same manner as above, except that DMT-MM was not used.

[0108] (2) NT-proBNP was detected by latex immunoturbidimetry in the same manner as in (2) of <Test Example 3> above, except that NT-proBNP peptide (manufactured by Eurofins) prepared by the Fmoc synthesis method was serially diluted in a 1% BSA-containing PBS solution (standard substance solution) as the detection sample for NT-proBNP by agglutination method. The antibody-immobilized latex particles used were the combinations of antibody a(+) and antibody b(+) (antibody a(+) × antibody b(+)), antibody a(-) and antibody b(+) (antibody a(-) × antibody b(+)), and antibody a(-) and antibody b(-) (antibody a(-) × antibody b(-)) prepared in (1) above. Figure 6 shows a graph illustrating the relationship between the NT-proBNP peptide concentration [pg / mL] and the change in absorbance [ΔOD × 10,000] for each combination.

[0109] As shown in Figure 6, in combinations of antibody-immobilized latex particles carrying antibody a and antibody-immobilized latex particles carrying antibody b, high detection sensitivity was obtained in both cases: when the latex particles were covalently bonded to each antibody and when they were physically adsorbed. However, comparing the two, it was confirmed that even better detection sensitivity was achieved when the latex particles were covalently bonded to at least one of the antibodies, and more preferably to both antibodies.

[0110] As described above, the present invention makes it possible to provide an NT-proBNP detection method that can detect the N-terminal precursor fragment of type B natriuretic peptide (NT-proBNP) with high sensitivity, as well as an antibody composition, antibody-immobilized particles, and a kit for use therein.

Claims

1. A method for detecting the N-terminal fragment of a type B natriuretic peptide precursor (NT-proBNP) in a sample, comprising the step of contacting the sample with antibody-immobilized particles, wherein the antibody-immobilized particles comprise insoluble particles and anti-NT-proBNP antibodies supported on the insoluble particles, and the insoluble particles are at least one selected from the group consisting of latex particles and metal or other colloidal particles, and the anti-NT-proBNP antibody is a combination of a first antibody that recognizes an epitope located at positions 12 to 30 of the amino acid sequence of NT-proBNP and a second antibody that recognizes an epitope located at positions 33 to 55 of the amino acid sequence of NT-proBNP, wherein the first antibody and the second antibody may be supported on the same insoluble particle or on separate insoluble particles.

2. The NT-proBNP detection method according to claim 1, wherein the first antibody is reactive to a peptide containing positions 12 to 30 of the amino acid sequence of NT-proBNP, and substantially inactive to a peptide consisting of positions 5 to 19 of the amino acid sequence of NT-proBNP and a peptide consisting of positions 21 to 35 of the amino acid sequence of NT-proBNP.

3. The NT-proBNP detection method according to claim 1, wherein the second antibody is reactive to a peptide containing positions 33 to 55 of the amino acid sequence of NT-proBNP, and substantially inactive to a peptide consisting of positions 31 to 45 of the amino acid sequence of NT-proBNP and a peptide consisting of positions 43 to 57 of the amino acid sequence of NT-proBNP.

4. The NT-proBNP detection method according to claim 1, wherein at least one of the bindings between the insoluble particles and the first antibody, and the binding between the insoluble particles and the second antibody, is a covalent bond.

5. An antibody composition for use in the NT-proBNP detection method according to any one of claims 1 to 4, comprising a first antibody that recognizes an epitope located at positions 12 to 30 of the amino acid sequence of NT-proBNP.

6. The antibody composition according to claim 5, wherein the first antibody is reactive to a peptide comprising positions 12 to 30 of the amino acid sequence of NT-proBNP, and substantially inactive to a peptide comprising positions 5 to 19 of the amino acid sequence of NT-proBNP and a peptide comprising positions 21 to 35 of the amino acid sequence of NT-proBNP.

7. Antibody-immobilized particles for use in the NT-proBNP detection method according to any one of claims 1 to 4, comprising insoluble particles and an anti-NT-proBNP antibody supported on the insoluble particles, wherein the insoluble particles are at least one selected from the group consisting of latex particles and metal or other colloidal particles, and the anti-NT-proBNP antibody is a combination of a first antibody that recognizes an epitope located at positions 12 to 30 of the amino acid sequence of NT-proBNP and a second antibody that recognizes an epitope located at positions 33 to 55 of the amino acid sequence of NT-proBNP, wherein the first antibody and the second antibody may be supported on the same insoluble particle or on separate insoluble particles.

8. The antibody-immobilized particle according to claim 7, wherein the first antibody is reactive to a peptide comprising positions 12 to 30 of the amino acid sequence of NT-proBNP, and substantially inactive to a peptide comprising positions 5 to 19 of the amino acid sequence of NT-proBNP and a peptide comprising positions 21 to 35 of the amino acid sequence of NT-proBNP.

9. The antibody-immobilized particle according to claim 7, wherein the second antibody is reactive to a peptide comprising positions 33 to 55 of the amino acid sequence of NT-proBNP, and substantially inactive to a peptide comprising positions 31 to 45 of the amino acid sequence of NT-proBNP and a peptide comprising positions 43 to 57 of the amino acid sequence of NT-proBNP.

10. The antibody-immobilized particle according to claim 7, wherein at least one of the bindings between the insoluble particle and the first antibody, and the binding between the insoluble particle and the second antibody, is a covalent bond.

11. A kit for use in the NT-proBNP detection method according to any one of claims 1 to 4, comprising a first antibody that recognizes an epitope located at positions 12 to 30 of the amino acid sequence of NT-proBNP, and a second antibody that recognizes an epitope located at positions 33 to 55 of the amino acid sequence of NT-proBNP.

12. The kit according to claim 11, wherein the first antibody is reactive to a peptide comprising positions 12-30 of the amino acid sequence of NT-proBNP, and substantially inactive to a peptide comprising positions 5-19 of the amino acid sequence of NT-proBNP and a peptide comprising positions 21-35 of the amino acid sequence of NT-proBNP.

13. The kit according to claim 11, wherein the second antibody is reactive to a peptide comprising positions 33 to 55 of the amino acid sequence of NT-proBNP, and substantially inactive to a peptide comprising positions 31 to 45 of the amino acid sequence of NT-proBNP and a peptide comprising positions 43 to 57 of the amino acid sequence of NT-proBNP.

14. The kit according to claim 11, further comprising insoluble particles, wherein the insoluble particles are at least one selected from the group consisting of latex particles and metal colloidal particles.

15. The kit according to claim 14, wherein the first antibody, the second antibody, and the insoluble particles are antibody-immobilized particles in which the first antibody and the second antibody are supported on the same or separate insoluble particles.

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